| Background Genital tract Chlamydia trachomatis（CT）infection is the most common sexually transmitted disease in the world.Most of the patients are asymptomatic infections,but untimely diagnosis and treatment often lead to serious complications,such as cervicitis,infertility,ectopic pregnancy,blocked fallopian tubes,and premature birth,which seriously threaten human reproductive health.Currently,macrolides,tetracyclines are the main treatment medicine for CT infection.However,as the report of clinical treatment failure increase in recent years,new methods or new drugs to treat CT infection are urgently needed.Our team found that berberine chloride（BBR）has in vitro anti-CT activity,but the mechanism has not been reported in domestic and foreign literature.In this study,the inhibitory effect of BBR on CT was investigated by the cell infection model and animal infection model.We also preliminarily investigated the mechanism of BBR inhibiting CT infection by combining transcriptomic analysis.Objective 1.Clarifying the inhibitory effect of BBR on CT infection from the cellular level.2.Investigating the inhibitory effect of BBR on CT infection from the mouse infection model.3.Preliminary studying the mechanism of BBR inhibiting CT infection combined with transcriptomics.Method 1.The concentration of BBR at the cellular level was determined by Cell Counting Kit 8 assay.The effect of BBR on CT infection in different serotypes of CT（D,E,F,L1）and different cell lines（Hela,Vero,Mc Coy）were investigated by immunofluorescence staining,progeny infection titer,Western blotting and transmission electron microscopy.2.Exploring the effect of BBR in inhibiting CT infection in vivo by establishing a mouse model of chlamydia vaginal tract infection.3.Analysis of the gene expression of control group and BBR-treated group by RNAseq technology.GO functional enrichment analysis and KEGG pathway enrichment analysis were implemented for differentially expressed genes.The transcription and expression levels of related genes were detected by RT-q PCR and Western blotting,and the mechanism of BBR inhibiting CT infection was preliminarily explored.Results 1.Here,we demonstrated that BBR significantly decreased the size and number of inclusions,as well as the number of infectious progeny.The suppressive effect of BBR was observed across the Chlamydia serotypes D,E,F,and L1 and across Hela,Mc Coy,and Vero host cells（P<0.05）.The inhibitory effects of BBR on Chlamydia trachomatis infection were dose-dependent and time-dependent.When combined with azithromycin,BBR exerted a synergistic suppressive effect on Chlamydia infection.2.Significant differences in the number of infectious progeny from vaginal swabs of mice were recorded after BBR alone treatment for 3 and 7 days（P<0.05）.There was no significant difference between azithromycin alone treatment for 3 days and treatment control（P>0.05）.The number of infectious progeny showed that the azithromycin + BBR combined treatment for 3 days and 7 days were statistically different from the treatment control（P<0.05）.3.A total of 2969 genes with differential expression through transcriptome sequencing of the positive infection group and BBR-treated group.GO functional analysis showed that the differential genes were mainly enriched in oxidative stress,regulation of extrinsic apoptotic signaling and cell adhesion factor binding pathway.KEGG pathway analysis revealed that cell cycle,Apoptosis,NF-κB signaling pathway,carbon metabolism and other pathways were significantly enriched.4.Western blotting results showed that the expression of protein and phosphorylated protein of Eph A2 in the BBR-treated group was down-regulated,and the expression of ERK phosphorylated protein was down-regulated.Conclusions 1.BBR has a strong suppressive effect on CT infection across different Chlamydia serotypes and different host cells in cell culture.2.BBR inhibited Chlamydia infection in a mouse model.3.The inhibition mechanism of BBR might be related to the down-regulation of phosphorylated ERK and phosphorylated Eph A2 expression. |
