Effect of Estradiol and Progesterone on the Expression of TLR4 and Inflammatory Cytokines in Canine (Canis lupus familiaris) Endometrial Stromal Cells Induced by LPS
NI Jun, QIU Jing-Qi, LI Jin, FENG Yi-Qiu, LIU Fei-Yan, RUI Rong*
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Abstract:Pyometra is a common reproductive disease of the female dogs (Canis lupus familiaris). Hormonal disorder and bacterial infections are main predisposing factors. The endometrium is the target organ for hormone response. A large amount of inflammatory cytokines will produce when endometrial cells are abnormally activated. However, the immunoregulatory effects of steroid hormones on canine endometrial stromal cells (ESCs) in an inflammatory environment are still poorly understood. To study the regulation of estradiol (E2) and progesterone (P4) on the secretion of Toll like receptor 4 (TLR4) and related inflammatory cytokines in canine ESCs in vitro. The peripheral blood serum of pyometra and healthy canine were collected, and the concentration of E2 and P4 were detected by ELISA; canine endometrial ESCs were isolated by collagenase enzymolysis and identified by immunofluorescence. The detected E2 (0.2 nmol/L) and P4 (0.1 μmol/L) were added to the cell culture medium, then the effects of E2 and P4 on cell growth in vitro were determined by the method of 3- (4,5-dimethylthiazol-2-yl) - 2,5-diphenyltetrazolium bromide (MTT); then the cells were treated with 1 μg/mL lipopolysaccharide (LPS), and qPCR was performed to detect the gene expression of TLR4 and inflammatory cytokines in canine ESCs induced by LPS at 6, 12, and 24 h. The results showed that the cells isolated were ESCs with a high purity rate after immunofluorescence identification. E2 had no significant effect on the ESCs, while P4 and combination of E2 and P4 could significantly stimulate proliferation of ESCs in vitro. Compared with the control group, the gene expression of TLR4 and inflammatory cytokines were significantly up-regulated after LPS stimulation (P<0.05). Compared with the LPS group, P4 alone and the combination of two hormones significantly down-regulated the gene expression of TLR4 and inflammatory cytokines caused by LPS stimulation (P<0.05). This experiment proves that E2 and P4 play an important role in ESCs. This study provides basic data for clarifying the molecular mechanism of pyometra and the effects of E2 and P4 on endometrial innate mucosal immunity.
倪珺, 邱靖淇, 李瑾, 冯忆秋, 刘飞燕, 芮荣. 雌二醇和孕酮对LPS诱导的犬子宫内膜基质细胞TLR4及炎性因子表达的影响[J]. 农业生物技术学报, 2022, 30(10): 1936-1943.
NI Jun, QIU Jing-Qi, LI Jin, FENG Yi-Qiu, LIU Fei-Yan, RUI Rong. Effect of Estradiol and Progesterone on the Expression of TLR4 and Inflammatory Cytokines in Canine (Canis lupus familiaris) Endometrial Stromal Cells Induced by LPS. 农业生物技术学报, 2022, 30(10): 1936-1943.
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