Study On The Mechanism Of Fuyanshu Capsule By Regulating The TLRs/MyD88 Pathway Through KLF4 And A Systematic Review Of Expelling Dampness And Dissipating Blood Stasis Therapy Of Chinese Patent Medicine For SPID

Objective1.To provide evidence for the development of the ‘Clinical Application Guidelines for Traditional Chinese Medicines to Treat Sequelae of Pelvic Inflammatory Diseases’ and to provide a new method for re-evaluation of Chinese patent medicines on the market through the Evidence-based evaluation study of evaluating the effectiveness and safety of expelling dampness and dissipating blood stasis therapy of Chinese patent medicine for SPID.2.To re-evaluate the effect and investigate the mechanisms of Chinese patent medicine Fuyanshu capsule through observating the inflammatory pathological changes,inflammation scores,the expression of IL-6 and IL-1β,and the relative expression changes of KLF4、TLR2、TLR9、MyD88、NF-κB in uterus of SPID rats in different groups.Method1.A Systematic Review of Expelling Dampness and Dissipating Blood Stasis Therapy of Chinese Patent Medicine for SPID.We electronically searched CENTRAL,MEDLINE,EMBASE,CNKI,VIP,Wanfang,CBM,clinical trial registration platform,and domestic and international guidebook databases from the inception to December 2019.We included clinical trials of expelling dampness and dissipating blood stasis therapy of Chinese patent medicine for the treatment of SPID.End Note X8 software was used for document management.Two evaluators independently performed literature screening,data extraction,and methodological quality evaluation.Cochrane Collaboration Bias Risk Assessment Tool for RCTs,MINORS score for Non-randomized clinical trials,and NOS scale for observational clinical studies.Quantitatively synthesized research data was analyzed using Revman 5.3.5 software for meta-analysis.The GRADE(The Grading of Recommendations Assessment,Development and Evaluation)method was used to evaluate the evidence of the effectiveness and safety,and the quality of theevidence was divided into high,medium,low,and extremely low levels.2.Study on Anti-inflammatory Mechanism of Fuyanshu Capsules Regulating TLRs / MyD88 Pathway Through KLF4.We injected mixed bacteria liquid to establish a rat SPID model at first.Then we divided these rats into five groups randomly:model group,Kangfuyan capsule group(KFY),Fuyanshu(FYS)low-dose group,FYS middle-dose group,and FYS high-dose group.There were also sham operation group.There were 6 rats in each group.The sham operation group and the model group were orally administered with distilled water,and the KFY group and the FYS groups were orally administered with a suspension of the corresponding drug.Rats in each group were administrated intragastrically for 21 days.They were killed and laparotomy was performed to remove uterine tissue.We executed the rats and removed the uterus.Wedetermined the content of IL-6 and IL-1β by ELISA method.HE staining was used to observe the morphology of rat uterine tissue.RT-PCR method was used to determine the expression of TLR2,TLR9,MyD88,KLF4 mRNA,and Western Blot method was used to determine the relative expression levels of TLR2,TLR9,MyD88,and KLF4 proteins,and the expression of NF-κB was observed by immunohistochemistry.The correlation between KLF4 and key factors of the TLRs / MyD88 pathway was explored by Pearson method.Results1.The results of system review(1)Search results: We retrieved 10324 original articles according to the search strategy and 116 articles were finally included.The literature of the 18 proprietary Chinese medicines is as follows: Jingangteng preparation(n=22),Huangtengsu tablets(n=3),Penyanjing preparation(n=5),Huahong preparation(n=2),Fukeqianjin preparation(n=41),Gongyanping preparation(n=5),Pulingpenyankang granule(n =2),Kangfuyan capsule(n=12),Fule preparation(n=3),Fuyanshu capsules(n=4),Kangfuyan capsule(n=1),Jinji capsule(n=1),Fuyankang tablet(n=3),Fuyanjing capsules(n=2),Fuyanxiao capsule(n=3),Fuyankangfu preparation(n=3),Jinying capsule(n =3),Jinxiang capsule(n=1).(2)Methodological quality: A total of 109 RCTs were included in the study,8studies were rated as High risk,5 were rated as Low risk,and 96 studies were rated as unknown because of the lack of relevant information.4 non-randomized controlled trials were included,3 studies were rated 5 points,and 3 studies were rated 7-8 points.3 observational studies were included,and the NOS scale scores were 4-5.(3)Quality of evidence: The quality of evidence for 18 Chinese patent medicine is as follows: Fukeqianjin preparation,Jingangteng preparation,Kangfuyan capsule,Jinying capsule were rated B-level,Fuyankangfu preparation,Huahong preparation,Kangfuyan capsule,Penyanjing granule,Fuyanshu capsule were rated C-level,Gong yanping preparation,Fuyankang tablet,Huangtengsu tablet,Fule preparation,Pulingpenyankang granule,Jinji capsule,Fuyanjing capsule,Fuyanxiao capsule,Jinxiang capsule were rated D-level.2.The results of the mechanisms of Fuyanshu Capsules:(1)Description of pathological morphology of uterine tissue: In the sham operation group,the structure of the endometrium,myometrium and adventitia was clear,the epithelial cells were short columnar,and the arrangement was regular;the lamina propria cells were tightly arranged,and the uterine glandular structure was normal.In the model group,the uterine cavity wall was thin,the lamina propria decreased,the epithelial cells were degenerated and necrotic to varying degrees,the uterine gland epithelium was necrotic,a large amount of necrotic cell debris and inflammatory cells were infiltrated in the glandular cavity,anda large number of inflammatory cells infiltrated the lamina propria,partly involving the muscle layer,and some lamina propria were slightly edema.In the KFY group,the endometrial epithelial cells were mildly degenerative and necrotic,and a small number of inflammatory cells invaded the lamina propria.In the low-dose FYS group,the uterine cavity wall was thin,the lamina propria was reduced,the epithelial cells were mildly degenerated and necrotic,and the inflammatory cells infiltrated in the lamina propria.In the middle-dose group of FYS,the uterine cavity wall was thin,and the lamina propria decreased.The inflammatory cells infiltrated in the lamina propria,and the lamina propria was slightly edema.In the FYS high-dose group,a small amount ofepithelial cells degeneration and necrosis of the endometrium,a small amount of infiltration of inflammatory cells in the lamina propria,and a slight edema of the lamina propria.(2)Inflammation score of uterine tissue: The score of model group were significantly higher than that of sham operation group(P<0.01).Compared with the model group,the score of KFY group was significantly reduced(P<0.01),the scores of middle and high dose groups were reduced(P<0.05),and the score of low dose group was not significantly different(P>0.05).There was no significant difference between the scores of FYS dose groups and KFY groups(P>0.05).(3)IL-6: The expression of IL-6 in the model group was significantly higher than that of the sham operation group(P <0.01).Compared with the model group,the expression of IL-6 in the high-dose FYS group was significantly reduced(P <0.01).The expression of IL-6 in KFY group,FYS low and middle dose groups had a decreasing trend(P> 0.05).There was no significant difference between the KFY group and the FYS groups(P> 0.05).(4)IL-1β : The expression of IL-1β in the model group was significantly higher than that ofthe sham operation group(P <0.01).Compared with the model group,the expression of IL-1β in the KFY group and the FYS high-dose group was reduced(P<0.05).The expression of IL-1β in the FYS low and middle dose groups had a decreasing trend(P> 0.05).There was no significant difference between the KFY group and FYS groups(P> 0.05).(5)KLF4:The expression of KLF4 mRNA and protein in the model group were lower than those of the sham operation group(P <0.05).Compared with the model group,the expression of KLF4 mRNA in the KFY group and FYS groups had an increasing trend(P> 0.05).There was no significant difference between the groups(P> 0.05).The expression of KLF4 protein in KFY group and FYS high-dose group was significantly increased(P <0.01),and it was increased in FYS low and middle dose groups(P>0.05).There was no significant difference between KFY group and FYS middle and high dose groups(P> 0.05).(6)TLR2:The expression of TLR2 mRNA and protein in the model group wassignificantly higher than that of the sham operation group(P <0.01).Compared with the model group,the expression of TLR2 mRNA and protein in the KFY group and FYS high-dose group was significantly reduced(P <0.05).There was a downward trend in the FYS low and middle dose groups(P> 0.05).There was no significant difference in TLR2 mRNA expression between the KFY group and FYS dose groups(P>0.05).There was a significant difference in the expression of TLR2 protein between the KFY group and the FYS low and middle dose groups(P <0.05).(7)TLR9:The expression of TLR9 mRNA and protein in the model group were higher than those of the sham operation group(P <0.05).Compared with the model group,the expression of TLR9 mRNA and protein in the KFY group was reduced(P<0.05),and the expression of TLR9 protein was significantly reduced in the FYS high dose group(P <0.01).The expression of TLR9 mRNA in the low,middle and high dose groups of FYS and the expression of TLR9 protein in the low and middle dose groups of FYS all showed a decrease(P> 0.05).There was no significant difference in TLR9 mRNA and protein between KFY group and FYS groups(P> 0.05).(8)MyD88:The expression of MyD88 mRNA and protein in model group were higher than those of sham operation group(P <0.05).The expression of MyD88 mRNA was decreased in KFY group and FYS high dose group(P<0.05).MyD88 protein was significantly reduced in the KFY group,the middle and high dose groups of FYS(P <0.01).MyD88 mRNA was decreased in the low and middle dose groups of FYS,and MyD88 protein was decreased in the low dose group of FYS(P> 0.05).There was no difference in MyD88 mRNA between the KFY group and the FYS group(P> 0.05).There was asignificant differencein MyD88 protein betweenthe KFY group and the FYS low dose group(P <0.05).(9)NF-κB: The expression level of NF-κB protein in the model group was higher than thatof sham operationgroup(P<0.05).The expression level of NF-κB in the KFY group,FYS middle and high dose groups was reduced compared with the model group(P <0.05).There was a significant difference between the KFY group and the FYS low dose group(P <0.05).There was no significant difference between the KFY group and the FYS high dose group(P> 0.05).(10)Results of correlation analysis: KLF4 protein was negatively correlated with TLR2 protein,TLR9 protein,MyD88 protein,NF-κB protein、IL-6、IL-1β(P<0.01 or0.05).Conclusions1.Chinese patent medicine can alleviate chronic pelvic pain,improve vaginal secretions,and improve pelvic inflammatory signs.It may reduce pelvic inflammatory mass,reduce pelvic effusion,improve the quality of life of patients,reduce the recurrence rate,and do not increase the incidence of adverse reactions.The quality of evidence is low,ranging from medium quality(grade B)to very low quality(grade D).2.Fuyanshu capsule can alleviate inflammation of uterine tissue in SPID model rats and the anti-inflammatory mechanism may be related to up-regulation of KLF4 expression,regulation of TLRs/MyD88 signaling pathway,and down-regulation of inflammatory cytokines IL-1β and IL-6.3.As one of the upstream regulatory factors of TLRs/MyD88,KLF4 has a negative regulatory effect on the signaling pathway and may be one of the key targets for the anti-inflammatory effect of Fuyanshu Capsule.4.The exploration of the efficacy mechanism of Chinese patent medicines combined with the systematic evaluation of Chinese patent medicines,which are mutually confirmed,can provide ideas for the evaluation of Chinese patent medicines in the future.