miR-490-3p过表达对大鼠非酒精性脂肪性肝病的改善作用及其机制

doi:10.13481/j.1671-587X.20210620

摘要/Abstract

摘要： 目的

探讨miR-490-3p过表达对大鼠非酒精性脂肪性肝病（NAFLD）的改善作用，并初步分析其作用机制。

方法

32只雄性SD大鼠随机分为对照组、模型组、阴性对照组（mimic-NC组）和miR-490-3p模拟物组（miR-490-3p mimic组），每组8只。采用高脂饲料喂养复制NAFLD大鼠模型，对照组大鼠不进行任何处理，mimic-NC组和miR-490-3p mimic组大鼠分别尾静脉注射含无意义序列和miR-490-3p mimic质粒的腺病毒。采用实时荧光定量PCR（RT-qPCR）法检测各组大鼠肝组织中miR-490-3p表达水平，HE染色和油红O染色观察各组大鼠肝组织病理形态表现，ELISA法检测各组大鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）、白细胞介素1β（IL-1β）、丙二醛（MDA）、超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GSH-Px）水平，RT-qPCR法检测各组大鼠肝组织中Toll样受体4（TLR4）和核因子κB（NF-κB）mRNA表达水平，Western blotting法检测各组大鼠肝组织中TLR4和NF-κB蛋白表达水平。

结果

与对照组比较，模型组大鼠血清ALT、AST、TNF-α、IL-6、IL-1β和MDA水平和肝组织中TLR4 mRNA及蛋白、NF-κB mRNA表达水平均升高（P<0.05），血清SOD和GSH-Px水平及肝组织中miR-490-3p表达水平均降低（P<0.05）；与模型组比较，miR-490-3p mimic组大鼠血清ALT、AST、TNF-α、IL-6、IL-1β、MDA水平和肝组织中TLR4 mRNA及蛋白、NF-κB mRNA表达水平均降低（P<0.05），血清SOD和GSH-Px水平和肝组织中miR-490-3p表达水平升高（P<0.05）。HE染色，对照组大鼠肝细胞排列均匀；模型组和mimic-NC组大鼠肝细胞中堆积大量脂肪，门管区伴有明显炎性细胞浸润，部分肝细胞呈坏死，并出现纤维化病变；miR-490-3p mimic组大鼠肝组织病变程度较模型组减轻。油红O染色，对照组大鼠肝组织中未见明显红染脂肪滴；模型组和mimic-NC组大鼠肝组织中可见明显红染的脂肪滴，数量多且体积大；miR-490-3p mimic组大鼠肝组织中脂肪滴体积和数量均明显减少。

结论

miR-490-3p过表达可减轻大鼠NAFLD损伤，其机制可能与阻滞TLR4/NF-κB信号通路、减轻炎症反应和改善氧化应激有关。

关键词: miR-490-3p, 非酒精性脂肪性肝病, 炎症反应, 氧化应激, Toll样受体4/核因子κB信号通路

Abstract: Objective

To explore the improvement effect of miR-490-3p over-expression on non-alcoholic fatty liver disease （NAFLD） of the rats， and to preliminarily analyze its action mechanism.

Methods

A total of 32 male SD rats were randomly divided into control group， model group， negative control group （mimic-NC group），and miR-490-3p mimic group （miR-490-3p mimic group），and there were 8 rats in each group. The rats were fed with high-fat diet to replicate the NAFLD rat models. The rats in control group were given nothing，and the rats in mimic-NC and miR-490-3p mimic groups were injected with adenovirus containing meaningless sequence and miR-490-3p mimic plasmid through tail vein， respectively. The expression levels of miR-490-3p in liver tissue of the rats in various groups were detected by Real-time fluorescence quantitative （RT-qPCR）method. The serum levels of alanine aminotransferase （ALT），aspartate aminotransferase （AST）， tumor necrosis factor-α （TNF-α）， interleukin-6 （IL-6）， interleukin-1β （IL-1β）， malondialdehyde （MDA）， superoxide dismutase （SOD）， glutathione peroxidase （GSH-Px） in liver tissue of the rats in various groups were detected by ELASA method. The pathomorphology of liver tissue of the rats in various groups were detected by HE staining and Oil red O staining.The expression levels of Toll-like receptor 4 （TLR4） and nuclear factor κB （NF-κB） mRNA in liver tissue of the rats in various groups were detected by RT-qPCR method.The expression levels of TLR4 and NF-κB proteins in liver tissue of the rats in various groups were detected by Western blotting method.

Results

Compared with control group， the serum levels of ALT， AST， TNF-α， IL-6， IL-1β and MDA， the expression levels of TLR4 mRNA and protein and NF-κB mRNA in liver tissue of the rats in model group were increased（P<0.05）；while the serum levels of SOD and GSH-Px and expression level of miR-490-3p in liver tissue were decreased （P<0.05）. Compared with model group， the serum levels of ALT， AST， TNF-α， IL-6， IL-1β， and MDA and the expression levels of TLR4 mRNA and protein and NF-κB mRNA in liver tissue of the rats in miR-490-3p mimic group were decreased（P<0.05）， while the levels of serum SOD and GSH-Px， and the expression level of miR-490-3p in liver tissue were increased （P<0.05）.The HE staining results showed that the hepatocytes of the rats in control group were evenly arranged， a large amount of fat was accumulated in the hepatocytes in model group and mimic-NC group， and there was obvious inflammatory cell infiltration in the portal area， and some hepatocytes were necrotic and fibrotic； the degree of liver lesions in miR-490-3p mimic group was less than that in model group. The oil red O staining results showed that there were no obviously red stained fat droplets in liver tissue of the rats in control group，and the obviously red stained fat droplets were found in liver tissue of the rats in model group and mimic-NC group， with large quantity and volume； the volume and quantity of fat droplets in liver tissue of the rats in miR-490-3p mimic group were significantly reduced.

Conclusion

Over-expression of miR-490-3p can relieve the NAFLD damage，and its mechanism may be related to blocking the TLR4/NF-κB signaling pathways， reducing the inflammation response，and improving the oxidative stress.

Key words: miR-490-3p, non-alcoholic fatty liver disease, inflammatory response, oxidative stress, Toll-like receptor 4/nuclear factor κB signaling pathway

中图分类号:

R575.5

龚杰,雷泽华,张远威,黄雄,杜波,王志旭. miR-490-3p过表达对大鼠非酒精性脂肪性肝病的改善作用及其机制[J]. 吉林大学学报(医学版), 2021, 47(6): 1495-1501.

Jie GONG,Zehua LEI,Yuanwei ZHANG,Xiong HUNANG,Bo DU,Zhixu WANG. Improvement effect of miR-490-3p over-expression on non-alcoholic fatty liver disease in rats and its mechanism[J]. Journal of Jilin University(Medicine Edition), 2021, 47(6): 1495-1501.

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参考文献 20

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Group	ALT	AST
Control	36.94±2.71	89.17±5.38
Model	102.88±17.76^*	171.36±15.33^*
mimic-NC	99.14±10.77	167.53±12.61
miR-490-3p mimic	44.26±3.37^△	106.09±7.67^△

Group	TNF-α	IL-6	IL-1β
Control	40.02±11.34	36.59±4.47	15.81±2.34
Model	76.80±9.69^*	120.15±12.09^*	29.73±4.23^*
mimic-NC	72.90±10.30	116.59±9.66	27.39±3.43
miR-490-3p mimic	45.29±15.06^△	51.56±6.41^△	20.95±1.49^△

Group	MDA[m_B/（μmol·g^－1）]	SOD[λ_B/（U·mg^－1）]	GSH-Px[λ_B/（U·mg^－1）]
Control	5.07±0.54	151.79±10.18	648.49±73.46
Model	10.80±1.17^*	80.12±6.43^*	403.14±46.89^*
mimic-NC	10.21±0.78	85.65±5.86	428.61±55.39
miR-490-3p mimic	6.29±0.46^△	107.16±5.66^△	605.62±62.55^△

Group	TLR4		NF-κB
Group	mRNA	Protein	mRNA	Protein
Control	0.83±0.14	0.11±0.02	0.64±0.17	0.38±0.09
Model	3.58±0.29^*	0.94±0.06^*	4.21±0.34^*	0.74 ±0.69^*
mimic-NC	3.46±0.25	0.89±0.05	4.18±0.39	0.69±0.12
miR-490-3p mimic	2.24±0.22^△	0.39±0.03^△	2.76±0.26^△	0.51 ±0.14^△

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