Apo E^(-/-)动脉粥样硬化小鼠肝脏胆固醇代谢相关miRNA-mRNA网络构建及化瘀祛痰方的调控作用

Construction of miRNA-mRNA network related to cholesterol metabolism in liver of ApoE^(-/-) AS mice and the regulatory mechanism of Huayu Qutan Formula

目的:利用mRNA转录组学与miRNA测序技术构建ApoE^(-/-)动脉粥样硬化(AS)小鼠胆固醇代谢相关miRNA-mRNA分子网络,阐明ApoE^(-/-)小鼠脂代谢紊乱分子机制及化瘀祛痰方调控作用。方法:10只C57BL/6J小鼠为空白对照组,20只ApoE^(-/-)小鼠予高脂饲料喂养8周后分为模型组和中药组,每组10只。中药组予化瘀祛痰方20 g/kg体质量灌胃,其他两组予等量蒸馏水灌胃,给药8周后取材。生化分析仪检测小鼠血清血脂水平,HE染色观测肝脏组织形态学变化、油红O染色观测肝脏脂质沉积情况,mRNA转录组学与miRNA测序技术构建ApoE^(-/- )AS小鼠胆固醇代谢相关miRNA-mRNA分子网络,qRT-PCR验证表达趋势并明确化瘀祛痰方的干预作用。结果:与空白对照组比较,模型组小鼠血清TC、TG和LDL-C水平显著上升,HDL-C水平显著下降(P<0.01);肝脏细胞排列紊乱且肿胀,脂肪空泡变性严重,肝脏油红着色脂滴存在;胆固醇代谢相关基因SREBP2、PCSK9、LDLR、HMGCR、CYP7A1表达下调,qRT-PCR验证结果一致(P<0.01,P<0.05)。在调控他们的miRNAs中,有71个差异miRNAs,其中13个下调,58个上调,总体呈现负调控趋势,71个miRNAs与5个靶基因形成了一个分子调控网络,其中9个miRNAs对3个以上靶基因具有负调控作用,qRT-PCR验证结果一致(P<0.01,P<0.05)。与模型组比较,中药组TC、TG和LDL-C水平显著降低(P<0.01);肝脏细胞肿胀与脂肪空泡样变性明显改善,油红着色脂滴明显减少;SREBP2、PCSK9、LDLR和CYP7A1mRNA表达显著上调(P<0.01,P<0.05),miR-677-5p和miR-7043-3p显著下调(P<0.05)。结论:胆固醇代谢相关miRNA-mRNA分子网络可能在ApoE^(-/-)小鼠脂代谢紊乱中发挥关键作用,化瘀祛痰方可能通过调控PCSK9、SREBP2、LDLR和CYP7A1改善胆固醇稳态失衡,上游机制可能与其调控miR-677-5p和miR-7043-3p有关。

Objective:The miRNA-mRNA molecular network related to cholesterol metabolism in ApoE^(-/-)atherosclerotic mice was constructed by mRNA transcriptome and miRNA sequencing technology to clarify the molecular mechanism of lipid metabolism disorder in ApoE^(-/-) mice and the regulatory effect of Huayu Qutan Formula.Methods:Ten C57BL/6J mice were used as blank control group,and 20 ApoE^(-/-) mice were fed with high-fat diet and divided into model group and traditional Chinese medicine group,10 mice in each group.After 8 weeks,the traditional Chinese medicine group was given the Huayu Qutan Formula 20 g/kg by intragastric administration,while the other two groups were given the same amount of distilled water by intragastric administration,and the samples were collected after 8 weeks.Serum lipid levels of mice were detected by biochemical analyzer,liver histological and morphological changes were observed by HE staining,and liver lipid deposition was observed by oil red O staining.mRNA transcriptome and miRNA sequencing technology were used to construct miRNA-mRNA molecular network related to cholesterol metabolism in ApoE^(-/-) AS mice.qRT-PCR was used to verify the expression trend of partial differences in mRNAs and miRNAs,and to clarify the intervention effect of Huayu Quphan Formula.Results:Compared with blank control group,serum TC,TG and LDL-C levels in model group were significantly increased,while HDL-C levels were significantly decreased (P<0.01);Liver cells were disordered and swollen,with serious fat vacuolar degeneration,and liver oil red staining lipid droplets were present;The expressions of cholesterol metabolism-related genes SREBP2,PCSK9,LDLR,HMGCR,and CYP7A1 were down-regulated,and the results of qRT-PCR were consistent (P<0.01,P<0.05).Among the miRNAs regulating them,there were 71 differential miRNAs,of which 13 were down-regulated and 58were up-regulated,showing a general negative regulation trend.The 71 miRNAs formed a molecular regulation network with5 target genes,among which 9 miRNAs had negative regulation effects on more than 3 target genes.The qRT-PCR verification results were consistent with the sequencing results.Compared with model group,the levels of TC,TG and LDL-C in traditional Chinese medicine group were significantly decreased (P<0.01).The swelling of liver cells and vacuolar degeneration were significantly improved,and the lipid droplets of oil red staining were significantly reduced.The mRNA expressions of SREBP2,PCSK9,LDLR and CYP7A1 were significantly up-regulated (P<0.01,P<0.05),miR-677-5p and miR-7043-3p were significantly down-regulated (P<0.05).Conclusion:Cholesterol metabolism-related miRNA-mRNA molecular network may play a key role in lipid metabolism disorders in ApoE^(-/-) mice.Huayu Qutan Formula may improve cholesterol homeostasis imbalance by regulating PCSK9,SREBP2,LDLR and CYP7A1.The upstream mechanism may be related to its regulation of miR-677-5p and miR-7043-3p.