The Pharmacodynamics Effects And Molecular Machanisms Of Pulsatilla Decoction For Vulvovaginal Candidiasis Via Dectin-1 Signaling Pathway

Vulvovaginal candidiasis(VVC)is one of the most common vaginitis that affecting 75%of women at least once in their lifetime.The clinical symptoms of VVC are represented by obvious pruritus and burning pain in the vulvae and leucorrhea like curd.As a result,VVC can markedly compromise the quality of life in affected females.Large reseaches have showed that Dectin-1 is the main Pattern Recognition Receptor(PRR)that recognize the?-glucan,a component of fungal cell wall.At the onset of Candida invasion,PRR(Dectin-1)recognizes the?-glucan and triggers intracellular response.VVC is belonged to“Daixia”and“Yinyang”in Tradtional Chinese Medicine(TCM).TCM theorized that VVC is mainly caused by overflow of damp-heat and Pulsatilla Decoction is effective in correcting the dysregulation of heat and dampness.Therefore,it is widely used in China to treat VVC and has showed satisfactory effects in clinical practice.ObjectiveTo detect the expression of cytokine related to Dectin-1 signaling pathway in VVC patient.To investigate the anti-C.albicans activity of Pulsatilla decoction in vitro.To establish the VVC mouse model,study the effect and molecular mechanism on Dectin-1 signal pathway of Pulsatilla decoction for VVC.Methods1.Clinical researchFifty VVC patients and thirty healthy people were included in the research.The basic information and vaginal lavage fluid were collected.Then the level of cytokine related to Dectin-1 signaling pathway include IL-2,IL-6,IL-10,IL-22 and IFN-?were detected by ELISA.2.The identification of active substances in Pulsatilla decoctionReflux condensation method was used to obtain the Pulsatilla decoction water extract.Establish HPLC parameters to identify the active substances in Pulsatilla decoction:Esculin,Aesculetin,Phellodendrine hydrochloride,Berberini Hydrochloride and Anemoside B4.3.The research of anti-C.albicans activity of Pulsatilla decoction in vitroCollete the vaginal secretion of VVC patients,then isolate the C.albicans strain by Chromagar medium.To detect the minimum inhibitory concentration and minimum bactericidal concentration by broth microdilution method.4.The effect of Pulsatilla decoction for VVC mouse model(1)Make the mouse model of VVC:The 8-week,BALB/c with Raettin were selected.Pseudoestrus was induced during infection by injecting subcutaneously estradiol benzoate in the lower abdominal area of animal on3 days before inoculation(-Day 3).The estradiol treatments were repeated one day before infection(-Day 1).Infection of the vaginal canal was performed by inoculating the mice intravaginally on the next day(Day 0).Assess the model by lavage fluid culture and Grain stain,PAS stain of vaginal tissue.(2)A total of 90 mice were equally randomized to six groups:Control group,Model group,PD low dose group(PD-L),PD medium dose group(PD-M),PD high dose group(PD-H)and Fluconazole group(FLUC).The weight was recorded every day.Serum,vaginal lavage fluid and vaginal tissues were collected on Day 4,Day 7,Day 14 after treatment.(1)Collect vaginal lavage fluid to culture in the medium to calculated the fungal load.(2)Collect vaginal lavage fluid for Grain stain,evaluate the infection status of vaginal lavage fluid.(3)Collect vaginal tissue,fix it by paraformaldehyde and make pathological section,evaluate the adhesion of C.albicans on vaginal epithelium.(4)Vaginal tissues were fixed by glutaraldehyde and preprocessed.The microscopice images were obtained by scanning electron microscope(SEM).(5)Evaluate the situation of inflammation by HE stain.(6)Detect the serum levels of IL-1??IL-12(p70)?IL-23 by Luminex.5.The molecular mechanisms on Dectin-1 signal pathway in the treatment of Pulsatilla decoction for VVC(1)Detect the m RNA expression of Dectin-1,Syk,CARD9 and NF-?B by RT-qPCR in vaginal tissue.(2)Detect the protein expression of Dectin-1,Syk,CARD9 and NF-?B by Western Blot in vaginal tissue.(3)Make the vaginal tissues to pathological section,and detect the protein expression of Dectin-1,Syk,CARD9 and NF-?B by immunohistochemistry.(4)Detect the levels of IL-1??IL-2?IL-10?IFN-?by ELISA in vaginal tissue.Rusults1.Clinical researchThere was no statistical difference on the baseline information.The levels of IL-2?IL-22?IFN-?on model group were higher than control group(P<0.05).The level of IL-10 between two group was pretty close,however there was no stastitical difference.The level of IL-6 is lower than control group,however there was no stastitical difference.2.The identification of active substances in Pulsatilla decoctionThere were Esculin,Aesculetin,Phellodendrine hydrochloride,Berberini Hydrochloride and Anemoside B4 in Pulsatilla decoction.A hypersil gold C₁₈(250mm×4.6mm,5?m)column was used.The mobile phase consists of acetonitrile and 0.1%orthophosphoric acid.Ultra-violet spectra was in 201nm.3.The anti-C.albicans activity of Pulsatilla decoction in vitro(1)The MIC value:(1)The MIC of Fluconazole for standard strain was 0.5?g/ml.The MIC of Pulsatilla decoction for standard strain was 3.0926mg/ml.(2)The median MIC of Fluconazole for clinical strain was 0.5?g/ml.Eighteen strains were sensitive for Fluconazole(90%),two strains susceptible-dose dependengt for Fluconazole(10%).The median MIC of Pulsatilla decoction for clinical strains was 3.0963mg/ml.The 85%of Clinical strains of MIC value were less than 7.8125mg/ml.(2)The MBC value:(1)The MBC of Fluconazole for standard strain was 1?g/ml.The MBC of Pulsatilla decoction for standard strain was 15.625mg/ml.(2)The median MBC of Fluconazole for clinical strain was 2?g/ml.(3)The median MBC of Pulsatilla decoction for clinical strain was 15.625mg/ml.4.The effect of Pulsatilla decoction for VVC mouse model(1)There was no effect of Pulsatilla decoction for mice in the experimental process.After be infected,the weights of Model group were lower than other groups,howerver,it was significantly lower than PD-H group on Day 1 and Fluconazole group Day 4(P<0.05).There was no difference on–Day3,-Day 1,Day 0,Day 7 and Day 14 in all groups.(2)Pulsatilla decoction decreased the vaginal clony unit.The vaginal colonization in Model group persisted at 10⁴-10⁵level after inoculation.Compared with the Model group,all three PD groups markedly reduced the fungal load from Day 7 onwards(P<0.01).(3)Pulsatilla decoction decreased the hypha in vaginal lavarage.In the Model group,large number of hyphae was shown to intertwine with epithelial cell across all time points while the amount of hyphae in all three PD groups gradually decreased over time in Grain stain.There was no visible hyphae in PD-H group on Day 14.(4)Pulsatilla decoction inhibited the adhesion of microorganism in vaginal issue.The Model group exhibited severe infections marked by numerous hyphae and with parts of hyphae immersing in epithelium during the whole treatment course in PAS stain.With PD treatment,the visible microorganisms gradually decreased at variable speeds.On the Day 14,SEM images showed that there were large C.albican yeast and hyphae in the vaginal epithelium in model group.Scattered adhesions with varying degrees were observed in there PD groups.(5)Pulsatilla decoction alleviate the inflammation and tissue damage and decrease the levels of IL-12 and IL-23.HE stain on Day 14 showed that vaginal tissues in the Model group was characterized by severe neutrophil infiltration and microabscess in the mucosa.The degree of inflammation in PD was significantly mitigated with reduced neutrophil invasion and majority of tissue structure remained intact.The levels of IL-12 and IL-23 were higher in model group compared with control group(P<0.05),however,that were decreased in PD groups.5.The molecular mechanisms on Dectin-1 signal pathway in the treatment of Pulsatilla decoction for VVC(1)Pulsatilla decoction decreased the m RNA expression of Dectin-1?CARD9?NF-?B.Model group exhibited significantly higher level of Dectin-1,CARD9 and NF-?B expression than Control group(P<0.05).After intervention by Pulsatilla decoction,the above three genes were down-regulated.The expression level of Syk remained unchanged regardless of the treatment.(2)Pulsatilla decoction down-regulated the protein expression of Dectin-1 signal pathway.(1)Western-blot:expression of Syk,CARD9 and NF-?B were significantly upregulated in Model group while PD and Fluconazole treatments downregulate these protein expressions at variable levels,especially for NF-?B(P<0.01).(2)Immunohistochemistry:for the respect of location:Dectin-1,CARD9 and NF-?B were significantly elevated in mucosa layer and submucosa layer while Syk protein was mainly expressed in submucosa layer.For the respect of expression level,the above four proteins were all upregulated in Model group,however treatments of PD remarkably reduced the levels by various degrees with statistical significance.(3)Pulsatilla decoction decreased the levels of IL-1??IL-10 in vaginal tissue.The levels of IL-1?and IL-10 in model group was higher than control group(P<0.01).Compared with model group,PD groups could down-regulated the expression of IL-1?and IL-10.There was no statistic significance of IL-2and IFN-?levels among groups.Conclusion1.Pulsatilla decoction used in this research was included Esculin,Aesculetin,Phellodendrine hydrochloride,Berberini Hydrochloride and Anemoside B4.2.Pulsatilla decoction was with the anti-fungi activity in vitro.3.Pulsatilla decoction could down-regulate the fungal load in vaginal lavage fluid,inhibit the adhesion of C.albicans on vaginal epithelium,improve the inflammation status of vaginal epithelium,decrese the inflammation levels of IL-12 and IL-23 in serum.Pulsatilla decoction down-regulate the Dectin-1signal pathway from gene and protein levels.Thus,regulating Dectin-1 pathway may be the important mechanism of Pulsatilla decoction for VVC.