The Regulatory Role of RAS in LPS-induced Inflammatory of bMECs
ZHANG Xiang-Jun, ZHOU Xue-Zhang*
College of Life Science, Ningxia University / Key Laboratory of the Ministry of Education for the Conservation and Utilization of Special Biological Resources of Western China, Yinchuan 750021, China
Abstract：The aim of this study is to explore the renin-angiotensin system's (RAS) expression and location in bovine mammary epithelial cells (bMECs), and its regulatory role in LPS-induced inflammatory injury. After treating bMECs with different concentrations of LPS, CCK-8 method was used to examine cell viability, and qPCR was used to detect IL-1β, IL-6, IL-8, TNF-α, ACE2, ACE, MasR和AT1R mRNA expression. Enzyme linked immunosorbent assay (ELISA) was used to test the content of AngⅡ and Ang 1-7 protein in the supernatant. Western blot and cellular immunofluorescence were used to verify the expression and location of ACE2 protein in bMECs. The results showed that ACE2 was expressed and localized in the cell membrane and cytoplasm of bMECs. Producing obvious inflammatory damage by LPS treatment of bMECs for 24 h, the expression of IL-1β, IL-6, IL-8, TNF-α, ACE, AT1R gene, and AngⅡ proein increased with the LPS concentration. The expression of ACE2 and MasR mRNA firstly increased and then decreased, while the expression of Ang 1-7 protein decreased along with the LPS concentration. This study indicates the presence of RAS members such as ACE2, ACE, MasR, AT1R, AngⅡ and Ang 1-7 in bMECs, and the ACE2/MasR/Ang 1-7 and ACE/Ang Ⅱ/AT1R pathways involved in the regulation of bMECs inflammatory injury induced by LPS. This experiment lays the foundation for studying the regulatory role of RAS in mastitis and discovering new targets for the action of anti-inflammatory drug.
张想军, 周学章. RAS系统在LPS诱导bMECs炎性损伤过程中的调控作用[J]. 农业生物技术学报, 2021, 29(10): 1958-1967.
ZHANG Xiang-Jun, ZHOU Xue-Zhang. The Regulatory Role of RAS in LPS-induced Inflammatory of bMECs. 农业生物技术学报, 2021, 29(10): 1958-1967.
[1] 刘小倩, 刘颖, 张源淑, 等. 2017. ACE2在奶牛乳腺中的表达定位及其抗炎性损伤作用的研究[J]. 畜牧兽医学报, 48(03): 552-560. (Liu X Q, Liu Y, Zhang Y S, et al.2017. Expression localization of angiotensin converting enzyme 2 (ACE2) in dairy cow mammary gland and its anti-inflammation effect[J]. Chinese Journal of Animal and Veterinary Sciences, 48(03): 552-560.) [2] 赵凌, 潘阳阳, 余四九, 等. 2018. 金黄色葡萄球菌和大肠杆菌感染BMECs对细胞因子和受体表达及细胞凋亡的影响[J]. 农业生物技术学报, 26(12): 2132-2141. (Zhao L, Pan Y Y, Yu S J, et al.2018. Effect on the expression of cytokine, receptor and apoptosis in BMECs by Staphylococcus aureus and Escherichia coli infected[J]. Journal of Agricultural Biotechnology, 26(12): 2132-2141.) [3] Batlle D, Wysocki J, Soler M J, et al.2012. Angiotensin-converting enzyme 2: Enhancing the degradation of angiotensin II as a potential therapy for diabetic nephropathy[J]. Kidney International, 81(6): 520-528. [4] Bernardi S, Toffoli B, Zennaro C, et al.2012. High-salt diet increases glomerular ACE/ACE2 ratio leading to oxidative stress and kidney damage[J]. Nephrology Dialysis Transplantation, 27(5): 1793-1800. [5] Crowley S D, Rudemiller N P.2017. Immunologic effects of the renin-angiotensin system[J]. Journal of the American Society of Nephrology, 28(5): 1350-1361. [6] Donoghue M, Hsieh F, Baronas E, et al.2000. A novel angiotensin-converting enzyme-related carboxypeptidase (ACE2) converts angiotensin I to angiotensin 1-9[J]. Circulation Research, 87(5): E1-9. [7] Farag E, Sessler D I, Ebrahim Z, et al.2017. The renin angiotensin system and the brain: new developments[J]. Journal of Clinical Neuroscience, 46: 1-8. [8] Hamming I, Cooper M E, Haagmans B L, et al.2007. The emerging role of ACE2 in physiology and disease[J]. Journal of Pathology, 212(1): 1-11. [9] Hennighausen L, Lee H K.2020. Activation of the SARS-CoV-2 receptor Ace2 through JAK/STAT-dependent enhancers during pregnancy[J]. Cell Reports, 32(13): 108199. [10] Hikmet F, Mear L, Edvinsson A, et al.2020. The protein expression profile of ACE2 in human tissues[J]. Molecular Systems Biology, 16(7): e9610. [11] Jin H Y, Song B, Oudit G Y, et al.2012. ACE2 deficiency enhances angiotensin II-mediated aortic profilin-1 expression, inflammation and peroxynitrite production[J]. PLoS One, 7(6): e38502. [12] Kurihara T, Ozawa Y, Shinoda K, et al.2006. Neuroprotective effects of angiotensin II type 1 receptor (AT1R) blocker, telmisartan, via modulating AT1R and AT2R signaling in retinal inflammation[J]. Investigative Ophthalmology & Visual Science, 47(12): 5545-5552. [13] Li Y, Cao Y, Zeng Z, et al.2015. Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis prevents lipopolysaccharide-induced apoptosis of pulmonary microvascular endothelial cells by inhibiting JNK/NF-kappaB pathways[J]. Scientific Reports, 5, 8209. [14] Li Y, Zeng Z, Cao Y, et al.2016. Angiotensin-converting enzyme 2 preventslipopolysaccharide-induced rat acute lung injury via suppressing the ERK1/2 and NF-kappaB signaling pathways[J]. Scientific Reports, 6: 27911. [15] Mizuiri S, Hemmi H, Arita M, et al.2008. Expression of ACE and ACE2 in individuals with diabetic kidney disease and healthy controls[J]. American Journal of Kidney Diseases, 51(4): 613-623. [16] Murphy T J, Alexander R W, Griendling K K, et al.1991. Isolation of a cDNA encoding the vascular type-1 angiotensin II receptor[J]. Nature, 351(6323): 233-236. [17] Paz Ocaranza M, Riquelme J A, García L, et al.2020. Counter-regulatory renin-angiotensin system in cardiovascular disease[J]. Nature Reviews Cardiology, 17(2): 116-129. [18] Patel S, Rauf A, Khan H, et al.2017. Renin-angiotensin-aldosterone (RAAS): The ubiquitous system for homeostasis and pathologies[J]. Biomed Pharmacother, 94: 317-325. [19] Paul M, Poyan Mehr A, Kreutz R.2006. Physiology of local renin-angiotensin systems[J]. Physiological Reviews, 86(3): 747-803. [20] Santos R A, Ferreira A J, Verano-Braga T, et al.2013. Angiotensin-converting enzyme 2, angiotensin-(1-7) and Mas: New players of the renin-angiotensin system[J]. Journal of Endocrinology, 216(2): R1-r17. [21] Simões e Silva A C, Silveira K D, Ferreira A J, et al.2013. ACE2, angiotensin-(1-7) and Mas receptor axis in inflammation and fibrosis[J]. British Journal of Pharmacology, 169(3): 477-492. [22] Tigerstedt R, Bergman P G.1898. Niere und Kreislauf[J]. Skandinavisches Archiv Für Physiologie, 8(1): 223-271. [23] Tipnis S R, Hooper N M, Hyde R, et al.2000. A human homolog of angiotensin-converting enzyme. Cloning and functional expression as a captopril-insensitive carboxypeptidase[J]. Journal of Biological Chemistry, 275(43): 33238-33243. [24] Simões E Silva A C, Teixeira M M.2016. ACE inhibition, ACE2 and angiotensin-(1-7) axis in kidney and cardiac inflammation and fibrosis[J]. Pharmacological Research, 107: 154-162. [25] Wang K, Liu X, Xiao H, et al.2017. The correlation between inflammatory injury induced by LPS and RAS in EpH4-Ev cells[J]. International Immunopharmacology, 46: 23-30. [26] White A J, Cheruvu S C, Sarris M, et al.2015. Expression of classical components of the renin-angiotensin system in the human eye[J]. Journal of the Renin-Angiotensin-Aldosterone System, 16(1): 59-66. [27] Wong M H, Chapin O C, Johnson M D.2012. LPS-stimulated cytokine production in type I cells is modulated by the renin-angiotensin system[J]. American Journal of Respiratory Cell and Molecular Biology, 46(5): 641-650. [28] Wosten-van Asperen R M, Lutter R, Specht P A, et al.2011. Acute respiratory distress syndrome leads to reduced ratio of ACE/ACE2 activities and is prevented by angiotensin-(1-7) or an angiotensin II receptor antagonist[J]. Journal of Pathology, 225(4): 618-627. [29] Yaguchi S, Ogawa Y, Kawakita T, et al.2015. Tissue renin-angiotensin system in lacrimal gland fibrosis in a murine model of chronic graft-versus-host disease[J]. The Journal of Cornea and External Disease, 34(Suppl 11): S142-152. [30] Yang T, Xu C.2017. Physiology and pathophysiology of the intrarenal renin-angiotensin system: An Update[J]. Journal of the American Society of Nephrology, 28(4): 1040-1049.
