Study On The Mechanism Of Medicine-separated Moxibustion Regulating Spinal Cord Autophagy In The Treatment Of Chronic Inflammatory Visceral Pain Through MTOR Signal Pathway

ObjectiveTo explore the central mechanism of medicine-separated moxibustion in the treatment of chronic inflammatory visceral pain from the point of view of spinal cord PI3K/AKTmTOR and AMPK-mTOR signal pathway regulating autophagy,so as to provide scientific experimental basis for elucidating the analgesic mechanism of medicineseparated moxibustion in the treatment of chronic inflammatory visceral pain.Methods1.The rats were randomly divided into normal group,model group,medicineseparated moxibustion group and pseudo-medicine-separated moxibustion group;the rat model was established by TNBS enema;the medicine-separated moxibustion group was treated with aconite cake-separated moxibustion on bilateral Tianshu and Qihai points after modeling;the moxa on aconite cake was not ignited in the sham-medicine-separated moxibustion group;the normal group was a blank control;the analgesic effect of medicine-separated moxibustion on IBD visceral pain was observed by AWR,MWT and TWL scores.HE staining method was used to observe the histopathological changes of rat colon under light microscope,and Western blot method,immunofluorescence staining method and Real-time PCR method were used to detect the expression of spinal cord autophagy-related proteins LC3-Ⅱ,p62,Beclin-1 and ATG7 and mRNA.To explore the regulatory effect of medicine-separated moxibustion on spinal cord autophagy.2.The rats were randomly divided into normal group,model group,PI3K inhibitor group,DMSO group,medicine-separated moxibustion group and sham medicineseparated moxibustion group;the rat model was established by TNBS enema;the medicine-separated moxibustion group was treated with aconite cake-separated moxibustion on bilateral Tianshu and Qihai points after modeling;the PI3K inhibitor group was treated with intrathecal injection of PI3K specific inhibitor LY294002;while the moxibustion group did not ignite the moxa on the aconite cake;the DMSO solvent control group was treated with intrathecal injection of 5%DMSO.The normal group was set as the blank control group.The analgesic effect of herbal-separated moxibustion was observed by AWR,MWT and TWL scores;the expression of pPI3K,pAKT,pmTOR,ATG13 protein and mRNA in spinal cord of rats in each group was detected by Western blot and Real-time PCR methods;the interaction of mTOR with p62 and ATG7 in spinal cord tissue was detected by immunoprecipitation method;the expression of autophagy-related proteins LC3-Ⅱ,p62,Beclin-1 and ATG7 in spinal cord tissue of rats in each group was detected by Western blot method.IL-1 β and TNF-αprotein expression in spinal cord tissue of rats in each group was detected by ELISA method.To explore the regulatory effect of medicine-separated moxibustion on IBD visceral pain rats from the point of view of regulating spinal cord autophagy-related PI3K/AKT-mTOR signal pathway.3.The rats were randomly divided into medicine-separated moxibustion group,sham medicine-separated moxibustion group,AMPK activator group,normal saline group,normal group and model group;the rat model was established by TNBS enema;the medicine-separated moxibustion group was treated with aconite cake-separated moxibustion on bilateral Tianshu and Qihai points after modeling;the AMPK activator group was treated with intrathecal injection of AMPK specific activator AICAR,while the moxa on aconite cake was not ignited.0.9%saline was injected intrathecally in the saline control group,and the normal group was set as the blank control group.AWR,MWT and TWL scores were used to observe the analgesic effect of medicine-separated moxibustion on IBD visceral pain,Western blot and Real-time PCR methods were used to detect the expression of pAMPK,pmTOR,ATG13 protein and mRNA in spinal cord of rats in each group,Western blot method was used to detect the expression of autophagyrelated proteins LC3-Ⅱ,p62,Beclin-1 andATG7.ELISA method was used to detect the expression of IL-1β and TNF-α protein in spinal cord tissue of rats in each group.To explore the regulatory effect of medicine-separated moxibustion on IBD visceral pain rats from the point of view of regulating spinal cord autophagy-related AMPK-mTOR signal pathway.Results1.Pain behavior detection(1)AWR detection:The AWR score of the model rats was significantly higher than that of the normal group,and the AWR score of the rats treated with medicine-separated moxibustion,PI3K inhibitor and AMPK activator was significantly lower than that of the model group,and there was no significant difference in the AWR score between the pseudo-medicine-separated moxibustion group,DMSO solvent control group,normal saline group and the model group.(2)MWT detection:The mechanical foot withdrawal threshold of model rats was significantly lower than that of normal group,and the MWT of medicine-separated moxibustion,PI3K inhibitor and AMPK activator group was significantly higher than that of model group,while MWT of rats in pseudo-medicine-separated moxibustion group,DMSO solvent control group and normal saline group had no significant change compared with model group.(3)TWL detection:Compared with the normal group,the thermal foot reflex latency of the model rats was significantly shortened,and compared with the model group,the TWL of rats in the medicine-separated moxibustion group,PI3K inhibitor and AMPK activator group were significantly prolonged compared with the model group,while there was no significant change in TWL in the sham medicine-separated moxibustion group,DMSO solvent control group and normal saline group.2.Effect of Herbal Cake-separated moxibustion on Autophagy related protein and mRNA in Spinal Cord tissue of Rats with IBD visceral pain:(1)Compared with the normal group,the expressions of LC3-Ⅱ,Beclin-1 and ATG7 protein in spinal cord of rats with IBD visceral pain were significantly down-regulated;compared with the model group,the expressions of LC3-Ⅱ,Beclin-1 and ATG7 protein in spinal cord of rats in herbal-separated moxibustion group,PI3K inhibitor group and AMPK activator group were significantly up-regulated;compared with the normal group,the expression of p62 protein in spinal cord tissue of model group was significantly upregulated.Compared with the model group,the expression of p62 protein in the spinal cord of rats in the medicine-separated moxibustion group,PI3K inhibitor group and AMPK activator group was significantly down-regulated,while compared with the model group,there was no significant difference in the expression of LC3-Ⅱ,Beclin-1,p62 and ATG7 in the spinal cord of rats in the sham-separated medicine moxibustion group,DMSO group and normal saline group.(2)Compared with the normal group,the expressions of LC3-Ⅱ,Beclin-1 and ATG7 mRNA in the spinal cord of rats with IBD visceral pain were significantly down-regulated;compared with the model group,the expressions of LC3-Ⅱ,Beclin-1 and ATG7 mRNA in the herbal-separated moxibustion group,PI3K inhibitor group and AMPK activator group were significantly up-regulated;compared with the normal group,the expression of p62 mRNA in the spinal cord of the model group was significantly up-regulated.Compared with the model group,the expression of p62 mRNA in the spinal cord of rats in the medicine-separated moxibustion group,PI3K inhibitor group and AMPK activator group was significantly down-regulated,while compared with the model group,there was no significant difference in the expression of LC3-Ⅱ,Beclin-1,p62 and ATG7 mRNA in the spinal cord of rats in the sham-separated medicine moxibustion group,DMSO group and normal saline group.3.Effect of Herbal Cake-separated moxibustion on IL-1β and TNF-α protein expression in Spinal Cord of Rats with IBD visceral pain:(1)Compared with the normal group,the expression of IL-1 β protein in spinal cord of rats with IBD visceral pain was significantly increased,and compared with the model group,the expression of IL-1 β protein in spinal cord of rats in medicine-separated moxibustion group,PI3K inhibitor group and AMPK activator group decreased significantly,while compared with model group,there was no significant difference in IL-1 β protein expression in spinal cord of rats in sham-separated medicine moxibustion group,DMSO group and normal saline group.(2)Compared with the normal group,the expression of TNF-α protein in spinal cord of rats with IBD visceral pain was significantly increased,and compared with the model group,the expression of TNF-α protein in spinal cord of rats in medicine-separated moxibustion group,PI3K inhibitor group and AMPK activator group decreased significantly,while compared with model group,there was no significant difference in TNF-α protein expression in spinal cord of rats in sham-separated medicine moxibustion group,DMSO group and normal saline group.4.Regulatory effect of Medicine-separated moxibustion on related Indexes of PI3K/AKT-mTOR signal Pathway in Spinal Cord of Rats with IBD visceral pain:(1)Compared with the normal group,the expression of pPI3K,pAKT and pmTOR protein in the spinal cord of rats in the sham-separated medicine moxibustion group,DMSO group and model group increased significantly,while the expression level of ATG13 protein in the spinal cord decreased significantly.Compared with the model group,the expression levels of pPI3K,pAKT and pmTOR protein in spinal cord tissue of rats in medicine-separated moxibustion group and PI3K inhibitor group were significantly decreased,while the expression level of ATG13 protein in spinal cord tissue was significantly up-regulated.(2)Compared with the normal group,the expression of pPI3K,pAKT and pmTOR mRNA in the spinal cord of rats in the sham-separated medicine moxibustion group,DMSO group and model group increased significantly,while the expression level of ATG13 mRNA in the spinal cord decreased significantly.Compared with the model group,the expression levels of pPI3K,pAKT and pmTOR mRNA in spinal cord tissue of rats in medicine-separated moxibustion group and PI3K inhibitor group were significantly decreased,while the expression level of ATG13 mRNA in spinal cord tissue was significantly up-regulated.5.Regulatory effect of Medicine-separated moxibustion on related Indexes of AMPK-mTOR signal Pathway in Spinal Cord of Rats with IBD visceral pain:(1)Compared with the normal group,the expression of pmTOR protein in the spinal cord of rats in the sham-separated medicine moxibustion group,DMSO group and model group increased significantly,while the expression level of pAMPK and ATG13 protein in the spinal cord decreased significantly.Compared with the model group,the expression levels of pmTOR protein in spinal cord tissue of rats in medicine-separated moxibustion group and PI3K inhibitor group were significantly decreased,while the expression level of pAMPK and ATG13 protein in spinal cord tissue was significantly up-regulated.(2)Compared with the normal group,the expression of pmTOR mRNA in the spinal cord of rats in the sham-separated medicine moxibustion group,DMSO group and model group increased significantly,while the expression level of pAMPK and ATG13 mRNA in the spinal cord decreased significantly.Compared with the model group,the expression levels of pmTOR mRNA in spinal cord tissue of rats in medicine-separated moxibustion group and PI3K inhibitor group were significantly decreased,while the expression level of pAMPK and ATG13 mRNA in spinal cord tissue was significantly up-regulated.Conclusion1.Medicine-separated moxibustion can up-regulate the expression of spinal cord autophagy-related proteins LC3-Ⅱ,Beclin-1,ATG7 protein and mRNA,and downregulate p62 protein and mRNA expression in rats with chronic inflammatory visceral pain,which can regulate rat spinal cord cell autophagy.Medicine-separated moxibustion may play an analgesic effect by promoting spinal cord cell autophagy in rats with chronic inflammatory visceral pain.2.Spinal cord PI3K/AKT-mTOR and AMPK-mTOR signal pathways may be involved in the formation of chronic inflammatory visceral pain by regulating autophagy.Intrathecal injection of PI3K inhibitor and AMPK activator could down-regulate the expression of mTOR phosphorylated protein and mRNA,up-regulate the expression of spinal cord autophagy-related proteins LC3-Ⅱ,Beclin-1 and ATG7,and exert analgesic effect on rats with chronic inflammatory visceral pain.3.Herbal-separated moxibustion may relieve chronic inflammatory visceral pain by regulating the activation of PI3K/AKT-mTOR signal pathway and AMPK-mTOR signal pathway,inhibiting the expression of mTOR and regulating spinal cord cell autophagy.