虾青素对牛子宫内膜细胞炎症损伤的保护作用

doi:10.3864/j.issn.0578-1752.2019.17.012

中国农业科学 ›› 2019, Vol. 52 ›› Issue (17): 3049-3058.doi: 10.3864/j.issn.0578-1752.2019.17.012

• 畜牧·兽医·资源昆虫 • 上一篇下一篇

虾青素对牛子宫内膜细胞炎症损伤的保护作用

张辰^1,²,谭秀文²,魏晨²,张相伦²,靳青²,刘桂芬²,刘晓牧²,万发春^1,²()

1 山东师范大学生命科学学院,济南 250014
2 山东省农业科学院畜牧兽医研究所/山东省畜禽疫病防治与繁育重点实验室/山东省肉牛生产性能测定中心/山东省畜禽健康养殖工程技术中心,济南 250100

收稿日期:2018-12-10 接受日期:2019-05-09 出版日期:2019-09-01 发布日期:2019-09-10
通讯作者: 万发春
作者简介:张辰,Tel：18505440868;E-mail：zcsdnu@163.com。|谭秀文,Tel：18615638322;E-mail： lafta@sina.com。
基金资助:
国家自然科学基金(31802062);现代农业肉牛牦牛产业技术体系建设专项资金(CARS-37);山东省农业科学院农业科技创新工程(CXGC2018E10)

Protective Effect of Astaxanthin on Inflammatory Injury of Endometrial Cells in Bovine

ZHANG Chen^1,²,TAN XiuWen²,WEI Chen²,ZHANG XiangLun²,JIN Qing²,LIU GuiFen²,LIU XiaoMu²,WAN FaChun^1,²()

1 College of Life Sciences, Shandong Normal University, Jinan 250014
2 Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences/Shandong Key Lab of Animal Disease Control and Breeding/ Shandong Provincial Testing Center of Beef Cattle Performance/Shandong Provincial Engineering Technology Center of Animal Healthy Breeding, Jinan 250100

Received:2018-12-10 Accepted:2019-05-09 Online:2019-09-01 Published:2019-09-10
Contact: FaChun WAN

摘要/Abstract

摘要：

【目的】利用虾青素较强的抗氧化特性,通过探讨黏膜屏障重塑对脂多糖（LPS）诱导的牛子宫内膜细胞炎症的影响,为牛子宫内膜细胞炎的预防和治疗提供理论基础。【方法】培养液中添加1 μg·mL ^-1 LPS,培养子宫内膜细胞6 h,检测培养液中炎症因子TNF-α和IL-6含量,建立细胞的体外炎症模型。在此基础上去掉培养液,重新加入含1×10 ^-6 mol·L ^-1 虾青素的新鲜培养液,继续培养细胞24 h。对照组为不添加LPS或AST,LPS组为仅添加LPS,AST组为添加LPS和AST。采用ELISA法检测培养液中TNF-α和IL-6炎症因子分泌量以及细胞SOD和CAT酶活性,利用流式细胞仪检测细胞内活性氧簇（ROS）水平,利用免疫荧光染色检测细胞紧密连接蛋白Claudin、CDH1、TJP1的分布情况,利用荧光定量RT-PCR法和Western Blot法分别检测Claudin、CDH1、TJP1基因的mRNA表达水平和蛋白表达水平。【结果】利用1 μg·mL ^-1 LPS培养子宫内膜细胞6 h,检测发现培养液中炎症因子IL-6和TNF-α分泌水平显著高于对照组（P小于0.05）,说明LPS诱导子宫内膜细胞产生炎症反应。与对照组相比,细胞内ROS水平显著增加（P小于0.05）,SOD和CAT酶活性都显著降低（P小于0.05）,说明LPS诱导的子宫内膜细胞炎症造成了氧化损伤。加入1×10 ^-6 mol·L ^-1 虾青素培养细胞24 h后,发现培养液中IL-6和TNF-α因子的分泌水平显著低于LPS组（P小于0.05）,同时ROS阳性细胞比率显著下降（P小于0.05）,SOD和CAT酶活性都显著升高（P小于0.05）。LPS组与对照组相比,细胞膜处Claudin、CDH1、TJP1蛋白的荧光信号减弱,这3种紧密连接蛋白的mRNA和蛋白表达水平也都显著降低（P小于0.05）。加入1×10 ^-6 mol·L ^-1虾青素培养24 h后,与LPS组相比,在细胞边缘和细胞核内可以检测到荧光信号很强的Claudin、CDH1、TJP1蛋白,而且这3种紧密连接蛋白的mRNA和蛋白表达水平也都显著升高（P小于0.05）。【结论】虾青素通过降低子宫内膜细胞因炎症反应产生的氧化损伤,减轻炎症导致的子宫内膜细胞紧密连接结构损伤,对子宫内膜的物理免疫屏障起保护作用,为牛子宫内膜炎的预防和治疗提供理论借鉴意义。

关键词: 牛, 虾青素, 子宫内膜细胞, 炎症因子, 紧密连接蛋白

Abstract:

【Objective】 The purpose of this study was to examine whether astaxanthin with strong antioxidant properties could protect endometrial cells against lipopolysaccharide (LPS)-induced inflammatory injury through mucosal barrier remodeling, so as to provide a theoretical basis for the prevention and treatment of bovine endometritis. 【Method】 The endometrial cells were cultured in the medium supplemented with 1 μg·mL ^-1 LPS for 6 h, then the inflammatory factors (such as TNF-α and IL-6) in the cultured medium were detected to establish an inflammation model in vitro. On this basis, the cultured medium was removed and fresh culture medium containing 1×10 ^-6 mol·L ^-1 astaxanthin was exchanged, and endometrial cells were further cultured for 24 hours. The control group was not added with LPS or AST, the LPS group was only added with LPS, and the LPS+AST group was added with LPS and AST. The inflammatory factors of TNF-α and IL-6 in the cultured medium and the activities of cellular SOD and CAT enzymes were detected by ELISA. The levels of reactive oxygen species (ROS) in the cells were detected by flow cytometry, and the distribution of tight junction protein, such as Claudin, CDH1 and TJP1, were observed by immunofluorescence staining. The mRNA and protein expression were examined by using fluorescence quantitative RT-PCR and Western Blot, respectively. 【Result】 The endometrial cells were cultured in the medium supplemented with 1 μg·mL ^-1 LPS for 6 h, the production of IL-6 and TNF-α in the cultured medium were significantly higher than those in the control group (P＜0.05), indicating that LPS induced inflammation of endometrial cells. Compared with the control group, the level of intracellular ROS significantly increased (P＜0.05), and the activities of cellular SOD and CAT enzymes significantly decreased (P＜0.05), which indicated that LPS-induced inflammation in endometrial cells caused oxidative damage. When endometrial cells were further cultured in the medium containing 1×10 ^-6 mol·L ^-1 astaxanthin for 24 h, the levels of IL-6 and TNF-α in cultured medium were significantly lower than those in LPS group (P＜0.05), the percentage of ROS positive cells decreased significantly (P＜0.05), and the activities of SOD and CAT enzymes increased significantly (P＜0.05). Compared with the control group, the fluorescence signals of Claudin, CDH1 and TJP1 proteins in the LPS group were weakened, and the levels of the mRNA and protein expression of tight junction proteins also decreased significantly (P＜0.05). However, when compared with the LPS group, Claudin, CDH1 and TJP1 proteins with strong fluorescence signals were detected in cell margin and nucleus in LPS+AST group, and the levels of mRNA and protein expression also significantly increased (P＜0.05).【Conclusion】 Astaxanthin reduced the oxidative damage which caused by inflammatory of endometrial cells, relived the structure damage of tight junction proteins in endometrial cells, which might protect the physical immune barrier of endometrium and provided theoretical reference for the prevention and treatment of bovine endometritis.

Key words: bovine, astaxanthin, endometrial cells, inflammatory factors, tight junction protein

张辰, 谭秀文, 魏晨, 张相伦, 靳青, 刘桂芬, 刘晓牧, 万发春. 虾青素对牛子宫内膜细胞炎症损伤的保护作用[J]. 中国农业科学, 2019, 52(17): 3049-3058.

ZHANG Chen, TAN XiuWen, WEI Chen, ZHANG XiangLun, JIN Qing, LIU GuiFen, LIU XiaoMu, WAN FaChun. Protective Effect of Astaxanthin on Inflammatory Injury of Endometrial Cells in Bovine[J]. Scientia Agricultura Sinica, 2019, 52(17): 3049-3058.

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基因 Gene	引物序列 Primer sequence	序列号 Accession number	产物长度 Length
β-actin	F:CACCGCAAATGCTTCTAGGC R:TGTCACCTTCACCGTTCCAG	NM_173979.3	186
Claudin	F: AAGACGACGAGGCACAGAAG R: CGGGGTCATGGGGTCATAGA	NM_001001854.2	137
CDH1	F: CGTGACCGACCAGAATGACA R: GGCTGGGATTGTGTAACCGA	NM_001002763.1	166
TJP1	F: CTGCTGCCAAAGAAGGCTTG R: CGGATTCTACGATGCGACGA	XM_010817146.1	177

虾青素对牛子宫内膜细胞炎症损伤的保护作用

Protective Effect of Astaxanthin on Inflammatory Injury of Endometrial Cells in Bovine

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