Pharmacological Evaluation of Phenolic Acids in Neonatal Streptzotocin (nSTX)-induces Type 2 Diabetes Mellitus Associated Complications

Abstract

Diabetes mellitus is a metabolic disease characterized by severe and persistent hyperglycemia as a result of increased insulin resistance and decreased secretion from pancreatic cells. It is one of the world's top causes of death. Improper treatment or poor hyperglycemic control frequently leads to chronic complications such as diabetic nephropathy, neuropathy, cardiomyopathy, and liver injury. The neonatal streptozotocin-diabetic rat model (nSTZ) is a well-known and widely utilized animal model to induce type 2 diabetes mellitus (T2DM). Phenolic acids, a subclass of plant phenolics, are found to be beneficial for cancer, diabetes, and cardiovascular diseases. Syringic acid (SA) and vanillic acid (VA) have been reported to possess strong antioxidant, anti-diabetic, anti-inflammatory, anti-glycating, nephroprotective, hepatoprotective, neuroprotective, cardioprotective activities etc. The present study aimed to investigate the safety of SA and VA as well as their protective effect on T2DM-associated diabetic complications in Wistar rats. A subacute study in rats was carried out to assess the safety of these phenolic acids when used for a long time. A limit test was performed for both SA and VA by the administration of a single dose of 2000 mg/kg p.o, and rats were observed for 14 days for any toxic signs and mortality. Based on the findings of the limit test, 1000 mg/kg was considered to be a safe dose for subacute toxicity studies. For subacute toxicity study, animals were divided in 3 groups [in each group n= 12 (6 male and 6 female rats)]; Control group; Treatment group received SA and VA (1000 mg/kg/day, p.o for 14 days); Satellite group received SA and VA (1000 mg/kg/day, p.o for 14 days) and observed for another 14 days post treatment. Physical parameters such as food intake (FI), water intake (WI) and body weight (BW) were measured every week. Serum biochemistry, hematological evaluations, relative body organ weight, and histopathological studies were performed at the end of the study period. In conclusion, SA and VA were found to be safe in subacute toxicity studies. A neonatal diabetes model was established, and the dose of STZ was validated to get the fasting blood glucose (FBG) level to > 150 mg/dl in adult rats. STZ at 110 mg/kg i.p was administered in split form. At the age of 8 weeks, cardinal symptoms of diabetes, such as hyperglycemia, polydipsia, polyphagia, polyuria, and a reduction in BW, were observed. Animals were divided into 9 groups (in each group n=6): Normal control (NC); NC+ SA (50 mg/kg p.o); NC+ VA (100 mg/kg p.o); n-STZ diabetic control (DC); DC+ SA (25 mg/kg p.o); DC+ SA (50 mg/kg p.o); DC+ VA (50 mg/kg p.o); DC+VA (100 mg/kg p.o); DC+ MET (200 mg/kg p.o). As per the experimental design, SA and VA have to be administered daily for 10 weeks, from the 8th week to the 18th week postnatal. To assess the development of diabetes, diabetic complications and progression of treatments in nSTZ rats, biochemical markers in serum and behavioural parameters (cold allodynia and thermal hyperalgesia) were assessed throughout the study period. The effects of SA and VA on T2DM-associated diabetic complications (nephropathy, neuropathy, cardiomyopathy, liver injury) were assessed with the help of various physical and cardiac hypertrophic parameters, biochemical estimations in serum and urine, oxidative stress, Na+/K+ ATPase level in the tissue homogenates and organ damage were assessed by histopathological study in the form of scores by a blind observer in all understudy body organs (kidney, liver, sciatic nerve and heart) at the end of treatment. Chronic SA and VA treatment reduced hyperglycemia, polydipsia, polyphagia, polyuria, relative renal weight, relative hepatic weight, and cardiac hypertrophic parameters. Furthermore, dyslipidemia was restored along with a significant reduction in inflammatory markers, biomarkers for cardiac cell injury, renal functional markers, biomarkers of hepatic cell injury, and oxidative stress in treatment groups. In all treatment groups, Na+/K+ ATPase activity was increased in renal, hepatic, cardiac, and sciatic nerve homogenates. In addition, histopathological scores were reduced in treatment groups as compared to diabetic control rats. In conclusion, SA and VA appear to have anti-oxidative stress, anti-inflammatory, and anti-hyperglycemic effects which might play role in the prevention of T2DM and T2DM-related complications. However, there is a need to explore further mechanisms of action of VA and SA through gene expression studies and target-specific studies, and also a need to evaluate the chronic safety to propose it for further human studies.