Effects Of Butyl Alcohol Extract Of Baitouweng Decoction And Three Main Components On Chemotaxis Of Neutrophils In Vulvovaginal Candidiasis Mice

Objective:The aim of this thesis is to investigate the effect of the Butyl Alcohol Extract of Baitouweng Decoction?BAEB?and its three main components,esculin hydrate,berberine hydrochloride and Pulsatilla saponin on neutrophil chemotaxis in Vulvovaginal Candidiasis?VVC?.Methods:1 A mouse VVC model was constructed by injecting Candida Albicans?C.Albicans?at a dose of 2×10⁶ CFU per mouse using a small-gauge syringe into a mouse vagina after an estrous stage of the mouse was induced by the chemical substance estradiol benzoate.The crude extract BAEB was administrated at three doses according to the mouse weight:20mg/kg,40mg/kg,and 80mg/kg,respectively.After the treatment,morphology and numbers of Candida Albicans in the vaginal secretions of mice were checked by microscopy examination of Gram-stained smear and colony counting,respectively.Pathological changes of mouse vaginal tissue were examined by HE staining;the expression levels of LDH,IL-6,and TNF-?in mouse vaginal lavage fluid were analyzed by ELISA;the numbers of neutrophil infiltration in vaginal secretions of mice were counted by microscopy examination of Wright-stained cells.Furthermore,IL-8 and MIP-2 gene expression levels and protein levels in mouse vaginal tissues were analyzed using q RT-PCR and ELISA.2 Neutrophils were induced by treating human promyelocytic leukemia cells?HL60?with DMSO?1.3%V/V?.Five days after treatment,a morphological examination assay of DMSO-treated cells was carried out under an inverted microscopy.The CD11b expression of the cells was analyzed by flow cytometry.The effects of esculin,berberine hydrochloride,and Pulsatilla saponin B4 on the cell proliferation and migration were detected by CCK8 assay and Transwell assay,respectively.The effects of these drugs on the production and polarization of protein F-actin in the cells were also examined by flow cytometry and laser confocal microscopy.PI3K/AKT signaling pathway was checked by western blot.Results:1 In this study,it has been shown that BAEB can significantly inhibit the mycelium formation of Candida Albicans and decrease its number in the vagina of mice,reduce the pathological damage of the vaginal mucosa due to Candida infection and inflammatory cells infiltrated in the vagina.Compared with the control group,the expression levels of inflammatory factors LDH,IL-6 and TNF-??p<0.05,p<0.01,p<0.001?in the experimental group were significantly reduced after treatment with BAEB,which suggests that BAEB has a better therapeutic effect on VVC.Further investigation found that the chemokines IL-8 and MIP-2 were significantly down-regulated in the experimental group,and decreased by 1.9 times and1.5 times?p<0.05?,respectively,compared with the control.Also,the morphological microscopic examination results showed that the number of neutrophil infiltration in the vaginal secretions from the experimental group was significantly reduced when compared with the control?p<0.001?.2 To further identify the mechanism,we induce the neutrophil by treating HL-60cells with DMSO.Five days after DMSO treatment,the neutrophil can be observed.The nucleus of HL-60 cells changed from a small and round regular shape to an irregular shape with a lobulated nucleus after DMSO induction.Also,flow cytometry analysis showed that the CD11b expression in these cells increased significantly.Together,these results show that DMSO can induce neutrophils in HL-60 cell lines.Next,to examine the effects of esculin hydrate,berberine hydrochloride,and Pulsatilla saponin B4 on neutrophil chemotaxis,we test the effect of different concentrations of these drugs on neutrophil chemotaxis.The results showed that 100?g·m L^-1 esculin,8?g·m L^-1berberine hydrochloride,and 80?g·m L^-1 Pulsatilla saponin B4 can inhibit the chemotaxis of neutrophils and significantly reduce F-actin expression.Western blot validated berberine hydrochloride can reduce phosphorylated PI3K/AKT protein expression,suggesting the PI3K/AKT signaling pathway could play an important role in the regulation of neutrophil chemotaxis?p<0.05?.Conclusion:BAEB has the evident therapeutic effect on VVC.The main mechanism is to inhibit the chemotaxis of neutrophils to the vagina by down-regulating F-actin expression and the PI3K/AKT signaing pathway.