To Explore The Mechanism Of Xiaoyou Decoction In The Treatment Of HR-HPV Complicated With LSIL Based On Autophagy

Objective:1.To explore the molecular mechanism of Xiaoyou decoction in the treatment of HR-HPV combined with LSIL by network pharmacology and molecular docking technology.2.To explore the mechanism of Xiaoyou decoction in the intervention of HR-HPV based on autophagy.3.To observe the clinical efficacy of Xiaoyou decoction in the treatment of HR-HPV combined with LSIL.Methods:1.TCMSP database was used to screen the active ingredients of Xiaoyou decoction,Pubchem database was used to query the corresponding SMILE structures,and Swiss Target Prediction database was applied to project the corresponding targets of candidate ingredients.GeneCards was used to screen the targets of HR-HPV and LSIL,and the intersection targets of them were input into STRING 11.0 database to obtain the protein interaction relationship.Cytoscape 3.7.2 was used to construct the PPI network,DAVID database was used for GO and KEGG enrichment analysis,Autodock TooLs 1.5.6 and Pymol were used for molecular docking study.2.Human cervical squamous cell SiHa in logarithmic growth phase was selected.Three siRNA and one negative control NC targeting human HPV16 E6 and HPV16 E7 genes were designed and synthesized,respectively,and transfected into cells.Using real-time PCR to detect the expression of HPV16 E6/E7 mRNA.Western blot was used to detect the expression of HPV16 E6 and HPV16 E7 protein in each group.Different concentrations of Xiaoyou decoction(0.5g/L,lg/L,2g/L,4g/L,8g/L)were added.Using western blot to detect the expression of LC3 Ⅱ/Ⅰ and Beclin-1 protein in SiHa cells transfected with HPV16 E6/E7 siRNA at different time points(24h,48h,72h).3.A total of 62 patients with HR-HPV persistent infection complicated with LSIL who met the criteria of this study were selected and random divided into the treatment group(n=31)and the blank control group(n=31).The treatment group was given Xiaoyou decoction for vaginal irrigation,and the control group was not treated.The course of treatment was 3 months,and the HR-HPV infection,TCT-DNA changes,lesion outcome,vaginal microecology changes,and TCM syndrome score changes before and after treatment were analyzed and compared 3 months after drug withdrawal.The lessions were followed up for 6 months after drug withdrawal.Results:1.A total of 421 active ingredients were screened out and 1033 active targets were obtained.A total of 1795 HR-HPV targets and 1419 LSIL-related targets were identified,and the intersection of the two targets was 863.A total of 209 intersection targets were identified between Xiaoyou decoction and HR-HPV combined with LSIL.The effective compounds of Xiaoyou decoction on HR-HPV combined with LSIL were naringenin,isorhamnetin,pinifloride,emodin,tetrandrine,ivy saponin,astilbin,soy isoflavone,hydroxynoscapine.The core targets were MAPK3,STAT3,MAPK1,SRC,TP53,AKT1,PIK3CA,JUN,EP300,CTNNB1.The biological processes mainly involved were positive regulation of transcription of RNA polymerase Ⅱ promoter,positive regulation of transcription,positive regulation of cell proliferation,negative regulation of transcription of RNA polymerase Ⅱ promoter,DNA templating,and signal transduction.The cell components mainly involved were nucleus,cytoplasmic matrix,cytoplasm,nucleoplasm,and cell membrane.The main molecular functions involved were protein binding,identical protein binding,ATP binding,enzyme binding,and protein kinase activity.The main pathways include cancer pathway,proteoglycan in cancer,endocrine resistance,EGFR tyro sine kinase inhibitor resistance,PI3K-Akt signaling pathway,T cell receptor signaling pathway,human papillomavirus infection,PD-L1 expression and PD-1 checkpoint pathway in cancer,cell autophagy and other biological processes.The active components of Xiaoyou decoction in the treatment of HR-HPV combined with LSIL act on the autophagy pathway of cells,such as isobutyryl shikonin,isovaleryl shikoni n,naringenin,β-acetoxyisovaleryl akkanin and isorhamnetin.The results of molecular docking showed that the compound isobutyryl-shikonin had the strongest binding to 7N8T.2.There was no significant difference in the mRNA expression level of human cervical squamous cell SiHa after transfection with HPV16 E6 and HPV16 E7 siRNA cells by Real-time PCR(P>0.05)between non-transfection group(Control)and negative control transfection group(NC).The mRNA expression levels of the nontransfection group and the negative control transfection group were significantly different from those of HPV 16 E6/E7 siRNA-1 transfection group,HPV 16 E6/E7 siRNA-2 transfection group and HPV 16 E6/E7 siRNA-3 transfection group,respectively(P<0.05).The mRNA expression level of HPV 16 E6/E7 siRNA-3 transfection group was significantly different from that of HPV 16 E6/E7 siRNA1 transfection group and HPV 16 E6/E7 siRNA-2 transfection group(P<0.05).Western blot analysis was performed for the nontransfection group(Control),negative control transfection group(NC),HPV 16 E6/E7 siRNA transfection group and HPV 16 E6/E7 after adding different concentrations of Xiaoyou decoction for 24h,48h and 72h siRNA+Xiaoyou decoction liquid group 1(0.5g/L),HPV 16 E6/E7 siRNA+Xiaoyou decoction liquid group 2(1g/L),HPV 16 E6/E7 siRNA+Xiaoyou decoction liquid group 3(2g/L),HPV 16 E6/E7.There were significant differences in Beclin-1 and LC3Ⅱ/Ⅰprotein levels between siRNA+Xiaoyou decoction group 4(4g/L)and HPV 16 E6/E7 siRNA+Xiaoyou decoction group 5(8g/L)(P<0.05).3.There was no significant difference in HR-HPV type between the two groups before treatment.After 3 months treatment,HR-HPV outcome(HR-HPV negative,HR-HPV persistent positive,HR-HPV subtype change),TCT-DNA status(NILM,ASC-US,LSIL),analysis of DNA aneuploidies(no aneuploidies,little aneuploidies,lots of aneuploidies)in the two groups were significantly higher than those in the control group(P<0.05).Lesion outcome(lesion regression,lesion persistence,lesion progression)were significantly different(P<0.05).There were significant differences in vaginal microecology(pH,amine taste test,cleanliness)between the two groups after treatment(P<0.05).After treatment,there were significant differences in TCM syndrome scores(the main syndrome volume,color quality,pruritus vulvae and total score)between the two groups(P<0.05).There were no significant differences in TCM syndrome scores(the combined syndrome of red urine,sticky and uncomfortable stool,dry mouth without thirst,small abdominal distension or pain)between the two groups(P>0.05).There was a significant difference in the outcome of lesions between the two groups after 6 months of treatment(P<0.05).Conclusion:1.To investigate the mechanism of Xiaoyou decoction in the treatment of HR-HPV combined with LSIL through multi-components,multi-targets and multi-pathways,especially the components and targets of the autophagy pathway.2.Xiaoyou Decoction can promote the expression of autophagyrelated proteins Beclin-1 and LC3Ⅱ/Ⅰ in HPV-positive cervical cancer SiHa cells in a concentration and time-dependent manner,and induce autophagy in HPV-positive cervical cancer SiHa cells.3.Xiaoyou decoction can significantly improve the negative conversion rate of HR-HPV,the negative conversion rate of TCT abnormal patients,the regression rate of HR-HPV combined with LSIL lesions,and improve the vaginal microecological environment of patients.