Exploring the Mechanism of Huayu Qutan Formula in Improving Endothelial-to-mesenchymal transition in ApoE-/-AS Mice through ApoA-Ⅰ/Akt1/β-catenin/TGF-β Pathway
Objective To explore the mechanism of Huayu Qutan Formula in Improving Endothelial-to-mesenchymal transi-tion in ApoE-/-AS Mice through ApoA-Ⅰ/Akt1/β-catenin/TGF-β Pathway.Methods 24 ApoE-/-mice were randomly di-vided into model group,Huayu Qutan Formula group and simvastatin group,with 8 mice in each group;Eight C57BL/6J mice were used as the blank control group.ApoE-/-mice were fed with high-fat feed to prepare an AS model,while C57BL/6J mice were fed with regular feed.After 12 weeks,the blank control group and model group were given equal volume of physiological sa-line by gavage.The Huayu Qutan formula group was given 20 g/(kg·d)of Huayu Qutan formula by gavage,while the simvastatin group was given 2.275 mg/(kg·d)of simvastatin suspension by gavage once a day for 4 weeks.Using a fully automated bio-chemical analyzer to detect serum lipid levels;Hematoxylin eosin staining(HE)was used to observe the pathological tissue mor-phology of the aorta;Immunofluorescence assay was used to detect the degree of EndMT in the aorta;Real time quantitative PCR(RT-qPCR)was used to detect the mRNA expression levels of aortic transcription factor E-box binding zinc finger protein 1(ZEB1)and TWIST;Western blot for detecting the expression levels of liver ApoA-Ⅰ and aorta α-Smooth muscle actin(α-SMA),VE cadherin,ZEB1,TWIST,Akt1,p-Akt1 β-Catenin,p-β-Catenin,TGF-β1,Smad2 and p-Smad2 proteins.Re-sults Compared with the blank control group,the serum levels of total cholesterol(TC),triglycerides(TG),and low-density lip-oprotein cholesterol(LDL-C)in the model group mice were significantly increased,while the levels of high-density lipoprotein cholesterol(HDL-C)were significantly decreased(P＜0.05);The inner wall of the aorta is uneven and smooth,and a large number of foam cells can be seen gathering to form atherosclerotic plaques.The intima of the vessel where the plaques attach is obviously thickened and the structure is disordered,and detached plaque fragments can be seen in the lumen;The positive cells ofα-SMA and VE-Cadherin double staining in the aorta were significantly increased,and the expression level of α-SMA protein was significantly increased,the expression level of VE-cadherin protein significantly decreased(P＜0.05);The mRNA and protein expression levels of aortic transcription factor ZEB1 and TWIST were significantly increased(P＜0.05);The expression levels of TGF-β1 and p-Smad2 proteins in the aorta were significantly increased(P＜0.05),ApoA-Ⅰ in the liver,p-Akt1 and p-β-cateninin the aortawere significantly reduced(P＜0 05).After intervention with the Huayu Qutan formula,the serum levels of TC,TG,and LDL-C in mice significantly decreased,while the levels of HDL-C significantly increased(P＜0.05);The inner wall of the aorta is not smooth,with a small amount of endothelial cells shedding and a significant reduction in wall thickness;The positive cells of α-SMA and VE-Cadherin double staining in the aorta were significantly reduced,and the expression level of α-SMA protein was significantly reduced,the expression level of VE-cadherin protein significantly increased(P＜0.05);The mRNA and protein expression levels of aortic transcription factor ZEB1 and TWIST were significantly reduced(P＜0.05);The expression levels of TGF-β1 and p-Smad2 proteins in the aorta were significantly reduced(P＜0.05),ApoA-Ⅰ in the liver,p-Akt1and p-β-cateninin the aortawere significantlyincreased(P＜0 05).Conclusion The Huayu Qutan formula can significantly improve EndMT in ApoE-/-AS mice,and its mechanism of action may be related to the regula-tion of ApoA-Ⅰ/Akt1/β-catenin/TGF-β Pathways,regulation of EndMT related transcription factors,and thus regulation of EndMT gene expression.
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