Therapy And Mechanism Of Vaginal Microbiota Transplantation And Combination Of Vaginal Probiotics In The Vaginal Bacterial Infections

Background and Aims:Variety of vaginal diseases was caused by the imbalance of the species or their quantities of microflora in the vaginal microecological system.Currently,the treatment of vaginal bacterial infections is mainly based on the use of antibiotics,which can significantly change the microflora of the vagina,such as dysbacteriosis,resulting in poor treatment and higher relapse rates of vaginal bacterial infections.Numerous researches have shown that the health status of the human body possesses a strong relationship with the intestinal flora.Bifidobacteria and Lactobacilli existing in intestine play important roles in the digestion,immune regulation,and the prevention and treatment of chronic diseases.In light of the similar physiological state and flora composition between vaginal tract and the gut environment system,as well as the similar pathogenesis and treatment of Clostridium difficile infection(CDI)and bacterial vaginosis(BV),our group proposed the concept of vaginal microbiota transplantation(VMT)for the treatment of bacterial vaginosis.This method aims to utilize the normal human vaginal flora or probiotic combinations isolated from normal human vaginal tract,and translated them into a laboratory-constructed rat bacterial vaginosis model to restore and reconstruct the balance of vaginal flora.Method:(1)The isolation of microorganisms from vaginal secretions of healthy women.From the vaginal secretions,preliminary screening of vaginal microorganisms by bacterial morphology,colony morphology,Gram stain,and 16 S rRNA sequencing technology were used to identify strains.(2)Evaluation of the probiotic properties of isolates strains in vitro.To further study the probiotic characterization of the selected strains,their acid and bile resistance,anti-oxidation ability,inhibition of pathogenic bacteria growth,cell adhesion,and colonization resistance were evaluated by in vitro experiments.(3)Establishment of the rat vaginitis model.Experimental rats were divided into five groups: negative control group(C group,N=5),model group(M group,N=5),metronidazole treatment group(MA group,N=5),probiotic combination therapy group(MP group,N=5),vaginal flora transplantation treatment group(MV group,N=5).Escherichia coli,Staphylococcus aureus and ?-Streptococcus sp.were mixed at a ratio of 2:1:1.Rat uterine tissue was used for HE staining after modeling to made sure of the success of modeling.Then,drugs were used to treat the mice for another 7 days,and then resumed for three days.(4)Rat vaginitis model evaluation after vaginal microbiota transplantation treatment.At recovery period,uterus tissues were dissected,and the HE staining for detection the pathophysiological changes,and IHC staining was used for detection the expression of Caspase-3,Caspase-8 and FasL.Then,IL-4,IL-1? and TNF-? inflammatory cytokines in the uterus tissue were measured using a commercial Elisa kit and q-PCR at protein level and gene level,respectively;High-throughput sequencing and q-PCR were used to determine the microbial diversity and numbers of vaginal flora in rats.Result:(1)From the vaginal secretions of healthy women,we isolated 10 strains named as L.helveticus,L.crispatus,L.acidophilus,L.gasseri,L.salivarius,P.stutzeri,S.capitis,E.faecalis,S.anginosus,and S.epidermidis.L.helveticus,L.crispatus,L.acidophilus,L.gasseri,and L.salivarius were selected for subsequent experiments.(2)Evaluation results of probiotic properties showed that L.helveticus,L.crispatus,L.acidophilus,L.gasseri and L.salivarius were compatible with the degree of acid tolerance to the vaginal acidic environment but were not tolerant to the pressure of bile salts;In addition,most strain possessed sound scavenging ability of superoxide radicals,ferrous irons Ion,hydroxyl radicals,1,1-diphenyl-2-picrylhydrazyl(DPPH)and total reducing power;All isolates had successfully inhibited growth of vaginosis pathogens such as Candida albicans,Staphylococcus aureus,E.coli,beta-hemolytic streptococcus;the adhesion results indicated that the selected strains could adhere to Hela cells with a high number,and could greatly inhibit the adherence of pathogens to Hela cells.(3)Anatomical observation of the uterus tissue.Compared with the C groups,the uterus morphology,color and uterine wall in M group were thin,and the uterine inflammation disappeared but the individual samples appeared uterine deformity and atrophy in MA group,and the uterine morphology in MP and MV group restored to the normal level.HE results were consistent with the morphology observation.IHC results showed that the expression of Caspase-3,Caspase-8,and FasL were down-regulated in recovery period.Moreover,compared with the C group,the level of mRNA and protein of IL-4 in the uterus of the M group was significantly decreased,while the mRNA and protein levels of IL-1? and TNF-? were significantly increased.The results showed that both MP and MV groups had significant protective effects(p<0.05).The results of high-throughput sequencing showed that VMT had effectively sustained the microbial diversity after treatment,and q-PCR results indicated that VMT had reduced the number of Enterococci and Enterobacteriaceae,while increased the number of Lactobacilli.Conclusion:(1)The vaginal probiotics combination and VMT have a positive therapeutic effect on bacterial vaginal infections;(2)The vaginal probiotics combination and VMT can reduce the apoptotic factors of Caspase-3,Caspase-8 and FasL,raise the level of anti-inflammatory cytokines IL-4,and decrease the levels of proinflammatory cytokines IL-1? and TNF-?;(3)High-throughput sequencing results show that although metronidazole has a positive effect on the treatment of bacterial vaginosis,it reduces vaginal inflammation but also disrupt the diversity of vaginal flora.The vaginal probiotics combination and VMT not only have a positive effect on reducing vaginal inflammation,but also maintain or restore the diversity of bacterial vaginal inflammation;(4)For the first time,the concept of VMT is put forward,which provides a theoretical basis and data support for the development of female health drugs.