Study On Bacteriostatic Effect Of Rhein On Chlamydia Trachomatis And Its Preliminary Mechanism

BackgroundUrogenital tract Chlamydia trachomatis(Ct)infection is one of the most common sexually transmitted diseases in the world.Many people infected with Ct are asymptomatic,but untreated urogenital chlamydia infection can lead to serious complications,such as prostatitis,pelvic inflammatory disease and infertility.Currently,antibiotics are the main treatment for Ct infection.However,as the risk of antibiotic resistance increases year by year,there are more and more reports of clinical failure.New treatment therapies are urgently needed.Our team found that rhein can inhibit Ct.However,there was no report in domestic and foreign that the inhibitory effect and the mechanism of rhein on the growth of Ct.In this study,the inhibition effect of rhein on the growth of Ct was investigated by the cell infection model and animal infection model.The mechanism of rhein on Ct was preliminarily investigated by RNA-seq transcriptome analysis.Objective1.Clarifying the effect of rhein on Ct infection from the cellular level.2.Explaining the effect of rhein on Ct infection from the mouse infection model.3.To investigate the potential mechanism of rhein on Ct infection by RNA-seq transcriptome analysis.Method1.The cytotoxicity of rhein was examined by Cell Counting Kit 8 assay.Immunofluorescence staining,progeny infection titer,western blot and transmission electron microscopy were used to observe the effects of rhein on four serotypes of Ct(D,E,F,L1)infection and the effects of rhein on Ct infection in different cell lines.2.Investigating the anti-chlamydia effect of rhein in vivo by establishing a Balb/c mouse model of Ct infection.3.To investigate the potential pathway of rhein by exposed rhein at different stages of Ct infection.4.Extracting RNA of control and rhein-treated group for transcriptome sequencing at 48 h post of infection.GO and KEGG enrichment analysis of differentially expressed genes was implemented.Selecting differential genes and using Real-Time PCR detects their mRNA expression.The changes in protein expression were detected by western blot.Results1.At the cellular level,rhein showed significant inhibitory effects on the growth of Ct in multiple serovars of Ct,including D,E,F,and L1,and in various host cells,including HeLa,McCoy and Vero.A few aberrant RBs were observed by transmission electron microscopy in HeLa cells infected with Ct serovar D and treated with 40 μM rhein,compared with many small,mature EB particles within the inclusion of DMSO-treated cells at 48 h post-infection(pi).The inhibitory effect of rhein has dose-effect and timeeffect.A greater inhibitory effect on Ct was observed when rhein was combined with AZM compared with rhein and AZM in vitro.2.In a mouse infection model,the number of infectious progeny from vaginal swabs of mice showed that the AZM + rhein for 3 days and 7 days was statistically different from the positive control group.There were no statistical differences between the positive control group and rhein alone for 3 days or azithromycin alone for 3 days.And azithromycin alone for 7 days is statistically different from the positive control group.3.Rhein and Ct were co-incubated for 12,24,36 and 48 hours,respectively.There was no significant difference in infectivity and inclusion area between the rhein-treated groupand the control group,suggesting that rhein had no direct inactivation effect on Ct.The infected cells were treated with rhein at different stages of Ct infection(24 hours before infection,2 hours of adsorption infection,46 hours post-adsorption infection).It was found that the infectivity,inclusion area and progeny titer of rhein-treated were significantly different from those of the positive control group,suggesting that rhein played an inhibitory role during 46 hours post-adsorption infection.4.Through the transcriptome sequencing of the positive infection group and rhein-treated group.1940 differential genes were obtained;Go functional analysis genes were mainly enriched in adhesion junctions and other protein bindings,cell division regulation,and oxidative stress;KEGG pathway analysis revealed that PI3K-AKT signaling pathway and carbon metabolism are significantly enriched.The expression of the p-Akt protein in the rhein-treated group was down-regulated compared with that in the infection group.Conclusions1.Rhein has a significant inhibitory effect on Ct in the cell infection model.2.Rhein and azithromycin can synergistically inhibit Ct infection both in vivo and in vitro.3.Rhein can inhibit Ct infection by interacting with the host.By RNA-seq analysis,the inhibition mechanism of rhein might be related to the down-regulation of the PI3K-AKT pathway.