| Neuropathic pain(NPP)is a common clinical pain disease.It is challenging for its treatment.NPP is a class of diseases caused by long-term changes in the mechanisms of synaptic plasticity in the spinal cord,subcortex,and cortical regions.The World Congress of the International Association for the Study of Pain defined neuropathic pain as "pain caused by or caused by primary injury or dysfunction of the nervous system".Its manifestations are mainly characterized by spontaneous pain,hyperalgesia,induced pain and paresthesia.In the general population,compared with various diseases,the prevalence of NPP has reached a higher proportion of 8%,and the number of NPP patients in China has been nearly 100 million person-times.As a relatively specific type of pain,it affects a large number of people,around 7-10%of the world.Due to the high incidence of NPP,which has seriously affected people’s quality of life,people gradually pay attention to it,and medical researchers are constantly exploring it,in order to find a good treatment and improve the quality of life of the affected population.The Anterior Cingulate Cortex(ACC)plays an important role in the development of neuropathic pain,and enhanced neuronal excitability in the Spinal Dorsal Horn(SDH)induces Central sensitization(CS).CS plays an important role in the production and maintenance of NPP.The Panx1-Src-NMDAR association is a new important pathway of CS.Previous studies have found that Ligusticum chuanxiong can play a certain role in CS of neuropathic pain model rats by interfering with the Panx1-Src-NMDAR-2B signal pathway,and ligusticum Chuanxiong can also significantly improve central hyperalgesia response of NPP,but its mechanism remains unclear.NPP is a chronic disease,and its treatment is mainly based on western medicine,such as antidepressants and antiepileptic drugs.However,western medicine does not have a good therapeutic effect in the short term.However,longterm medication is prone to drug dependence,drug resistance,safety and other problems.With the national attention to traditional Chinese medicine and the progress and development of traditional Chinese medicine in recent years,the curative effect of traditional Chinese medicine in the treatment of NPP is more significant than that of western medicine without obvious side effects,so it is of great significance to explore the treatment of NPP by traditional Chinese medicine.Therefore,our research group will continue to conduct in-depth research,taking Grin2b tm1.1(Grin2a)model mice as the research object,to explore the effect and influence of Ligusticum chuanxiong on NPP at the level of ultrafine matter and molecular genes.Objective:To determine whether Grin2b tm1.1(Grin2a)mice can be used as NPP model mice.To determine which type of Grin2b tm1.1(Grin2a)mice on Panx1-Src-NMDAR signaling pathway is affected by CRE.To explain the mechanism of action of CRE in the treatment of NPP,to partially clarify the scientific connotation of clinical analgesic effect of Chuanxiong xiong,and to provide theoretical basis and methodological support for the search for effective NPP Chinese medicine preparation.Methods:Chapter One:Comparison of wild and heterozygous types of Grin2b tm1.1(Grin2a)mice(1)Behavioral tests were carried out by Von Frey Hairs,Cold spray,electronic tenderness,light tail lash,and hot plate test,to observe the differences of mechanical pain threshold,Cold pain sensitivity,tail tenderness,tail heat radiation,and foot heat pain in wild-type and heterozygous mice.(2)The difference of neural tissue in ACC and SDH was observed by hematoxylin and eosin staining(HE)and Nissl staining.(3)The dynamic changes of amino acid neurotransmitters such as Glu,D-Ser and Gly in the extracellular fluid of ACC were detected and compared between the two groups of rats by microdialysis experiment combined with HPLC.(4)The protein levels of Panx1,Src and NMDAR-2B in ACC and SDH were compared by Western Blot.(5)The gene levels of Panx1,Src and NMDAR-2B in ACC and SDH were compared by RT-PCR.Chapter Two:Effect of CRE on wild-type Grin2b tm1.1(Grin2a)mice(1)The effect of CRE on the analgesic activity of wild-type mice was investigated by behavioral test Von Frey Hairs.(2)The effects of CRE on the histology of ACC and SDH of wild-type mice were observed by HE and Nissl staining.(3)The effects of CRE for dynamic changes of Glu,D-Ser and Gly in extracellular fluid of ACC of wild-type mice were detected by microdialysis combined with HPLC.(4)Western Blot was used to investigate the changes of Panx1Src-NMDAR related protein molecules of CRE in ACC and SDH of wild-type mice.(5)The effects of CRE on the molecular changes of Panx1-Src-NMDAR related genes in ACC and SDH of wild-type mice were explored by RT-PCR.Chapter Three:Effect of CRE on heterozygous Grin2b tm1.1(Grin2a)mice(1)The effect of CRE on the analgesic activity of heterozygous mice was investigated by behavioral test Von Frey Hairs;(2)The effects of CRE on the histological changes of ACC and SDH of heterozygous mice were observed by HE and Nissl staining.(3)The effects of CRE for dynamic changes of Glu,D-Ser and Gly in extracellular fluid of ACC of heterozygous mice were detected by microdialysis combined with HPLC.(4)Western Blot was used to investigate the changes of Panx1Src-NMDAR related protein molecules of ethanol extract of CRE in the ACC and SDH of heterozygous mice.(5)The effects of CRE on the molecular changes of Panx1-Src-NMDAR related genes in ACC and SDH of heterozygous mice were explored by RT-PCR.Results:The chapter One is the comparison between wild-type and heterozygous type(1)Behavior:Compared with wild-type group,mechanical pain threshold of mice in heterozygous group was significantly decreased(P<0.01);heterozygous type and the wild-type were not sensitive to cold pain,tenderness pain,heat radiation pain and heat pain,and there was no statistical difference.(2)HE and Nissl staining:HE staining showed that there is no obvious difference in ACC and SDH with the two types of mice.The number of neuron cells is equal,full in shape,complete in structure,orderly in arrangement,clear in outline,and no interstitial space or cavity is formed.The cytoplasm and nucleus are evenly stained,the nucleus is in the center,the nucleoli are clearly visible,and there is no nuclear shrinkage,cell infiltration and expansion and necrosis.The Nissl staining of results show that there is no obvious difference in ACC and SDH of the two regions of mice.The number of neuron cells is large,the structure is regular and full in shape,the outline is clear,and the arrangement is uniform and orderly.Blue-purple Nissl bodies were seen in the cytoplasm,which were uniformly arranged without scattered vacuoles.No loss of neurons,infiltration of inflammatory cells and necrosis lesions were observed.(3)Changes of Glu,D-Ser and Gly amino acid contents in extracellular fluid of brain:Compared with wild-type mice,the content of Glu in extracellular fluid in ACC area of heterozygous mice was significantly increased at 60 min,20 min,140 min and 220-300 min after administration(P<0.05,P<0.01).D-ser content was significantly increased 20 min before administration and 260 min after administration(P<0.05,P<0.01),Gly content was significantly increased 60 min before administration and 20-100 min after administration(P<0.05).(4)Western Blot technique:Compared with the wild-type,the protein levels of Panx1,Src and NMDAR-2B in ACC and SDH regions of heterozygous mice were significantly increased(P<0.05,P<0.01).(5)RT-PCR technique:Compared with wild-type,the gene levels of Panxl and Src in ACC and Src,NMDAR-2B in SDH region of spinal cord of heterozygous type mice were significantly increased(P<0.05,P<0.01).The chapter Two is the effect of ethanol extract of Ligusticum Chuanxiong on wild-type mice(1)Behavior:The CRE and Ifenprodil had no effect on the analgesic activity of wild-type mice.(2)In HE and Nissl staining:The CRE and Ifenprodil had no histomorphological effect on the ACC and SDH of spinal cord of wild-type mice.(3)The CRE and Ifenprodil had no effect on the contents of Glu,D-Ser and Gly amino acids in extracellular fluid of ACC region of wild-type mice.(4)The CRE and Ifenprodil showed no statistical difference in the protein levels of Panxl,Src and NMDAR-2B in ACC and SDH of wild-type mice.(5)The CRE and Ifenprodil had no significant effect on Panx1,Src and NMDAR-2B genes in ACC and SDH of wild mice,with no statistical significance.The chapter three is the effect of ethanol extract of Ligusticum Chuanxiong on heterozygous mice(1)Behavior:Compared with the heterozygous type mice,after continuous intraperitoneal injection of Ifenprodil for 10 d,the analgesic activity of heterozygous mice was significantly increased at 0,60-240 min after administration(P<0.05,P<0.01).The analgesic activity of the heterozygous type mice was significantly increased in the CRE-H group at 30,90-240 min after administration(P<0.05,P<0.01).The content of Glu in extracellular fluid of ACC of heterozygous mice was significantly decreased during 100-140 min and 220-300 min after treatment(P<0.05,P<0.01).The analgesic activity in CRE-M group was significantly increased during 120～240 min after administration(P<0.01),and that in CRE-L group was significantly increased during 60～120 min after administration(P<0.05,P<0.01).(2)HE and Nissl staining:The CRE and Ifenprodil had no histomorphological effect in ACC and SDH of heterozygous mice.(3)Changes of Glu,D-Ser and Gly amino acid contents in cerebrospinal fluid:Compared with the heterozygous type mice,Ifenprodil significantly decreased Glu content in ACC extracellular fluid of heterozygous mice 60-20min before administration and 220-300min after administration(P<0.05,P<0.01).CRE-H could significantly reduce Glu content in ACC extracellular fluid of heterozygous mice at 100-140 min and 220-300 min after treatment(P<0.05,P<0.01).CRE-M could significantly reduce the content of Glu in the extracellular fluid of ACC during 220260 min after treatment(P<0.01),and CRE-L could significantly reduce the content of Glu in the extracellular fluid of ACC during 220 min after treatment(P<0.05).The content of D-Ser in extracellular fluid of ACC was decreased by CRE-H at 20min before treatment(P<0.05).CRE-M could decrease the content of D-Ser in extracellular fluid at 20 min before treatment and 20-60 min,260-340 min after treatment(P<0.05,P<0.01).CRE-L could decrease the content of D-Ser in extracellular fluid of ACC at 20 min before treatment and 220-340 min after treatment(P<0.05,P<0.01).CRE-M could decrease the content of Gly in extracellular fluid in ACC during 20-60 min before treatment and 20-100 min,340 min after treatment(P<0.05,P<0.01).The content of Gly in extracellular fluid of ACC was also decreased by CRE-L at 20 min before treatment and 220 min after treatment(P<0.05).(4)Western Blot technique:Compared with heterozygous type,Ifenprodil and CRE-H,M and L significantly reduced Src and NMDAR-2B protein levels in SDH(P<0.01).The alcohol extract of Ligusticum chuanxiong had a tendency to decrease the protein levels of Panx1,Src and NMDAR-2B in ACC and SDH.(5)RT-PCR technique:Compared with the heterozygous type,Ifenprodil significantly reduced the gene levels of of Panx1 and Src in ACC as well as Src and NMDAR-2B in SDH of mice(P<0.01),CRE-H could significantly reduce the gene levels of Panx1 and Src in ACC of mice(P<0.05,P<0.01),and CRE-M could significantly reduce the gene levels of Panx1 and Src in ACC as well as Src and NMDAR-2B in SDH of mice(P<0.01).CRE-L significantly decreased Panx1 gene in ACC and Panx1 and Src gene in SDH(P<0.05,P<0.01),Conclusion:1.The results of the chapter one of the experiment showed that compared with wild-type mice,heterozygous Grin2btm1.1(Grin2a)mice significantly reduced mechanical pain threshold,contents of Glu,D-Ser and Gly amino acids in ACC,protein levels of Panx1,Src and NMDAR-2B in ACC and gene levels of Panx1 and Src in ACC and Src and NMDAR-2B in SDH.Therefore,heterozygous Grin2btm1.1(Grin2a)mice can be studied as neuropathic pain model mice.2.The wild-type mice showed no difference after drug intervention with CRE and Ifenprodil.3.The CRE and Ifenprodil could significantly improve the analgesic activity of heterozygous Grin2b tm1.1(Grin2a)mice.The CRE could significantly reduce the contents of Glu,D-Ser and Gly in extracellular fluid of ACC.Also significantly reduce Src and NMDAR-2B protein in SDH,the gene levels of Panx1 and Src in ACC,Src and NMDAR-2B in SDH.Therefore,the central analgesic effect of Chuanxiong might be through increasing mechanical pain threshold,increasing analgesic activity,inhibiting the release of Glu,D-Ser and Gly in the extracellular fluid of ACC,and decreasing the protein and gene expression of Panx1,Src and NMDAR-2B in ACC and SDH.The Panx1-Src-NMDAR signaling pathway is related to the regulation of spinal cord and brain. |
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