海藻糖芭樂汁對第二型尿病大鼠之腎臟及胰的保護效應

全文

(1)國立臺灣師範大學生命科學系碩士論文. 海藻糖芭樂汁對第二型糖尿病大鼠之腎臟及胰臟的保護效應 Protective Effects of Guava (Psidium guajava) Juice Combination with Trehalose in Kidney and Pancreas in T2DM rats. 研究生: 郭彥廷 Yen-Ting, Kuo 指導教授: 鄭劍廷教授 Chiang-Ting, Chien 中華民國 104 年 7 月.

(2) 誌謝轉眼之間，碩士生涯的兩年即將畫下句點。這不長不短的歲月，猶如一趟旅程，旅程中遇見許多幫助我達到終點的師長、朋友及親人。首先，最感謝的，是一路上指導我，為我指點迷津的鄭老師。大四那年，感謝老師收我進實驗室學習。考進碩士班後，老師即給予我自己一份研究計畫，指導我，從實驗計畫的設計，到實驗進行時的技巧、邏輯概念等等。直到今天，終於完成了碩士論文。很感謝老師，使我們在研究的路上能學習多元的實驗項目以及不匱乏經費的實驗環境，也感謝老師能讓我們發揮自己的實驗構想。一路上，老師都是那麼地有耐心，細心的引領著我們。忙碌的老師，即使揹著系主任的重擔，也仍然心繫實驗室大家的實驗進度，只要一有空就會關心學生們。真的非常幸運能在碩士生涯遇到這樣一位好老師。感謝我的口試委員們: 義守大學職能治療學系李秉家老師和台大醫學院內科徐世平老師，能在百忙中來參加我的口試，並且給予我論文編寫的指導，以及實驗上的建議。除了鄭老師，碩士的兩年，身邊有許多學長姊、學弟妹的幫忙。謝謝黃俊超學長一直以來對我的照顧，除了教我分生實驗，也常聽我 I.

(3) 發牢騷、幫我一起思考實驗上所遇到的困難。感謝李世斌學長時常給予實驗技巧的建議及指導。謝謝林煒哲學長，教導我螢光染色及給予我許多實驗上的建議。謝謝宋伊婷學妹，這位平時一起行動的好夥伴，一起覓食、一起在實驗後談心，一路上有你的相伴，真的非常感謝。謝謝孫鈴媗同學，當初陪我一起進入鄭老師的實驗室，一起闖關打怪。謝謝台大共儀中心林佩吟小姐，幫助我進行 HPLC 的實驗。另外，也謝謝碩士生涯中，一起度過兩年的同儕們。感謝葉雅欣、余禮儒及黃珮甄同學，雖然各自有各自的研究領域，在這兩年，彼此之間不吝嗇的互相打氣及給予研究建議都是非常溫暖且珍貴的。最後，還要謝謝一直以來給予我精神及經濟支持家人、在我身旁給我鼓勵及安慰的先傑以及 B308 研究室的全體夥伴。感謝你們陪我完成人生最後的學生生涯。這趟旅程，因為有你們大家才更豐富，我才能走到終點。. 郭彥廷. 謹誌於. 國立臺灣師範大學生命科學系氧化壓力生理實驗室 II.

(4) 中華民國 104 年 7 月. 中文摘要第二型糖尿病(Type 2 diabetes, T2DM)是目前廣泛流行的代謝症候群。研究預測第二型糖尿病的人口數，在西元 2030 年時會攀升至 3.66 億。芭樂 (Psidium guajava)，被發現具有抗氧化(anti-oxidation)、抗發炎(anti-inflammation)及抗糖尿病(anti-diabetes)的特性。並且也被發現在第一型糖尿病的大鼠上，可以保護胰臟的 β 細胞，免於受到氧化壓力損害。然而，很少研究是利用芭樂的可食部位探討其對第二型糖尿病之間的作用機制。所以我們利用芭樂可食部位製成芭樂汁 (40%)，以研究此主題。為了提升芭樂汁的適口性，我們在果汁當中添加了海藻糖(Trehalose)。海藻糖是一種雙醣，目前已被應用為細胞冷凍保存的工具。先前的研究指出它可以防止阿茲海默症中，類澱粉蛋白的累積。以及亨丁頓氏舞蹈症中，多聚谷氨酰胺的形成而產生抗氧化之作用。本研究之主要目的在評估芭樂汁(40%)添加海藻糖(5%) 對於第二型糖尿病大鼠在腎臟與胰臟的保護作用效應。利用 Nicotinamide (NA)及 Streptozotocin (STZ)腹腔注射以誘發 Wistar 品系雌性大鼠第二型糖尿病。誘發成功後，分為六組進行實驗，分別為 CON, DM, T1, T2, T5 和 B1。每日灌餵芭樂汁，連續四周。灌餵之劑量如下: T1, T2, T5: 4, 8, 20 ml/kg BW 芭樂汁含 5%海藻糖。 B1: III.

(5) 4 ml/kg BW 芭樂汁不含 5% 海藻糖。紀錄葡萄糖耐受性試驗(OGTT)、血清胰島素、糖化血色素與換算之胰島素阻抗和分泌量。並測量腎臟活體自由基，而後進行犧牲，收取組織進行免疫組織染色、螢光染色、西方墨點法及離體血清自由基測試。我們亦以 LC/MS 的方式定量芭樂有效成份。結果顯示，芭樂汁中含有高量的槲皮素，且槲皮素與芭樂汁可清除 H2O2 and HOCl。而本研究也發現，海藻糖可清除 H2O2，但無法清除 HOCl。第二型糖尿病(DM 組)會增加大鼠之氧化壓力及發炎反應。相較之下，T1, T2, T5 組在灌餵海藻糖芭樂汁之後，表現出較低程度的氧化壓力及發炎指標，如 IL-1β, Caspase 3 及 4-HNE。第二型糖尿病(DM 組)增加胰島素阻抗和降低胰島素分泌量。相較之下，T1, T2, T5 組在灌餵海藻糖芭樂汁之後，會降低第二型糖尿病(DM 組)所增加之腎臟和胰臟氧化壓力及發炎反應包括降低 IL-1β, Caspase 3 及 4-HNE 之表現。而且會降低胰島素阻抗和部份增加胰島素分泌量。此外，我們發現在離體血清自由基測試中，B1 組的血清自由基較 T1, T2, T5 組高，其中 T2 及 T5 組統計達顯著差異(P < 0.05)。免疫組織染色及螢光染色結果也有相同趨勢。這結果表示海藻糖芭樂汁對於腎臟及胰臟的保護功效，較單獨芭樂汁佳。總結，海藻糖可以提升芭樂汁在第二型糖尿病中的保護功效。將 IV.

(6) 兩者合併攝取，可降低腎臟及胰臟的氧化壓力及發炎反應。關鍵字: 芭樂、海藻糖、第二型糖尿病、氧化壓力、發炎. V.

(7) Abstract Type II diabetes is one of the most epidemic metabolic syndrome. It was predicted that people with T2DM would rise to 366 million in 2030. Guava (Psidium guajava) has been reported to provide anti-oxidation, anti-inflammation, and anti-diabetes. Besides, it protected β cells from the damage of oxidative stress in type I DM rats. However, there were few studies which report the mechanism between the edible proportion of guava and the T2DM. Therefore, we utilized guava juice to investigate its effect on T2DM. To improve the palatability, we added one kind of sugar, trehalose. Trehalose is a disaccharide, which has been used in cryopreservation of cells. In addition, it was found to avoid β-amyloid formation and polyglutamine (polyQ) in Alzheimer disease and Huntington’s disease indicating its protective function. This study is to discover the protective mechanism of guava juice (40%) combination with trehalose (5%) on the pathophysiology of kidney and pancreas in T2DM rats. T2DM was induced in female Wistar rats by intraperitoneal administration of nicotinamide and streptozotocin and combination with high fructose diets for 8 weeks. After successful induction (> 230 mg/dL), the rats were divided into 6 groups, CON, DM, T1, T2, T5, B1, and were fed with different dosage of guava juice combination with or without trehalose for 4 weeks (Dose: T1, T2, T5: 4, 8, 20 ml/kg BW guava juice with 5% trehalose; B1: 4 ml/kg BW guava juice without trehalose).OGTT, plasma insulin, HbA1c, Homeostasis model assessment of IR (HOMA-IR, an indicator of insulin resistance) and HOMA-β (an index of the function of β cell in VI.

(8) pancreas and insulin secretion) were determined. We also measured the kidney reactive oxygen species (ROS) in vivo. The oxidative and inflammatory degrees were measured by immunohistochemistry stain, fluorescent stain, serum free radical value and western blotting. We also measured the active component of guava juice with LC/MS analysis. We found high content of quercetin existing in the guava juice. Quercetin and guava juice could scavenge H2O2 and HOCl, whereas trehalose can selectively reduce H2O2, not HOCl in the in vitro study. The results showed that the rats in group DM had elevated the degree of oxidative stress and inflammatory levels. In contrast, rats treated with oral intake of trehalose and guava juice in group T1, T2, T5 showed less expression of oxidative and inflammatory indicators, such as IL-1β, Caspase 3 and 4-HNE compared to DM group. Consistently, in the measurement of serum free radical levels, we found that rats in T1, T2 and T5 have significantly (P < 0.05) lower free radicals counts than B1 and DM groups. The results of immunohisotchemic and fluorescent stain also showed that oral intake of guava juice with trehalose in T1, T2, T5 rats had less (P < 0.05) oxidative damage, autophagy and apoptosis in the kidney and pancreas than B1 rats. In conclusion, trehalose supplement seems to provide the additively protective effect of guava juice in T2DM. Combination with trehalose and guava juice not only increases palatability, but also protects pancreas and kidney against oxidative and proinflammatory damages in T2DM. Key words: Guava, Trehalose, Type II diabetes, Oxidative stress, Inflammation. VII.

(9) 目錄誌謝............................................................................................................. I 中文摘要...................................................................................................III Abstract .................................................................................................... VI Abbreviations ..............................................................................................1 1. Introduction ..........................................................................................3 1-1 Type 2 diabetes (T2DM)............................................................3 1-2 Insulin insensitivity....................................................................3 1-3 Diabetic nephropathy .................................................................4 1-4 Reactive oxygen species (ROS) ................................................4 1-5 Guava (Psidium guajava) ..........................................................5 1-6 Trehalose ....................................................................................6 1-7 Goal............................................................................................7 2. Methods and materials .........................................................................8 2-1 Animals ......................................................................................8 2-2 Induction of T2DM ....................................................................8 2-3 Animal Treatment ......................................................................9 2-4 Determine Blood Glucose .......................................................10 2-5 Determine Insulin Levels.........................................................10 2-6 HOMA-IR and HOMA-β ........................................................ 11 2-7 Glucose Tolerance Test ............................................................ 11 2-8 Determine HbA1c ....................................................................12 2-9 Metabolic Parameter ................................................................12 2-10 Assay of ROS Level .............................................................13 2-11 Immunohistochemistry (IHC) ..............................................14 2-12 Masson’s Stain .....................................................................15 2-13 TUNEL Stain ........................................................................16 2-14 Fluorescent Stain ..................................................................18 2-15 Western Blot .........................................................................19 2-16 Guava Extraction ..................................................................20 2-17 LC/MS ..................................................................................21 3. Results ................................................................................................22 3-1 Guava Juice in vitro ROS Levels ............................................22 3-2 Trehalose in vitro ROS Levels.................................................22 3-3 Quercetin Content In Guava Extraction ..................................22 VIII.

(10) 3-4 Guava Extraction in vitro ROS Levels ....................................23 3-5 Oral Guava Juice Tolerance Test .............................................23 3-6 Intra Venous Glucose and Trehalose Tolerance Test ...............23 3-7 Oral Glucose Tolerance Test ....................................................24 3-8 Blood Glucose Changes...........................................................24 3-9 Insulin Levels...........................................................................25 3-10 HOMA-IR ............................................................................26 3-11 HOMA-β ..............................................................................26 3-12 HbA1c Levels.......................................................................26 3-13 Metabolic Parameter ............................................................27 3-14 Renal in vivo ROS Levels ....................................................27 3-15 Serum in vitro ROS Levels ..................................................27 3-16 HE Stain ...............................................................................28 3-17 Masson’s Stain .....................................................................28 3-18 Fluorescent Stain ..................................................................29 3-19 IHC Stain ..............................................................................30 3-20 TUNEL Stain........................................................................31 3-21 Western Blot .........................................................................32 4. Discussion ..........................................................................................33 5. References ..........................................................................................39 6. Figures and Tables .............................................................................44. IX.

(11) Abbreviations 4-HNE. 4-Hydroxynonenal. AGE. Advanced Glycation End Products. AUC. Area Under Curve. BW. Body Weight. CL. Chemiluminescence. CKD. Chronic Kidney Disease. FBG. Fasted Blood Glucose. GEE1. Guava juice ethanol extraction of Thailand guava. GEE2. Guava juice ethanol extraction of pearl guava. GWE1. Guava juice water extraction of Thailand guava. GWE2. Guava juice water extraction of pearl guava. HbA1c. Glycated Hemoglobin A1c. HE Stain. Hematoxylin And Eosin Stain. HOMA. Homeostatic Model Assessment. HRP. Horseradish Peroxidase. IHC. Immunohistochemistry. IL-1β. Interleukin-1β. IL-6. Interleukin-6. IVGTT. Intra Venous Glucose Tolerance Test. i.p.. Intraperitoneal. i.v.. Intravenous. MCLA. 2-Methyl-6-(4-Methoxyphenyl)-3,7-Dihydroimid 1.

(12) azo-(1,2-A)-Pyrazin-3-One Hydrochloride NF-κB. Nuclear Factor Kappa-B. NOX. NADPH Oxidase. OGTT. Oral Glucose Tolerance Test. PKC. Protein Kinase C. PMT. Photomultiplier. PVDF. Polyvinylidene Fluoride. RAGE. Receptor For AGE. ROS. Reactive Oxygen Species. STZ. Streptozotocin. T2DM. Type 2 Diabetes. 2.

(13) 1. Introduction 1-1. Type 2 diabetes (T2DM) T2DM is a kind of metabolic disorders, which has become a major problem both in developed and developing countries (Attila Hunyadi et al., 2012). Although there have been so many researches of T2DM, the number of people with diabetes mellitus is still increasing all over the world (Tomonori Nakamura et al., 2006). According to the research of Sarah Wild et al. (2004), people with T2DM would probably rise to 366 million in 2030. In addition, in the area of Asia, the increasing rate of people with T2DM shows no sign of slowing (Kun-Ho Yoon et al., 2006). Based on the investigation of National Health Research Institutes of Taiwan in 2007, the cost of diabetes-related medical expenses ranked fifth. The prevalence was rising as well. Among all aged groups, male over 65-year-old had the highest prevalence (27.7%). Therefore, the importance of prevention and therapy of T2DM still can’t be ignored.. 1-2. Insulin insensitivity Insulin insensitivity in T2DM is related to pancreas function decline over time. In normal situation, insulin is the major hormone that regulates blood glucose. β cells in pancreas adjust insulin action at different situations. For example, insulin action decreases while insulin secretion increases (Michael Stumvoll, et al.2005). Insulin resistance is one of the features of T2DM, happening at the early or intermediate stage. It happens when diabetic genes, adipokines, 3.

(14) inflammation, hyperglycemia, free fatty acids and other factors make β cells dysfunction. These factors make insulin works less than expected. Then, glucose uptake from muscle decreases, and glucose production from liver increases, and fatty acids generated from lipolysis increased. As a result, blood glucose and fatty acids in circulation are raised. Conversely, insulin resistance and secretion get worsen (Michael Stumvoll, et al.2005). 1-3. Diabetic nephropathy In addition of the damage of hyperglycemia, a lot of complications occur in diabetic patients, including of atherosclerosis, diabetic retinopathy, and diabetic nephropathy (Tomoko Kakehi and Chihiro Yabe-Nishimura, 2008). Among these complications, chronic kidney disorders (CKD) is a major complication. Additionally, diabetes had become the most common cause of end stage renal failure (Jay C. Jha et al., 2014). Diabetic nephropathy is characterized by an abnormal level (30 mg/day or 20 g/min) of albumin in urine, referred to as microalbuminuria (Molitch ME et al., 2004). Furthermore, it leads to proteinuria and end-stage renal failure. Chronic hyperglycemia is the most important factor of renal hypertrophy, glomerulosclerosis, and tubulointerstitial fibrosis, which are the common morphologies demonstrated in end-stage renal failure (Tomoko Kakehi and Chihiro Yabe-Nishimura, 2008).. 1-4. Reactive oxygen species (ROS) Increasing ROS which augments oxidative stress in the tissue, are generated by hyperglycemia (Tomoko Kakehi and Chihiro 4.

(15) Yabe-Nishimura, 2008). Oxidative stress has been considered to be the major factor to diabetic nephropathy. Both intracellular and extracellular hyperglycemia leads to ROS generation, and causes tissue damage. High glucose level in extracellular increases the formation of advanced glycation end products (AGE), and it furthermore interacts with the receptor for AGE (RAGE). Similarly, intracellularly, increased glucose level promotes the activity of protein kinase C (PKC) and NADPH oxidase (NOX). These pathways elevate ROS generation and expression of some transcription factors, such as nuclear factor kappa B (NF-κB) (Nigel A. Calcutt et al., 2009).. 1-5. Guava (Psidium guajava) Guava is a kind of tropical fruits, popular in Asia, including Taiwan (Chia-Yun Lin and Mei-Chin Yin, 2012). It has been reported that 5.

(16) guava has properties of anti-oxidative, anti-inflammantory and anti-diabetic effect (Thomas Eidenberger et al., 2013), due to abundant of vitamin C , flavonoids and polyphenolic compounds (Gema Flores et al., 2013). These properties have made guava obtain a lot of attention. In a previous study, guava extractions can decrease ROS, interlukin-6 (IL-6), tumor necrosis factor-α, and interlukin-1β (IL-1β) levels in kidney of type 1 diabetic mice (Chia-Yun Lin and Mei-Chin Yin, 2012). However, T2DM is responsible for over 90% of overall diabetic cases. There were researches involved the interactions between gauva leaf extraction and T2DM (Thomas Eidenberger et al., 2013), but few of which reported the relationship between the edible proportion of guava and the T2DM. 1-6. Trehalose Besides guava, we are also very interested in a kind of disaccharide, trehalose. This disaccharide is non-reducing and links two glucose unit in an a,a-1,1-glycosidic linkage (Qiaofang Chen and Gabriel G. Haddad, 2004). Trehalose is a naturally occurring disaccharide which presents in a wide variety of organisms, including plants, bacteria, yeast, and invertebrates (Cheng Xu et al.,2013). This sugar plays a role of protection in these organisms. It helps cell against environment stress, such as heat, cold, desiccation, dehydration, and oxidation by preventing protein denaturation (Sovan Sarkar et al., 2007). Because of its unique property, trehalose has been used in 6.

(17) cryopreserved mammalian cells, Huntinton’s disease (Liu, R et al., 2005), and Alzheimer’s disease (Sovan Sarkar et al., 2007). For example, the study showed that trehalose can greatly improve the survival of mammalian cells during cryopreservation (Ali Eroglu et al., 2000). 1-7. Goal The goal was to explore the effects and mechanisms of guava juice combined with trehalose on the pathophysiology of kidney and pancreas in rats induced T2DM.. 7.

(18) 2. Methods and materials 2-1. Animals The experiments were performed with 7-week old female Wistar rats. These rats were purchased from BioLASCO Taiwan Co. Ltd. (Taipei) and were housed at the Experimental Animal Center, National Taiwan Normal University, at a constant temperature and with a consistent light cycle (light from 07:00 to 18:00 O'clock). Food and water were provided ad libitum. All surgical and experimental procedures were approved by National Taiwan Normal University Animal Care and Use Committee and were in accordance with the guidelines of the National Science Council of Republic of China (NSC 1997). Room temperature was kept at 25 ±2oC. Animals were randomly assigned to 6 groups: control (CON) group; type II DM (DM) group; T2DM with 4 ml/kg BW of guava juice with 2 ml/kg BW of trehalose (T1); T2DM with 8 ml/kg BW of guava juice with 4 ml/kg BW of trehalose (T2); T2DM with 20 ml/kg BW of guava juice with 1 ml/kg BW of trehalose (T5) and T2DM with 4 ml/kg BW of guava juice without trehalose (B1), as clearly described below.. 2-2. Induction of T2DM Group CON consumed standard diet and normal water. The other 5 groups were induced to be T2DM by consuming high fructose diet for 8 weeks and then intraperitoneal (i.p.) injection of nicotinamide (NA) (Sigma, Missouri, USA) (230mg/kg) and streptozotocin (STZ) (Sigma, Missouri, USA) (65mg/kg). The high fructose includes 21% 8.

(19) fructose water and 60% fructose diet (TD. 89247, Harlan Teklad). The nutrition information of 60% fructose diet is showed in the following column. After consuming high fructose diet for 8 weeks, the rats were injected NA and STZ, and these rats with fasted blood glucose (FBG) higher than 230 mg/dL were recognized to be T2DM.. 2-3. % by weight. % kcal from. Protein. 18.3. 20.2. Carbohydrate. 60.4. 66.8. Fat. 5.2. 12.9. Animal Treatment Different treatments among these groups are showed in the columns below. Different groups of rats were given different doses of 40% guava juice by gavage. The treatment of guava juice lasted for 4 weeks.. 9.

(20) Group. CON DM T1. T2. T5. B1. T2DM. -. . . . . . Treatment. -. -. Guava. Guava. Guava. Guava. juice with juice with juice with juice. Guava juice -. trehalose. trehalose. trehalose. only. -. 4. 8. 20. 4. -. +. +. +. -. dose (ml/Kg BW) 5%. -. trehalose in guava juice. 2-4. Determine Blood Glucose We collected the blood sample from the tail vein. Blood glucose concentrations (mg/dL) were determined by Bayer Ascencsia ELITE XL (Bayer Healthcare, Whippany, USA). Fasted blood glucose was measured after fasted overnight (14 hour).. 2-5. Determine Insulin Levels Insulin levels were determined by Mercodia Rat Insulin ELISA kit (Mercodia, Uppsala, Sweden). We prepared enzyme conjugate 1X solution and washed buffer 1X solution. We pipetted 10μL of each samples and calibrators duplicated into the coated plate. We then added 100μL enzyme conjugate 1X solution to each well, and incubated on a plate shaker for 2 hours (700-900 rpm) at 18-25o C. We inverted the plate to discard the reaction volume, added 350 μL wash buffer to each well and then discarded the volume. We 10.

(21) repeated this step 5 times. After then, we added 200 μL of substrate TMB to each well, and incubated for 15 min at 18-25o C. Finally, we added 50 μL of stop solution to stop reaction. After mixing, we read optical density at 450 nm within 30 min and calculated the results. Data of this experiment were displayed by area under curve (AUC) (min × μg/L). 2-6. HOMA-IR and HOMA-β Homeostasis model assessment of IR (HOMA-IR) is an indicator of insulin resistance in diabetic patients (Akira Katsuki et al., 2001). On the other hand, HOMA-β represents the function of β cell in pancreas and insulin secretion index. HOMA-IR is calculated by the formulae: [fasted insulin (μU/mL) fasting blood glucose (mg/dL)] /405. HOMA-β is calculated by the formulae: (360fasted insulin (μU/mL)/(fasting blood glucose (mg/dL) minus 63). We calculated and recorded these 2 values at week 0 (the beginning of the 4 week guava juice treating experiment) and week 4 (the end of the 4 week guava juice treating experiment).. 2-7. Glucose Tolerance Test 2-7-1 Intra Venous Glucose Tolerance Test (IVGTT) After fasted overnight (14 hour), the animals were anaesthetized with avertin (200mg/kg BW, i.p.) (Sigma, Missouri, USA). The carotid and jugular veins were intubated with PE50 tube to sample the blood sample for blood glucose testing and to inject glucose (0.5 g/kg). In this experiment, we also injected trehalose (0.5 g/kg) to 11.

(22) compare the capacity to lift blood glucose with glucose. The values of blood glucose were recorded before the injection, and 1, 5, 10, 20, 30, 50, and 75 min after it. 2-7-2 Oral Glucose Tolerance Test (OGTT) OGTT were performed after fasted overnight (14 hour). An oral glucose load (2 g/kg BW) was treated by gavage. Blood samples were collected to test blood glucose levels from the tail vein. These samples were collected before the gavage (0 min) and 30, 60, 90 and 120 min after it. We also determined the tolerance of guava juice to determine blood glucose change after consumption of guava juice. 2-8. Determine HbA1c HbA1c,. glycated. hemoglobin. A1c,. was. measured. by. Cation-Exchange HPLC (HLC-723 G7 HPLC analyzer, Tosoh bioscience, Belgium). We collected whole blood samples, and diluted the samples with elution buffer and hemolysis Wash Solution. We injected diluted samples in columns. Depending on different charge on each hemoglobin N-terminal, various electronegativity were separated sequentially. After glycated hemoglobin washed out, we detected the absorbance at the 450 nm wavelength. We calculated the ratio of area of the peak (mVsec) and total area (SUM of area of every peak) (mVsec) and expressed in percentage. A1c (%) = A1c Area/Total Area. 2-9. Metabolic Parameter After treatment of guava juice for 4 weeks, animals were placed individually in the metabolic cage for 24 hours. Food consumption, 12.

(23) water consumption, and urine volume were recorded from this experiment. Water consumption was measured by a 100 mL bottle, which had calibration on it. 2-10 Assay of ROS Level 2-10-1 in vitro ROS Level The ROS was measured in a completely dark chamber of the Chemiluminescence. Analyzing. System. (CLD-110,. Tohoku. Electronic In. Co., Sendai, Japan), which can detect the chemiluminescence (CL) emitted by luminal-amplified ROS. CL emitted was first amplified by photomultiplier (PMT) (Tohoku Electronic Industry, Miyagi, Tokyo), and then the intensity was defined by CLD-110. The assay was performed in duplicate for each sample and was expressed as ROS counts/10 s. We turned on the cooler to cool down the photomultiplier (PMT) to 5oC. We pipetted 200 μL of tested samples (guava juice and trehalose) into the chamber, and the signals detection was recorded for 50 seconds as a baseline. We then pipetted 500 μL of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione;. Sigma,. Missouri,. USA), a kind of CL enhancer, and the detection was recorded for 50 seconds. We added H2O2 (34.5-36.5 %, 1000 times diluted) or HOCl (0.5%, 10000 times diluted) at 100 μL, as an exogenous ROS source, and then we evaluated the potential of tested samples to scavenge these ROS levels. We also measured the ROS levels from the collected serum (0.2 mL) by luminal-amplification before sacrifice. 2-10-2 in vivo ROS Level 13.

(24) Renal ROS levels of rats were determined according to our previous reports (Chien et al., 2001). The machine CLD-110 to record in vivo ROS level is the same with the one to record in vitro ROS levels. The way to detect CL from kidney was continually injected (0.01 mL/min) 0.4 mg/mL MCLA (2-methyl-6-(4-methoxyphenyl)-3, 7-dihydroimidazo-(1, 2-a)- pyrazin-3-one hydrochloride) from the femoral vein. MCLA generated CL by reaction with superoxide O2–. Animals were first anesthetized by urethane. Under anesthesia, the trachea and femoral vein were intubated (PE 100 for trachea, PE 50 for femoral vein). The left kidney was exposed, and the animal was placed in a dark box. The box was shielded with a plate that excluded other photon emission from other sources. There was a window left unshielded at the position of exposed kidney and the position under reflector, which reflected the photons from the exposed kidney surface onto the detector area. The detection was recorded for 7200 seconds in total. The first 300 seconds was recorded as a baseline level. Then, MCLA started to be injected, and detection was recorded until 7200 seconds. After this experiment, animals were sacrificed, and the tissues were collected for the other experiments. 2-11 Immunohistochemistry (IHC) Tissue sections were deparaffinized in xylene and rehydrated in an ethanol. We immersed slides in xylene for 5 minutes at room temperature and repeated using fresh xylene for second 5 minutes incubation. We immersed slides in 100% ethanol for 5 minutes at 14.

(25) room temperature and repeated using fresh 100% ethanol for 5 minutes. We immersed slides in 90% ethanol for 3 minutes, and then 75% ethanol for 3 minutes, and then 50% ethanol for 3 minutes at room temperature. Then the tissue sections were submitted to antigen retrieval step. The Buffer solution used for heat-induced epitope retrieval was sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0). After 15 minutes of antigen retrieval step at 90oC, the sections were blocked for non-specific binding with 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at 37o C and incubated with the primary antibodies for 18 hours at 4°C. Primary antibodies included mouse anti IL-1β (1:1000; Cell Signaling Technology, Denver, MA, USA), rabbit anti Caspase-3 (1:500; Cell Signaling Technology, Denver, MA, USA), and rabbit anti 4-HNE (1:500; Alpha Diagnostic, San Antonio, TX, USA). Tissue sections were washed with PBS three times and then were incubated with secondary antibodies HRP-conjugated rabbit anti-mouse IgG for 1 hour at room temperature. After washing with PBS for 3 times, we immersed slides in DAB (ImmPACT DAB Peroxidase Substrate; Vector, Burlingame, California, USA) for 3-5 seconds, washed with ddH2O, and immersed slides in hematoxylin for 5 minutes. The sections were dehydrated in ethanol series and were mounted in mounting medium (Leica, Wetzlar, Germany). 2-12 Masson’s Stain These sections were brought to water with xylene and ethanol and mordanted in Bouin’s solution 60°C for 1 hour. These sections were 15.

(26) washed in running tap water for removal of the picric acid. These sections were stained in Weigert’s iron hematoxylin working solution (mixture of Hematoxylin A and Weigert’s Hematoxylin B at a 1:1 ratio) for 10 minutes. These sections were washed in running tap water for 5 minutes, rinsed in distilled water, and mordanted in Biebrich Scarlet-Acid Fuchsin solution for 5 minutes. After washing with. distilled. water,. these. sections. were. incubated. in. Phosphotungstic/phosphomolybdic acid for 10 minutes. The sections were then transfered directly into Aniline blue for 5 minutes, rinsed in distilled water and immersed in 1% Acetic acid for 1 minute. Finally, these sections were dehydrated in ethanol series and mounted in mounting medium. 2-13 TUNEL Stain We used BioVision’s Apo-BrdU-IHCTM Kit to performed a two-color TUNEL stain (Terminal deoxynucleotide transferase dUTP Nick End Labeling) (catalog #K403-50; 50 assays; stored at -20oC) assay for labeling DNA breaks to detect apoptotic cells by immunohistochemistry. We immersed slides in xylene for 5 minutes at room temperature and repeated using fresh xylene for second 5 minutes incubation. We immersed slides in 100% ethanol for 5 minutes at room temperature and repeated using fresh 100% ethanol for second 5 minutes. We immersed slides in 90% ethanol for 3 min, 75% ethanol for 3 min, and then 50% ethanol for 3 minutes at room temperature. We immersed slides into 1X PBS and dried the glass slide. We diluted enough Proteinase K at 1:100 in 10 mM Tris with 16.

(27) pH 8. We covered the entire specimen with 100 µl proteinase K and incubated at room temperature for 20 minutes. We rinsed slides with 1X PBS, gently tapped off excess liquid and carefully dried the glass. We diluted 30% H2O2 1:10 in methanol. We covered the entire specimen with 100 µl of 3% H2O2 and incubated at room temperature for 5 minutes. We rinsed the slide with 1X PBS, gently tapped off excess liquid and carefully dried the glass slide. We diluted only enough 5X Reaction Buffer as needed 1:5 with dH2O. We covered the entire specimen with 100 µl of the 1X Reaction Buffer and incubated at room temperature for 10 to 30 minutes. We carefully blotted the 1X Reaction Buffer from the specimen and immediately applied 50 µl of Complete Labeling Reaction Mixture onto each specimen. We covered the specimen with a piece of Parafilm cut slightly larger than the specimen. We placed slides in a humid chamber and incubate at 37°C for 1 to 1.5 hours, then removed Parafilm cover slip and rinsed slide with PBS. We gently tapped off excess liquid and carefully dried the glass. We covered the entire specimen with 100 µl of Blocking Buffer. We incubated at room temperature for 10 minutes, blotted the Blocking Buffer from the specimen, and immediately covered specimen with 100 µl of Antibody Solution. We incubated with the Antibody Solution in the dark for 1-1.5 hours at room temperature and rinsed slide in PBS. We gently tapped off excess liquid and carefully dried the glass. We covered the entire specimen with 100 µl of Blocking Buffer and diluted only enough of the 200X Conjugate 1:200 in Blocking 17.

(28) Buffer. Then, we carefully blotted the Blocking Buffer from the specimen and immediately applied 100 µl of diluted conjugate to the specimen. We incubated at room temperature for 30 minutes. Five minutes before concluding incubation, we prepared DAB solution by dissolving one tablet of DAB and one tablet of H2O2/Urea in one ml of tap H2O. Rinse slides with 1X PBS. We gently tapped off excess liquid and carefully dried the glass slide. We covered the entire specimen with 100 µl of DAB solution and incubated at room temperature for 15 minutes. We rinsed slides with H2O and blotted and immediately covered the entire specimen with 100 µl of Methyl Green Counterstain solution for incubation at room temperature for 3 minutes. We pressed the edge of the slide against an absorbent towel to draw off most of the counterstain. We dipped slides 2 times briefly into 100% ethanol and blotted slides briefly on an absorbent towel. We dipped slides into xylene and wiped excess xylene from back of slide and around specimen. Finally, we mounted a glass cover slip using a mounting media over the specimen. 2-14 Fluorescent Stain Tissue sections were deparaffinized in xylene and rehydrated in an ethanol series. Then the tissue sections were submitted to antigen retrieval step. The Buffer solution used for heat-induced epitope retrieval was sodium citrate buffer (10 mM Sodium citrate, 0.05% Tween 20, pH 6.0). After 15 minutes of antigen retrieval step, sections were blocked for non-specific binding with 5% bovine serum albumin (Sigma-Aldrich, St. Louis, MO,USA) for 1 hour at 18.

(29) room temperature and incubated with the primary antibodies, including bs-2912R-Cy5 (rabbit anti MAP1A/MAP1B LC3 B polyclonal antibody, Cy5 conjugated) (1: 500,Woburn, MS, USA) and bs-0081R-FITC (rabbit anti-Caspase-3 polyclonal antibody, FITC conjugated) (1: 500,Woburn, MS, USA) for 18 hours at 4℃. The nuclear stain was dyed with Hoechst33342 (1:1000; Sigma-Aldrich, St. Louis, MO, USA) for 1 hour at room temperature. Tissue sections were washed with PBS three times. After washing with PBS, tissue sections were mounted with mounting medium (Leica, Wetzlar, Germany). The slides were then scanned by Leica TCS SP3 laser confocal microscope (Leica) to obtain the confocal images. 2-15 Western Blot Tissues were grinded to powders in liquid nitrogen. Then the tissue powders were lysed in Lysis Buffer (Cell Signaling Technology, Denver, MA, USA) supplemented with protease inhibitor (Roche) for 10 minutes at 4℃. The tissue homogenate was centrifuged at 14000 rpm for 30 minutes. After centrifugation, the supernatant was collected into a new eppendorf. The concentration of protein was measured by Protein Assay Dye Reagent Concentrate (Bio-Rad, Hercules, CA, USA). Forty μg protein samples were mixed with 1X sample buffer and were boiled for 5 minutes. Protein samples were resolved. in. 10%. SDS-polyacrylamide. gel. electrophoresis. (SDS-PAGE) and transferred to PVDF membrane (Millipore, Billerica, MA, USA). Then the blot was blocked with Hyblock 19.

(30) (Hycell, Taipei, Taiwan) for 1 minute, and incubated with primary antibodies overnight at 4℃. After washing three times with TBST, the. blot. was. incubated. with. horseradish. peroxidase. (HRP)-conjugated secondary antibodies at room temperature for 1 hour. Detection of signals was performed by Immobilon Western Chemiluminescent HRP Substrate (Millipore). Primary antibodies included Caspase-1, LC3β (MBL, Naka-ku, Nagoya, Japan), Bax, Bcl-2, Beclin-1 (Cell Signaling Technology, Denver, MA, USA), and β-actin (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies included HRP-conjugated rabbit anti-mouse IgG, HRP-conjugated donkey anti-goat IgG, and HRP-conjugated goat anti-rabbit IgG (all for 1:10000; all from Sigma Aldrich St. Louis, MO, USA). 2-16 Guava Extraction The guava juice used in this study was made by 2 kinds of guava harvested in Taiwan, Thailand guava (G1) and pearl guava (G2). Fifty gram edible portion (seeds portion was removed) of fresh fruits was blended with 150 mL H2O or 50% ethanol. After storing the extraction mixture at room temperature (25oC) for 24 hours, we filtered the extraction through gauze, and then Whatman No. 1 filter paper (Chia-Yun Lin and Mei-Chin Yin, 2012). Strata C18-E column (6 mL) was first rinsed by 12 mL methanol, and then rinsed by 12 mL ddH2O. We added the 24 mL water extraction or 48 mL ethanol extraction (2 times diluted by ddH2O) in the column. After 20.

(31) all the extraction dripping off, we washed the column by 12 mL ddH2O. We rinsed out the final concentrated extraction by 12 mL methanol. Guava water extraction of G1 (GWE1), G2 (GWE2) and guava ethanol extraction of G1 (GEE1), G2 (GEE2) were stored at -20oC. 2-17 LC/MS We used Ultimate 3000 HPLC system comprising a HPLC pump, diode array absorbance detector to analyze tested samples (guava juice extractions) at the range from 200 to 600 nm (Thermo Finnigan, San Jose, USA). Separation was carried out using a 150_2.0 mm i.d. 4 mm Synergi RP-Max column (Phenomenex, Macclesfield, UK) eluted with a gradient over 40 min of 10–80% acetonitrile in 0.1% aqueous formic acid at a flow rate of 0.2 ml/min. After passing through the flow of the diode array detector, the column eluate was directed to a LXQ ion trap mass spectrometer fitted with an electrospray interface (Thermo Finnigan). The analyses utilized the negative ion mode as this provided the best limits of detection for quercetin. Capillary temperature was set at 325°C, sheath gas and auxiliary gas were 30 and 15 arb, respectively, and source voltage was 3.5 kV.. 21.

(32) 3. Results 3-1 Guava Juice in vitro ROS Levels In this experiment, we used H2O2 and HOCl as exogenous ROS sources to evaluate the scavenging ROS ability of guava juice. Our results showed that guava juice showed highly and dose-dependently scavenging H2O2 and HOCl ability from concentration 5 % to 40 %. (Figure 1) 3-2 Trehalose in vitro ROS Levels As shown in figure 2, the ability of trehalose to scavenge ROS seemed to be selectively. Trehalose selectively and dose-dependently decreased H2O2 CL counts from concentration 10%-50% (Figure 2A). In contrast, trehalose did not affect HOCl counts from concentration 10%-50% (Figure 2B). 3-3 Quercetin Content In Guava Extraction We tested quercetin content, a kind of anti-oxidative flavonoid we were interested in, in our guava juice. In the results of LC/MS, the concentration of quercetin and other materials in guava juice extractions were shown in the Figure 3. The highest content in the guava extraction was quercetin. The concentrations of quercetin were 182.3 ng/mL in GWE1, 133.4 ng/mL in GWE2, 223.1 ng/mL in GEE1 and 244.5 ng/mL in GEE2 (Figure 4). According to guava concentration and volume, we calculated the content of quercetin in our guava juice sample was 0.633 μg/mL. In our study, each group of rats had consumed different dose of quercetin per day. T1 and B1 rats consumed 2.5 μg/kg BW, while T2 rats consumed 5.0 μg/kg BW and 22.

(33) T5 consumed 12.6 μg/kg BW per day. 3-4 Guava Extraction in vitro ROS Levels Water extract of Thailand guava (GWE1) and pearl guava (GWE2) and ethanol extract of Thailand guava (GEE1) and pearl guava (GEE2) were used to evaluate their ROS scavenging ability. Our results showed that water extraction and ethanol extraction of two kinds of guava displayed high ability to scavenge H2O2 (Figure 5A) and HOCl ROS (Figure 5B). Using ddH2O as a reference control, the increased H2O2 and HOCl counts were significantly (P < 0.05) decreased by four kinds of guava extraction. GWE1 and GWE2 had a significantly efficient potential (P < 0.05) than GEE1 and GEE2 in scavenging H2O2 and HOCl activity. Water extract of guava had a higher ROS scavenging ability than ethanol extract. 3-5 Oral Guava Juice Tolerance Test As shown in Figure 6, to determine three kinds of guava juice on blood glucose levels, we treated 4 ml/kg BW guava juice containing 12% trehalose, 8.8% sucrose, or 40% guava juice (without sugar supplement) in normal animals. The data showed that these 3 kinds of guava juice did not increase blood glucose dramatically. Additionally, there was no significant difference among them. This result represented guava juice, at these doses, had no effect on blood glucose. According to our data, 12% trehalose and 8.8% sucrose supplement did not affect the blood glucose levels. 3-6 Intra Venous Glucose and Trehalose Tolerance Test To evaluate the alterations of blood glucose level in response to 23.

(34) intravenous (i.v.) glucose or trehalose, we administered 0.5 g/kg body weight glucose or trehalose via an intravenous route. The blood glucose level significantly elevated to 302.4 mg/dL at 1 min after i.v. glucose (Figure 7). However, the blood glucose was not significantly elevated at 1 min after i.v. trehalose. After 1st minute of glucose administration, the elevated blood glucose level started to decrease. However, at 75th minute, the blood glucose was still higher than fasted blood glucose (FBG) at the time of beginning. On the other hand, normal animals treated trehalose did not show significant changes in blood glucose. There was only a slight increasing of blood glucose during 1st to 10th minute. The significant (P < 0.05) differences between i.v. glucose and trehalose were still evident during 1st to 50th minute. 3-7 Oral Glucose Tolerance Test In CON rats, blood glucose changes were kept in a relatively small range (Figure 8). No matter in week 0, 2 or 4, values of blood glucose in CON were never over 150 mg/dL. In other 5 groups, there were no different among them. Values of blood glucose were highest in 30 and 60 min, and ranged from 500 to 600 mg/dL in these 5 groups with T2DM. Blood glucose wasn’t lowered by the treatment of Guava juice and trehalose in this study. 3-8 Blood Glucose Changes We compared FBG (0 min) and blood glucose at the end (120 min) of OGTT test at week 4 (Figure 9). In CON rats, values of blood glucose at 0 and 120 min were both below 100 mg/dL. There was no 24.

(35) difference between 0 min and 120 min. It meant that blood glucose dropped down at 120 min, and the value was equal to FBG. In DM rats, 0 min blood glucose and 120 min was significantly different (P <0.05). Blood glucose couldn’t decline at the end of OGTT test. In DM rats, the burden of glucose couldn’t be normally regulated by the function or secretion of insulin. In other groups (T1, T2, T5 and B1) of rats, the blood glucose response was still higher at 120 min as compared to the values at 0 min and this response was similar to DM group. There was no statistical difference among DM, T1, T2, T5 and B1 groups. It was indicated that treatment of guava juice helped control blood glucose in OGTT. And it might associate with adjustment of insulin function or secretion, which had been tested by insulin levels and HOMA values. 3-9 Insulin Levels The insulin level in response to guava juice and trehalose in T2DM was indicated in Figure 10. Insulin levels were indicated by the index of area under curve (AUC = min  μg/L). AUC of insulin levels were 66.6±2.1 min  μg/L in CON. Due to the impairment of  cells in T2DM, AUC declined to 21.9±1.4 min  μg/L in DM. AUC was elevated in T1 (36.0±0.5 min  μg/L), T2 (38.2±1.1 min μg/L), and T5 (41.5±1.8 min  μg/L), though there were no significant differences vs. DM. AUC of B1 was 28.9±0.8 min  μg/L. Guava juice combined with trehalose significantly preserved the function of insulin secretion in T2DM rats.. 25.

(36) 3-10 HOMA-IR HOMA-IR values of CON rats were about 2 at week 0 and week 4 (Figure 11). In DM rats, this value was elevated (up to 4.9±0.2) and remained stable from week 0 to week 4. In T1, T2, T5 and B1 rats, HOMA-IR were all lower at week 4 than week 1. And in T2 and T5, these changes were significantly (P < 0.05) decreased. The dose of guava juice and trehalose treated in T2 and T5 rats may not only help preserve the function of insulin secretion, but also lower the value of insulin-resistance. 3-11 HOMA-β In CON rats, the values of HOMA-β were about 20 at week 0 and week 4 (Figure 12). All the rats with T2DM had lower HOMA-β values (below 5) compared to CON. The low HOMA-β values in DM rats still remained between week 0 and week 4. However, the other 4 groups (T1, T2, T5 and B1) had a trend to elevate the HOMA-β value at week 4 compared to week 1. Among these groups, T1 was statistically different (P < 0.05). 3-12 HbA1c Levels HbA1c level of DM rats was twice of CON rats. In T2 and T5 rats, HbA1c levels were slightly lowered, though there were no statistically differences (Figure 13). HbA1c could tell the status of blood glucose control in the last 3 month, while our experiment lasted only 1 month. This might be the reason why there were no statistically differences among DM rats and other rats which had been treated guava juice and trehalose. 26.

(37) 3-13 Metabolic Parameter Diabetes was associated with reducing weight, increasing water intake, increasing food consumption and increasing urine volume (3p of diabetes: polydipsia, polyphagia and polyuria). In our metabolic cage experiments, T2DM caused lower body weight in DM rats. And also, higher food intake and higher water intake could be observed. The bottle used to measured water consumption was 100 mL. Hence, the maximum of total water consumption was 100 mL. T1, T2 and T5 rats had higher body weight than DM (P <0.05), while B1 rats showed no difference compared with DM (Table 1). 3-14 Renal in vivo ROS Levels Renal CL count was highest in DM rats. It meant the kidney of T2DM could be injured by high ROS level. And after consuming guava juice, T1, T2, T5 and B1 had lower CL counts (Figure 14). 3-15 Serum in vitro ROS Levels We collected serum before sacrificed for ROS test. Serum of DM had higher ROS level significantly higher than in CON (P <0.05) (Figure 15). It meant DM may cause high ROS in blood, which circulates throughout the body. In contrast, after consumption of guava juice, CL counts of T1, T2, T5 and B1 rats were all significantly reduced than those in DM (P < 0.05). Among them, T1, T2 and T5 had lower ROS levels than B1. Guava lowered DM-enhanced ROS level. The addition of trehalose in the guava juice seemed to enforce the ability of guava juice to lower ROS. Although the difference between T1 and 27.

(38) B1 was not significant, T2 and T5 had significantly lower serumal ROS than B1 (P < 0.05). 3-16 HE Stain To examine the tissue abnormality, we histologically evaluated pancreas and kidney tissues by HE Stain. In pancreatic section, we found that DM rats had smaller islet of Langerhans diameters (Figure 16-A). It represented islet shrinkage. Besides, pancreas of DM rats showed irregular arrangement. The boundary of cells were blurred or vanished and cells were necrotic. Larger sizes of islet were found in T1, T2, T5, and B1 rats compared to DM rats. Additionally, the arrangement of pancreatic cells in T1, T2, T5, and B1 rats became more regular than DM rats. In renal sections, hemorrhage presented in DM rats (Figure 16-B). Also, neutrophils occurred in renal section of DM rats, representing inflammation. T1, T2, T5 and B1 rats showed less hemorrhage and neutrophils gathering, which meant the inflammatory damage of T2DM on kidney had been rescued. Additionally, T1, T2 and T5 rats showed less of these characters than B1 rats. 3-17 Masson’s Stain Masson’ Stain could stain sclerotic area in a blue color. Masson’s stain recipe produced blue collagen and bone. Collagen IV accumulation occurred in kidney of type 1 diabetes rats, associated with glomerulosclerosis and mesangial expansion in diabetic nephropathy (Jay C. Jha, et al. 2014). In this study, we found that sclerosis occurred mostly in renal tubules in our T2DM rats rather 28.

(39) than glomeruli (Figure 17-B). T1, T2, T5 and B1 rats showed less blue area in renal sections compared to DM (Figure 17-A). Among these groups, T2 and T5 rats displayed relatively less sclerotic area. 3-18 Fluorescent Stain We examined these two types of programmed cell death, apoptosis and autophagy, by fluorescent stains. Green fluorescent represented caspase 3-mediated apoptosis, whereas red fluorescent represented LC3-B-mediated autophagy. In the renal section, we found high expression of caspase 3 and LC3-B in cytoplasm of DM group compared to CON group (Figure 18). We could also found the colocalization of these 2 proteins in the renal tubules of DM kidney. These results implicated that T2DM causes two types of programmed cell death, apoptosis and autophagy, in the kidney. The level of expressions of caspase 3 and LC3-B proteins were markedly reduced in T1, T2, T5 and B1. According to our results, the treatment of 4, 8 and 20 mL/kg BW guava juice with trehalose could further reduce cell death in the kidney than 4 mL/kg BW guava juice alone. DM could cause autophagy and apoptosis in the pancreatic cells (M. Masini et al., 2009; Alexandra E et al., 2003). In this study, we found higher caspase 3 and LC3 B expressions in pancreatic section of DM rats (Figure 19). Moreove, caspase 3 showed higher than LC3 B. It means apoptosis might occur more often than autophagy in T2DM of this kind of inducement. T1, T2 and T5 decreased the expression of caspase 3 and LC3-B in the pancreatic section of T2DM rats. B1 29.

(40) showed more caspase 3 and LC3-B expression than T1, T2 or T5. This result implicated that the addition of trehalose in guava juice confers further protection against DM-induced pancreatic injury. 3-19 IHC Stain To evaluate oxidative injury, inflammatory cytokines and apoptosis levels in the kidney, we performed specific IHC stain. Oxidative stress indicated by 4-HNE, a production of lipid peroxidation, expression was higher in DM rats compared to CON (Figure 20). Rats with guava juice combination with (T1, T2, or T5) or without trehalose (B1) had less 4-HNE expression. B1 rats had the highest level of 4-HNE expression compared to T1, T2 and T5. DM rats displayed higher expression of proinflammatory cytokines, IL-1β, in the kidney when compared to CON (Figure 21). The other 4 groups treated with guava juice (T1, T2, T5 and B1) decreased renal IL-1β expression than DM rats (P <0.05). Similarly, B1 showed the high IL-1β expression and was statistically higher than T1, T2 and T5 (P <0.05). The expression of caspase 3 in the kidney displayed a similar pattern to 4-HNE and IL-1β. DM rats had higher caspase 3 levels compared to CON indicating a higher apoptosis level in T2DM (Figure 22). T1, T2, T5 and B1 rats had lower caspase 3 expressions (P <0.05). B1 had the highest expression among these 4 groups. Pancreatic 4-HNE expression was higher in DM rats compared to CON (Figure 23). Rats with guava juice combination with (T1, T2, or T5) or without trehalose (B1) had less pancreatic 4-HNE expression. 30.

(41) B1 rats had the highest level of pancreatic 4-HNE expression compared to T1, T2 and T5. DM rats displayed higher expression of proinflammatory cytokines, IL-1β, in the pancreas compared to CON (Figure 24). The other 4 groups treated with guava juice (T1, T2, T5 and B1) statistically reduced pancreatic IL-1β expression than DM rats (P < 0.05). Similarly, B1 showed the high IL-1β expression and was statistically higher than T1 and T2 in the pancreas (P < 0.05). The expression of caspase 3 in the pancreas showed a similar pattern to 4-HNE and IL-1β. DM rats had higher pancreatic caspase 3 levels compared to CON indicating a higher apoptosis level in T2DM (Figure 25). T1, T2, T5 and B1 rats reduced pancreatic caspase 3 expressions (P < 0.05) compared to DM. B1 had the highest expression among these 4 groups. In summary, treatment of guava juice with trehalose on T2DM rats lowered oxidative, proinflammatory and apoptosis levels in the kidney and pancreas. The supplement of trehalose in guava juice enhanced the anti-oxidative, anti-inflammatory and anti-apoptotic effects, which could be proved by higher oxidative, pyroptosis and apoptosis levels shown in B1 rats vs. T1 group. 3-20 TUNEL Stain We used TUNEL stain to quantify the apoptotic formation. The brown color appeared in the nucleus where the DNA fragmentation occurred. In renal tubules, T2DM caused a lot of apoptosis formation in DM rats as compared to CON (Figure 27). This result was well correlated 31.

(42) with the IHC stain and fluorescent stain. In group B1, the TUNEL positive cells were still highly expressed in the renal tubules. In T1, T2 and T5 rats, the TUNEL positive cells significantly decreased (P < 0.05). In addition, T2DM caused apoptosis formation in the pancreas. DM showed highest expression of TUNEL positive cells in the pancreatic section among all groups, while T1, T2, T5 and B1 groups statistically decreased TUNEL positive cell number in the pancreas (Figure 26). 3-21 Western Blot We investigated the effect of guava juice and trehalose on autophagy, apoptosis and pyroptosis related proteins in the kidney and pancreas of T2DM. Compared to the control kidney and pancreas, the expressions of autophagy related protein, Beclin-1 and LC3-B, were significantly higher in DM rats and were lowered in T1, T2 and T5 (P < 0.05) (Figure 28, 30, 32). Caspase 1, an inflammation and pyroptosis related protein, was higher in DM, but not significantly (Figure 31). Bax and Bcl-2 were apoptosis related proteins. Bax was positively related to apoptosis while Bcl-2 inhibited apoptosis. Bax expression was higher in DM (P <0.05). The ratio of Bcl-2 and Bax showed the balance between them. Although there was no significantly difference, the expression still showed a trend that T1, T2 had a higher ratio. DM had a lower ratio.(Figure 29, 33). 32.

(43) 4. Discussion This study proved that guava juice combined with trehalose had an efficiently protective function against T2DM-induced renal and pancreatic dysfunction. This evidence is supported by the improved parameters in HOMA-β, metabolic parameters, levels of oxidative stress, inflammation and programmed cell death in the renal and pancreatic tissues of T2DM rats. Genetic disposition, aging, obesity and dietetic life style were the major causes of T2DM (K. Srinivasan, et al., 2005).The animal model we used in this study was relatively similar to the lifestyle of human nowadays. High calories and high fats diet were the main dietetic life style causing metabolic syndromes and chronic diseases. High fructose diet used in this study mimics this lifestyle of human. It had been proved that high fructose is associated with insulin resistance, hyperglycemia and hypertension (Hariom Yadav, et al., 2006). It induced lipogenesis from liver and produces great amount of triglyceride which furthermore reduced insulin sensitivity (Wilfried P. Bieger, et al., 1984). Traditional T2DM models, such as yellow A(vy/-) mice, db/db mice, were trans gene and spontaneous induced animals. They had the advantages of stability and time saving, but also cost a lot. T2DM development in these kinds of animals was predominantly genetic, unlike human population, and also unlike the pattern of human clinical situation (K. Srinivasan, et al., 2005). A traditional and easy way. to. induce. T1DM. was. STZ. injection.. STZ. is. a. glucosamine-nitrosurea compound. STZ entered pancreas β cell by 33.

(44) glucose transport system and damages DNA by alkylation. It caused insulin secretion damage and cell death (Nigel A. Calcutt, et al., 2009). In contrast, NA supplied NAD, which would be consumed because of DNA damage induced by STZ (Tomonori Nakamura et al., 2006). Animals administered STZ and NA appeared a closer phase to T2DM (Pellegrino Masiello, et al., 1998). That is the reason why we used this model in our study. Previous study had revealed the anti-oxidative property of guava and its’ leaf (Hui-Yin Chen and Gow-Chin Yen, 2007). In our study, the anti-oxidative ability of guava juice was tested by a chemiluminescence analyzing system and the anti-oxidative ability was efficient in scavenging H2O2 and HOCl and was consistent with the previous finding. Beside, this ability has been preserved after orally consumed. The result of in vitro serum ROS levels showed that the anti-oxidative property of guava scavenged ROS. The result of 4-HNE IHC stain in renal and pancreatic sections also showed the same finding. In addition, we found the addition of trehalose seemed to improve the anti-oxidative property of guava. In previous study, growing Candida albicans cells from trehalose-deficient mutant were extremely sensitive to severe oxidative stress exposure (H2O2), while in wild-type cells, H2O2 exposure induced intracellular accumulation of trehalose and a higher survival rate after the same exposure (Qiaofang Chen and Gabriel G. Haddad, 2004). The researchers also thought trehalose could be a promising free radical scavenger to mammals’ organs (Qiaofang Chen and Gabriel G. Haddad, 2004). In our study, serum ROS levels were 34.

(45) very low, especially in T2 and T5 rats, and even lower than B1 rats. Although there was no significantly difference between B1 and T1, T1 still showed lower ROS level than B1. This finding was consistent with the result of 4-HNE stain. We suggest that guava juice through the high content of quercetin and trehalose itself to reduce the ROS-induced oxidative injury. This result might support the inference of the research of Qiaofang Chen and Gabriel G. Haddad in 2004. Except ROS level, what was the difference between guava juice only and guava juice combined with trehalose? We compared T1 to B1 to observe whether trehalose really had any effect on T2DM. We found that, according to the result of HOMA-β, T1 rats had a better control on β cell function than B1. T1 rats had slightly higher HOMA-β at week 4 than week 0, while B1 stayed the same value. However, this protective function didn’t last on HOMA-IR and insulin levels. HOMA-IR could be a useful method not only diagnosing insulin resistance, but also for follow-up during the treatment of patients with T2DM (Akira Katsuki et al., 2001). Based on our data, there was no difference of HOMA-IR between T1 and B1. The addition of trehalose didn’t increase in sensitivity of insulin between T1 and B1. Along the increasing dose of guava juice, insulin secretion had slightly increased, and HOMA-IR had reduced. These results indicated the addition of trehalose helps conserve β cell function, but not insulin resistance. However, the guava juice might help increase the sensitivity of insulin with the dose more 8 ml/kg BW/day (T2 and T5 groups). Diabetic nephropathy was associated with structural abnormalities 35.

(46) including glomerulosclerosis and specifically mesangial expansion (Jay C. Jha et al., 2014). Result of Masson’s stain revealed collagen accumulation condition in kidney. Collagen accumulation was associated with sclerosis. The renal sections showed that most sclerosis happened at tubule area, rather than in the glomerular area. DM-induced sclerotic injury was rescued by treatment of guava juice in T2 and T5 rats. In addition, treatment of guava juice protected hemolysis and inflammation in the kidney and cell arrangement in pancreas. The most basic characteristic of diabetes was hyperglycemia. However, in this study, there is no obvious improvement of blood glucose. The blood glucose difference among DM rats and other rats treated guava juice and trehalose was very small. In a previous research, guava showed the properties of antihyperglycemia (Chin-Shiu Huang et al., 2011). However, the animal model was different with our study. Nevertheless, our animal model was more likely at the late phase of T2DM, because it lacked insulin compensation of β cells, which happened at the primary phase. In this study, we defined T2DM successfully induced as FBG over 230 mg/dL. This condition seemed to be stricter than others. For example, in a previous study, T2DM was defined with FBG over 11.1 mmol/L, which is equal to 200 mg/dL, in rats injected STZ and NA (Sibel Tas et al., 2007). Furthermore, in a mouse study with STZ and NA injection, plasma insulin levels stay the same with control mice (Tomonori Nakamura et al., 2006). Due to these differences, the damage of T2DM in our rats could be relatively 36.

(47) irreparable. In this study, we investigated three kinds of programed cell death, including apoptosis, autophagy and pyroptosis. In respect of apoptosis, stains of caspase 3 of both renal and pancreatic sections showed that, treatment of guava juice prevented cells from apoptosis. And the TUNEL stain showed the same trend on renal, but pancreatic sections. In pancreas, TUNEL stain showed that apoptosis still happened in B1, but apoptosis was prevented in other groups treated with trehalose, indicating that trehalose had the potential to prevent apoptosis in pancreas. In the respect of autophagy, we noticed an autophagy related protein, Beclin-1, increasing in DM rats. Beclin-1 was a protein which. had a central role in autophagy and increased during periods of cell stress (R Kang et al., 2011). The Beclin-1 expression was lower in kidney as shown by the result of western blot. The LC3-B is a protein required for the formation of the autophagosome. In brief, guava juice. itself could help protect cell from apoptosis, pyroptosis and autophagy, the addition of trehalose helped enhanced the protective function from apoptosis in pancreas. Some indexes in this study showed that T5, which groups of rats had consumed the highest dose of guava juice and trehalose per day, didn’t receive the best protective function. For example, the values of water intake, Bax expression in kidney and HOMA-β in T5 were not better than T1 and T2. Logically speaking, we expected that the protective intensity of guava juice and trehalose would be strengthened as the dose rose. However, fruit juice was still had some 37.

(48) differences from fruit itself. Intake of fruit was inversely associated with incidence of T2DM. Commonly fruit juice was filtered, so the fiber was relatively lower than fruit. Lack of fiber, fruit juice had become a large sugar load. Frequent consumption of fruit juices may contribute to a higher dietary glycemic load which has been positively associated with DM (Lydia A et al., 2008). Therefore, in our study,. we found when the dose of guava juice was up to 20 ml/kg BW/ day with trehalose 1 g/kg BW/day may be overloading. The dose consumed by T2 rats may have the best protective function to T2DM. In conclusion, the properties of anti-oxidative, anti-inflammatory and antidiabetic effect of guava were undoubtable. The addition of trehalose helped prevent cell death from apoptosis, conserve β cell function and enhanced the anti-oxidative ability.. 38.

(49) 5. References Bazzano, L. A., Li, T. Y., Joshipura, K. J., & Hu, F. B. (2008). Intake of fruit, vegetables, and fruit juices and risk of diabetes in women. Diabetes care. Bieger, W. P., Michel, G., Barwich, D., Biehl, K., & Wirth, A. (1984). Diminished insulin receptors on monocytes and erythrocytes in hypertriglyceridemia. Metabolism, 33(11), 982-987. Butler, A. E., Janson, J., Bonner-Weir, S., Ritzel, R., Rizza, R. A., & Butler, P. C. (2003). β-cell deficit and increased β-cell apoptosis in humans with type 2 diabetes. Diabetes, 52(1), 102-110. Calcutt, N. A., Cooper, M. E., Kern, T. S., & Schmidt, A. M. (2009). Therapies for hyperglycaemia-induced diabetic complications: from animal models to clinical trials. Nature Reviews Drug Discovery, 8(5), 417-430. Chen, Q., & Haddad, G. G. (2004). Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals. Journal of Experimental Biology, 207(18), 3125-3129. Sarah, W., Gojka, R., Anders, G., Richard, S., & Hilary, K. (2004). Global prevalence of diabetes. Diabetes care, 27(5), 1047-1053. Chen, H. Y., & Yen, G. C. (2007). Antioxidant activity and free radical-scavenging capacity of extracts from guava (Psidium guajava L.) leaves. Food Chemistry, 101(2), 686-694. Chien, C. T., Lee, P. H., Chen, C. F., Ma, M. C., Lai, M. K., & Hsu, S. M. (2001). De novo demonstration and co-localization of free-radical production and apoptosis formation in rat kidney subjected to ischemia 39.

(50) reperfusion.Journal of the American Society of Nephrology, 12(5), 973-982. Eidenberger, T., Selg, M., & Krennhuber, K. (2013). Inhibition of dipeptidyl peptidase activity by flavonol glycosides of guava (Psidium guajava L.): A key to the beneficial effects of guava in type II diabetes mellitus. Fitoterapia, 89, 74-79. Eroglu, A., Russo, M. J., Bieganski, R., Fowler, A., Cheley, S., Bayley, H., & Toner, M. (2000). Intracellular trehalose improves the survival of cryopreserved mammalian cells. Nature biotechnology, 18(2), 163-167. Flores, G., Dastmalchi, K., Wu, S. B., Whalen, K., Dabo, A. J., Reynertson, K. A., Foronjy, R. F., D’Armiento, J.M. & Kennelly, E. J. (2013). Phenolic-rich extract from the Costa Rican guava (Psidium friedrichsthalianum) pulp with antioxidant and anti-inflammatory activity. Potential for COPD therapy. Food chemistry, 141(2), 889-895. Huang, C. S., Yin, M. C., & Chiu, L. C. (2011). Antihyperglycemic and antioxidative. potential. of. Psidium. guajava. fruit. in. streptozotocin-induced diabetic rats. Food and Chemical Toxicology, 49(9), 2189-2195. Hunyadi, A., Martins, A., Hsieh, T. J., Seres, A., & Zupkó, I. (2012). Chlorogenic acid and rutin play a major role in the in vivo anti-diabetic activity of Morus alba leaf extract on type II diabetic rats. PLOS ONE, e50619. Jha, J. C., Gray, S. P., Barit, D., Okabe, J., El-Osta, A., Namikoshi, T., Thallas-Bonke, V., Wingler, K., Szyndralewiez, C., Heitz, F., Touyz, R. M., Cooper, M. E., Schmidt, H. H. W. & Jandeleit-Dahm, K. A. (2014). 40.

(51) Genetic targeting or pharmacologic inhibition of NADPH oxidase Nox4 provides renoprotection in long-term diabetic nephropathy. Journal of the American Society of Nephrology, ASN-2013070810. Kakehi, T., & Yabe-Nishimura, C. (2008, July). NOX enzymes and diabetic complications. In Seminars in immunopathology (Vol. 30, No. 3, pp. 301-314). Springer-Verlag. Kang, R., Zeh, H. J., Lotze, M. T., & Tang, D. (2011). The Beclin 1 network. regulates. autophagy. and. apoptosis.. Cell. Death. &. Differentiation, 18(4), 571-580. Liu, R., Barkhordarian, H., Emadi, S., Park, C. B., & Sierks, M. R. (2005). Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42. Neurobiology of disease, 20(1), 74-81. Katsuki, A., Sumida, Y., Gabazza, E. C., Murashima, S., Furuta, M., Araki-Sasaki, R.,Hori, Y., Yano. Y. & Adachi, Y. (2001). Homeostasis model assessment is a reliable indicator of insulin resistance during follow-up of patients with type 2 diabetes. Diabetes care, 24(2), 362-365. Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D., Novelli, M. & Ribes, G. (1998). Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes, 47(2), 224-229. Masiello, P., Broca, C., Gross, R., Roye, M., Manteghetti, M., Hillaire-Buys, D., Novelli, M. & Ribes, G. (1998). Experimental NIDDM: development of a new model in adult rats administered streptozotocin and nicotinamide. Diabetes, 47(2), 224-229. 41.

(52) Masini, M., Bugliani, M., Lupi, R., Del Guerra, S., Boggi, U., Filipponi, F., Marselli, L., Masiellom, P. & Marchetti, P. (2009). Autophagy in human type 2 diabetes pancreatic beta cells. Diabetologia, 52(6), 1083-1086. Molitch, M. E., Defronzo, R. A., Franz, M. J., Keane, W. F., Mogensen, C. E., Parving, H. H., & Steffes, M. W. (2004). Nephropathy in diabetes. Diabetes care, 27, S79-83. Nakamura, T., Terajima, T., Ogata, T., Ueno, K., Hashimoto, N., Ono, K., &. Yano,. S.. (2006).. Establishment. and. pathophysiological. characterization of type 2 diabetic mouse model produced by streptozotocin and nicotinamide.Biological and Pharmaceutical Bulletin, 29(6), 1167-1174. Srinivasan, K., Viswanad, B., Asrat, L., Kaul, C. L., & Ramarao, P. (2005). Combination of high-fat diet-fed and low-dose streptozotocin-treated rat: a model for type 2 diabetes and pharmacological screening. Pharmacological Research, 52(4), 313-320. Sarkar, S., Davies, J. E., Huang, Z., Tunnacliffe, A., & Rubinsztein, D. C. (2007). Trehalose, a novel mTOR-independent autophagy enhancer, accelerates the clearance of mutant huntingtin and α-synuclein. Journal of Biological Chemistry, 282(8), 5641-5652. Stumvoll, M., Goldstein, B. J., & van Haeften, T. W. (2005). Type 2 diabetes: principles of pathogenesis and therapy. The Lancet, 365(9467), 1333-1346. Tas, S., Sarandol, E., Ayvalik, S. Z., Serdar, Z., & Dirican, M. (2007). Vanadyl sulfate, taurine, and combined vanadyl sulfate and taurine. 42.

(53) treatments in diabetic rats: effects on the oxidative and antioxidative systems. Archives of medical research, 38(3), 276-283. Xu, C., Li, X., Wang, F., Weng, H., & Yang, P. (2013). Trehalose prevents neural tube defects by correcting maternal diabetes-suppressed autophagy. and. neurogenesis.. American. Journal. of. Physiology-Endocrinology and Metabolism,305(5), E667-E678. Yadav, H., Jain, S., & Sinha, P. R. (2007). Antidiabetic effect of probiotic dahi containing Lactobacillus acidophilus and Lactobacillus casei in high fructose fed rats. Nutrition, 23(1), 62-68. Yoon, K. H., Lee, J. H., Kim, J. W., Cho, J. H., Choi, Y. H., Ko, S. H., ... & Son, H. Y. (2006). Epidemic obesity and type 2 diabetes in Asia. The Lancet,368(9548), 1681-1688.. 43.

(54) 6. Figures and Tables CON. DM. T1. T2. T5. B1. Body weight (g). 305.0±13.0. 258.8±9.4a. 308.0±7.1b. 295.4±6.9b. 288.8±9.7b. 265.0±7.3a. Food intake (g/day). 24.1±1.2. 32.20±2.7a. 22.5±3.2. 26.2±2.2. 33.2±2.8a. 34.1±1.0a. Water intake (ml/day). 42.0±1.9. 100±0.0a. 87.6±4.3ab. 79.8±7.4ab. 100.0±0.0a. 96.8±2.8a. Urine (g/day). 18.8±3.5. 86.4±9.2a. 95.9±5.8a. 81.6±10.1a. 80.0±9.2a. 98.9±5.9a. Kidney(mg)/BW(g). 3.7±0.1. 6.1±0.2a. 6.0±0.1a. 5.4±0.4a. 5.3±0.1a. 5.5±0.1a. Table 1. Data was expressed as mean ± SEM. DM. n=3-5 each group. a P <0.05 vs. CON. b P <0.05 vs. DM.. 44.

(55) A.. B.. Figure 1. Different concentration of guava juice on scavenging ROS levels of H2O2 (A) and HOCl (B) in vitro. Using ddH2O as a reference control, the increased H2O2 and HOCl counts were significantly and dose-dependently decreased in 5%-40% of guava juice. 40% of guava juice displayed the strongest scavenging ROS activity. All data was expressed as mean ± SEM. n=3-5 in each group. * P < 0.05 vs. CON.. 45.

(56) A.. B.. Figure 2. Different concentration of trehalose on scavenging ROS levels of H2O2 (A) and HOCl (B) in vitro. Using ddH2O as a reference control, the increased H2O2, but not HOCl were significantly and dose-dependently decreased in 10%-50% of trehalose. n=3-5 in each group. H2O: ddH2O; T10: 10% trehalose in ddH2O; T20: 20% trehalose dissolved in ddH2O; T30, 30% trehalose in ddH2O; T50, 50% trehalose in ddH2O. All data was expressed as 46.

(57) mean ± SEM. n=3-5 in each group. * P < 0.05 vs. CON.. 47.

(58) Figure 3. Typical Chromatograph of Quercetin content in the Extract of Guava by HPLC Analysis. The chromatograph of the standard mixture is shown in the left panel, whereas the chromatograph of the water guava exraction was indicated in the right panel. Five chemicals including Ellagic acid, Ferulic acid, Rosmarinic acid, Quercetin and Narinegenin were detected in the smaples.. 48.

(59) Figure 4. Quercetin content in the extract of Guava juice by HPLC analysis. The concentrations of quercetin were 182.3 ng/mL in GWE1, 133.4 ng/mL in GWE2, 223.1 ng/mL in GEE1 and 244.5 ng/mL in GEE2. Water extract of Thailand guava, GWE1; Water extract of pearl guava, GWE2; ethanol extract of Thailand guava, GEE1; ethanol extract of pearl guava, GEE2.. 49.

(60) A.. B.. Figure 5. Different kinds of guava extraction on scavenging ROS levels of H2O2 (A) and HOCl (B) in vitro. Water extract of Thailand guava (GWE1) and pearl guava (GWE2) and ethanol extract of Thailand guava (GEE1) and pearl guava (GEE2) were used to evaluate their ROS scavenging ability. Using ddH2O as a reference control, the increased H2O2 and HOCl counts were significantly (P < 0.05) decreased by four kinds of guava extraction. GWE1 and GWE2 had a significantly efficient potential (P < 0.05) than GEE1 and GEE2 in scavenging H2O2 and HOCl activity. All data was expressed as mean ± SEM. n=3-5 in each group. * P < 0.05 vs. CON.. 50.