目的 探讨微小RNA-129-5p（miR-129-5p）调控Rho激酶（RhoA）/Rho蛋白相关卷曲螺旋激酶（ROCK）通路对代谢相关的脂肪性肝病（MAFLD）大鼠肝损伤的影响。 方法 收集28例2024年6月至12月期间于我院进行治疗的MAFLD患者为MAFLD组，另收集28例健康体检者作为对照组；qRT-PCR法检测MAFLD组和对照组血清中miR-129-5p、RhoA、ROCK mRNA表达。双荧光素酶报告基因实验验证miR-129-5p与RhoA的互作；构建MAFLD大鼠，将造模成功的大鼠随机分为MAFLD组、阴性对照（NC agomir）组（尾静脉注射NC agomir）、miR-129-5p激动剂（miR-129-5p agomir）组（尾静脉注射miR-129-5p agomir）、溶血磷脂酸（LPA）（尾静脉注射miR-129-5p agomir+1 mg/kg的LPA），每组12只，另选择12只正常大鼠为NC组，NC组和MAFLD组注射等量生理盐水。检测各组血清中肝功能、血脂、炎症因子以及肝组织中TC、TG的表达；qRT-PCR法检测MAFLD大鼠肝组织miR-129-5p、RhoA、ROCK mRNA表达；HE染色和油红O染色检测肝组织病理学；TUNEL染色检测肝组织细胞凋亡；Western blot检测肝组织中RhoA、ROCK蛋白表达。 结果 与对照组比较，MAFLD组血清中miR-129-5p表达降低，RhoA和ROCK mRNA表达升高（P<0.05）。miR-129-5p可以靶向负调控RhoA。NC组肝脏组织正常；MAFLD组和NC agomir组肝脏组织可见肝细胞脂肪变性、形态明显肿胀且呈气球样变及呈空泡化和大量红色脂肪滴；miR-129-5p agomir组有少量肝细胞轻度脂肪变性，胞质内可见微小的圆形空泡和脂肪滴明显减少；LPA组肝细胞脂肪变性进一步加重，脂肪滴进一步增多。MAFLD组肝脏和血清中TC、肝脏和血清中TG、LDL-C、AST、ALT、TNF-α、IL-6、IL-1β、RhoA mRNA和蛋白、ROCK mRNA和蛋白、凋亡率高于NC组，HDL-C、miR-129-5p低于NC组（P<0.05）；miR-129-5p agomir组肝脏和血清中TC、肝脏和血清中TG、LDL-C、AST、ALT、TNF-α、IL-6、IL-1β、RhoA mRNA和蛋白、ROCK mRNA和蛋白、凋亡率低于MAFLD组、NC agomir组，HDL-C、miR-129-5p高于MAFLD组、NC agomir组（P<0.05）；LPA组肝脏和血清中TC、肝脏和血清中TG、LDL-C、AST、ALT、TNF-α、IL-6、IL-1β、RhoA mRNA和蛋白、ROCK mRNA和蛋白、凋亡率高于miR-129-5p agomir组，HDL-C低于miR-129-5p agomir组（P<0.05）。 结论 miR-129-5p可能通过靶向负调控RhoA/ROCK通路缓解MAFLD引起的肝损伤。
Abstract
Objective To explore the effect of microRNA-129-5p (miR-129-5p) on liver injury in metabolic dysfunction-associated fatty liver disease (MAFLD) rats by regulating the Rho kinase (RhoA)/Rho-associated coiled coil kinase (ROCK) pathway. Methods A total of 28 patients with MAFLD who were treated in our hospital from June to December 2024 were selected as MAFLD group, while another 28 healthy individuals undergoing physical examinations were selected as control group. The expression levels of miR-129-5p, RhoA, and ROCK mRNA in the serum of MAFLD group and control group were detected using qRT-PCR method. The dual luciferase reporter gene assay was used to verify the interaction between miR-129-5p and RhoA. MAFLD rats were constructed, and successfully modeled rats were classified into MAFLD group, NC agomir group (tail vein injection of NC agomir), miR-129-5p agomir group (tail vein injection of miR-129-5p agomir), and LPA (tail vein injection of miR-129-5p agomir+1 mg/kg LPA) randomly, each with 12 rats. Another 12 normal rats were considered as NC group. NC group and MAFLD group were injected with equal amounts of physiological saline. The serum liver function, blood lipids, inflammatory factors, and the expression of TC and TG in liver tissue were measured. QRT-PCR was used to detect miR-129-5p, RhoA, and ROCK mRNA in liver tissue of MAFLD rats. HE staining and Oil Red O staining were used to detect liver tissue pathology. TUNEL staining was applied to detect apoptosis of liver tissue cells. Western blot was applied to detect RhoA and ROCK proteins in liver tissue. Results Compared with control group, the expression of miR-129-5p in the serum of MAFLD group decreased, while the expressions of RhoA and ROCK mRNA increased (P<0.05). miR-129-5p could target negative regulation of RhoA. The liver tissue of NC group was normal. The liver tissues of MAFLD group and NC agomir group showed hepatic steatosis, prominent swelling in morphology, balloon like changes, vacuolization, and many red fat droplets. The rats of miR-129-5p agomir group showed a small amount of mild hepatic steatosis, with small circular vacuoles and prominently reduced lipid droplets observed in cytoplasm. The hepatic steatosis in LPA group further worsened, and the number of fat droplets was further increased. The rats in MAFLD group had higher TC in liver and serum, TG, LDL-C, AST, ALT, TNF-α, IL-6, IL-1β, RhoA mRNA and protein, ROCK mRNA and protein in liver and serum, and apoptosis rate than NC group, and lower HDL-C and miR-129-5p than the NC group (P<0.05). The rats in miR-129-5p agomir group had lower TC in liver and serum, TG, LDL-C, AST, ALT, TNF-α, IL-6, IL-1β, RhoA mRNA and protein, ROCK mRNA and protein in the liver and serum, and apoptosis rate than MAFLD group and NC agomir group, and higher HDL-C and miR-129-5p than MAFLD group and NC agomir group (P<0.05). The LPA group had higher TC in liver and serum, TG, LDL-C, AST, ALT, TNF-α, IL-6, IL-1β, RhoA mRNA and protein, ROCK mRNA and protein in liver and serum, and apoptosis rate than miR-129-5p agomir group, and lower HDL-C than miR-129-5p agomir group (P<0.05). Conclusions MiR-129-5p may alleviate liver injury caused by MAFLD by targeting negative regulation of RhoA/ROCK pathway.
关键词
微小RNA-129-5p /
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Rho蛋白相关卷曲螺旋激酶 /
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Rho激酶 /
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代谢相关的脂肪性肝病 /
肝损伤
Key words
MicroRNA-129-5p /
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Rho-associated coiled-coil kinase /
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Rho kinase /
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Metabolic dysfunction-associated fatty liver disease /
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Liver injury
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