Neuroprotective Potential of a Lanosteryl Triterpene from Protorhus longifolia Stem Bark (Benrh.) Engl in high fat diet fed and STZ -induced diabetic neuropathy in rats

Chronic hyperglycemia, observed in the diabetic state, is responsible for various diabetes related complications including development of diabetic nephropathy. Diabetic neuropathy development and progression is linked to various hyperglycemia-induced cellular and tissue damaging factors such as advanced glycation end product (AGE) formation, oxidative stress and inflammation. Activation of these pathophysiological pathways alters the antioxidant defense of nerve cells and cause protein malfunction, resulting in loss of neurotrophic support. Currently, there is no specific treatment for neuropathy and the current antidiabetic drugs fail to prevent development of long-term diabetic complications. Therefore, researchers have renewed their interest in the exploration of natural products as alternative potential remedies for the disease treatment. The present study aimed at investigating the neuroprotective potential of lanosteryl triterpene (RA-3) from Protorhus longifolia stem bark in high fat diet (HFD) fed and streptozotocin (STZ) -induced diabetic neuropathy in rats. The triterpene was isolated from the chloroform extract of P. longifolia stem bark using silica gel column chromatography. The chemical structure of RA-3 was confirmed by spectral data analysis. The in vitro anti-glycation activity of the triterpene was investigated using BSA and haemoglobin-fructose assays. The HFD fed and STZ- induced diabetic neuropathy in the rat model was used to evaluate the diabetic neuroprotective potential of RA-3. The animals were initially divided into two major groups, the normal fed and HFD fed rats for 28 days. The HFD group rats were then injected (intraperitoneally) with a low dose of STZ (30 mg/kg body weight) to induce diabetes. The diabetic rats were further randomly divided into three groups: diabetic control, positive control treated with metformin (100 mg/kg) and RA-3 (100 mg/kg) treated group. The drugs were orally administered daily for 28 days. The tail flick method was used to assess the responses of the rats to cold and hot allodynia following 28 days of treatment with the respective drugs. On the last day of the experimental period, all rats were euthanized and blood (serum), sciatic nerve and brain tissues were collected for analysis of biochemical parameters. v The in vitro results revealed the anti-protein glycation potential of RA-3, accompanied by reduced fructosamine content and enhanced protection of protein thiol groups. Elevated fasting plasma glucose and serum fructosamine levels, as well as a significant reduction in the sciatic nerve levels of nerve growth factor (NGF), were detected in the non-treated diabetic group. However, treatment of the diabetic rats with RA-3 effectively lowered the fasting blood glucose and serum fructosamine while increasing the NGF levels. Relatively reduced sensation to both hot and cold allodynia, along with reduced activity of acetylcholinesterase, were also detected in the RA-3-treated diabetic rats when compared to the increased parameters in the untreated diabetic group. Treatment of the diabetic rats with RA-3 further exhibited increased tissue (sciatic nerve) antioxidant status, superoxide dismutase and reduced glutathione content accompanied by reduction in malondialdehyde levels. Relatively lower levels of pro-inflammatory markers (cyclooxygenase-2, tumor necrosis factor alpha, interleukin-6 and transforming growth factor beta-1) were also observed in both the serum and sciatic nerve tissue of the RA-3 treated diabetic rats. The levels of these inflammatory markers were elevated in the diabetic control group. The results obtained indicate that RA-3 possesses anti-diabetic neuroprotective potential. Its mechanism of action could be linked to its antihyperglycemic, antioxidant and anti-inflammatory activities.
A thesis submitted in fulfillment of the requirement for Masters degree in the Department of Biochemistry and Microbiology, Faculty of Science and Agriculture, University of Zululand,2019.
Neuroprotective Potential, Protorhus longifolia Stem Bark