AIM OF STUDY: To investigate the potential protective effects of L. flavescens in pancreatic β cells through inhibition of apoptosis and autophagy cell death mechanisms in in vitro and in vivo models.
MATERIALS AND METHODS: L. flavescens leaves were extracted using solvent in increasing polarities: hexane, ethyl acetate, methanol and water. All extracts were tested for INS-1 β cells viability stimulated by streptozotocin (STZ). The extract which promotes the highest cell protective activity was further evaluated for insulin secretion, apoptosis and autophagy signaling pathways. Then, the acute toxicity of extract was carried out in SD rats according to OECD 423 guideline. The active extract was tested in diabetic rats where the pancreatic β islets were evaluated for insulin, apoptosis and autophagy protein.
RESULTS: The methanolic extract of L. flavescens (MELF) was found to increase INS-1 β cells viability and insulin secretion against STZ. In addition, MELF has been shown to inhibit INS-1 β cells apoptosis and autophagy activity. Notably, there was no toxicity observed in SD rats when administered with MELF. Furthermore, MELF exhibited anti-hyperglycemic activity in diabetic rats where apoptosis and autophagy protein expression was found to be suppressed in pancreatic β islets.
CONCLUSION: MELF was found to protect pancreatic β cells function from STZ-induced apoptosis and autophagy in in vitro and in vivo.
Methods: 2,2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric reducing antioxidant power assay (FRAP) were applied to evaluate the antioxidant activity of carob. In vitro cytotoxicity of carob was conducted on human hepatocytes (WRL68) and rat pancreatic β-cells (RIN-5F). Acute oral toxicity of carob was conducted on a total of 18 male and 18 female Sprague-Dawley (SD) rats, which were subdivided into three groups (n = 6), namely: high and low dose carob-treated (CS5000 and CS2000, respectively) as well as the normal control (NC) receiving a single oral dose of 5,000 mg kg-1 carob, 2,000 mg kg-1 carob and 5 mL kg-1 distilled water for 14 days, respectively. Alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, total bilirubin, creatinine and urea were assessed. Livers and kidneys were harvested for histopathology. In vitro inhibitory effect against α-amylase and α-glucosidase was evaluated. In vivo glycemic activity was conducted on 24 male SD rats which were previously intraperitoneally injected with 55 mg kg-1 streptozotocin (STZ) followed by 210 mg kg-1nicotinamide to induce type 2 diabetes mellitus. An extra non-injected group (n = 6) was added as a normal control (NC). The injected-rats were divided into four groups (n = 6), namely: diabetic control (D0), 5 mg kg-1glibenclamide-treated diabetic (GD), 500 mg kg-1 carob-treated diabetic (CS500) and 1,000 mg kg-1 carob-treated diabetic (CS1000). All groups received a single oral daily dose of their treatment for 4 weeks. Body weight, fasting blood glucose (FBG), oral glucose tolerance test, biochemistry, insulin and hemostatic model assessment were assessed. Pancreases was harvested for histopathology.
Results: Carob demonstrated a FRAP value of 3191.67 ± 54.34 µmoL Fe++ and IC50 of DPPH of 11.23 ± 0.47 µg mL-1. In vitro, carob was non-toxic on hepatocytes and pancreatic β-cells. In acute oral toxicity, liver and kidney functions and their histological sections showed no abnormalities. Carob exerted an in vitro inhibitory effect against α-amylase and α-glucosidase with IC50 of 92.99 ± 0.22 and 97.13 ± 4.11 µg mL-1, respectively. In diabetic induced rats, FBG of CS1000 was significantly less than diabetic control. Histological pancreatic sections of CS1000 showed less destruction of β-cells than CS500 and diabetic control.
Conclusion: Carob pod did not cause acute systemic toxicity and showed in vitro antioxidant effects. On the other hand, inhibiting α-amylase and α-glucosidase was evident. Interestingly, a high dose of carob exhibits an in vivo antihyperglycemic activity and warrants further in-depth study to identify the potential carob extract composition.
MATERIALS AND METHODS: Male Wistar rats were used for the experiments. Blood glucose (BG), urea, blood pressure (BP), and heart rate (HR) were analyzed before and 48 h after STZ injection. Further, these parameters were monitored up to 3 months of diabetes induction. Subsequently, the inflammatory markers (C-reactive protein, tumor necrosis factor-alpha, and nitrate) and oxidative stress markers were estimated after 3 months of diabetes induction in the kidney homogenate. Histological analysis of renal tissue was also carried out.
RESULTS: Linear elevation of BG, urea, mean arterial pressure (MAP), and HR was observed up to 3 months of diabetes induction. In the same manner, inflammatory and oxidative stress markers were also found to be significantly increased. Notably, the histological analysis revealed the signs of nephropathy such as increased mesangial cell number, thickness of basement membrane, and renal artery. Inflammatory and oxidative stress markers positively correlated with elevated BP and BG, but the correlation was better with BP rather than BG.
CONCLUSION: Hypertension has a strong implication in the increased oxidative stress and inflammation of diabetic kidney at the very early stage of diabetes mellitus.