Study On The Effect Of The Butyl Alcohol Extract Of Baitouweng Decoction On Improving The Vaginal Mucosal Barrier Of Vulvovaginal Candidiasis Mice And The Main Component Of Berberine In Inhibiting The Adhesion Of Candida Albicans To The Vaginal Epithelium

Objective:To study the effect of Butyl Alcohol Extract of Baitouweng Decoction（BAEB）on the integrity of vaginal mucosal epithelial barrier in mice with vulvovaginal candidiasis（VVC）and to further explore the mechanism of berberine hydrochloride,the main component of BAEB,on the adhesion of Candida albicans to human vaginal epithelial cells in vitro.Methods:1.SPF female Kunming mice were subcutaneously injected with estradiol benzoate3 times every other day to induce pseudoestrus;and they were randomly divided into 6groups（blank control group,fluconazole group,model group,baeb high,medium and low dose groups）;except the blank control group,mice in other groups were inoculated with Candida albicans SC5314 standard strain with 20 ul bacterial solution(2×10⁶CFU·ml^-1)per mouse Albicans to the vagina of mice,suspended upside down for 5minutes,continuous 7 days to build VVC model.Then BAEB（80 mg/kg,40 mg/kg,20 mg/kg）was given by gavage according to corresponding groups for 7 days.After BAEB treatment,Gram staining was used to observe the morphology of Candida albicans in vaginal secretions of mice;plate method was used to detect the vaginal fungal load of mice after baeb treatment;HE（hematoxylin-eosin staining）staining was used to observe the pathological changes of vaginal mucosa;MT（Masson’s trichrome staining）,HE and PAS（periodic acid Schiff staining）were used to observe the integrity of the vaginal mucosal epithelial barrier;The expressions of mucin-1,Mucin-4 andβ-defensin2 were detected by immunohistochemistry;The dynamic expressions of mucin-1,Mucin-4 andβ-defensin 2 were detected by Western blot on day 0,3 and 7 days.2.CCK8 detection and inverted microscope morphological observation showed that2×10⁵ cells/ml is the optimal growth density of A431 cells,and the cell spread is large,the plasmodesmata is tight,the folds are prominent,and there are very few floating objects;The results of Gram staining showed that Candida albicans adhered to A431 cells with the largest amount of 2×10⁷CFU·ml-1 at 2 h at the early adhesion time and different bacterial concentrations（p<0.0001）;The levels of IL-2 and IL-4 in the supernatant of cells stimulated by Candida albicans and the dynamic ratio of IL-2/IL-4 were detected by ELISA;The dynamic expression of ICAM-1 on cell membrane stimulated by Candida albicans was observed by immunofluorescence;The dynamic changes of adhesion molecules ICAM-1 and mucin-1 and Mucin-4 in A431cells stimulated by Candida albicans were detected by Western blotting.Results:1.Gram staining and plate coating showed that the amount of Candida albicans hyphae in the vagina of the model group was more denser,longer and higher than that of the blank control group;HE staining,MT staining and PAS staining showed that the structure of vaginal mucosa in the model group was seriously damaged,the keratinized layer fell off,the epithelial cells proliferated and the inflammatory infiltration intensified;The expressions of mucin-1,Mucin-4 andβ-defensin 2 on the surface of vaginal epithelial cells were significantly increased by immunohistochemistry and Western blot（p<0.0001）;After VVC mice were treated with BAEB,compared with the model group,the formation of hyphae in the vagina of BAEB（high,medium,and low dose groups）mice was significantly inhibited,and the fungal load was significantly reduced（p<0.05）;Inflammation and infiltration are correspondingly reduced,vaginal mucosal tissue damage is gradually improved,the epithelial barrier is repaired and its integrity is restored;the mucin-1,mucin-4 andβ-defensin 2 protein levels on the surface of vaginal epithelial cells are significantly down-regulated（p<0.001）.2.CCK8 detection and inverted microscope morphological observation showed that2×10⁵ cells/ml is the optimal growth density of A431 cells,and the cell spread is large,the plasmodesmata is tight,the folds are prominent,and there are very few floating objects.The results of Gram staining showed that Candida albicans adhered to A431 cells at 2×10⁷CFU·ml-1 at the early adhesion time and different bacterial concentrations（p<0.0001）.The ELISA test showed that the secretion of the pro-inflammatory factor IL-2was increased compared with the normal group cell model group,and the increase was extremely large,and the secretion of the anti-inflammatory factor IL-4 was also increased,but the magnitude was very small;and the pro-inflammatory factor IL-2 after the intervention of berberine hydrochloride secretion was significantly down-regulated,and the anti-inflammatory factor IL-4 secretion was significantly up-regulated.The results of immunofluorescence showed that the expression of adhesion molecule ICAM-1 was significantly increased compared with the normal group（p<0.0001）and the expression of ICAM-1 was the highest at 2 h;the expression of ICAM-1 after the intervention of berberine hydrochloride significantly reduced（p<0.0001）.Western blotting results showed that the expressions of adhesion molecules ICAM-1,mucin-1 and mucin-4 were significantly increased compared with the normal group cell model group,and had a short-term time dependence;while ICAM-1 after the intervention of berberine hydrochloride the expressions of mucin-1 and mucin-4 were significantly lower than the model group.Conclusion:This experiment successfully constructed a mouse vulvovaginal candidiasis（Vulvovaginal Candidiasis,VVC）model,and found that the treatment of VVC mice by BAEB is closely related to the repair of the vaginal epithelial barrier and the remodeling of the mucosal integrity,and its mechanism of action may be through it is achieved by regulating the expression of the body’s innate immune-related immune molecules mucin-1,mucin-4 and beta defensin 2.In vitro experiments have shown that the main component of BAEB,berberine hydrochloride,can inhibit the early adhesion and damage of Candida albicans to vaginal epithelial cells,and down-regulate adhesion-related molecules ICAM-1 and mucin-1 and mucin-4 when the concentration is lower than its MIC.The expression of further clarified the role and mechanism of BAEB against VVC from the perspective of vaginal epithelial cells and related immune molecules.