Protective Effects and Mechanisms of Exogenous Addition or siRNA Interference with ACE2 on Oxidative Stress and Inflammatory Injury in NAFLD Cells Caused by Oleic Acid
XU Jia-Jing, WANG Huan-Huan, MA Chang, CHEN Xi-Wen, WANG Gong-Min, LIU Ying, ZHANG Yuan-Shu*
Key Laboratory of Animal Physiology and Biochemistry, Ministry of Agriculture and Rural Affairs/College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China
Abstract：Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease characterized by the production of large amounts of lipid deposits in the liver, which poses a serious health risk to humans. In recent years, there has been a gradual increase in the incidence of fatty liver disease in pets and farm animals, which has resulted in serious economic losses to the farming industry. Angiotensin-converting enzyme 2 (ACE2) has been shown to positively regulate anti-inflammatory, anti-injury and anti-fibrotic effects in a variety of diseases, including fatty liver. The previous study of our group found that ACE2 might exert some anti-injury effects on NAFLD induced by exogenous stimuli, but its exact protective mechanism is not clear. In this study, normal rat (Rattus norvegicus) hepatic stromal BRL-3A cells were induced with 0.025~0.200 mmol/L oleic acid (OA) for 24 h to establish a non-alcoholic fatty liver disease (NAFLD) cell model, and hepatocellular injury markers were measured to determine the optimal induction conditions. Subsequently, the cells were treated with either exogenous ACE2 active protein or siRNA targeting ACE2, after which lipid deposition was observed through staining, and changes in parameters including total anti-oxidative capacity (T-AOC), alanine aminotransferase (ALT), aspartate aminotransferase (AST), and inflammatory factors were detected. Western blot detected the protein expression of cellular ACE2, and mas receptor (MasR); ELISA detected the content of Ang Ⅱ and Ang 1-7 in the supernatant of the cells, and clarified the effect of ACE2 on NAFLD cellular damage. Results showed that: 1) NAFLD cell model was successfully established, analyzed by oil red staining to detect the changes of triglyceride (TG) content and reactive oxygen species (ROS) in hepatocytes, and the induction condition of NAFLD cell model was selected to be 0.100 mmol/L oleic acid for 24 h, and 3 different concentrations of OA (low, medium, and high (0.025, 0.100, and 0.200 mmol/L)) were determined for the follow-up test. 2) Compared with the control group, ACE2 protein expression was significantly down-regulated in the high concentration OA-treated group (P<0.05), and there was no significant difference in ACE2 expression in the low- and medium-concentration groups, the expression levels of ACE2 and MasR in the group treated with the addition of ACE2-activated proteins were significantly elevated (P<0.05), and after the transfection of siRNA-ACE2, the expression level of ACE2 was significantly reduced (P<0.05), and MasR expression level was reduced but the difference was not significant; 3) In medium concentration OA treatment, ACE2 and MasR expression was up-regulated, Ang 1-7 and Ang Ⅱ levels were increased, lipid accumulation was increased, oxidative stress was aggravated, antioxidant capacity was weakened, inflammatory factors and NO content were extremely significantly elevated (P<0.01), iNOS activity was extremely significantly enhanced (P<0.01), and anti-inflammatory factor content was extremely significantly reduced (P<0.01); 4) Compared with the OA-treated group, the ACE2 active protein-treated group cellular ACE2, MasR, and Ang 1-7 levels were significantly or extremely significantly up-regulated (P<0.05 or P<0.01) and Ang Ⅱ levels were down-regulated. The relative intracellular lipid droplet area and TG content were significantly reduced (P<0.05), while ALT, AST viability and T-AOC were enhanced to different degrees, and oxidative stress and inflammatory response were reduced.Transfection with siRNA-ACE2 treatment reversed the above changes. The experimental results suggested that treatment with ACE2-activated protein could alleviate cellular lipid deposition, attenuated cellular oxidative stress and inflammatory response, and had a certain alleviating effect on OA-induced NAFLD in rat hepatocytes. The mechanism was mainly realized by degrading Ang Ⅱ, activating Ang 1-7/Mas, and acting on the NO/iNOS signaling pathway. Thus, this study revealed a mechanism by which ACE2 alleviates NAFLD at the cellular level and provide an important theoretical foundation for developing ACE2 as a potential therapeutic target for NAFLD.
徐佳靖, 王换换, 马畅, 陈希文, 王公民, 刘颖, 张源淑. 外源添加或siRNA干扰ACE2对油酸致NAFLD细胞氧化应激和炎性损伤的保护作用及机制[J]. 农业生物技术学报, 2025, 33(12): 2677-2690.
XU Jia-Jing, WANG Huan-Huan, MA Chang, CHEN Xi-Wen, WANG Gong-Min, LIU Ying, ZHANG Yuan-Shu. Protective Effects and Mechanisms of Exogenous Addition or siRNA Interference with ACE2 on Oxidative Stress and Inflammatory Injury in NAFLD Cells Caused by Oleic Acid. 农业生物技术学报, 2025, 33(12): 2677-2690.
[1] 汪亮, 张霞. 2010. 肾素-血管紧张素系统在非酒精性脂肪肝发病中的作用[J]. 重庆医学, 39(22): 3139-3141+3149. (Wang L, Zhang X.2010. The role of the renin-angiotensin system in the pathogenesis of nonalcoholic fatty liver disease[J]. Chongqing Medical Journal, 39(22): 3139-3141+3149.) [2] 王艳霞, 张伟, 韩东宁, 等. 2011. 血管紧张素转化酶2(ACE2)对大鼠肾氧化应激损伤的保护作用及其机制[J].农业生物技术学报, 19(05): 881-886. (Wang Y X, Zhang W, Han D N, et al.2011. Protective effect and mechanism of angiotensin converting enzyme 2(ACE2) on renal oxidative stress injury in rats[J]. Journal of Agricultural Biotechnology, 19(05): 881-886.) [3] Ahmadian E, Pennefather P S, Eftekhari A, et al.2016. Role of renin-angiotensin system in liver diseases: An outline on the potential therapeutic points of intervention[J]. Expert Review of Gastroenterology & Hepatology, 10(11): 1279-1288. [4] Ayoub M A, Vijayan R.2021. Hemorphins targeting G protein-coupled receptors[J]. Pharmaceuticals (Basel), 14(3): 225. [5] Benhamouche-Trouillet S, Postic C.2016. Emerging role of miR-21 in non-alcoholic fatty liver disease[J]. Gut: An International Journal of Gastroenterology and Hepatology, 65(11): 1781-1783. [6] Byrne C D, Targher G.2015. NAFLD: A multisystem disease[J]. Journal of Hepatology, 62(1 Suppl): S47-S64. [7] Cao X, Yang F Y, Shi T T, et al.2016. Angiotensin-converting enzyme 2/angiotensin-(1-7)/Mas axis activates Akt signaling to ameliorate hepatic steatosis[J]. Scientific Reports, 6: 21592. [8] Cohen J B, Hanff T C, Bress A P, et al.2020. Relationship between ACE2 and other components of the renin-angiotensin system[J]. Current Hypertension Reports, 22(7): 44 [9] 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. [10] Elshafei A, Khidr E G, El-Husseiny A A, et al.2021. RAAS, ACE2 and COVID-19; a mechanistic review[J]. Saudi Journal of Biological Sciences, 28(11): 6465-6470. [11] Kudo H, Yata Y, Takahara T, et al.2009. Telmisartan attenuates progression of steatohepatitis in mice: Role of hepatic macrophage infiltration and effects on adipose tissue[J]. Liver International, , 29(7): 988-996. [12] Liu B, Wu R, Zhang W, et al.2012. Fosinopril improves liver fibrosis by upregulating ACE2/Angiotensin-(1-7) axis activation in rats with nonalcoholic steatohepatitis[J]. Latin American Journal of Pharmacy, 31(4): 588-596. [13] Pacher P, Beckman J S, Liaudet J, et al.2007. Nitric oxide and peroxynitrite in health and disease[J]. Physiological Reviews, 87(1): 315-424. [14] Paz Ocaranza M, Riquelme A J, García L, et al.2020. Counter-regulatory renin-angiotensin system in cardiovascular disease[J]. Nature Reviews Cardiology, 17(2): 116-129. [15] Reitman S, Frankel S.1957. A colorimetric method for the determination of serum glutamic oxalacetic and glutamic pyruvic transaminases[J]. American Journal of Clinical Pathology, 1(1957): 56-63. [16] Serfozo P, Wysocki J, Gulua G, et al.2020. Ang Ⅱ (Angiotensin Ⅱ) conversion to Angiotensin-(1-7) in the circulation is POP (prolyloligopeptidase)-dependent and ACE2 (angiotensin-converting enzyme 2)-independent[J]. Hypertension, 75(1): 173-182. [17] Smith P K, Krohn R I, Hermansonet G T, et al.1985. Measurement of protein using bicinchoninic acid[J]. Analytical Biochemistry, (1985): 76-85. [18] Suzuki Y, Ruiz-Ortega M, Lorenzo O, et al.2003. Inflammation and angiotensin Ⅱ[J]. The International Journal of Biochemistry & Cell Biology, 35(6): 881-900. [19] Tan J J, Wen S, Dong X Y, et al.2025. D-Psicose mitigates NAFLD mice induced by a high-fat diet by reducing lipid accumulation, inflammation, and oxidative stress[J]. Frontiers in Nutrition, 12: 1574151. [20] Turner A J, Hooper N M.2002. The angiotensin-converting enzyme gene family: Genomics and pharmacology[J]. Trends in Pharmacological Sciences, 23(4): 177-183. [21] Vickers C, Hales P, Kaushik V, et al.2002. Hydrolysis of biological peptides by human angiotensin-converting enzyme-related carboxypeptidase[J]. Journal of Biological Chemistry, 277(17): 14838-14843. [22] 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(2017): 23-30. [23] Warner F J, Lew R A, Smith I A, et al.2005. Angiotensin-converting enzyme 2 (ACE2), but not ACE, is preferentially localized to the apical surface of polarized kidney cells[J]. Journal of Biological Chemistry, 280(47): 39353-39362. [24] Wei Y, Clark S E, Morris E M, et al.2008. Angiotensin Ⅱ-induced non-alcoholic fatty liver disease is mediated by oxidative stress in transgenic TG (mRen2) 27 (Ren2) rats[J]. Journal of Hepatology, 49(3): 417-428. [25] Wu Y, Ma K L, Zhang Y, et al.2016. Lipid disorder and intrahepatic renin-angiotensin system activation synergistically contribute to non-alcoholic fatty liver disease[J]. Liver International, 36(10): 1525-1534. [26] Xue H, Zhou L, Yuan P, et al.2012. Counteraction between angiotensin Ⅱ and angiotensin-(1-7) via activating angiotensin type I and Mas receptor on rat renal mesangial cells[J]. Regulatory Peptides, 177(1-3): 12-20. [27] Yang J, Yang Y, Tan X Y, et al.2025. Unlocking the potential of the ACE2/Ang-(1-7)/Mas axis in liver diseases: From molecular mechanisms to translational applications[J]. Diabetes, Obesity & Metabolism, 27(8): 4069-4082. [28] Yang M, Ma X Y, Xuan X P, et al.2020. Liraglutide attenuates non-alcoholic fatty liver disease in mice by regulating the local renin-angiotensin system[J]. Frontiers in Pharmacology, 11: 432.
