Abstract:
This study aimed to investigate the protective effect and its underlying mechanism of n-butanol extract of Pulsatilla Decoction(BEPD) containing medicinal serum on vaginal epithelial cells under Candida glabrata stimulation via the epidermal growth factor receptor/mitogen activated protein kinase( EGFR/MAPK) pathway based on transcriptomics. A vulvovaginal candidiasis(VVC) mouse model was established first and transcriptome sequencing was performed for the vaginal mucosa tissues to analyze the gene expression differences among the control, VVC model, and BEPD intervention groups. Simultaneously, BEPD-containing serum and fluconazole-containing serum were prepared. A431 cells were divided into the control, model, blank serum, fluconazole-containing serum, BEPD-containing serum, EGFR agonist and EGFR inhibitor groups. Additionally, in vitro experiments were conducted using BEPD-containing serum, fluconazole-containing serum, and an EGFR agonist and inhibitor to investigate the intervention mechanisms of BEPD on C. glabrata-induced vaginal epithelial cell damage. Cell counting kit-8(CCK-8) assay was utilized to determine the safe concentrations of C. glabrata, drug-containing serum, and compounds on A431 cells. Enzyme-linked immunosorbent assay(ELISA)was employed to measure the expression levels of interleukin(IL)-1β, IL-6, granulocyte-macrophage colony-stimulating factor(GMCSF), granulocyte CSF(G-CSF), chemokine(C-X-C motif) ligand 20(CCL20), and lactate dehydrogenase(LDH). Gram staining was used to evaluate the adhesion of C. glabrata to vaginal epithelial cells. Flow cytometry was utilized to assess the effect of C.glabrata on A431 cell apoptosis. Based on the transcriptomics results, immunofluorescence was performed to measure the expressions of p-EGFR and p-ERK1/2 proteins, while Western blot validated the expressions of p-EGFR, p-ERK1/2, p-C-Fos, p-P38, Bax and Bcl-2 proteins. Sequencing results showed that compared with the VVC model, BEPD treatment up-regulated 1 075 genes and downregulated 927 genes, mainly enriched in immune-inflammatory pathways, including MAPK. Mechanistically, BEPD significantly reduced the expression of p-EGFR, p-ERK1/2, p-C-Fos and p-P38, as well as the secretion of IL-1β, IL-6, GM-CSF, G-CSF and CCL20, LDH release induced by C. glabrata, and the adhesion of C. glabrata to A431 cells, suggesting that BEPD exerts a protective effect on vaginal epithelial cells damaged by C. glabrata infection by modulating the EGFR/MAPK axis. In addition, BEPD downregulated the pro-apoptotic protein Bax expression and up-regulated the anti-apoptotic protein Bcl-2 expression, leading to a reduction in C. glabrata-induced cell apoptosis. In conclusion, this study reveals that the intervention of BEPD in C. glabrata-induced VVC may be attributed to its regulation of the EGFR/MAPK pathway, which protects vaginal epithelial cells.
摘要:
本研究旨在探讨白头翁汤正丁醇提取物(BEPD)含药血清通过基于转录组学的表皮生长因子受体/丝裂原活化蛋白激酶(EGFR/MAPK)通路对光滑念珠菌刺激下阴道上皮细胞的保护作用及其机制。首先建立外阴阴道念珠菌病(VVC)小鼠模型,对阴道粘膜组织进行转录组测序,分析对照之间的基因表达差异,VVC型号,和BEPD干预组。同时,制备含BEPD的血清和含氟康唑的血清。A431细胞分为对照,模型,空白血清,含氟康唑的血清,含BEPD的血清,EGFR激动剂和EGFR抑制剂组。此外,使用含BEPD的血清进行体外实验,含氟康唑的血清,和EGFR激动剂和抑制剂,以探讨BEPD对光滑梭菌引起的阴道上皮细胞损伤的干预机制。细胞计数试剂盒-8(CCK-8)测定用于确定光滑梭菌的安全浓度,含药血清,和A431细胞上的化合物。采用酶联免疫吸附试验(ELISA)检测白细胞介素(IL)-1β的表达水平,IL-6,粒细胞-巨噬细胞集落刺激因子(GMCSF),粒细胞CSF(G-CSF),趋化因子(C-X-C基序)配体20(CCL20),和乳酸脱氢酶(LDH)。革兰氏染色用于评估光滑梭菌对阴道上皮细胞的粘附。流式细胞术用于评估光滑梭菌对A431细胞凋亡的影响。根据转录组学结果,免疫荧光检测p-EGFR和p-ERK1/2蛋白的表达,而Westernblot验证了p-EGFR的表达,p-ERK1/2,p-C-Fos,p-P38、Bax和Bcl-2蛋白。测序结果表明,与VVC模型相比,BEPD治疗上调1075个基因和下调927个基因,主要富含免疫炎症途径,包括MAPK。机械上,BEPD显著降低p-EGFR的表达,p-ERK1/2,p-C-Fos和p-P38,以及IL-1β的分泌,IL-6,GM-CSF,G-CSF和CCL20,光滑梭菌诱导的LDH释放,以及光滑梭菌对A431细胞的粘附,提示BEPD通过调节EGFR/MAPK轴对光滑梭菌感染损伤的阴道上皮细胞具有保护作用。此外,BEPD下调促凋亡蛋白Bax表达,上调抗凋亡蛋白Bcl-2表达,导致光滑梭菌诱导的细胞凋亡减少。总之,这项研究表明,BEPD对光滑梭菌诱导的VVC的干预可能归因于其对EGFR/MAPK通路的调节,保护阴道上皮细胞。