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%
9
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.
% by weight % kcal from
Protein 18.3 20.2
Carbohydrate 60.4 66.8
Fat 5.2 12.9
2-3 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.
10 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
11
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: (360fasted 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
12
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,
13
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
14
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
15
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
16
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
17
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
18
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
19
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
20
(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
21
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.
22
3. Results