Effects Of Dihydromyricetin On The Comorbid Diabetic Neuropathic Pain And Depressive Behavior In Rats Mediated By P2x₇ Receptor

Background:Diabetic neuropathic pain?DNP?is a common complication of diabetic neuropathy.It is usually characterized by burning pain of lower extremity skin.The incidence of pain or nerve injury symptoms in diabetic patients increases with age and course of disease.Severe diabetic neuropathic pain can also be associated with anxiety and depression?DP?.Compared with other complications of diabetes,diabetic neuropathic pain is a more important determinant of depression,and may aggravate depressive symptoms.Depression and diabetic neuropathic pain can promote each other's progress,and the comorbidity of diabetic neuropathic pain and depression would greatly impact on the quality of life of patients.At present,there is no effective treatment for the comorbidity of diabetic neuropathic pain and depression.P2X₇ is a member of purinergic receptor family.ATP can induce pain by activating P2X₇ receptor,plays an important role in neuroinflammatory pathway and participates in the occurrence of depressive symptoms.P2X₇ receptors on glial cells of the nervous system are important targets for comorbid diabetic neuropathic pain and depression.Dihydromyricetin?DHM?has many pharmacological effects,such as antioxidation,antiinflammatory,and can weaken nerve injury.Objective:In this study,we established a rat model of diabetic neuropathic pain combined with depression to observe the effect of DHM on the comorbiddiabetic neuropathic pain and depression,to explore its possible mechanism,to broaden and deepen the understanding of the role and the mechanism of DHM.The project will explore a new method for preventing and treating the comorbid diabetic neuropathic pain and depression.Methods:The SD rats were randomly divided into six groups:normal control group?control group?,normal and dihydromyricetin group?control+DHM group?,model group?DNP+DP group?,model and dihydromyricetin group?DNP+DP+DHM group?,model and P2X₇ shRNA group?DNP+DP+P2X₇ shRNA?and model and scramble shRNA group?DNP+DP+scramble shRNA?.The model of diabetic neuropathic pain combined with depression was established.The pain behavior of rats was monitored by by mechanical withdrawal threshold?MWT?and thermal withdrawl latency?TWL?.The depressive behavior of rats was monitored by sugar water preference test?SPT?,forced swimming test?FST?and open-field test?OFT?.The expression of P2X₇ receptor,TNF-?and IL-1?in dorsal root ganglion?DRG?and spinal cord?SPI?were detected by real-time fluorescence quantitative PCR?qPCR?and Western blot.The expression of P2X₇ receptor in DRG and spinal cord was also tested by immunohistochemical method.The co-expression of P2X₇receptor and GFAP in DRG and spinal cord was detected by immunofluorescence double labeling method.ELISA was used to detect the changes of serum inflammatory cytokines TNF-?and IL-1?in each group.The changes of phosphorylation level of ERK1/2 in DRG and spinal cord were measured by Western blot.Results:1.The model of diabetic neuropathic pain with depression was successfully established.Compared with the control group,the values of MWT,TWL,SPT and OFT in DNP+DP group were significantly lower,and immobility time of FST was significantly higher?p<0.01?.After treatment with DHM or P2X₇ shRNA,the MWT,TWL,SPT and OFT levels of rats in DNP+DP+DHM group and DNP+DP+P2X₇ shRNA group were significantly higher than those of the control group?p<0.01?,and immobility time of FST was decreased significantly?p<0.01?.2.The detection results of P2X₇ receptor:the expression of P2X₇ receptor in DRG and SPI was detected by qPCR,Western blot,immunohistochemistry and immunofluorescence double labeling.The results showed that the expression level of P2X₇ receptor in DRG and SPI of DNP+DP group was significantly higher than that of control group?p<0.01?.After the treatment of DHM or P2X₇ shRNA,the expression level of P2X₇ receptor in DRG and SPI of DNP+DP+DHM group and DNP+DP+P2X₇ shRNA group were significantly lower than that of DNP+DP group?p<0.01?.3.The detection results of TNF-?and IL-1?:the results showed that the levels of TNF-?and IL-1?in serum,DRG and SPI of rats in DNP+DP group were significantly higher than those in Control group?p<0.01?.After the treatment of DHM or P2X₇ shRNA,the TNF-?and IL-1?levels of DNP+DP+DHM group and DNP+DP+P2X₇ shRNA group were significantly lower than those of DNP+DP group?p<0.01?.4.The detection results of p-ERK1/2:Western blot was used to detect the expression of p-ERK1/2 in DRG and SPI of rats in each group.The results showed that there was no significant difference between total ERK1/2 and?-actin in each experimental group?p>0.05?.Compared with the control group,p-ERK1/2 to total ERK1/2 in DNP+DP group increased significantly,showed the level of phosphorylatedERK1/2wasincreasedsignificantly?P<0.01?.ERK1/2phosphorylation levels in DNP+DP+DHM group and DNP+DP+P2X₇ shRNA group were significantly lower than those in DNP+DP group?p<0.01?.Conclusion:The pain and depression-like behavior of rats with the comorbidity of diabetic neuropathic pain and depression can be alleviated by DHM treatment.The effect of DHM treatment was consistent with that of P2X₇ shRNA.The possible mechanism is that DHM may reduce the expression of P2X₇ receptor in DRG and spinal cord,down-regulate the phosphorylation level of ERK1/2,and the levels of inflammatory factors TNF-?and IL-1?were dropped.