| Purpose:Through the establishment of ApoE-/-mice model fed with high fat,the miRNA-m RNA molecular network of cholesterol metabolisation-related differential genes was constructed by using total transcriptional and miRNA sequencing technology,and the molecular mechanism of ApoE-/-mouse lipid metabolism disorder and the regulation effect of Huayu Qutan Fang was elucidated.Material and method:Ten C57BL/6J mice were blank control group,and 20 ApoE-/-mice were model group and TCM group.Mice in the blank control group were fed with normal mouse diet every day,while mice in the model group and the traditional Chinese medicine group were fed with high-fat diet every day.After 8 weeks,they were randomly divided into model group and traditional Chinese medicine group.The traditional Chinese medicine group was given the prescription of removing phlegm and removing blood stasis,containing the crude drug 20g/kg body mass gavage,Rats in the blank control group and the model group were given the same amount of distilled water by gavage.During the administration period,the three groups of rats were fed the same diet,and the samples were collected after 8 weeks of administration.Serum cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C)and high density lipoprotein cholesterol(HDL-C)were measured by biochemical analyzer.Histomorphological changes of liver were observed by HE staining,and lipid deposition was observed by oil red O staining.m RNA of differentially expressed genes related to cholesterol metabolism in mouse liver of the model group was analyzed by full transcriptome analysis,and miRNA of differentially expressed genes regulating cholesterol metabolism were screened by miRNA sequencing technology.Real-time fluorescence quantitative PCR was used to verify the expression trend of partial differential m RNA and miRNA and to clarify the intervention effect of Huayu Qutan Fang.Results: 1.Changes of blood lipid levels in each groupCompared with the blank control group,serum TC,TG and LDL-C levels in the model group increased significantly,while HDL-C levels decreased significantly(p<0.01).Compared with the model group,the levels of TC,TG and LDL-C decreased significantly after TCM treatment(p<0.01),while the levels of HDL-C increased but had no statistical significance(p> 0.05).2.Histomorphological changes and lipid deposition in the liver of mice in each groupThe results of HE staining showed that the morphological structure of the liver in the normal control group was normal,with clear morphological structure and uniform size of liver cells and clear nucleoli.The liver cells in the model group were disordered and swollen,and the vacuolar degeneration of fat was severe.After the treatment of removing phlegm and removing blood stasis,the liver cell swelling and vacuolar degeneration were significantly improved.The results of oil red O staining showed that there were no lipids in the liver of the blank control group.In the model group,there were fat droplets in the liver of mice.After the treatment of removing phlegm and removing blood stasis,ApoE-/-mice liver oil red staining lipid drops were significantly reduced.3.Expression of m RNA related to cholesterol metabolism in the liver of ApoE-/-mice and its regulation of miRNAThe total transcriptional results showed that,compared with the blank control group,the expressions of cholesterol metabolism related genes SREBP2,PCSK9,LDLR,HMGCR and CYP7A1 in the liver of the model group were down-regulated.Among the miRNA regulated by them,there were 72 differential miRNA,among which 14 were down-regulated and 58 were up-regulated,showing an overall negative regulatory trend.72 miRNA formed a molecular regulatory network with 5 target genes,among which 9 miRNA regulated more than 3 target genes.Mir-351-5p and mmo-mir-676-3p were able to regulate 4 genes,namely HMGCR,LDLR,PCSK9,SREBP2 and LDLR,PCSK9,HMGCR and CYP7A1,respectively.4.Effect of decoction for removing blood stasis and removing phlegm on m RNA and miRNA expression of cholesterol metabolism related genes in ApoE-/-mouse livermRNA validation results showed that the m RNA expressions of SREBP2,HMGCR,LDLR,CYP7A1 and PCSK9 in the model group were significantly down-regulated compared with the blank control group(p<0.05,p<0.01).Compared with the model group,m RNA expressions of SREBP2,LDLR,CYP7A1 and PCSK9 in the traditional Chinese medicine group were significantly up-regulated(p<0.05,p<0.01),and HMGCR changes were not significant.miRNA validation results showed that compared with the blank control group,miR-1933-3p,miR-335-3p,miR-351-5p,miR-485-5p,miR-5114,miR-676-3p,miR-677-5p,miR-7043-3p and miR-7091-3p were significantly up-regulated in the model group(p<0.05,p<0.01),consistent with the sequencing results.Compared with the model group,miR-677-5p and miR-7043-3p in the TCM group were significantly down-regulated(p<0.05).Conclusion: 1.Huayu Qutan Fang can effectively improve the blood lipid and liver lipid depositionlevel of ApoE-/-mice.2.The miRNA-m RNA molecular network associated with cholesterol metabolism may play a key role in the disorder of lipid metabolism in ApoE-/-mice.3.Huayu Qutan Fang may improve the imbalance of cholesterol homeostasis by regulating PCSK9,SREBP2,LDLR and CYP7A1,and the upstream mechanism may be related to the regulation of mir-677-5p and mir-7043-3p. |
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