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
23
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
24
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
25
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.
26
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. 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.
27
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
28
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
29
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
30
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.
31
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
32
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)
33