Effect And Mechanism Of Palmatine On P2X4 Receptor Expression In PC12 Cells Pain Model Induced By Substance P

Objective:Trigeminal neuralgia（TN）is one of the chronic peripheral neuropathic pain,which seriously affects the quality of patient’s life.The annual incidence rate has been reported as 0.015%,it mostly occurs in middle-aged and elderly people,and higher in females than in males,gradually increases with age.The pathological mechanism of trigeminal neuralgia is still unclear,and there are many therapeutic methods.Although the current treatment is effective,there are still limitations and deficiencies.A large number of studies have shown that P2X4 receptor is involved in the generation and maintenance of neuropathic pain and plays a key role in the pathogenesis of neuropathic pain（NPP）.Previous laboratory studies also suggested that P2X4 could mediate the pathological changes of trigeminal neuralgia.Fibriuretinin is a natural isoquinoline alkaloid,also known as Palmatine,which has a wide range of pharmacological effects,anti-inflammatory and antiviral effects.Studies have shown that Palmatine can effectively relieve chronic pain in rats with trigeminal neuralgia.However,there is no report on the effect of Palmatine on P2X4receptor-mediated trigeminal neuralgia associated neuropathic pain.In this study,the damage pain model of nerve cells induced by substance P was established.At the cellular level,this project intends to investigate whether P2X4receptor involved in nerve cell damage induced by substance P,and to observe the effect and possible mechanism of Palmatine on P2X4 receptor-mediated nerve cell injury,so as to provide a new approach and basis for the prevention and treatment of chronic neuropathic pain related to trigeminal neuralgia.Methods:In this study,a cell pain model was established using PC12 cells co-cultured with the substance P.The experiments groups were divided into PC12 cells normal group（Ctrl）,Palmatine group（Palmatine）,substance P group（SP）,substance P and Palmatine administration group（SP+Palmatine）,substance P and P2X4 sh RNA administration group（SP+P2X4 sh RNA）,substance P and scramble sh RNA administration group（SP+scramble sh RNA）.The experiments included as follows:（1）Screening of Substance P concentration:CCK-8 was used to detect the effects of substance P（0.1 n M,1 n M,10 n M,100 n M,and 1000 n M）on cell survival,and the most appropriate modeling concentration of substance P was initially selected.（2）Screening of Palmatine concentration:CCK-8 detected the effects of different concentrations of Palmatine（1μM,25μM,50μM,100μM）on cell survival.Under the conditions of initial concentration of substance P（100 nm）,real-Time PCR was used to detect the m RNA expression of inflammatory factor（TNF-α）to screen out the drug concentration of Palmatine.（3）CCK-8 detected the effect of Palmatine on the survival rate of PC12cells cultured with substance P.（4）Screening of the optimal P2X4 receptor interference sequence:real-time PCR was used to detect the relative expression of P2X4 m RNA in PC12 cells（Control group,Scramble sh RNA group,P2X4 sh RNA-1group,P2X4 sh RNA-2 group and P2X4 sh RNA-group 3）.Then the optimal P2X4sh RNA sequence was selected.（5）Real-time PCR was used to detect the m RNA expression changes of P2X4 receptor,BDNF and Trk B in PC12 cells.（6）Western blot was used to detect the expression of P2X4,BDNF/Trk B,p38 MAPK receptor,Caspase-1,NLRP3 inflammasome,GSDMD and ASC in PC12 cells.（7）BBcell Probe ^TMF3 fluorescence probe was used to detect the change of intracellular calcium ion concentration in PC12 cells.（8）Enzyme Linked immunosorbent assay（Elisa）was used to detect the release of TNF-α,IL-1βand IL-18 levels in culture supernatant of PC12 nerve cell culture.（9）The expression of P2X4 and BDNF receptors in PC12 cells in each group was detected by immunofluorescence double-labeled method.Results:（1）Screening of substance P modeling concentration:CCK-8 test results showed that compared with Ctrl group,the survival rate of substance P treated with 0.1,1 and10 nmol/L was above 90%.The survival rate of cells treated with substance P at 100nmol/L was about 80%（P<0.01）.The survival rate of cells treated with 1000 nmol/L substance P decreased to below 60%（P<0.001）.Therefore,we initially selected 100nmol/L as the optimal modeling concentration of substance P.（2）Screening of Palmatine concentration:CCK-8 showed that there was no significant difference in cell survival rate in 0,1,25 and 100μmol/L Palmatine groups compared with Ctrl group（P>0.05）.Compared with Ctrl group,the survival rate of 50μmol/L anthotenin group was significantly increased（P<0.05）;The m RNA expression of inflammatory factor（TNF-α）detected by real-time PCR showed that compared with Ctrl group,the m RNA expression of TNF-αin substance P treatment group was significantly increased（P<0.001）.The m RNA expression of TNF-αwas decreased when the treatment concentration of Palmatine was 1μmol/L and 25μmol/L,compared with that of substance P only（P<0.01）.The P2X4 m RNA expression was significantly decreased at 50 and 100μmol/L,compared with the group treated with substance P alone（P<0.001）,but there was no significant difference between the two groups.According to the results of cell activity assay and RT-PCR,50μmol/L of Palmatine was selected as the drug concentration.（3）CCK-8 showed that substance P had certain damage to PC12 cells,and the protective effect of Palmatine on PC12 cell damage induced by substance P was observed.（4）Optimal P2X4 receptor interference sequence screening:real-time PCR results showed that the interference efficiency of the second P2X4 sh RNA sequence was the best.（5）Real-time PCR results showed that the m RNA expression levels of P2X4,BDNF and Trk B in SP group were significantly higher than those in Ctrl group（P<0.001）.Palmatine and P2X4 sh RNA could decrease significantly the expression in SP group（P<0.001）.while Scramble sh RNA did not have the same effect（P>0.05）.（6）Western blot results showed that the protein expression levels of P2X4,BDNF/Trk B,p-p38 MAPK,Caspase-1,NLRP3 inflammassome,GSDMD and ASC in SP group were significantly increased compared with control group（P<0.01）.The expression levels of above proteins in SP+Palmatine group and SP+P2X4 sh RNA group were significantly lower than those in SP group（P<0.001）.SP+Scramble sh RNA group had no significant difference compared with SP group（P>0.05）.（7）ELISA results showed that compared with control group,the contents of IL-1β,IL-18 and TNF-αin supernatant of PC12 cells in SP group were increased,and the difference was statistically significant（P<0.05）,while decreased significantly after application of Palmatine and P2X4 sh RNA respectively in SP group（P<0.05）.（8）Intracellular calcium concentration results showed that the intracellular calcium concentration in SP group was increased compared with Ctrl group（P<0.001）.It can be blocked by Palmatine and P2X4 sh RNA respectively（P<0.001）.（9）Immunofluorescence results showed that P2X4 and BDNF receptor were co-expressed in PC12 cells.The co-expression of P2X4 and BDNF receptor in SP group was significantly higher than that in Ctrl group（P<0.001）.Compared with SP group,the expression level of SP+Palmatine group and SP+P2X4 sh RNA group were decreased（P<0.001）.SP+Scramble sh RNA group had no significant difference compared with SP group（P>0.05）.Conclusion:P2X4 receptor is involved in SP induced neuronl injury.Palmatine may be involved in the pathological changes of substance P induced cellular pain model by affecting the expression of P2X4-BDNF receptor and NLRP3/caspase-1 pyroptosis signaling pathway in PC12 neurons,have protective effect on PC12 cell injury.