诃子鞣酸对LPS诱导的小鼠子宫内膜炎的保护作用
Protective effect of chebulagic acid on LPS-induced endometritis in mice
张兰欣 1郭文瑞 1巩志国 1李姣姣 1王建宁 1刘鑫煜 1杨英 1李政忆 1李娟 1高瑞峰1
作者信息
- 1. 内蒙古农业大学兽医学院,内蒙古呼和浩特 010018;农业农村部动物疾病临床诊疗技术重点实验室,内蒙古呼和浩特 010018
- 折叠
摘要
为探究诃子鞣酸(chebulagic acid,CA)对脂多糖(lipopolysaccharide,LPS)诱导的小鼠子宫内膜炎的保护作用.本研究体外试验部分,使用MTT法检测CA对小鼠腹腔巨噬细胞活性的影响,通过ELISA和 Westen blot分别检测CA对LPS刺激的巨噬细胞促炎性细胞因子(TNF-α、IL-1β和IL-6)分泌水平和ERK、p38和p65磷酸化水平的影响;体内试验部分,通过ELISA分析CA对LPS刺激后小鼠子宫组织TNF-α、IL-1β和IL-6分泌水平的影响,使用组织免疫荧光法检测小鼠子宫损伤相关因子(HMGB1)的表达水平,通过HE染色法观察小鼠子宫组织的病理变化,使用血常规法分析小鼠全血中白细胞数、中性粒细胞数和单核细胞数的变化.结果显示,CA质量浓度为12.5、25.0、50.0 mg/L对巨噬细胞活性均无显著影响,因此后续试验结果与CA的细胞毒性无关.CA可显著下调受到LPS刺激的巨噬细胞中的TNF-α,IL-1β和IL-6分泌水平,且具有浓度依赖性.此外,CA可显著下调受LPS诱导的巨噬细胞中ERK、p38和p65的磷酸化水平.CA可显著下调LPS刺激小鼠后子宫组织中TNF-α、IL-1β和IL-6的分泌水平;LPS刺激可增加小鼠全血中的白细胞数、中性粒细胞数和单核细胞数,而使用CA治疗可使其显著降低;相比于LPS刺激组,CA治疗组子宫组织中HMGB1的表达水平显著降低;LPS刺激小鼠子宫后导致子宫角出血和水肿,使用CA治疗后子宫角出血程度和水肿程度均有所降低.同时,HE病理切片显示LPS刺激组小鼠子宫组织中出现大量的嗜中性粒细胞浸润,子宫内膜上皮发生增生、水肿、坏死及脱落,CA(100 mg/kg)治疗后,子宫组织基本恢复正常,具有浓度依赖性.以上结果表明,CA可通过抑制小鼠腹腔巨噬细胞中MAPK和NF-κB通路的激活,下调巨噬细胞和子宫组织中炎症介质的表达水平从而减轻LPS诱导的小鼠子宫内膜炎.
Abstract
This study aims to investigate the protective effect of chebulagic acid(CA)on lipopo-lysaccharide(LPS)-induced endometritis in mice.In the in vitro experiment part of this study,MTT assay was used to assess the effect of CA on the cell activity of mouse peritoneal macropha-ges.The effects of CA on the secretion of pro-inflammatory cytokines(TNF-α,IL-1β and IL-6)and the phosphorylation levels of ERK,p38 and p65 in LPS-stimulated macrophages were detected by ELISA and Western blot,respectively.In the in vivo test,the secretion levels of pro-inflammatory cytokines(TNF-α,IL-1β and IL-6)in the mouse uterine tissues after LPS stimulation and treat-ment by CA were analyzed by ELISA method,the expression level of mouse uterine injury-related factor(HMGB1)was detected by tissue immunofluorescence method,and the pathological changes of mouse uterine tissues were observed by HE staining.The changes of white blood cell count,neu-trophil count and monocyte count in whole blood of mice were analyzed by routine blood test.The experimental results showed that the concentrations of CA at 12.5,25,50 mg/L had no significant effect on macrophage activity,so the subsequent results were not related to the cytotoxicity of CA.CA significantly downregulated the levels of TNF-α,IL-1β and IL-6 secretion in LPS-induced mac-rophages in a concentration-dependent manner.In addition,CA significantly downregulated the phosphorylation levels of ERK,p38,and p65 in LPS-stimulated macrophages.CA significantly downregulated the expression levels of TNF-α,IL-1β and IL-6 in uterine tissue after LPS stimula-tion in mice.LPS stimulation increased the number of leukocytes,neutrophils,and monocytes in whole blood of mice,while CA treatment significantly reduced the number of leukocytes.Compared with the LPS-stimulated group,the expression level of HMGB1 in uterine tissue of the CA-treated group was significantly decreased.LPS stimulation of the mouse uterus resulted in hemorrhage and edema in the uterine horn,and the degree of hemorrhage and edema was reduced after CA treat-ment.At the same time,HE pathological sections showed that many neutrophil infiltrates appeared in the uterine tissue of the LPS-stimulated group,and the endometrial epithelium exhibited hyper-plasia,edema,necrosis,and exfoliation.After CA(100 mg/kg)treatment,the uterine tissue basi-cally returned to normal in a concentration-dependent manner.In conclusion,CA can reduce LPS-induced endometritis in mice by inhibiting the activation of MAPK and NF-κB pathways in perito-neal macrophages and down-regulating the expression of inflammatory mediators in macrophages and uterine tissues.
关键词
子宫内膜炎/诃子鞣酸/脂多糖/炎症介质/炎症信号通路/高迁移率蛋白1/小鼠Key words
endometritis/chebulagic acid/lipopolysaccharide/inflammatory mediators/inflammatory signaling pathway/high-mobility group box protein 1/mice引用本文复制引用
基金项目
国家自然科学基金(32160856)
内蒙古自治区高等学校青年科技英才支持计划(NJYT24056)
内蒙古自治区自然科学基金(2023MS03035)
内蒙古自治区自然科学基金(2023LHMS03015)
出版年
2024
NETL
NSTL
万方数据