Changes in pancreatic histology, insulin secretion and oxidative status in diabetic rats following treatment with Ficus deltoidea and vitexin

The leaves of F. deltoidea var. deltoidea were collected from Forest Research Institute Malaysia, Kepong, Malaysia in January 2015. The sample was then deposited at the Herbarium Unit, Universiti Kebangsaan Malaysia, Bangi and identified by Mr. Sani Miran with a voucher number Herbarium UKMB-40315. The leaves were washed thoroughly and over-dried at 37 ± 5 °C. The dried leaves were finely powdered using an electric grinder. For extraction, 100 g of powdered leaves was soaked in 1 L absolute methanol for three days at room temperature [16]. Liquid extracts were concentrated using a rotary evaporator at 40 °C and subjected to freeze drying for 48 h. The extraction yield calculated was 10.6%.The extracts were kept in tightly closed glass containers and stored at −20 °C until further use.

The animal use and experimental protocols involved in the study were approved by the Universiti Putra Malaysia, Animal Care and Use Committee with an approval number: UPM/IACUC/AUP-R090/2014. A total of 30 male Sprague Dawley rats of four-week-old (mean body weight, 100 ± 5 g) were procured from Chenur Supplier Sdn. Bhd., Serdang, Selangor. The rats were housed at Laboratory Animal Facility and Management (LAFAM), Universiti Teknologi MARA, Puncak Alam, Selangor. The animals were acclimatized upon arrival for a week and were housed at a density of three per cage in a temperature controlled room (22 ± 1 °C and a 12 h light/dark cycle). The blood glucose levels and body weights of all animals were measured at the beginning of the study. The rats were identified with a cage card indicating project number, dose level, group, and animal number. They had access to standard rat chow (Gold Coin Holdings, Kuala Lumpur, Malaysia) and water ad libitum.

Diabetes-like hyperglycaemia was induced experimentally in rats through intraperitoneal injection of 0.5 ml STZ (Sigma-Aldrich, Deisenhofen, Germany) at a dosage of 60 mg/kg of body weight (b.w.) [20]. After a week, animals with fasting blood glucose levels >11 mmol/L were considered diabetic [21].

The rats were divided into five groups of six rats per treatment group. The treatment group were normal control rats received saline (NC), diabetic control rats received saline (DC), Diabetic rats treated with 1000 mg/kg b.w. of metformin (DMET) [22], diabetic rats treated with 1000 mg/kg b.w. of F.deltoidea (DFD) [16], diabetic rats treated with 1 mg/kg b.w. of vitexin (DV) [11].

At the end of the experiment, all animals were fasted overnight. Animals were then anesthetized with ketamine (80 mg/kg) and xylazine (8 mg/kg), followed by terminal exsanguination. Blood samples (10-15 ml) were collected via cardiac puncture from the rats into plain red-top tube containing no anticoagulants (BD Vacutainer®, USA). The blood samples were then centrifuged at 4000  g for 15 minutes, and serum was stored in aliquots at −80 °C. Triglycerides in serum were determined using an automatic analyser (Hitachi 911, Boehringer-Mannheim, Germany). Meanwhile, pancreas was carefully excised, rinsed in ice-cold saline and stored in 10% formalin for tissue characterization.

The intraperitoneal glucose tolerance test (IPGTT) and intraperitoneal insulin tolerance test (IPITT) were performed at the end of 8 weeks treatment on all experimental groups. These tests were performed with minor modifications following the method described by Abdollahi [23]. The rats were fasted for 8 h prior to testing. For the IPGTT, glucose (2 g/kg) was injected intraperitoneally at time 0. Following day, the rats were intraperitoneally injected with insulin (1.5 IU/kg) for the IPITT. Blood glucose measurements from the tails were performed at 0, 30, 60 and 120 min. The blood glucose concentration versus time (minutes) was plotted and the area under curve (AUC) was calculated following the trapezoidal rule.

The levels of serum insulin were determined by Enzyme Linked Immunosorbent Assay kit specific for rat insulin (Cloud-Clone Corp., Houston, USA) as described by Zhang et al. [24]. Serum was allowed to thaw and 50 μl serum was pipetted into duplicate wells followed by 50 μl of enzyme conjugate solution. This mixture was incubated for 1 h on a plate shaker (800 rpm) at 37 °C. The plate was inverted on an absorbent paper after the final wash. Afterward, 90 μl of substrate solution was pipetted and incubated at 37 °C. Finally, the reaction was stopped 15 min later by adding 50 μl of stop solution to each well. The optical density was read at 450 nm with microplate reader (Epoch Microplate Spectrophotometer, BioTek, USA). The values for the calibrators were used to plot a calibrator curve from which the values for the samples were extrapolated.

Three indirect indexes for the assessment of insulin sensitivity were calculated using serum insulin, glucose and triglyceride at the end of the experimental period. The HOMA index uses the formula described by Matthews et al. [25] while the quantitative insulin sensitivity check index (QUICKI) is based on the logarithmic transformation. Accordingly, McAuley’s index [26] was calculated based on the increase of triglycerides levels and insulin according to the following equations:

The paraffin-embedded pancreas was sectioned at 4 μm using a semiautomated microtome (RM2155; Leica Micro-systems). The tissue sections were then mounted on glass slides using a hot plate (HI1220; Leica Microsystems). Afterward, the tissue sections were deparafinized by xylene and rehydrated by different graded ethanol dilution (100%, 90%, and 70%). The sections were stained with hematoxylin and eosin (H&E). All slides were examined using light microscopy (Motic BA410, Wetzlar, Germany) equipped with a digital camera (Moticam Pro 285A, Wetzlar, Germany) under a magnification of X200.

The amylin level in the serum was determined by using Rat Amylin Enzyme Immunoassay Kit (RayBiotech Incorporation, USA). Standards and reagents were prepared carefully according to the procedure in the instruction manual prior the assay. One hundred microliters of anti-amylin antibody was added to each well and incubated overnight at 4 °C. The solution was then discarded and washed four times by using 1X wash buffer. A 100 μl of each standard, sample and blank (assay diluent) were added in triplicates to the wells. The wells were then covered and incubated for overnight at 4 °C. The solutions were discarded and washed four times. An amount of 100 μl HRP-streptavidin solution was added to each well and incubated for 45 min at room temperature with gentle shaking. One hundred microliters of TMB one-step substrate reagent was added to each well and allowed to incubate for 30 min at room temperature in the dark with gentle shaking. Finally, 50 μl of stop solution was added to each well and the absorbance was immediately read at 450 nm wavelength using microplate reader (Epoch 2 microplate spectrophotometer, BioTek Instruments, Inc., Vermont, USA).

The pancreatic tissue was homogenized in 10% (w/v) homogenizing buffer (50 mM Tris-HCl, 1.15% KCl pH 7.4) using a Teflon pestle (Glass-Col, USA) at 900 rpm. The homogenates were centrifuged at 9000 g in a refrigerated centrifuge (4 °C) for 10 min to remove nuclei and debris. The supernatant obtained was used for biochemical assays and FT-IR analysis. Protein concentration was estimated by the method of Lowry [27], using bovine serum albumin as the standard.

The levels of MDA equivalents were determined in pancreas by TBARS assay kit (Cayman, MI, USA) as describe by Hardwick et al. [28]. The absorbance was determined spectrophotometrically at wavelength of 540 nm using a spectrophotometer.

Glutathione peroxidase (GPx) activity was measured using assay kit (Cayman, MI, USA). The experimental procedures were carried out according to the manufacturer’s instructions [29]. The measurement of GPx activity is based on the principle of a coupled reaction with glutathione reductase (GR). The oxidized glutathione (GSSG) formed after reduction of hydroperoxide by GPx is recycled to its reduced state by GR in the presence of NADPH. The oxidation of NADPH is accompanied by a decrease in absorbance at 340 nm. One unit of GPx was defined as the amount of enzyme that catalyzes the oxidation of 1 nmol of NADPH per minute at 25 °C.

Superoxide dismutase (SOD) activity was determined using assay kit (Cayman, MI, USA). This kit utilizes a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD was defined as the amount of enzyme needed to produce 50% dismutation of superoxide radical.

Lipids from the serum and pancreas were extracted according to the methods described by Hajjar et al. [30]. According to this method, 1 ml of serum were thawed at room temperature for 30 min and extracted using the Folch method [31] (chloroform:methanol, 2:1, v/v) containing butylated hydroxytoluene as antioxidant. Then, fatty acids methyl esters (FAME) were prepared using 0.66 N potassium hydroxide (KOH) in methanol and 14% methanolic boron trifluoride (BF₃) (Sigma Chemical Co. St. Louis, Missouri, USA). The FAME were separated with an Agilent 7890A Series GC system (Agilent Technologies, Palo Alto, CA, USA) using a 30 m × 0.25 mm ID (0.20 μm film thickness) Supelco SP-2330 capillary column (Supelco, Inc., Bellefonte, PA, USA). The fatty acid proportions are expressed as percentage of total identified fatty acids. One microlitre of FAME was injected by an auto sampler into the chromatograph, equipped with a split/splitless injector and a flame ionization detector (FID) detector. The injector temperature was programmed at 250 °C and the detector temperature was 300 °C. The column temperature program initiated runs at 100 °C, for 2 min, warmed to 170 °C at 10 °C/min, held for 2 min, warmed to 200 °C at 7.5 °C/min, and then held for 20 min to facilitate optimal separation. Identification of fatty acids was carried out by comparing relative FAME peak retention times of samples to standards obtained from Sigma (St. Louis, MO, USA).

Infrared spectroscopic experiments were performed using a Bruker 66 V FT-IR spectrometer (Bruker Corp., MA, USA) that was equipped with a focal plane array detector. Twenty microliter of each homogenate sample was then deposited on a liquid cell (demounted cell) using a pipette according to the method reported by Demir et al. [32]. All individual FT-IR spectra were recorded over the range 4000-400 cm⁻¹ at room temperature. In order to resolve the overlapped absorption components in FT-IR spectra, second derivative spectra were calculated using Savitzky–Golay algorithm. All spectra processing was performed by using OPUS 7.0 software (Bruker Optics, GmbH). The peaks of each spectrum curve were then fitted and calculated. The region enriched vibration changes were compared to existing literature database towards providing chemical information on the targeted tissues. Absorptions belonging to fatty acyl chains, proteins and carbohydrates of biological samples are basically available in the 3020-2800 cm⁻¹, 1700-1500 cm⁻¹ and 1200-900 cm⁻¹ spectral intervals, respectively [33].

The fasting blood glucose, IPGTT and IPITT data sets were analysed using repeated measures ANOVA. Meanwhile, one-way ANOVA analyses were done on insulin levels, insulin sensitivity indexes, oxidative stress marker, antixioxidant enzymes serum and pancreas fatty acids compositions data sets to investigate the differences among the treated groups. In both cases Duncan’s multiple comparison test was employed to elucidate significant means. Results were presented as the mean ± 1 SD. All analysis was performed at 95% confidence level.

The means and standard deviations of FBG in entire groups are given in Table 1. The FBG concentrations in the DC animals were increased by 50.65% compared to initial values, confirming the validity of the diabetogenic dose of STZ in this study. It is noticeable that the treatment for 8 weeks with either metformin or F. deltoidea or vitexin resulted in a significant reduction in FBG. The FBG decreased by 38.03% and 47.85% following F. deltoidea and vitexin treatment, respectively as compared to pre-treatment values. All treated diabetic groups depict significant changes at week 6 of treatment.

As depicted in Table 2, serum insulin levels, HOMA-B, QUICKI and McAuley index was alleviated markedly in the DC rats compared to the NC rats. It was also found that STZ associated with a significant increase in serum triglyceride and HOMA-IR index. Notably, F. deltoidea increased the secretion of insulin. Adhering to three indirect indexes used to predict insulin sensitivity, both F. deltoidea and vitexin had a significant impact in lowering insulin resistance. Triglyceride levels also decreased in both the DFD and DV rats. Nevertheless, both treatments failed to improve the HOMA-B scores.

The histopathology of rat pancreas was shown in Fig. 2. Microscopic investigation of pancreas sections of NC rats showed the normal appearance of islets of Langerhans. The islets appeared lightly stained than the surrounding acinar cells. The acinar cells are formed of pyramidal cells with basal nuclei and apical acidophilic cytoplasm (Fig. 2a). However, the DC rats showed pathological changes of both exocrine and endocrine components. The acinar cells were swollen and small vacuoles were observed in almost all acinar cells. Interlobular ducts were lined with flattened epithelium. Islet β-cells are almost entirely lost in STZ-treated rats (Fig. 2b). Similar findings were obtained in the DMET rats. In fact, both the DC and DMET pancreas associated with different intensity of eosin as compared to the NC rats (Fig. 2c). On the other hand, DFD and DV groups depicted evidence of cellular regeneration among the islets of Langerhans (Fig. 2d and e). Atrophic change of the acinar cells was less severe and the border between exocrine and endocrine portions became more distinct.

Table 3 summarizes the results for the effect of F. deltoidea and vitexin on pancreas lipid peroxidation and the activities of antioxidant enzymes. There was a significant decrease in the activity of SOD and GPx upon injection of STZ, reflecting the depletion of endogenous antioxidant enzymes activities in serum. Strikingly, administration of F. deltoidea to diabetic rats resulted in a significant increase in the SOD and GPx values relative to the DC group. Although GPx markedly increased following vitexin treatment, the SOD activity remained low throughout the studied period. Nevertheless, the DFD and DV groups associated with a significant reduction in pancreatic TBARS.

As shown in Fig. 3, serum amylin levels were significantly increased in the DC group. However, metformin and F. deltoidea treatments significantly reduced the levels of serum amylin.

Table 4 lists the percentages of total fatty acids in the serum. It was demonstrated that the total serum saturated fatty acid (SFA), monounsaturated fatty acid (MUFA), and polyunsaturated fatty acids (PUFA) were roughly similar across the treatment groups. However, there were significant differences (P < 0.05) in the serum fatty acid profiles of DV group. The DV animals had more total n-3 PUFA in their serum as compared to the DC and NC animals. The serum docosahexaenoic acid (DHA) was clearly the highest in the DV groups.

It is noticeable that the injection of STZ had insignificant effect on the total SFA and PUFA (Table 5). However, the DC group clearly had the lowest level of total MUFA. Similarly, the DV animals experienced significant decline of total pancreas MUFA at the end of the trial. Oleic acid was significantly decreased in both groups. However, the total n-3 PUFA levels in the pancreas of DV animals was more than three-fold and two-fold higher than those found in the NC and DC animals, respectively.

The FT-IR spectra changes in response to alterations in tissue and serum glucose upon F. deltoidea and vitexin treatment were investigated. In general, all animal groups had strong bands at 1660 cm⁻¹, arising from C = O stretching vibration of the peptide group and another broad peak at 3100-3600 cm⁻¹ corresponding to stretching of the hydroxyl groups (Fig. 4a). As shown in Fig. 4b, the most obvious differences between experimental groups were found in the range of 1200 and 1000 cm⁻¹. This peak indicates the presence of sugar.

The second derivative spectra below shows that the glucose peak intensities at 1080 and 1036 cm⁻¹ increased in the DC rats with high blood glucose. The intensity of glucose peak was nevertheless decreased among all treated groups (Fig. 5b-d), which is consistent with the reduction of FBG concentrations (Table 1). This data indicates a possible correlation between the peak intensities at 1080 and 1036 cm⁻¹ and the blood glucose concentration obtained by conventional methods. Unexpectedly, the IR spectra of serum from the DMET and DV rats elicited a greater increase in the intensity of fructose peak at 1057 cm⁻¹. However, lesser intensity of fructose band was observed in the IR spectra of DFD group.

The FT-IR spectra of pancreas tissues from different groups are shown in Fig. 6a. For detailed analysis of IR spectra, the region was divided into two distinct frequency ranges, namely 3000-2800 cm⁻¹ (Fig. 6b) and 1500-1050 cm⁻¹ (Fig. 6c). It was noticed that the IR spectra of pancreas are different at three regions which were 3000-2800 cm⁻¹, 1450-1380 cm⁻¹, and 1200-1000 cm⁻¹. These observations imply that the structural and functional changes in pancreas are somehow related to lipid, protein and glucose specific absorption band.

The second derivative analysis showed that the most dominant peak in the region of 3000-2800 cm⁻¹ had decreased to levels far below normal in the DC and DMET groups. Interestingly, the spectra of pancreas from the DFD and DV rats are almost similar to that of the normal spectrum (Fig. 7c and d). These results suggest that lipid is involved in pancreatic regeneration.

It was found that the DC animals are associated with a lower intensity of band within 1440-1390 cm⁻¹ region (Fig. 8a), indicating the diminished of methyl groups for enhancing pancreatic β cell regeneration. Similar FT-IR spectra were obtained from the DMET groups as illustrated in Fig. 8b. However, second derivative spectra showed the reappearance of weak peak at 1422 and 1408 cm⁻¹ in the DMET group. As seen in Fig. 8c, the spectra of DFD group displayed a clearer reappearance of the methyl band with minor shifts. Meanwhile, similar spectral characteristics seen in the NC animals illustrated in the spectra of DV group (Fig. 8d).

Figure 9a demonstrates that the pancreatic tissues from NC animals depict the presence of five significant peaks in the region of 1190-1100 cm⁻¹. However, the intensity and frequency of the sugar band of the DC group was reduced and shifted especially between 1130 and 1106 cm⁻¹. Strikingly, the spectra of DFD and DV treated groups do contain some similarities to the NC group. Both groups displayed five peaks within the region, suggesting that glucose is important elements toward the regeneration of pancreatic β-cells.