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.
[1] 缪江霞,林景丹. 2002. 细胞因子与子宫内膜局部免疫[J]. 广东医学, (S1): 193-194. (Miu J X, Lin J D. 2002. Cytokines and endometrial local immunity[J]. Guangdong Medical Journal, (S1): 193-194.) [2] Aflatoonian R, Fazeli A.2008. Toll-like receptors in female reproductive tract and their menstrual cycle dependent expression[J]. Journal of Reproductive Immunology, 77(1): 7-13. [3] Borges A M, Healey G D, Sheldon I M.2012. Explants of intact endometrium to model bovine innate immunity and inflammation ex vivo[J]. American Journal of Reproductive Immunology, 67(6): 526-539. [4] Chen J, Wang W, Shi C, et al.2014. A comparative study of sodium houttuyfonate and 2-undecanone for their in vitro and in vivo anti-inflammatory activities and stabilities[J]. International Journal of Molecular Sciences, 15(12): 22978-22994. [5] Concannon P W.2011. Reproductive cycles of the domestic bitch[J]. Animal Reproduction Science, 124(3-4): 200-210. [6] Deshpande R, Khalili H, Pergolizzi R G, et al.1997. Estradiol down-regulates LPS-induced cytokine production and NFκB activation in murine macrophages[J]. American Journal of Reproductive Immunology, 38(1): 46-54. [7] Foerster K, He W, Manuel J, et al.2007. LPS-induced occult loss in mice requires FGL2[J]. American Journal of Reproductive Immunology, 58(6): 524-529. [8] Ghasemi F, Gonzalez-Cano P, Griebel P J, et al.2012. Proinflammatory cytokine gene expression in endometrial cytobrush samples harvested from cows with and without subclinical endometritis[J]. Theriogenology, 78(7): 1538-1547. [9] Goddard L M, Ton A N, Org T, et al.2013. Selective suppression of endothelial cytokine production by progesterone receptor[J]. Vascular Pharmacology, 59(1-2): 36-43. [10] Hagman R.2012. Clinical and molecular characteristics of pyometra in female dogs[J]. Reproduction in Domestic Animals, 47(Suppl 6): 323-325. [11] Herath S, Fischer D P, Werling D, et al.2006. Expression and function of Toll-like receptor 4 in the endometrial cells of the uterus[J]. Endocrinology, 147(1): 562-570. [12] Jones L A, Anthony J P, Henriquez F L, et al.2008. Toll-like receptor-4-mediated macrophage activation is differentially regulated by progesterone via the glucocorticoid and progesterone receptors[J]. Immunology, 125(1): 59-69. [13] King A E, Critchley H O.2010. Oestrogen and progesterone regulation of inflammatory processes in the human endometrium[J]. Journal of Steroid Biochemistry and Molecular Biology, 120(2-3): 116-126. [14] Laveti D, Kumar M, Hemalatha R, et al.2013. Anti-inflammatory treatments for chronic diseases: A review[J]. Inflammation & Allergy Drug Targets, 12(5): 349-361. [15] Paavonen T, Andersson L C, Adlercreutz H.1981. Sex hormone regulation of in vitro immune response. Estradiol enhances human B cell maturation via inhibition of suppressor T cells in pokeweed mitogen-stimulated cultures[J]. Journal of Experimental Medicine, 154(6): 1935-1945. [16] Pierro E, Minici F, Alesiani O, et al.2001. Stromal-epithelial interactions modulate estrogen responsiveness in normal human endometrium[J]. Biology of Reproduction, 64(3): 831-838. [17] Rider V.2006. Isolation of hormone responsive uterine stromal cells: An in vitro model for stromal cell proliferation and differentiation[J]. Methods in Molecular Medicine, 121: 57-67. [18] Santos R S, de Fatima L A, Frank A P, et al.2017. The effects of 17 alpha-estradiol to inhibit inflammation in vitro[J]. Biology of Sex Differences, 8(1): 30. [19] Silva L D, Onclin K, Verstegen J P.1995. Cervical opening in relation to progesterone and oestradiol during heat in beagle bitches[J]. Journal of Reproduction and Fertility, 104(1): 85-90. [20] Singh L K, Patra M K, Mishra G K, et al.2018. Endometrial transcripts of proinflammatory cytokine and enzymes in prostaglandin synthesis are upregulated in the bitches with atrophic pyometra[J]. Veterinary Immunology and Immunopathology, 205: 65-71. [21] Smith F O.2006. Canine pyometra[J]. Theriogenology, 66(3): 610-612. [22] Su L, Sun Y, Ma F, et al.2009. Progesterone inhibits Toll-like receptor 4-mediated innate immune response in macrophages by suppressing NF-kappaB activation and enhancing SOCS1 expression[J]. Immunology Letters, 125(2): 151-155. [23] Sugiura K, Nishikawa M, Ishiguro K, et al.2004. Effect of ovarian hormones on periodical changes in immune resistance associated with estrous cycle in the beagle bitch[J]. Immunobiology, 209(8): 619-627. [24] Verstegen J, Dhaliwal G, Verstegen-Onclin K.2008. Mucometra, cystic endometrial hyperplasia, and pyometra in the bitch: Advances in treatment and assessment of future reproductive success[J]. Theriogenology, 70(3): 364-374. [25] Wira C R, Rodriguez-Garcia M, Patel M V.2015. The role of sex hormones in immune protection of the female reproductive tract[J]. Nature Reviews Immunology, 15(4): 217-230. [26] Yu L, Wang L, Li M, et al.2010. Expression of toll-like receptor 4 is down-regulated during progression of cervical neoplasia[J]. Cancer Immunology Immunotherapy, 59(7): 1021-1028. [27] Zerbe H, Schuberth H J, Engelke F, et al.2003. Development and comparison of in vivo and in vitro models for endometritis in cows and mares[J]. Theriogenology, 60(2): 209-223. [28] Zughaier S M, Zimmer S M, Datta A, et al.2005. Differential induction of the toll-like receptor 4-MyD88-dependent and -independent signaling pathways by endotoxins[J]. Infection & Immunity, 73(5): 2940-2950.
