Efficacy Assessment And Molecular Mechanism Of Compatibility Of Yuanhu Zhitong Prescription For Treating Pain

BackgroundPain,one of the most common symptoms clinically,hearts the body and mind of patients and affects their normal life.Neuropathic Pain(NP)and Inflammatory Pain(IP)are the most representatives of pain.In our previous study,we have found the possible pharmacodynamic material basis of YZP,clarified the pharmacodynamic effect of pain,and found through pharmacokinetics that the possible compatibility mechanism of YH and BZ is to increase blood drug concentration.But the compatibility molecular mechanism of YH and BZ in pain is still unclear.According to above considerations,our study explores the pharmacodynamic evaluation and molecular mechanism of YH and BZ compatibility in the treatment of neuropathic pain and inflammatory pain to provide theory for clinical application and experimental basis of YZP.MethodsThe study is based on the pharmacodynamic evaluation and molecular mechanism of YZP and its components YH and BZ.Part 1,Study on the ethology that YZP and its components YH and BZ acting on the pain model.Pharmacodynamic evaluation was carried out in this part by the model chronic constriction injury of sciatic nerve(CCI)and Complete Freund's adjuvant(CFA).The time of administration was set according to the characteristics of the two kinds of pain to observe the effects of prescription and single drug on Thermal withdrawal latency(TWL)and the Mechanical withdrawal threshold(MWT)of the two models.In CCI group,YH,BZ and YZP were given respectively and continuously for 5 days after modeling 6 days to observe the changes of TWL and MWT on the affected side before modeling 1day and after modeling 1,3,5,7 days,the group-setting of CFA was the same as CCI.YH,BZ.and YZP were given respectively and continuously for 9 days after modeling 30 minutes to observe he changes of TWL and MWT on the affected side before modeling 1 day and after modeling 1,3,5,7 daysPart 2,The exploration of the molecular mechanism of YZP and its components YH and BZ in the treatment of pain.Based on the results of previous experiments and literature research,the affected spinal cord tissues modeling for 10 days(the 5th day of administration)of the CCI model and the affected spinal cord tissues modeling for 8 days(the 9th day of administration)of the CFA model were selected for the second RNA-seq generation sequencing.Sequencing results of the two models were used to screen differentially expressed genes after administration and GO enrichment analysis was performed on these different genes to explore the possible candidate genes of YH and BZ for synergistic analgesia and their biological processes and to investigate the direct action site YZP acting on and the pathways YZP involved in by searching for targets reversely according to the 14 blood components of YZP.ResultsPart 1,Study on the ethology that YZP and its components YH and BZ acting on the pain model.There were abnormal forms of foot crouching or ecstrophy on affected side except for the sham group after CCI model establishment and there is a downstream of MWT and TWL after modeling,which was significantly lower than before-operation.The difference between the model group and the sham group was at least P<0.05.On the 7th day after operation(the 2nd day after administration),there was a significant difference at least P<0.05 of MWT and TWL between the YZP group and YH group.There was no significant difference of the MWT and TWL after administration in BZ group.However,the overall pain threshold of YZP group was slightly higher than YH group.Part 2,The exploration of the molecular mechanism of YZP and its components YH and BZ in the treatment of pain.YZP and its components YH and BZ have twelve co-callback mRNAs in CCI model including Bst2,Car4,Ccdc69,Cdknlc,Dpt,Fam64a,Mki67,Mstlr,Siglecl,Tagln,Tmem14c and Tom112,The results of the GO enrichment analysis shows that YZP and its components involve in inflammatory reaction,body defense,injury response and metal ion transport and other biological processes related to immune regulation and vascular regulation.YZP and and its components had 11 co-callback mRNAs including Aatf,Alad,Ate1,Cntnap5c,Fam122b,LOC102723236,Mybl2,Myg1,Pknox1,Tmeml4c and Tmod2.The results of GO enrichment analysis showed that YZP and its components involve in protein hydrolysis,metabolism and vascular regulation and so on.The synergistic effect to pain of YH and BZ may be achieved by directly targeting some targets on these three signaling pathways of cAMP,PI3K-Akt and MAPK,YH may act on GCPR,JNK and p38,while BZ may act on BRAF,and they both may work together on PI3K,PDE4,RTK.CCI regulates Tlr8,Dcn,Comp,Bmmp6,Igf2,Illrn,and so on,CFA regulates Wnt16,Wnt5b,Hiflan,Itgb4,Col2a1,Thbs3,and so on after administration of YZP directly or indirectly relating to these three pathways.ConlusionThis study proves that YZP and its components YH can improve mechanical hypersensitivity and thermal allodynia induced by CCI and CFA models.The spliting dose of BZ has no effect to remove the pain on the two models.The effect of YZP is better than the single drug on a certain extent.The synergistic effect of YH and BZ contained in YZP on NP may be by acting on Bst2?Car4?Ccdc69?Cdkn1c?Dpt?Fam64a?Mki67?Mstlr?Siglecl?Tagln?Tmem14c?Tom112 of the spinal cord tissue and intervention on inflammatory response,body defense,damage response,metal ion transport,and biological processes associated with immune regulation and vascular regulation.And the intervention on IP may be by acting on Aatf?Alad?Atel?Cntnap5c?Fam122b?LOC102723236?Myb12?Mygl?Pknoxl?Tmeml 4c?Tmod2 of the spinal cord tissue and the related pathways such as proteolysis,metabolism,vascular regulation,etc.The synergistic effect to pain of YH and BZ may be achieved by directly targeting some targets on these three signaling pathways of cAMP,PI3K-Akt and MAPK,YH may act on GCPR,JNK and p38,while BZ may act on BRAF,and they both may work together on PI3K,PDE4,RTK.