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

Abstract

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

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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References 29

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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

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

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