Study On The Efficacy And Mechanism Of Yanghe Decoction In The Treatment Of Spinal Tuberculosis Based On CGAS-STING Pathway

Background:Yanghe decoction（YHT）is widely used in the treatment of bone tuberculosis as a classic formula for yin gangrene and phlegm.A large number of literature studies have shown that YHT has a good effect in the treatment of spinal tuberculosis,but the study of its mechanism is still rare.The cGAS-STING pathway,as a natural innate immune pathway and autophagy,plays a more extensive role in anti-bacteria.This topic is intended to explore the role of YHT in regulating macrophage autophagy by interfering with the cGAS-STING pathway to eliminate Mycobacterium tuberculosis,reduce cytotoxicity and inflammatory reaction,and enhance the level of innate immunity and adaptive immune response of the body,It provides a new direction for the treatment of spinal tuberculosis with traditional Chinese medicine.Study1 Meta analysis of the curative effect of YHT on patients with spinal tuberculosisObjective:This study uses the method of systematic evaluation to analyze and evaluate the clinical trial of YHT in the treatment of spinal tuberculosis patients in order to analyze the effectiveness and safety of YHT in the treatment of spinal tuberculosis.Methods:We searched the documents on the treatment of spinal tuberculosis with YHT from the establishment of the database to date in the database of CNKI,Wanfang Database,Vip Database,China Biomedical Literature Database,Pub Med,Web of Science,Embase,Cochrane Central Register of Controlled Trials（CENTRAL）,and screened the documents,and evaluated the quality of the included documents with the"risk of bias"tool in the Cochrane System Evaluation Manual,Use Rev Man 5.4 software to evaluate the clinical efficacy,pain relief,tumor necrosis factor TNF-αof intervention methods meta analysis was carried out for the influence of ESR and adverse reactions.Results:The 9 articles included in this study were rated B,and 549patients were included in the 9 studies.There were 274 patients in the control group and 275 patients in the YHT treatment group.META analysis results:（1）The effective rate of spine patients in the treatment group was significantly higher than that in the control group,with a statistically significant difference[OR=6.02,95%CI（2.93,12.38）,Z=4.88,P<0.001].（2）YHT can significantly reduce the pain intensity of patients with spinal tuberculosis,and the difference is statistically significant（MD=-0.72,95%CI（-0.91,-0.53）,Z=7.37,P<0.001）.（3）The treatment group supplemented with YHT can significantly reduce the TNF of patients with spinal tuberculosis-αThe difference was statistically significant[MD=-1.94,95%CI（-3.06,-0.81）,Z=3.38,P<0.001].（4）There was no significant difference in ESR level between the control group and the Yanghe Decoction treatment group[MD=-3.05,95%CI（-6.38,0.28）,Z=1.79,P=0.07].（5）There was no statistically significant difference in adverse reactions between the treatment group and the control group during treatment[OR=0.68,95%CI（0.37,1.24）,Z=1.26,P=0.21].Summary:YHT has obvious effect on the treatment of spinal tuberculosis.It can relieve the pain of the back and lower the tumor necrosis factor TNF-αThe effect of reducing inflammatory reaction is not obvious in improving erythrocyte sedimentation rate and reducing adverse reactions.There are few sample cases in this study,which need to be further confirmed by a large sample of high-quality RCT research.Study 2 Based on network pharmacology and molecular docking,explore the mechanism of YHT in the treatment of spinal tuberculosisObjective:Based on network pharmacology and molecular docking technology,to screen the core targets of YHT in the treatment of spinal tuberculosis,and predict the possible mechanisms involved,so as to provide a theoretical basis for the follow-up mechanism research.Methods:TCMSP database,HERB database and BATMAN-TCM database were used to screen the active drug components and effective drug targets of YHT in the treatment of spinal tuberculosis,and combined with Gene Cards database to predict the target;After enrichment and analysis of GO and KEGG with R software,the network of traditional Chinese material-active components-intersection gene-disease was established,the protein interaction grid was carried out with STRING database,and the molecular docking experiment was completed with Autodock vina software.Results:There are 471 effective active ingredients,640 drug targets,1119 disease targets,and Hub genes screened from YHT for treating spinal tuberculosis,including IL-6、IL-1β、TNF-α。As the main target of YHT in the treatment of spinal tuberculosis.According to GO/KEGG enrichment analysis,YHT may achieve the effect of treating spinal tuberculosis by regulating AGE-RAGE signal pathway,cytoplasmic DNA sensing pathway（cGAS-sting pathway）,HIF-1,TNF and other signal pathways to reduce inflammatory reaction,regulate autophagy,oxygen metabolism,immune response and other ways;Molecular docking suggests that the active ingredients in YHT,such as quercetin and linolenic acid,can interact with IL-6 and TNF-α、IL-1βGood combination.Summary:YHT may achieve the effect of treating spinal tuberculosis through multiple targets and channels,providing a theoretical basis for the follow-up treatment of spinal tuberculosis with YHT.Study3 Clinical Effect of YHT on Spinal Tuberculosis of Yang Deficiency TypeObjective:To observe the clinical efficacy of YHT in treating spinal tuberculosis through clinical research,and provide reliable scientific basis for YHT in treating spinal tuberculosis.Methods:40 patients with mild spinal tuberculosis of Yang-deficiency type were randomly divided into two groups:the control group（20 cases）was treated with anti-tuberculosis therapy,and the treatment group（20 cases）was treated with YHT+anti-tuberculosis therapy.The observation time was 3months.The therapeutic effect indexes of the two groups were observed at 4time points:before treatment（T₀）,1 month（T₁）,2 months（T₂）,and 3 months（T₃）.The general data,pain score（VAS score）,waist dysfunction score（ODI score）,inflammation index:ESR,C-reactive protein,TCM syndrome score,TCM curative effect,and adverse reaction rate of the two groups were compared.At the two time points before treatment（T₀）and 3 months after treatment（T₃）,the proportion of T cell subgroup,inflammatory factor IL-6,TNF-αin serum were measured Horizontal expression was compared after detection.Results:（1）Clinical indicators:In general,there was no statistical significance between the two groups（P>0.05）,indicating comparability.VAS scores in both groups were improved compared with before treatment.VAS scores in the treatment group were significantly lower than those in the control group at T₁and T₂time points（P<0.05）,while VAS scores in the two groups tended to be stable at T₃time point（P>0.05）.The ODI score of the two groups was improved compared with that before treatment,and the ODI score of the treatment group was significantly lower than that of the control group at the time points of T₁,T₂and T₃（P<0.05）,the difference was statistically significant.In terms of ESR,both groups were improved compared with before treatment.At T₁and T₂,ESR level of the treatment group was significantly lower than that of the control group（P<0.05）,while at T₃,ESR level of the two groups tended to be stable and there was no significant difference（P>0.05）.In terms of C-reactive protein,both groups were improved compared with before treatment,C-reactive protein in treatment group was significantly lower than control group at T₁and T₂time points（P<0.05）,and C-reactive protein level in treatment group tended to be stable at T₃time point（P>0.05）.In terms of TCM syndrome scores,both groups improved after treatment,and TCM syndrome scores in the treatment group were significantly lower than those in the control group at time points T₁,T₂and T₃（P<0.05）,the difference was statistically significant.In terms of the efficacy of TCM and the incidence of adverse reactions,the effective efficiency of TCM in the control group was 30%and the incidence of adverse reactions was 25%.In the treatment group,the effective rate of TCM was 55%,and the incidence of ADR was 15%.（2）T cell subsets:The level of T lymphocyte subsets in both groups was significantly improved after treatment;At T₃time point,CD4+,CD4+/CD8+levels in the treatment group were significantly higher than those in the control group,and CD8+levels were significantly lower than those in the control group（P<0.05）,and the difference was statistically significant.（3）Inflammatory cytokines IL-6 and tumor necrosis factor TNF-α:the expression levels of IL-6 and TNF-αwere significantly decreased in both groups after treatment;The expression levels of IL-6 and TNF-αin the treatment group were significantly lower than those in the control group（P<0.05）,and the difference was statistically significant.Summary:YHT combined with standardized anti-tuberculosis treatment for spinal tuberculosis has a significant effect in improving clinical symptoms,symptoms of traditional Chinese medicine,reducing inflammatory reaction,and improving the immune level of patients.Study4 Correlation analysis of expression of key factors of cGAS-STING signaling pathway before and after treatment of spinal tuberculosisObjective:To explore the correlation between the treatment of spinal tuberculosis with YHT and the innate immune response pathway cGAS-STING through experiments.Methods:Fasting peripheral blood of 40 patients with spinal tuberculosis included in the experiment was collected at T₀and T₃time points in the morning,and PBMC monocytes were extracted from peripheral blood.Part of peripheral blood and PBMC cells extracted were stored at ultra-low temperature for reserve.Key molecules in cGAS-STING pathway in monocytes were detected by PCR.The difference between the two groups was compared.Serum levels of IFN-αand IFN-βwere detected by ELISA.Results:Compared with the control group,the levels of cGAS,STING,TBK1 and IRF3 in monocytes and IFN-αand IFN-βin serum in the treatment group were significantly increased after treatment（P<0.05）,and the difference was statistically significant.Summary:YHT may up-regulate the key molecules in the cGAS-STING pathway to produce a large number of interferin factors,thus enhancing the adaptive immune response of the body to achieve the effect of treating spinal tuberculosis.Study 5 Cell experiment in vitroObjective:Through cell experiment in vitro,it is verified that YHT may regulate macrophage autophagy by interfering with cGAS-STING pathway,eliminate Mycobacterium tuberculosis to achieve anti-tuberculosis effect,reduce inflammatory reaction and regulate immune response level of the body.Methods:Induce THP-1 cells to produce phagocytosis,establish THP-1macrophage model infected by M.tb for standby,silence the cell model with cGAS and store it for standby,complete the preparation of YHT medicated serum,screen out the optimal concentration,and divide the THP-1macrophage,si-cGAS macrophage,si-SCR macrophage infected by M.tb into groups,and use YHT medicated serum,3-MA RAPA was divided into A normal serum+M.tb infection group after intervention;B YHT containing serum+M.tb infection group;CYHT serum+3-MA autophagy inhibitor+M.tb infection group;D YHT containing serum+si-SCR+M.tb infection group;E YHT containing serum+si-cGAS+M.tb infection group;The expression of cGAS,STING,TBK1,IRF3,type I IFNsm RNA and autophagy-related genes ATG5,Beclin-1,LL-37 and LC3B m RNA in the cGAS-STING pathway were detected by PCR in the 6 groups infected with RAPA+si-cGAS+M.tb;Western blot was used to detect cGAS-STING pathway related proteins,autophagy and inflammation related proteins,and to compare the differences of the six groups of cells after intervention.Results:（1）The concentration of 20%is the best concentration of YHT medicated serum,which has strong bactericidal ability and little effect on cell viability,that is,the best concentration of YHT medicated serum designed in the follow-up experiment is 20%.（2）PCR results showed that the relative expression of cGAS,STING,TBK1,IRF3,type I IFNsm RNA,autophagy-related genes ATG5,Beclin-1,LL-37,LC3B m RNA in YHT group was significantly higher than that in Control group（P<0.05）;Compared with YHT group,the expression of ATG5,Beclin-1,LL-37 and LC3B m RNA in YHT+si-cGAS group decreased（P>0.05）,while the expression of ATG5,Beclin-1,LL-37 and LC3B m RNA in YHT+3-MA group decreased significantly（P<0.05）;There was no significant difference in the expression of ATG5,Beclin-1,LL-37 and LC3B m RNA in YHT+si-SCR group（P>0.05）;Compared with YHT+si-SCR group,there was no significant difference in the expression of cGAS,STING,TBK1,IRF3,type I IFNsm RNA in YHT+si-cGAS group（P>0.05）.（3）WB test results:Compared with the Control group,the expression of ATG5,Beclin-1,LL-37,LC3-Ⅱ/LC3-Ⅰprotein in YHT group,YHT+si-cGAS group and RAPA+si-cGAS group was significantly up-regulated（P<0.05）,the expressions of inflammation-related IL6 and TNF-αwere significantly decreased（P<0.05）;Compared with the Control group,the protein expression of cGAS,STING,TBK1,IRF3,type I IFNsm RNA in YHT,YHT+3-MA,YHT+si-SCR group was significantly up-regulated（A<B,A<C,A<D,P<0.05）;Compared with YHT+si-SCR group,there was no significant difference in the expression of cGAS,STING,TBK1,IRF3,type I IFNsm RNA protein in YHT+si-cGAS group（P>0.05）.（4）Compared with the Control group,the cell colony count in YHT group was significantly decreased（P<0.05）.Compared with YHT+3-MA group,colony count in YHT group was significantly decreased（P<0.05）.Compared with YHT+si-c GARs group,YHT+si-SCR（control interference si RNA）colony count was significantly reduced（P<0.05）.（5）Compared with the Control group,the number of autophagy and lysosome fusion was significantly increased in YHT group（P>0.05）,while the number of autophagy and lysosome fusion was less in YHT+3-MA group;Compared with YHT group,there was no significant difference in the number of autophagy and lysosome fusion between YHT+si-SCR and YHT group（P>0.05）,while the number of autophagy and lysosome fusion in YHT+si-cGAS group was significantly reduced（P<0.05）.Summary:YHT can promote autophagy of macrophages to clear Mycobacterium tuberculosis by up-regulating cGAS-STING pathway,reduce inflammatory response and regulate the body’s immune balance.