Mechanism Of N-butanol Extract Of Pulsatilla Decoction In Treatment Of Vulvovaginal Candidiasis Based On The Negative Regulation Of NLRP3 Inflammasome Via PKCδ/NLRC4/IL-1Ra Axis

1 Objective:This study aims to explore the mechanism of n-butanol alcohol extract of Pulsatilla decoction(BEPD)in the treatment of vulvovaginal candidiasis(VVC)mice from the perspective of PKCδ/NLRC4/IL-1Ra axis negatively regulating NLRP3 inflammasome.This study further explored the effect of BEPD-containing serum on PKCδ/NLRC4/IL-1Ra axis of A431 cells stimulated by Candida albicans in vitro,and to preliminarily elucidate the mechanism of BEPD in the treatment of VVC.2 Methods:In vivo experiments: SPF female C57BL/6 mice were randomly divided into 6groups: blank control group,VVC model group,BEPD high,medium and low dose group and fluconazole group.Except the blank control group,the mice in other groups were established VVC model by estrogen-dependent method for 7 d.On the 8th day,the mice in each group were given different concentrations of BEPD,fluconazole and normal saline by gavage for 7 d.During the experiment,the general state of the mice and the local signs of the vagina were observed daily,and the body weight of the mice in each group was recorded.The vaginal lavage fluid of the mice was collected on the 1st,3rd,7th and 14 th days of VVC infection and used to detect the following indicators: Gram staining was used to detect the morphology of Candida albicans in the vaginal lavage fluid of the mice;microdilution method was used to detect the fungal load of vaginal lavage fluid in each group;pap staining was used to detect the infiltration of neutrophils in vaginal lavage fluid of mice,and ELISA was used to detect the contents of IL-1β,IL-18 and LDH in vaginal lavage fluid of mice;On the 15 th day of modeling,the mice were sacrificed and the vaginal tissues of the mice;HE staining was used for vaginal histopathological analysis;immunohistochemistry(IHC)was used to analyze the expression and distribution of NLRP3,PKCδ,pNLRC4 and IL-1Ra in vaginal tissues;immunofluorescence(IF)was used to measure the expression and distribution of pNLRC4 and IL-1Ra in vaginal tissues;western blot(WB)was used to test the expression of NLRP3,PKCδ,pNLRC4 and IL-1Ra in vaginal tissues of mice;qRT-PCR was used to detect the expression of NLRP3,PKCδ,NLRC4 and IL-1Ra mRNA.In vitro experiments: different concentrations of A431 cells were inoculated into 6-well plates to observe the optimal concentration of A431 cell seed plates;5%,10%,15%,20% and 25% BEPD-containing serum and fluconazole-containing serum were added to well-grown A431 cells,respectively.The safe concentrations of BEPD-containing serum and fluconazole-containing serum on A431 cells were detected by CCK-8 method;Candida albicans with concentration of 4×107 CFU/mL,2×107 CFU/mL,8×106CFU/mL,4×106 CFU/mL,2×106 CFU/mL,1×106 CFU/mL,5×105 CFU/mL was added to the well-grown A431 cells to stimulate 1h,2h,4h;the optimal stimulation concentration of Candida albicans on A431 cells was detected by CCK-8 method;the adhesion of Candida albicans to A431 cells was observed by Gram staining;the effects of BEPD-containing serum on LDH,IL-1β and IL-6 in the supernatant of Candida albicans-stimulated A431 cells were detected by ELISA;the effect of BEPD-containing serum on the expression of PKCδ/NLRC4/IL-1Ra axis protein in A431 cells stimulated by Candida albicans was detected by WB;the effect of BEPD-containing serum on the expression of PKCδ/NLRC4/IL-1Ra axis mRNA in A431 cells stimulated by Candida albicans was detected by qRT-PCR;the expression of IL-1Ra protein in A431 cells stimulated by Candida albicans was detected by IF.3 Results:1.BEPD can significantly improve the local symptoms and signs and general state of VVC mice,reduce the fungal load,the number of neutrophils,the number of Candida albicans hyphae and the levels of inflammatory factors IL-1β,IL-18 and LDH in vaginal lavage fluid of VVC mice.2.BEPD can reduce the vaginal tissue inflammation of VVC mice,reduce the adhesion of Candida albicans to the vaginal tissue of VVC mice,increase the protein expression levels of NLRP3,PKCδ,pNLRC4 and IL-1Ra in the vaginal tissue of VVC mice,and increase the relative expression of NLRP3,PKCδ,NLRC4 and IL-1Ra mRNA in the vaginal tissue of mice.3.When A431 cells were stimulated with Candida albicans for 4 h,a large number of Candida albicans adhered to A431 cells,and the contents of IL-1β,IL-16 and LDH in the cell supernatant were significantly increased.The expression of PKCδ,pNLRC4 and IL-1Ra protein in A431 cells was significantly reduced,and the expression of PKCδ,NLRC4 and IL-1Ra mRNA was significantly reduced.The distribution and expression of IL-1Ra were reduced.When A431 cells stimulated by Candida albicans were treated with BEPDcontaining serum,Candida albicans adhered to A431 cells was significantly reduced,and the contents of IL-1β,IL-6 and LDH in cell supernatant were significantly reduced.The protein and mRNA expression of PKCδ,pNLRC4 and IL-1Ra in A431 cells increased significantly,and the distribution and expression of IL-1Ra increased.Conclusion:1.The therapeutic effect of BEPD on VVC mice may be related to the promotion of PKCδ/NLRC4/IL-1Ra axis to negatively regulate NLRP3 inflammasome.2.The serum containing BEPD has a significant protective effect on A431 cells stimulated by Candida albicans,and its mechanism may be related to the up-regulation of PKCδ/NLRC4/IL-1Ra axis.