| Objective: This study is aimed to offer some biological markers for intervention,diagnosis and treatment of nonalcoholic fatty liver disease in an early stage,as well as lay the foundation for further studies in the role of microRNAs in apoptosis.Expression of microRNAs spectrum was screened in rat's liver tissues.Methods : After 1 week's adoptive feeding,16 4-weeks-old weighing about 200 grams male SD rats were divided into control group(named E group)and model group(named Y group)randomly(8 rats in each group).E group was fed with normal diet,while Y group was fed with high fat diet containing sugar and fat.The animal model of nonalcoholic fatty liver disease was established after 12 weeks.Then the animals were sacrificed and the liver tissues were rapidly taken out to do HE staining to observe the severity of fatty degeneration,inflammatory activity and calculate histological score.Then blood was drawn off from abdominal aorta and automatic biochemical analyzer was used to measure the level of aspartate aminotransferase,aspartate aminotransferase,total cholesterol,total triglyceride and fasting plasma glucose with enzymatic method.The total RNA of each fresh liver tissue was extracted with Trizol.After qualified with NanoDrop 2000 and Agilent Bioanalyzer 2100,the sample was performed on microRNAs gene chip with Gene Chip miRNA 4.0 to screen the differentially expressed miRNA,and the miRNA was verified with real-time PCR.Results: 1?General observation: In the process of feeding, the animals of E group was live and spiritual with clean hair, and gained weight continually. On the other wise, the animal of Y group was dispirited and sluggish with dull hair, and gained weight slowly. 2?The index of liver: Compared with E group, the weight of rat and liver in Y group were no obviously changed(P>0.05), while the liver index was increased significantly(P<0.05). 3?Biochemical parameters: Compared with E group, the level of ALT, TG and TC were no significantly changed(P>0.05),while the number of AST was increased obviously(P<0.05).4?Liver tissue's HE staining: The liver tissues of Y group had severe fatty degeneration, even ballooning degeneration, and points or focal necrosis and inflammatory cell infiltration appeared. 5?micro RNAs gene chip: Compared with E group, there were 10 differentially expressed mi RNAs with its fold change surpassing 2 fold. rno-mi R-743a-3p, rno-mi R-741-3p, rno-mi R-101a-3p, rno-mi R-29b-3p and rno-mi R-871-3p were overexpressed in Y group, while rno-mi R-344 g, rno-mi R-183-5p, rno-mi R-32-3p, rno-mi R-182 and rno-mir-182 were downregulated in Y group. 6?Synthesizing mi RNAs gene chip results and combining with relevant references, mi R-182, mi R-29b-3p and mi R-741-3p were verified with real-time quantitative PCR. As the results manifestation, compared with E group, mi R-182, mi R-29b-3p and mi R-741-3p were overexpressed(P<0.05), which largely consistent with the results of microarray. Conclusion: 1?High sugar and high fat diet is the cause of nonalcoholic fatty liver disease and high sugar and fat diet is a safe and effective way to establish the animal model of NAFLD; 2?There are some significant changes in miRNA between control group and model group. It means that the differentially expressed micro RNAs may play some roles in the process of NAFLD, but its mechanism remains unclear;3?The result of microarray is largely same as that of real-time quantitative PCR. It means that the result of microarray is so reliable that it can be used to screen differentially expressing mi RNAs in NAFLD. 4?mi R-182, mi R-29b-3p and mi R-741-3p may take part in the pathogenesis of NAFLD, but its specific pathway and mechanism remains to be further explored. |
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