基于转录组学研究白头翁汤正丁醇提取物经EGFR/MAPK通路对光滑念珠菌刺激下阴道上皮细胞的保护作用机制  

Mechanism of protective effect of n-butanol extract of Pulsatilla Decoction on vaginal epithelial cells under Candida albicans stimulation through EGFR/MAPK pathway based on transcriptomics

作　　者：张佳萍 张婷 吴惠 吴大强[2] 邵菁[2] 刘婷婷[4] 汪天明 汪长中[2] ZHANG Jia-ping;ZHANG Ting;WU Hui;WU Da-qiang;SHAO Jing;LIU Ting-ting;WANG Tian-ming;WANG Chang-zhong(School of Pharmacy,Anhui University of Chinese Medicine,Hefei 230012,China;School of Integrated Chinese and Western Medicine,Anhui University of Chinese Medicine,Hefei 230012,China;Anhui Rural and Social Science and Technology Development Center,Hefei 230088,China;Department of Pharmacy,the First Affiliated Hospital of Anhui Medical University,Hefei 230022,China)

机构地区：[1]安徽中医药大学药学院,安徽合肥230012 [2]安徽中医药大学中西医结合学院,安徽合肥230012 [3]安徽省农村与社会科技发展中心,安徽合肥230088 [4]安徽医科大学第一附属医院药剂科,安徽合肥230022

出　　处：《中国中药杂志》2024年第11期3021-3030,共10页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金项目(82374173,81774034);安徽省重点研发项目(202104a07020020);安徽省高校自然科学研究重点项目(KJ2021A0590)。

摘　　要：基于转录组学研究白头翁汤正丁醇提取物(n-butanol extract of Pulsatilla Decoction, BEPD)含药血清通过调控表皮生长因子受体(EGFR)/丝裂原活化蛋白激酶(MAPK)通路对光滑念珠菌刺激下阴道上皮细胞的保护作用及机制。首先构建外阴阴道念珠菌病(VVC)模型小鼠,然后将该组小鼠连同空白对照组与BEPD干预组小鼠的阴道黏膜组织进行转录组测序分析基因表达差异,同时制备BEPD含药血清与氟康唑含药血清。将人阴道上皮细胞系(A431细胞)分为对照组、模型组、空白血清组、氟康唑含药血清组、BEPD含药血清组、EGFR激动剂组、EGFR抑制剂组。以细胞增殖与活性检测(CCK-8)法分别确定光滑念珠菌、含药血清及药物对A431细胞的安全浓度;酶联免疫吸附法(ELISA)检测细胞上清液白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、粒细胞集落刺激因子(G-CSF)、趋化因子配体20(CCL20)和乳酸脱氢酶(LDH)的水平;革兰染色观察光滑念珠菌黏附阴道上皮细胞;流式细胞术检测光滑念珠菌对A431细胞凋亡的影响;根据转录组学结果,以免疫荧光检测细胞p-EGFR、p-ERK1/2蛋白的表达,免疫印迹法检测细胞中p-EGFR、p-ERK1/2、p-CFos、p-P38、Bax、Bcl-2蛋白的表达。转录组测序显示,相对于模型组,BEPD治疗后出现1 075个基因上调和927基因下调,主要富集于包括MAPK等在内的免疫炎症相关通路。细胞学实验显示,BEPD含药血清能够降低细胞上清液中IL-1β、IL-6、GMCSF、G-CSF、CCL20炎症因子的水平,缓解由光滑念珠菌引起的细胞中LDH的释放,减少光滑念珠菌对A431细胞的黏附,降低光滑念珠菌诱导的细胞凋亡,下调促凋亡相关蛋白Bax和上调抗凋亡相关蛋白Bcl-2的表达,并显著下调p-EGFR、p-ERK1/2、p-C-Fos与p-P38的表达。该研究提示,BEPD对光滑念珠菌所致VVC的干预效应可能与其通过调节EGFR/MAPK通路保护阴道上皮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 1075 genes and downregulated 927 genes,mainly enriched in immune-inflammatory pathways,inc

关 键 词：白头翁汤正丁醇提取物含药血清 光滑念珠菌 阴道上皮细胞 EGFR/MAPK通路 凋亡 

分 类 号：R285[医药卫生—中药学]