| The high incidence of vulvovaginitis in prepubertal girls and the tendency for the condition to recur after antibiotic treatment are among the most common and challenging gynaecological problems in children and adolescents,and recurrent disease may lead to reproductive disorders in adulthood.Repeated antibiotic treatment over a long period of time may cause dysbiosis of the vaginal flora and pose other health risks.Previous clinical practice has shown that our Jie Tong Yin lotion is effective in treating this disease,with a low clinical recurrence rate.In order to investigate the clinical characteristics of vulvovaginitis in prepubertal girls and to explore the mechanism of action of traditional Chinese medicine,this study was designed to investigate the clinical efficacy and bioinformatics of the Jie Tong Yin lotion and to explore the effect of Jie Tong Yin lotion on the vaginal microecology and local immunity of prepubertal girls.ObjectivesStudy 1:To summarise the common causes,disease characteristics and TCM patterns of vulvovaginitis in prepubertal girls,to study the vaginal micro-ecological characteristics of girls between different patterns and to provide an objective reference basis for TCM pattern research.Study 2:To objectively evaluate the clinical efficacy of Jie Tong Yin lotion in the treatment of vulvovaginitis in pre-pubertal girls(damp-heat pouring downward Syndrome).Study 3:To conduct bioinformatics analysis of Jie Tong Yin lotion to find out the possible key targets and signalling pathways of Jie Tong Yin lotion in the treatment of vulvovaginitis in girls,so as to provide a theoretical basis for subsequent experimental studies.Study 4:To examine the vaginal micro-ecological flora of children before and after the treatment of vulvovaginitis,in order to explore the effect of Vaginal Vaginitis on the dysbiosis of the vaginal flora in girls with vulvovaginitis.Study 5:To examine the levels of vaginal immune cytokines in children before and after treatment with Jie Tong Yin lotion in order to explore the vaginal immune characteristics of girls with vulvovaginitis and the possible immunomodulatory effects of the herbal lotion.MethodsStudy 1:A single-centre,cross-sectional,descriptive study.A questionnaire on factors associated with the development of vulvovaginitis in prepubertal girls and a TCM evidence-based identification scale were developed to collect clinical data,identify and classify the evidence,and collect vaginal secretions from some of the affected children for vaginal microecological testing using 16s rRNA high-throughput sequencing to compare the vaginal microecological characteristics of different TCM evidence-based types.Study 2:A randomized,double-blind,placebo-controlled,clinical trial was used.Girls aged between 3 and 9 years with vulvovaginitis(damp-heat pouring downward syndrome)were used as the study subjects.The sitz bath was administered twice daily for 10 minutes for 14 days and the clinical efficacy of the treatment was evaluated at the end of the trial.Study 3:Using a network pharmacology study,the components and related targets of Jie Tong Yin lotion and the targets related to vulvovaginitis in girls were searched and a network topology analysis was conducted to predict and screen potential target proteins and related protein pathways for the treatment of vulvovaginitis in girls with Jie Tong Yin lotion.On this basis,molecular docking studies between the main active ingredients and the core targets of Jie Tong Yin lotion were conducted.Study 4:Vaginal secretions were collected from the children in Study 2 and collected again after the children in the test group had recovered.16S rRNA high-throughput sequencing was performed to compare the changes in vaginal microecology before and after treatment.Study 5:Vaginal swabs were collected from the children in Study 2 and collected again once after the children in the test group had healed.Vaginal IL-2,IL-4,IL-8,TGF-β1,TNF-αand IFN-y levels were measured by flow fluorescence assay and vaginal TLR2 and TLR4 levels were measured by enzyme-linked immunosorbent assay.The changes in vaginal immune cytokines before and after treatment were compared.ResultsStudy 1:A total of 428 questionnaires were qualified.Factors associated with the onset of vulvovaginitis in girls:respiratory infections,swimming/spas,urinary tract infections within 1 month prior to onset,30.74%cumulatively;poor diaphoretic hygiene habits,72.6%cumulatively;parental factors,51.6%cumulatively;59.3%of girls had manifestations of allergic diseases within the last 1 year.Clinical features of vulvovaginitis in girls:all children had vaginal discharge,inflammatory vestibular mucosal congestion was the main sign,itching and discomfort in 70.3%,flushing of the vulvar skin in 33.9%,urinary frequency and urgency in 11.4%and odour in 18.9%.The pathogenicity rate was 38.1%,with Haemophilus influenzae,Staphylococcus aureus,Escherichia coli and Streptococcus pyogenes being the most frequent pathogens.45.8%of the children(196 cases)also had a combination of other genitourinary disorders.In prepubertal girls,vaginitis of the vulva was most commonly associated with damp-heat pouring downward Syndrome,accounting for 77.1%of cases,followed by spleen deficiency and dampness trapped Syndrome,accounting for 18.0%of cases.The results of 16S rRNA sequencing indicated that the differences in the Alpha Diversity Ace Index and Chao1 Index between children with damp-heat pouring downward Syndrome(Group A)and spleen deficiency and dampness trapped Syndrome(Group B)were statistically different(P values<0.05),indicating that the species richness of Group A was higher than that of Group B.group was more dominated by bacteria of the phylum Aspergillus and Clostridium.The results of LEfSe analysis of samples between groups show Oscillospirales in group A and Anaerococcus,Prevotella-buccalis,Peptoniphilus,unclassified-Peptoniphilus in group B could be used as Biomarker.Study 2:81 cases in the test group and 38 cases in the control group participated in the statistical analysis.The total effective rate of the test group was 90.1%,with a cure rate of 85.2%and a healing rate of 75.3%,while the total effective rate of the control group was 52.6%,with a cure rate of 31.6%and a healing rate of 23.7%,with statistically significant differences between the two groups(P<0.01).At the end of the clinical trial,antibiotics were added in 13 cases in the trial group(16.0%)and 17 cases in the control group(44.7%),with a statistically significant difference in the rate of antibiotic use between the two groups(P<0.01).The disease recurrence rate was 7.40%in the test group and 13.12%in the control group.Study 3:The main active ingredients of Jie Tong Yin lotion were quercetin,muxicin and baicalin,involving possible key targets such as TPT53,AKT1,EGFR and IL-6,and signalling pathways such as IL-17,NF-κB and oestrogen as key pathways of action for the treatment of vulvovaginitis in girls,which were validated by molecular docking studies.Study 4:A total of 18 vaginal swab specimens were collected from 9 cured children in the test group before and after treatment.group A was the pre-treatment specimen and group B was the post-treatment specimen.After treatment,the abundance of Proteobacteria and Bacteroidota decreased;Haemophilus and Prevotella decreased.Peptostreptococcales-Tissierellales,Fusobacteriales(Clostridium)decreased after treatment,while Burkholderiales,Bifidobacteriales,Bifidobacterium Cyanobacteriales,and Lactobacillales increased in abundance after treatment.Study 5:A total of 29 pre-treatment vaginal swabs(Group A)and 20 post-treatment vaginal swabs(Group B)were collected.IL2 levels increased after treatment,from 46.38 pg/ml to 161.10 pg/ml,a statistically significant difference(P<0.01).IL4 decreased after treatment,from 197.72 pg/ml to 41.25 pg/ml,a statistically significant difference(P<0.05).TGF-β1 showed a decreasing trend after treatment,from 8.05 pg/ml to 4.36 pg/ml,but there was no statistically significant difference(P value 0.143).IL8,TNF-α and IFN-γ did not change significantly before and after treatment.After treatment,TLR2 levels in the vagina decreased from 16.81±1.47 pg/ml to 15.69±1.52 pg/ml,with a statistically significant difference(P<0.05),while TLR4 levels did not change significantly before and after treatment.ConclusionsThere are many factors involved in the development of vulvovaginitis in girls.Cultivating good hygienic habits and taking care of allergic factors can help prevent and treat the disease.All children had vaginal discharge and inflammatory vestibular mucosal congestion was the main sign.45.8%of the children also had other genitourinary disorders and the pathogenicity rate was 38.1%.Vulvovaginitis in prepubertal girls is most commonly associated with dampheat pouring downward Syndrome,with differences in vaginal microecology between the different syndromes.In the treatment of vulvovaginitis in prepubertal girls(damp-heat pouring downward Syndrome),the total effective rate was 90.1%,with a recovery rate of 85.2%and a cure rate of 75.3%,reducing the use of antibiotics.In the placebo lotion group,the total effective rate was 52.6%,with a recovery rate of 31.6%and a cure rate of 23.7%,confirming the need for hygiene measures.The bioinformatics study of Jie Tong Yin lotion suggests that this preparation may treat vulvovaginitis in girls through a multi-targeted,multi-pathway treatment.The treatment of girls with vulvovaginitis with Jie Tong Yin lotion has been associated with a reduction in clinical symptoms and a corresponding change in vaginal microecology,suggesting that Jie Tong Yin lotion has a role in regulating vaginal microecology.The intrinsic vaginal immune response in girls with vulvovaginitis was dominated by TLR2 activation and upregulation,and the cellular immunity was probably dominated by Th2 and Th17 type immune responses.The significant increase in IL-2 and decrease in IL-4 in the vagina after treatment may be related to the fact that the cleansing solution regulates the vaginal flora,up-regulates the Th1 cellular immune response and enhances the clearance of the infecting organism,while suppressing the Th2 immune response and decreasing the inflammatory response of the body. |
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