Clinical Efficacy And Mechanism Of Qinggan Sanjie Xiaoying Prescription In The Treatment Of Hashimoto's Thyroiditi

Objective:This project evaluates the clinical efficacy of Qinggan Sanjie Xiaoying Formula in treating Hashimoto’s thyroiditis by using a randomized,double-blind,placebo-controlled study method with multiple dimensions.It also explores the potential pathways and targets of Qinggan Sanjie Xiaoying Formula by integrative pharmacology methods and validates the results of integrative pharmacology through multi-target experiments.The aim is to elucidate the mechanism of action of Qinggan Sanjie Xiaoying Formula in treating Hashimoto’s thyroiditis.Methods:1 Clinical researchAccording to the inclusion criteria and exclusion criteria stipulated in the research plan,patients with Hashimoto’s thyroiditis with normal thyroid function were included.According to different intervention methods,they were divided into control group and traditional Chinese medicine group.The follow-up period was 3 months.TCM syndrome score,thyroid autoantibodies,thyroid function,goiter degree,thyroid disease syndrome(anxiety,depression,sleep disorder,fatigue,lower limb edema)score,thyroid volume(ultrasound examination),gland elasticity score(ultrasound examination),blood routine,liver function and renal function were used as observation indicators for multi-dimensional efficacy evaluation.2 Mechanism exploration2.1 Integrative Pharmacology ResearchBy using the method of integrative pharmacology,using TCMSP Chinese medicine component data platform,Uniprot database,etc.,combined with literature data,the main effective components and targets of Qinggan Sanjie Xiaoying Formula were screened.Then,GeneCards database and OMIM database were used to obtain the potential targets of Hashimoto ’s thyroiditis.The potential targets of syndrome types were obtained by TCMIP,and the disease targets were obtained after merging and deduplication.The compound targets were matched with the disease targets to obtain the common potential targets of compounddrug.The PPI network was constructed by using String database.KEGG signaling pathway analysis and GO enrichment analysis were performed using the Metascape platform.Finally,the ’target-pathway’ network was constructed using Cytoscape software.2.2 Laboratory researchAfter the clinical research was uncovered,20 patients treated with Qinggan Sanjie Xiaoying Formula and 20 healthy subjects were randomly selected as the research subjects,and their peripheral blood mononuclear cells(PBMC)and serum were extracted for experimental research.According to the IL-17 signaling pathway in the results of integrated pharmacology research,the targets of IL-1β,IL-6,TNF-α,IFN-γ,CXCL10 and MAPK1 enriched in the signaling pathway were selected.The real-time fluorescence quantitative PCR(qPCR)technology and enzyme-linked immunosorbent assay(ELISA)were used to detect the healthy people and patients with Hashimoto’s thyroiditis before and after the treatment of Qinggan Sanjie Xiaoying Formula.The results of integrated pharmacology were verified by multiple targets,which provided a basis for the mechanism of Qinggan Sanjie Xiaoying Formula in the treatment of Hashimoto ’s thyroiditis.Results:1 Clinical researchA total of 102 eligible subjects were included in this study,including 12 cases of shedding and 90 cases of final completion.Among them,46 cases in the TCM group and 44 cases in the control group.(1)Baseline informationThere was no significant difference in the baseline data(including general data and each observation index)between the two groups(P>0.05).(2)TCM Syndrome ScoreComparison within the group:Before and after the intervention,there was no significant change in TCM syndrome score in the control group,and the difference was not statistically significant(P>0.05).The TCM syndrome score of TCM group showed a significant downward trend,and the difference was statistically significant(P<0.01).Comparison between groups:After intervention,the TCM syndrome score of the TCM group was significantly lower than that of the control group,and the difference was statistically significant(P<0.01).(3)Thyroid autoimmune antibodyComparison within the group:Before and after intervention,TPOAb and TGAb in the control group had no significant change,and the difference was not statistically significant(P>0.05);TPOAb and TGAb in the TCM group showed a downward trend,and the differences were statistically significant(P<0.01).Comparison between groups:After intervention,the levels of TPOAb and TGAb in the TCM group were better than those in the control group,and the differences were statistically significant(P<0.01).(4)Thyroid functionComparison within the group:Before and after the intervention,TT3 decreased and FT4 increased in the TCM group,and TT3 increased in the control group.Although the above numerical differences are statistically significant,they are still within the normal range.(5)The degree of goiterComparison within the group:Before and after intervention,there was no significant change in the degree of goiter in the control group,and the difference was not statistically significant(P>0.05).The degree of goiter in TCM group showed a significant downward trend,and the difference was statistically significant(P<0.01).Comparison between groups:after intervention,the improvement of goiter in the TCM group was significantly better than that in the control group,and the difference was statistically significant(P<0.01).(6)Thyroid syndromeComparison within the group:Before and after the intervention,the HAMA anxiety and HAMA depression scores of the control group and the Chinese medicine group decreased,and the difference was statistically significant(P<0.05);the PSQI index,FSS index and edema score of the Chinese medicine group showed a downward trend,and the difference was statistically significant(P<0.05).Comparison between groups:After treatment,the improvement of HAMA anxiety and HAMA depression scores in the Chinese medicine group was better than that in the control group,and the difference was statistically significant(P<0.01).The PSQI index,FSS index and edema score of the Chinese medicine group were significantly better than those of the control group,and the difference was statistically significant(P<0.01).(7)Thyroid volumeIntra-group comparison:before and after the intervention,the right lobe volume,left lobe volume and isthmus thickness of the control group had no change,and the difference was not statistically significant(P>0.05).The right lobe volume,left lobe volume and isthmus thickness in the TCM group showed a downward trend,and the differences were statistically significant(P<0.05).Comparison between groups:After intervention,the right lobe volume,left lobe volume and isthmus thickness of the TCM group were significantly better than those of the control group,and the difference was statistically significant(P<0.01).(8)Thyroid gland elasticity scoreIntra-group comparison:Before and after intervention,there was no significant change in the glandular elasticity score in the control group,and the difference was not statistically significant(P>0.05).The gland elasticity score of the TCM group showed a downward trend,and the difference was statistically significant(P<0.05).Comparison between groups:After intervention,the decrease of glandular elasticity score in the TCM group was significantly better than that in the control group,and the difference was statistically significant(P<0.01).(9)Comprehensive efficiencyComparison between groups:After intervention,the total effective rates of TCM syndrome improvement in the control group and the TCM group were 27.27%and 80.43%,respectively.The total effective rate of the TCM group was significantly better than that of the control group,and the difference was statistically significant(P<0.05).The total effective rates of thyroid autoantibodies improvement in the control group and the TCM group were 36.36%and 78.26%,respectively.The total effective rate of the TCM group was significantly better than that of the control group,and the difference was statistically significant(P<0.05).(10)Safety evaluationAfter intervention,the blood routine,liver function and renal function of the TCM group did not change significantly.No adverse reactions occurred in all subjects.2 Mechanism exploration2.1 Integrative Pharmacology Research(1)Compound effective componentsThrough the database of TCM components,121 active components of Qinggan Sanjie Xiaoying Formula were mined,including quercetin,kaempferol,luteolin,beta-sitosterol,isorhamnetin,stigmasterol,and albiflorin,which were mainly related to antioxidant,antiinflammatory and immune regulation.(2)Core targetsThe targets of Qinggan Sanjie Xiaoying Formula were matched with HT disease targets,and 183 common targets of compound-HT disease were obtained.The PPI network diagram was constructed by string database.The potential core targets of Qinggan Sanjie Xiaoying Formula in the treatment of HT were SRC、RELA、AKT1、MAPK1、TP53、PIK3R1、TNF、MAPK14、ESR1、IL-6、FOS、CAV1、CXCL8、CCL2、PPARG、EGFR,etc.The above targets involve biological pathways such as inflammation,tumor,and vascular endothelium.(3)Pathway enrichment analysisGO and KEGG enrichment analysis showed that the core targets of compound-HT were mainly distributed in cell membrane microcapsules,plasma membrane rafts,membrane rafts,membrane micro-area structures and other cell structural components.They were mainly involved in the biological processes such as reactive oxygen species reaction,peptide reaction,inorganic reaction,and positive regulation of cell migration through protease binding,cell receptor binding,cytokine activation,and protein ligase binding.These biological processes are mainly through cancer-related pathways,AGE-RAGE signaling pathway,PI3K-Akt signaling pathway,apoptosis-related signaling pathway,lipid metabolism pathway,TNF inflammatory pathway,IL-17 signaling pathway,HIF-1 signaling pathway.2.2 Laboratory researchIn this study,a total of 20 patients with HT treated with Qinggan Sanjie Xiaoying Formula were randomly selected,and 20 healthy volunteers were included.(1)qPCR resultsCompared with healthy people,the expression levels of IL-6,TNF-α,IFN-γ,CXCL10 and MAPK1 mRNA in HT population were relatively increased,and the differences were statistically significant(P<0.01).The expression of IL-10 mRNA was increased,and the difference was statistically significant(P<0.05).After treatment with Qinggan Sanjie Xiaoying Formula,the levels of TNF-α and MAPK1 mRNA decreased,and the difference was statistically significant(P<0.05).The level of IL-6 mRNA decreased significan tly,and the difference was statistically significant(P<0.01).IL-1β,IFN-γ,CXCL10 mRNA showed a downward trend,but the difference was not statistically significant(P>0.05).(2)ELISA resultsCompared with healthy people,the levels of IL-1β,IL-6,TNF-α and CXCL10 in HT patients before treatment were significantly higher(P<0.01).The levels of IFN-y and MAPK1 increased,and the difference was statistically significant(P<0.05).After treatment with Qinggan Sanjie Xiaoying Formula,the levels of IL-1β and IL-6 decreased significantly(P<0.01),the levels of TNF-α and CXCL10 decreased significantly(P<0.05),and the levels of IFN-γ and MAPK1 decreased,but the difference was not statistically significant(P>0.05).Conclusion:Qinggan Sanjie Xiaoying Formula can effectively reduce the level of thyroid autoantibodies,improve the degree of goiter,improve clinical manifestations and quality of life(including emotional state,sleep disorders,fatigue,lower limb edema,etc.)in Hashimoto ’s thyroiditis patients with normal thyroid function.Its therapeutic mechanism is related to multiple pathways and multiple targets,mainly involving inflammatory response,oxidative stress,immune regulation and so on.Through the multi-target detection of cytokines enriched in the IL-17 signaling pathway,it was preliminarily verified that Qinggan Sanjie Xiaoying Formula can affect the targets of IL-1β,IL-6,TNF-α,IFN-γ,CXCL10 and MAPK1.The results are consistent with the exploration direction of integrative pharmacology,suggesting that this pathway may be one of the potential mechanisms of Qinggan Sanjie Xiaoying Formula in the treatment of Hashimoto’s thyroiditis,which has the significance of further research.