MNQ的一种衍生物对LPS体外诱导的牛卵巢卵泡颗粒细胞炎性损伤的保护作用  

Protective Effect of a Derivative of MNQ Against LPS-Induced Inflammatory Injuryin Bovine Ovarian Follicular Granulosa Cells in Vitro

作　　者：杨小峰 秦小伟[2] 吕丽华 YANG Xiaofeng;QIN Xiaowei;Lü Lihua(Department of Biology,Xinzhou Normal University,Xinzhou 034000,China;College of Animal Science,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]忻州师范学院生物系,忻州034000 [2]山西农业大学动物科学学院,太谷030801

出　　处：《畜牧兽医学报》2024年第5期2032-2041,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：山西省基础研究计划项目(20210302123393);“1331工程”提质增效建设计划项目子课题资助(2023WTS03)。

摘　　要：旨在采用凤仙花(Impatiens balsamina L)提取物2-甲氧基-1,4-萘醌(MNQ)的衍生物D_(19)消除体外暴露于脂多糖(LPS)中牛卵巢卵泡颗粒细胞(GCs)的炎症反应,并缓减由LPS引起的卵泡GCs功能性紊乱。本研究采集性成熟且健康的荷斯坦牛卵巢组织,分离并培养卵泡GCs。MTT法测定D_(19)和LPS对卵泡GCs存活率的影响。试验分为3组:对照组、LPS处理组和D_(19)联合LPS处理组,每组3个重复。qRT-PCR测定炎性因子和类固醇合成相关基因的相对表达量。TEM观察D_(19)对细胞炎性损伤的保护作用。ELISA检测培养液上清中雌二醇(E_(2))和孕酮(P_(4))的含量。结果表明,D_(19)对卵泡GCs作用12 h的最大安全浓度为64μmol·L^(-1)。LPS浓度为10μg·mL^(-1)时,作用12 h对卵泡GCs存活率影响不大,但炎性因子IL-6、IL-1β和TNF-α的相对表达量显著升高(P<0.01),D_(19)+L组与LPS组比较炎性因子的相对表达量却显著降低(P<0.01)。通过TEM观察表明D_(19)对LPS引起的细胞器结构性损伤具有保护作用。ELISA结果表明,LPS处理后培养液中E_(2)和P_(4)的含量显著降低(P<0.01),qRT-PCR检测到类固醇合成相关基因CYP19A1、CYP11A1、3β-HSD和STAR的相对表达量也显著降低(P<0.01)。但D_(19)联合LPS处理后,E_(2)和P_(4)分泌量以及类固醇合成相关基因的相对表达量均比LPS组显著升高(P<0.01)。本研究证明MNQ的衍生物D_(19)具有消除LPS体外诱导卵泡GCs炎症反应的功能,并能一定程度缓减LPS导致的卵泡GCs功能性紊乱。The aim of this experiment was to eliminate the inflammatory response of bovine ovarian follicular granulosa cells(GCs)exposed to lipopolysaccharide(LPS)in vitro and to attenuate LPS-induced functional disorders of follicular GCs using D_(19),a derivative of 2-methoxy-1,4-naphthoquinone(MNQ)from the extract of Impatiens balsamina L.Ovarian tissues were collected from sexually mature and healthy Holstein cattle,and follicular GCs were isolated and cultured.The effect of D_(19) and LPS on the survival of follicular GCs was determined by MTT assay.The experiment was divided into 3 groups:control,LPS-treated and D_(19)-combined LPS-treated groups,with 3 replicates in each group.The relative expression of inflammatory factors and genes related to steroid synthesis was determined by qRT-PCR.TEM was performed to observe the protective effect of D_(19) against cellular inflammatory injury.ELISA was performed to measure the levels of estradiol(E_(2))and progesterone(P_(4))in the supernatant of the culture fluid.The results showed that the maximum no-cytotoxic concentration of D_(19) acting on follicular GCs for 12 h was 64μmol·L^(-1).LPS concentration of 10μg·mL^(-1) had little effect on the survival of follicular GCs after 12 h of action,but the relative expression of the inflammatory factors IL-6,IL-1β,and TNF-αwas significantly higher(P<0.01),whereas the relative expression of the inflammatory factors in the D_(19)+L group was significantly lower compared with the LPS group(P<0.01).The protective effect of D_(19) against LPS-induced structural damage to organelles was demonstrated by TEM observation.ELISA results showed that the levels of E_(2) and P_(4) in the culture broth were significantly reduced after LPS treatment(P<0.01),and the relative expression of steroid synthesis-related genes CYP19 A 1,CYP11 A 1,3β-HSD,and STAR as detected by qRT-PCR was also significantly reduced(P<0.01).However,the amount of E_(2) and P_(4) secretion as well as the relative expression of steroid synthesis-related genes were sign

关 键 词：MNQ D_(19) LPS GCS 炎症反应 

分 类 号：S823.3[农业科学—畜牧学]