The Effects And Related-Mechanismsof Berberine In Ameliorating Neuropathic Pain In Diabetic Rats

Background: Diabetes mellitus（DM）is one of the most common chronic diseases presently with an incidence rate of 8.8%.Diabetes is usually accompanied by varieties of chronic complications.About 50% of diabetic patients will have different degrees of neuropathy,of which diabetic neuropathic pain（DNP）is the most common.DNP can cause severe physiological and psychological burden on patients and seriously affect their quality of life.Because of the complicated pathogenesis of DNP,latest research shows that oxidative stress,neuroinflammation and μ opioid receptor（MOR）may participate in the occurrence of neuropathic pain caused by diabetic neuropathy.Lipoic acid,statins,antioxidants and other drugs that inhibit the expression of inflammatory factors,as well as opioids can reduce the neuropathy to a certain extent and play a neuroprotective role in diabetic patients.However,it has said been that it is often difficult to obtain good results by using conventional chemosynthetic drugs.Therefore,it is important for clinical medical workers to find better drugs,especially natural drugs.Berberine is an isoquinoline alkaloid extracted from Coptis,phellodendron and barberry root.Berberine can reduce blood lipid,blood glucose,aids in antioxidation,inhibit inflammation and apoptosis.The study on C2C12 PC12 cells showed that Berberine does effectively inhibit the production of ROS and thus achieve the cell protection.In addition,Berberine can promote the recovery of colitis by inhibiting the inflammatory response of macrophages.Currently,berberine is being used in the clinical treatment of diabetes mellitus（DM）.Results showed that Berberine could alleviate the hyperalgesia of diabetic rats in a dose-dependent manner.However,its specific cellular and molecular mechanisms are not clear.When combined with the mechanism of DNP and molecular mechanism of Berberine’s cytoprotection,we hypothesized that Berberine might alleviate DNP by regulating oxidative stress,neuroinflammation or μ-opioid receptor level in rats.Therefore,this study intends to test the above hypothesis to further explore and reveal the effect of Berberine on DNP and its molecular mechanism.PART I.Diabetic Neuropathic Pain Model Construction and the therapeutic effects of Berberine on the DNPObjective: To establish a DNP rat model induced by streptozotocin（STZ）and determine whether the model is effective by blood glucose concentration and behavioral test,and further evaluate the therapeutic effects of berberine on DNP of rats.Methods: Male Wistar rats were randomly divided into two groups,normal control group（sham surgery group,citric acid sodium citrate buffer treatment）and diabetic model group（STZ）.The rats in the model group were injected intraperitoneally with a single dose of STZ（60mg / kg）.The rats in the control group were treated with the same dose of citrate sodium buffer.Fasting blood glucose level was measured 48 hours after administration and rats with blood glucose concentration greater than 16.7 mmol / L were selected as the study group for the diabetic model.Diabetic rats were weighed every 4 weeks after STZ injection and their mechanical and thermal pain thresholds were assessed by foot pressure test and tail flick test every 2 weeks.After the success of diabetic neuropathic pain model,DNP model animals were divided into model group,low dose group,moderate dose group and high dose group.The animals were given intraperitoneal injection of STZ + DMSO,5 mg / kg,20 mg / kg and 40 mg / kg of Berberine daily for 10 weeks.Blood glucose level and body weight were measured every 4 weeks.Foot pressure test and tail flick test were used to evaluate the mechanical pain threshold and thermal pain threshold of rats every two weeks.Results: After 48 hours of STZ injection,blood glucose of 78% of the rats was higher than 16.7 mmol / L.At the beginning of the 4th week after the STZ injection,the body weight of rats decreased significantly（P< 0.05）compared with the normal control group and the difference continued until the 12 th week.Compared with the model group,the blood glucose level of rats in the moderate dose（20 mg / kg）and high dose（40 mg / kg）of Berberine treatment groups were significantly lower（P< 0.01）.From the 4th week after the treatment,body weight of the moderate and high dose group was significantly higher than that of the model group（P< 0.05）.Compared with the model group,the moderate and high dose groups increased their reflexes to pressure stimulation（P< 0.001）,while the delayed time of tail flick reflex to heat stimulation was prolonged significantly（P< 0.001）and the difference lasted until the 12 th week.The relief effect of the high dose group on mechanical pain（P< 0.01）was better than that of the moderate dose group,but there was no significant difference in thermal pain.There was no significant difference in the above indexes between the low dose（5mg / kg）of Berberine treatment group and the model group.PART II.The,mechanisms ofalleviating DNP of rats by BerberineObjective: To evaluate the effects of Berberine on oxidative stress in DNP rats by detecting the contents of malondialdehyde（MDA）,hydrogen peroxide（H₂O₂）and catalase（CAT）activity in the spinal cord and dorsal root ganglia（DRG）after Berberine treatment and to infer whether Berberine can improve the oxidative stress state of DNP rats.to detect the expression of tumor necrosis factor-α（TNF-α）and interleukin-6（IL-6）in the spinal cord and DRG of DNP rats after Berberine treatment and to evaluate the level of neuroinflammation in rats,so as to verify whether Berberine can alleviate DNP by regulating neuroinflammation in rats.To detect the m RNA and protein expression of MOR in spinal cord and DRG of DNP rats after Berberine treatment and the ultrastructure of nerve axons,myelin sheath and capillaries in the spinal cord and brain of diabetic rats to explore the mechanism of berberine alleviating neuropathic pain in diabetic rats.Methods: After 10 weeks of Berberine treatment,spinal cord and DRG tissues of rats in the high dose treatment group were taken under anesthesia.The content of MDA,H₂O₂ and CAT activity in the spinal cord and DRG of rats were measured by spectrophotometry after tissue homogenization to evaluate the level of lipid peroxidation,active oxygen generation and total body oxidative stress;the protein expression levels of TNF-α and IL-6 were analyzed by Western blot to evaluate the level of inflammation;and the m RNA and protein expression levels of MOR were detected by QRT PCR and Western blot respectively.the spinal cord and DRG tissues of the high dose group were taken under anesthesia,and the m RNA and protein expression levels of MOR were detected by QRT PCR and Western blot respectively.Results: Compared with the model group,the level of MDA and the production of H₂O₂ in the spinal cord of rats in the high dose（40mg / kg）group decreased significantly（P< 0.05）,and the CAT activity increased significantly（P< 0.05）.At the same time,the level of MDA and the amount of H₂O₂ production of DRG in the treatment group were significantly lower than those in the model group（P< 0.05）,with CAT activity being significantly higher（P< 0.05）.compared with the model group,the protein expression level of TNF-α and IL-6 in the spinal cord of rats in the high dose（40 mg / kg）of Berberine treatment group decreased significantly（P< 0.05）and the protein levels of TNF-α and IL-6 in DRG of rats also decreased significantly（P< 0.05）.Compared with the model group,the expression of MOR m RNA（P< 0.05）and protein（P< 0.05）in the spinal cord of DNP rats had significantly increased by high dose（40 mg / kg）of Berberine treatment.At the same time,the m RNA（P< 0.05）and protein（P< 0.05）levels of MOR in DRG were significantly higher than those in the model group.Compared with the model group,high dose（40 mg / kg）berberine treatment improved the pathological ultrastructure of axons,myelin sheath and capillaries in spinal cord and brain.Conclusion:A single intraperitoneal injection of STZ cansuccessfully establish a diabetic rat model with DNP expression,which can be used as a DNP model for follow-up study.Berberine treatment can not only effectively reduce the STZ induced hyperglycemia and reduce weight loss,but also effectively inhibit the mechanical pain and thermal hyperalgesia in diabetic rats.Berberine has a dose-dependent effect on the treatment of DNP in rats,20 mg / kg and 40 mg / kg are the effective doses with 40 mg / kg being the best.Berberine up-regulates the expression of MOR in spinal and DRG,and effectively inhibits the oxidative stress and inflammation level,and the pathological ultrastructures of axons,myelin sheath and capillaries in spinal cord and brain were improved to DNP rats and alleviates DNP.