| Wandai decoction is a classic formula created by Qing-Zhu Fu,a famous doctor in Qing dynasty for the treatment of morbid leukorrhea disease.Based on the typical symptoms of vulvovaginal candidiasis(VVC),a common lower female reproductive tract infectious disease,it belongs to morbid leukorrhea disease.After searching the ancient and modern literature,the hypothesis Wandai decoction restores the vaginal microecology of VVC via mitochondrial protection was proposed.And the experiment designed to verify this hypothesis including clinical trial and basic research mainly focused on the microecology-microbiota in immunity microenvironmentmitochondrial STING pathway.Part 1:Methods:70 cases were recruited and randomized into Wandai decoction group and control group.Both group received Wandai decoction or fluconazole treatment,respectively.The primary outcome includes Test of cure(TOC),Clinical improvement(CI),Follow up(FU);the secondary outcome includes negative response of mycology,recurrent rate,changes of vaginal microbiota,metagenomics,metabolomics,the correlation between metagenomics and metabolomics,the alterations of IL-1α,IFN-α,STING,IRF3,MX2,the impairment of mitochondrial DNA,and the safety evaluation.Results:(1)30 cases were included to perform the data analysis due to that 3 were dropped off,2 removed in Wandai decoction group,and 2 were dropped off,3 removed in control group,respectively.(2)The primary outcome:The TOC is no significant difference(P>0.05)between Wandai decoction(53.3%,16/30)and control group(46.7%,14/30);The CI is found with significant difference(P<0.05)between Wandai decoction(90%,27/30)and control group(76.7%,23/30);The FU is determined with significant difference(P<0.05)between Wandai decoction(83.33%,25/30)and control group(53.33%,16/30).(3)The secondary outcome:during TOC visit,the negative response of mycology was no significant difference between Wandai decoction group(93.33%)and control group(83.33%);during the visit at the 3rd month,the recurrent rate presented with significant difference(P<0.01)between Wandai decoction group(6.67%)and control group(33.33%);vaginal microbiota:L.gasseri L.jensenii were increased in Wandai decoction group,while Prevortella_bivia_g_Prevotella,Atopobium_baginae_g_Atopobium increased in control group.Metagenomics results showed that the vaginal microbial community in the Wandai decoction group was CSTⅡ and Ⅴ(mainly L.gasseri and L.jensenii)after treatment;the vaginal microbial community in the control group was CSTⅢ and Ⅳ after treatment(mainly L.iners,Anaeroboc organism).The results of KEGG functional enrichment analysis showed that:in the cell cycle and meiotic functions of Candida,Wandai decoction group was significantly higher than the control group in the signaling pathways of cell cycle and meiosis(P<0.05).The results of CAZy functional analysis indicated that the Wandai decoction group was significantly higher than the control group in drug resistance and resistance function genes(P<0.05).Metabolomics results:Before and after treatment,the differential metabolites in Wandai decoction group were 59 compared with 43 in the control group.After treatment,the differential metabolites between Wandai decoction group and the control group increased to 120.Before and after treatment,40 differential metabolites were up-regulated and 3 differential metabolites were down-regulated in control group after treatment.In the Wandai decoction group,50 differential metabolites were upregulated and 9 differential metabolites were down-regulated.Compared between the two groups,53 metabolites were up-regulated and 67 metabolites were down-regulated after treatment.The results of KEGG metabolic pathway enrichment analysis of differential metabolites showed that Wandai decoction may be significantly better than fluconazole in improving local vaginal alpha linolenic acid,glycerophospholipid metabolism,mutual conversion of pentose and glucuronic acid,arachidonic acid and other metabolic pathways azoles.The results of the correlation analysis between metagenomics and metabolomics showed that the top 10 differential bacteria and differential metabolites between the groups before and after treatment were sorted according to the correlation value,and the top 5 differential bacteria correlated with each differential metabolite.The species are Lactobacillus_gasseri,Ureaplasma_parvum,Ureaplasma_urealyticum,Lactobacillus_paragasseri,Lactobacillus_jensenii.The top 5 differential metabolites associated with each species were Dilauryl 3,3’-thiodipropionate,Spirolide E,1-Oalpha-D-glucopyranosyl-1,2-nonadecandiol,DG(22:5(4Z,7Z,10Z,13Z,16Z)/22:6(4Z,7Z,10Z,13Z,16Z,19Z)/0:0),(25S)-5alpha-cholestan-3beta,4beta,6alpha,8beta,15alpha,16beta,26-heptol.Based on the results of the association analysis between the differential bacteria and differential metabolites,the KEGG signaling pathway enrichment analysis was performed on the differential metabolites and genes.The results showed that the differentially enriched signaling pathways in the control group before and after treatment were:NOD-like receptors(NOD-like receptors)signaling pathway,biosynthesis of amino acids(amino acid biosynthesis)signaling pathway.In the KEGG enrichment signaling pathways of differential genes and metabolites between groups after treatment,the following 4 signaling pathways were enriched:pentose and glucuronate interconversions(pentose and glucuronate interconversions)signaling pathway,glycerophospholopid metabolism signaling pathway,phenylpropanoid biosynthesis signaling pathway,biosynthesis of animo acids signaling pathway.Both Wandai decoction and fluconazole can increase the immune factor IFN-α in vaginal microenvironment,and there is no statistical difference between the two groups.The Wandai decoction group was better than the control group in reducing the local pro-inflammatory factor IL-1α in the vagina(P<0.05);the control group had an aggravating effect on mitochondria,and the Wandai decoction group did not show the effect of aggravating mitochondrial damage;the contents of STING,IRF3 and MX2 in the Wandai decoction group were significantly increased after treatment(P<0.05,P<0.01).After treatment with fluconazole,only the content of STING increased(P<0.01),and it had no significant effect on the changes of IRF3 and MX2(P>0.05).The measurement results of alanine aminotransferase,serum aspartate aminotransferase,blood urea nitrogen,and creatinine in VVC patients in the both group did not indicate abnormal results,and no adverse effect occurred.Conclusions:(1)The clinical efficacy of Wandai decoction group is better than control group in terms of alleviating or relieving VCC symptoms and signs,preventing VVC recurrence,and restoring vaginal microbial community structure;restoration of vaginal microecology in patients with VVC;(2)Wandai decoction could improve vaginal microecology;(3)Wandai decoction to improve vaginal microecology may be related to the influence of local α-linolenic acid,glycerophospholipid metabolism,mutual conversion of pentose and glucuronic acid,arachidonic acid and other metabolic pathways;(4)Wandai Decoction can restore vaginal microecology and reduce inflammation in VVC patients,which may be related to the activation of STING signaling pathway.Part 2:Methods:VVC mouse animal model were given Wandai decoction(high,medium,low dose),and fluconazole(positive control),antibiotic intervention.Combined with vaginal tissue HE staining,vaginal tissue epithelial cell electron microscopy,vaginal tissue apoptosis detection,vaginal lavage fluid inflammatory factors IL-6,IL-8,Il-10,mitochondrial DNA damage markers,STING signaling pathway related factors detection,the purpose was to explore the mitochondrial protective mechanism of Wandai decoction from the perspective of STING signaling pathway in the treatment of VVC.Results:(1)HE staining results showed that the VVC model was successfully established in the model group mice,which mainly showed that there was more neutrophil infiltration in the vaginal mucosa epithelium,part of the surface epithelial cells were necrotic and exfoliated,and there were lamina propria under the epithelium.More neutrophils were infiltrated,and the blood vessels in the lamina propria were slightly dilated and congested,the interstitial edema was obvious in some areas,and the tissue structure was loose.In the medium-dose group,the improvement of VVC inflammation was more significant.(2)Electron microscope results showed that:the degree of mitochondrial damage in the vaginal epithelial cells of the mice in the model group was relatively high,with severe mitochondrial damage,severe swelling,increased volume,loss of cristae fracture,partial vacuolization,and a small amount of membrane damage;In the high-and low-dose group,the degree of mitochondrial damage in the vaginal epithelial cells of the mice was moderate,some mitochondria were moderately or severely swollen,the matrix was partially dissolved,the cristae were partially lost,and local vacuoles were partially vacuolated.In the positive control group,the antibiotic group,and the normal group,the degree of mitochondrial damage in the vaginal epithelial cells of the mice was relatively low,with a small number of mild or slight swelling,uneven or shallow matrix,a few cristae fractures,and some cristae were neatly arranged.In the medium-dose group,the structure of some mitochondria in vaginal epithelial cells of mice was relatively good,no obvious swelling was observed,the matrix was uniform,and the cristae were neatly arranged.(3)TUNEL staining results of apoptotic cells:the number of apoptotic cells in the model group increased,compared with the normal group,P<0.01;although the number of apoptotic cells in the positive control group,high-dose group,and low-dose group decreased,compared with the model group,the number of apoptotic cells decreased,P>0.05;there was no significant difference between the positive control group and the high-dose group and the low-dose group,P>0.05.Compared with the model group,the antibiotic group and the middle-dose group,P<0.05.There was no significant difference between the antibiotic group and the middle-dose group,P>0.05.(4)Detection results of inflammatory factors in vaginal lavage fluid:the content of cytokines IL-6,IL-8 and IL-10 in the model group mice increased,compared with the normal group,P<0.0001;in the positive control group,the antibiotic group,the high-,medium-and low-dose group,the high contents of IL-6,IL-8 and IL-10 could be reduced,compared with the model group,P<0.01,or P<0.05;among the positive control group,the antibiotic group and the medium dose group,there was no significant difference,P>0.05;the medium dose group was found with significant different compared with the high dose and the low dose group,P<0.05.5)Detection results of mitochondrial damage marker 8-OHDG content:in model group the content of 8-OHDG was significantly increased,compared with the normal group,P<0.0001;the content of 8-OHDG in the positive control group,antibiotic group,highdose group,medium-dose group,and low-dose group could decrease,compared with the model group,P<0.01;There was no significant difference between the positive control group and the medium-dose group,P>0.05.Comparing the medium-dose group with the low-dose and highdose groups,P<0.05.(6)Detection results of related factors of STING signaling pathway:The contents of mitochondrial STING signaling pathway related factors I-IFN,STING,IRF3 and MX2 in vaginal lavage fluid of model group were lower than those of normal group,P<0.01.There was no significant difference in the contents of I-IFN,STING,IRF3 and MX2 between the antibiotic group and the model group,P>0.05.The content of I-IFN and STING in the positive control group was compared with the model group,P<0.05.The contents of I-IFN,STING,IRF3 and MX2 can be increased in the high-dose group,medium-dose group and low-dose group,compared with the model group,P<0.01 or P<0.05;high-dose group,low-dose group and positive control group comparison,the difference was not statistically significant,P>0.05.Comparing the medium-dose group with the positive control group,the difference was statistically significant,P<0.05;comparing the medium-dose group with the high-dose and lowdose groups,P<0.05.Conclusions:(1)The medium-dose group of Wandai decoction has more obvious effect on improving VVC inflammation.(2)The medium dose of Wandai decoction had better protective effect on VVC mitochondria;(3)the medium dose of Wandai decoction had stronger activation effect on STING signaling pathway. |
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