Study On The Mechanism Of Prunella Vulgaris Regulating Apoptosis Through JNK/P38MAPK Signaling Pathway In The Treatment Of Autoimmune Thyroiditis Based On Meta-analysis And Network Pharmacology

Background Autoimmune thyroiditis is a kind of immune system disease,which is not only a common cause of hypothyroidism,but also an important cause of precancerous lesions of thyroid papillary carcinoma.Currently,western medicine mostly uses symptomatic treatment,but there is still a lack of drugs that can effectively prevent the progression of the disease and have little adverse reactions.There have been relevant clinical reports and mechanisms of Prunella vulgaris in the treatment of autoimmune thyroiditis,but most of the clinical reports are single-center,small sample studies,which can’t provide high-quality evidence-based medical evidence for clinic,and the mechanism exploration mainly focuses on anti-inflammatory and immunomodulatory effect,which can’t form a perfect evidence system.Based on this,this study first applied systematic evaluation method to integrate the existing clinical studies to demonstrate the clinical efficacy of Prunella vulgaris in the treatment of autoimmune thyroiditis,and proved its effectiveness in the real world.Secondly,the network pharmacology method was used to find the main components and the Hub genes acting on the disease,and the molecular docking method was used to verify the possible theoretical mechanism.Finally, according to the results of network pharmacology,an animal experiment was set up to discuss the effects of Prunella vulgaris on immune regulation and thyroid cell apoptosis in rats with autoimmune thyroiditis,which enriched the theoretical system of Prunella vulgaris in the treatment of autoimmune thyroiditis and proved the effectiveness of clinical treatment.This study is divided into three parts:systematic evaluation,network pharmacology and animal experiment.Objective2.1 To evaluate the clinical efficacy and side effects of Prunella vulgaris in the treatment of autoimmune thyroiditis.2.2 To explore the possible pharmacological mechanism of Prunella vulgaris in the treatment of autoimmune thyroiditis by the method of network pharmacology.2.3 To evaluate the effect of Prunella vulgaris on the treatment of autoimmune thyroiditis in rats by animal experiment,and to explore its mechanism from the aspects of immune regulation and apoptosis.Methods3.1 A systematic review of Prunella vulgaris preparation in the treatment of autoimmune thyroiditis The work manual of Cochrane collaboration Network is used to formulate the retrieval strategy and evaluate the literature quality.Comprehensive search of four Chinese databases VIP,CNKI,WANFANG,CBM and four English databases PubMed,Cochrane library,Embase,SCI.thekey words included "Thyroiditis,Autoimmune","Thyroiditis,Lymphocytic","Prunella","Randomized Controlled Trial" and so on.The randomized controlled trials of Prunella vulgaris in the treatment of autoimmune thyroiditis published before February 3,2022 were searched.According to the inclusion criteria and exclusion criteria,the literature was screened,the data was extracted and the bias risk was assessed,and the relevant outcome indicators were statistically analyzed by Revman5.3 software.3.2 To explore the mechanism of Prunella vulgaris in the treatment of autoimmune thyroiditis based on network pharmacology The TCMSP was searched,and the effective components and targets of Prunella vulgaris were screened according to OB > 30% and DL > 0.18,and the target information was standardized by Uniport database.The related target genes of autoimmune thyroiditis were obtained by Gene Cards,OMIM and Pharm Gkb databases,the intersection targets of drugs and diseases were screened by R software,and the "drug-component-disease-target" network map was constructed by Cytoscape software.The intersection targets were analyzed by protein interaction network（PPI）through String platform,and the final core genes were obtained according to Cyto NCA score and Degree value.At the same time,the intersection targets were analyzed by GO and KEGG.Finally,the core active components of Prunella vulgaris were docked with the core genes of autoimmune thyroiditis by molecular docking technique.3.3 To explore the effect of Prunella vulgaris on immunomodulatory mechanism and apoptosis mechanism in rats with autoimmune thyroiditis60 SPF female SD rats were randomly divided into normal group,model control group,selenium yeast group,and Prunella vulgaris groups for low-dose,medium-dose and high-dose administrations,with 10 rats in each group.The autoimmune thyroiditis rat models established by high-iodine water（Na I）feeding combined with subcutaneous injection of porcine thyroglobulin（p Tg）and Freund’s adjuvant were then intragastrically given the corresponding drugs for 4 weeks.During the experiment,the state of the rats was dynamically observed,including general and behavioral conditions.After the experiment,the serum and thyroid tissues of the rats were taken.The contents of serum FT3,FT4,TSH,TPOAb,TGAb,INF-γ,IL-10,IL-17 and IL-35 were detected by ELISA.The histopathological changes of thyroid gland in rats were observed by HE staining.The contents of Th1,Th2,Th17 and Treg cells in peripheral blood of rats were detected by flow cytometry.The expression quantity of JNK and p38 MAPK genes in thyroid tissues was detected by q PCR.The protein contents of P-JNK and P-p38 MAPK in thyroid tissues were detected by Western blot.The expression of Bcl-2 and caspase-3in thyroid tissue were observed by immunohistochemistry.Results4.1 A systematic review of Prunella vulgaris preparation in the treatment of autoimmune thyroiditis A total of 16 studies were included.The results of Meta analysis showed that the total effective rate of Prunella vulgaris in the treatment of autoimmune thyroiditis was significantly higher than that in the control group [RR=1.20,95% CI（1.15,1.25）,P<0.00001].The improvement of TPOAb and TGAb in the treatment group was better than that in the control group [SMD=-1.53,95% CI（-2.32,-0.74）,P=0.0002;SMD=-1.91,95% CI（-2.91,-0.91）,P=0.0002].It was significantly superior to the control group in reducing the thyroid volume [the thickness of the right lobe of the thyroid WMD=-0.28,95% CI（-0.47,-0.08）,P=0.005;the thickness of the left lobe of the thyroid WMD=-0.27,95% CI（-0.49,-0.05）,P=0.02;and the thickness of the isthmus of the thyroid WMD=-0.12,95% CI（-0.14,-0.10）,P<0.00001].It was obviously superior to the control group in decreasing Th17/CD4+T[WMD=-1.31,95% CI（-1.50,-1.13）,P<0.00001].However,the incidence of adverse reactions in the Prunella vulgaris group was similar to that in the control group [RR=1.10,95% CI（0.49,2.49）,P=0.82].At the same time,the bias can be controlled according to the funnel chart of the total effective rate.4.2 To explore the mechanism of Prunella vulgaris in the treatment of autoimmune thyroiditis based on network pharmacology 11 active components such as quercetin and luteolin were obtained from TCMSP database.170 targets of non-repetitive action of Prunella vulgaris were obtained after annotations in Uniport database.After searching three major gene databases,5014 genes related to autoimmune thyroiditis were obtained.Through the construction of drug and disease intersection target network,a total of 143 intersection targets were obtained,and 13 core genes such as JUN and MAPK1 were obtained according to the Cyto NCA plug-in score.The 143 intersection targets were analyzed by GO and KEGG.GO enrichment analysis showed that there were 2284 biological processes,46 cellular components and 161 molecular functions,and KEGG enrichment analysis showed 172 signal pathways.Enrichment analysis showed that Prunella vulgaris could treat autoimmune thyroiditis by inhibiting inflammation,immune regulation and apoptosis.The corresponding proteins of JUN and MAPK1 core genes were docked with quercetin and luteolin of Prunella vulgaris,and the results showed that they had high affinity.4.3 To explore the effect of Prunella vulgaris on immunomodulatory mechanism and apoptosis mechanism in rats with autoimmune thyroiditis（1）In the experiment,serum TPOAb and TGAb of the model rats were significantly increased,and lymphocyte infiltration was observed in thyroid tissues,suggesting that the model of autoimmune thyroiditis was successfully established.（2）Compared with normal group,serum levels of FT3,FT4,TPOAb,TGAb,IFN-γ and IL-17 in model control group were significantly increased（P<0.05）,while serum levels of TSH,IL-10 and IL-35 were significantly decreased（P<0.05）.Compared with model control group,the levels of FT3,FT4,TPOAb,TGAb,IFN-γ and IL-17 in each dose Prunella vulgaris groups and selenium yeast group were significantly decreased（P<0.05）,while the levels of TSH,IL-10 and IL-35 were significantly increased（P<0.05）,and the curative effect of Prunella vulgaris was dose-dependent.Compared with selenium yeast group,Prunella vulgaris high-dose group had better improvement on FT3,FT4,TSH,TPOAb and TGAb（P<0.05）,and Prunella vulgaris medium-dose,high-dose groups had better improvement on IFN-γ,IL-17,IL-10 and IL-35（P<0.05）.（3）In the normal group,the thyroid structure was intact,the follicular cavity was full and filled with colloid,the epithelial cells were regular,and there was no lymphocyte infiltration.Compared with normal group,thyroid follicular cells were destroyed,the colloid components were lost,and a large number of lymphocytes were infiltrated in model control group.Compared with the model control group,the thyroid structure was recovered,the arrangement of follicular cells was regular,and only a little lymphocyte infiltration was found in the different dose groups and selenium yeast group,and the degree of recovery was related to the dose of Prunella vulgaris.Compared with selenium yeast group,medium and high dose groups of Prunella vulgaris had better effect on follicular destruction and lymphocyte infiltration.（4）Compared with normal group,the serum levels of Th1,Th17,Th1/Th2 and Th17/Treg in model control group were significantly increased（P<0.05）,while the levels of Th2 and Treg were significantly decreased（P<0.05）.Compared with model control group,the levels of Th1,Th17,Th1/Th2 and Th17/Treg in each dose Prunella vulgaris groups and selenium yeast group were significantly decreased（P<0.05）,while the levels of Th2 and Treg were significantly increased（P<0.05）,and the curative effect of Prunella vulgaris was dose-dependent.Compared with selenium yeast group,the improvement of Th1,Th2 and Th17/Treg in all dose groups was better（P<0.05）,and the improvement of Treg in medium and high dose groups was better（P<0.05）.（5）Compared with normal group,the expression levels of JNK and p38MAPK genes in model control group were significantly increased（P<0.05）.Compared with model control group,JNK and p38 MAPK gene expression levels were decreased in each dose Prunella vulgaris groups and selenium yeast group（P<0.05）.JNK and p38 MAPK were improved better in medium and high dose groups than in selenium yeast group（P<0.05）.（6）Compared with normal group,the proportions of P-JNK/Total JNK and P-p38 MAPK /Total MAPK in model control group were significantly higher（P<0.05）.Compared with model control group,the proportions of P-JNK/Total JNK and P-p38 MAPK /Total MAPK in each dose Prunella vulgaris groups and selenium yeast group were significantly decreased（P<0.05）.Compared with selenium yeast group,P-JNK /Total JNK and P-p38 MAPK /Total MAPK were improved better in medium and high dose groups of Prunella vulgaris（P<0.05）.（7）Compared with normal group,Bcl-2 in model control group were significantly decreased（P<0.05）,and Caspase-3 in model control group were significantly increased（P<0.05）.Compared with model control group,Bcl-2was significantly increased（P<0.05）in each dose Prunella vulgaris groups and selenium yeast group,and Caspase-3 was significantly decreased in each dose Prunella vulgaris groups and selenium yeast group（P<0.05）.Compared with selenium yeast group,the high dose of Prunella vulgaris group had better improvement on Bcl-2（P<0.05）,and the medium and high dose of Prunella vulgaris group had better improvement on Caspase-3（P<0.05）.Conclusions5.1 Prunella vulgaris is effective in the treatment of autoimmune thyroiditis.Prunella vulgaris can reduce the level of autoantibody,thyroid volume and Th17/CD4+T,and has little adverse reaction and immunomodulatory effect.5.2 Prunella vulgaris can treat autoimmune thyroiditis through multi-target,multi-component and multi-pathway.JUN and MAPK1 are the main Hub genes,and the results of molecular docking verification are credible.5.3 In this study,the rat model of autoimmune thyroiditis was successfully established,and it was found that pro-inflammatory factors such as Th1,Th17,Th1/Th2,Th17/Treg,IFN-γ and IL-17 were up-regulated and anti-inflammatory factors such as Th2,Treg,IL-10 and IL-35 were down-regulated in the rats.At the same time,by increasing the contents of JNK,p38 MAPK,P-JNK and P-p38 MAPK,inhibiting the expression of Bcl-2and promoting the expression of Caspase-3,thyroid lymphocyte infiltration and follicular cell apoptosis are further led to the increase of TPOAb and TGAb and the change of thyroid function.Prunella vulgaris capsule can improve the general physiological state of autoimmune thyroiditis model rats,reduce the levels of pro-inflammatory factors Th1,Th17,Th1/Th2,Th17/Treg,IFN-γ and IL-17,and increase the levels of anti-inflammatory factors Th2,Treg,IL-10 and IL-35.By decreasing the contents of JNK,p38 MAPK,P-JNK and P-p38 MAPK,increasing the expression of Bcl-2 and decreasing the expression of caspase-3,thyroid lymphocyte infiltration and follicular cell destruction in model rats were alleviated,TPOAb and TGAb levels were reduced,and thyroid function was improved,and it has the characteristics of dose dependence.