Effects Of Guanfu Base A On The Neuropathic Pain Mediated By P2Y₁₂ Receptor In Dorsal Root Ganglia

Background and Objective:Chronic neuropathic pain jeopardizes the physical and mental health of patients as it lasts for a long time and it is difficult to cure.The pathological mechanism of neuropathic pain is unclear that is lack of effective treatments.Therefore,the study of neuropathic pain becomes to be the focus in the field of pain.It has been reported that the P2Y₁₂ receptor upregulation is closely related to the occurrence and maintenance of pathological pain.Guanfu base A has anti-inflammatory effect.At present,there is no report about Guanfu base A on neuropathic pain transmission mediated by P2Y₁₂ receptor.In this study,Chronic constriction injure model was used to observe the effects of guanfu base A on the neuropathic pain mediated by P2Y₁₂ receptor in dorsal root ganglia.The findings from this study will provide the knowledge for developing novel strategy to prevent and treat neuropathic pain.Methods:The adult male Sprague-Dawley（SD）rats were randomly divided into control group（Ctrl）,sham-operated group（Sham）,CCI model group（CCI）,CCI rats treated with Guanfu base A group（CCI+GFA）.The changes of the paw thermal latency（TWL）and the mechanical withdrawal threshold（MWT）were observed.The expression levels of P2Y₁₂receptor protein and messenger RNA（m RNA）in the dorsal root ganglia（DRG）were detected by reverse transcription-polymerase chain reaction（RT-PCR）,immunohistochemistry and western blotting.Tumor necrois factorα（TNF-α）and P38 MAPK protein in DRG were detected by Western Blot.Results:1.The behavior results of CCI rats showed that at 5 days after surgery,mechanical allodynia and thermal pain threshold on surgical side limbs of CCI operated rats were much lower than sham-operated rats and control rats（p<0.01）.These changes were much obviously in CCI operated rats at 7 days after surgery（p<0.001）.Compared with the Ctrl and Sham rats,TWL and MWT in CCI rats decreased continuously during 7-14 days of post-operation（p<0.01）,but the TWL and MWT in CCI+GFA rats were significantly higher than CCI rats（p<0.001）.There were no significant changes in Ctrl,Sham and CCI+GFA groups during 14 days（p>0.05）.2.Western Blot and RT-PCR results showed that compared with Ctrl and Sham rats,the expression levels of P2Y₁₂ receptor protein and m RNA in DRG of CCI rats were significantly increased（p<0.001）,but those in CCI+GFA rats were significantly decreased compared to CCI rats（p<0.001）.No difference was found among Ctrl,Sham and CCI+GFA groups（p>0.05）.3.Immunohistochemistry results showed that the expression levels of P2Y₁₂receptor were significantly increased in CCI group than those in Ctrl and Sham groups（p<0.001）.Compared with CCI group,the expression levels of P2Y₁₂ receptor in CCI+GFA group were decreased（p<0.001）.There was no significant difference amonge Ctrl,Sham and CCI+GFA groups（p>0.05）;Double immunofluorescence test indicated that the P2Y₁₂ receptor and GFAP were co-expression on satellite glial cells in the DRG.The expression levels of P2Y₁₂ receptor and GFAP were increased in CCI group than those in Ctrl and Sham groups,the expression levels of P2Y₁₂ receptor and GFAP in CCI+GFA group were decreased compared with CCI group.4.Western Blot results showed that the expression levels of TNF-αin CCI group were significantly higher than those in Ctrl and Sham groups（p<0.001）.The expression levels of TNF-αin CCI+GFA were decreased compared with CCI group（p<0.001）.No significant difference was found among Ctrl,Sham and CCI+GFA groups（p>0.05）;The expression levels of phosphorylated p38 MAPK were significantly higher than those in Ctrl and Sham groups（p<0.001）.Compared wtih CCI group,the phosphorylated p38 MAPK expressions in CCI+GFA were lower（p<0.05）,but no significantly difference was found among Ctrl,Sham and CCI+GFA groups.Conclusion:Guanfu base A can reduce the upregulation of P2Y₁₂ receptor in DRG induced by neuropathic pain and inhibit the mechanical allodynia and thermal hyperalgesia.