| Atherosclerosis(AS)is a highly prevalent disease in today’s society,and is the main cause of the biggest killer of human health-ischemic heart disease-which is mostly treated with statins,but long-term use is more damaging to the liver and kidneys.In contrast,some special dietary components have good ameliorative and palliative effects on many chronic diseases.Therefore,the study of dietary intervention in the development of atherosclerosis is of great significance to safeguard human health.Wine is a fruit wine made from grapes through fermentation,which has a low alcohol content and contains a variety of bioactive components such as tannins,pigments and organic acids.However,there are few published reports on the health-promoting effects of wine supported by data.Therefore,in this study,the main polyphenolic components in Cabernet Sauvignon dry red wine were firstly detected by LC-MS/MS and high performance liquid chromatography,and their intervention effects and key targets on atherosclerosis were analyzed based on network pharmacology;then animal models were used to elucidate the anti-atherosclerotic efficacy and mechanism of Cabernet Sauvignon dry red wine,and the details and results of the study are as follows:1.Analysis of atherosclerosis-interfering effects and mechanisms of Cabernet Sauvignon dry red wine(CSDRW)based on LC-MS/MS combined with network pharmacology.We used LC-MS/MS and high performance liquid chromatography to screen the polyphenols in Cabernet Sauvignon dry red wine(CSDRW),and analyzed the atherosclerosis-interfering effects and targets of polyphenols in CSDRW by network pharmacology to establish a theoretical basis for the anti-atherosclerotic efficacy of CSDRW.The results showed that:(1)Analysis of polyphenols in CSDRW using non-targeted metabolomics techniques revealed that it contained 834 polyphenols.20 polyphenols with high relative content in wine were selected by reviewing the literature and based on the high relative content in wine,and further quantified using high performance liquid chromatography.The highest levels of Catechin(87.58±1.52 mg/L),Proanthocyanidin B1(64.05± 0.17 mg/L),Epicatechin(47.27 ± 0.81 mg/L)and Gallic acid(64.95 ± 0.22 mg/L)were found in the CSDRW.(2)Using the drug target prediction platform,we obtained 4935 atherosclerosis-related targets and 480 polyphenol-acting targets,and took the intersection to obtain 317 common targets,based on which we constructed an analysis map of atherosclerosis and key gene interactions.Through GO and KEGG enrichment analysis,the inflammatory pathways and lipid metabolic pathways of CSDRW intervention in atherosclerosis were uncovered,among which the key targets to improve atherosclerosis mainly involved AKT,TLR4,ABCA1,TLR9,IL-6,TNF-α,etc.2.The intervention effect of Cabernet Sauvignon dry red wine on atherosclerosis.In this experiment,a model of atherosclerosis was established using ApoE-/-mice as experimental animals,and the ameliorative effects of different doses of Cabernet Sauvignon dry red wine(CSDRW)on atherosclerosis were investigated.Negative control,model control,low(3.1 ml/kgbw)and high(6.2 ml/kgbw)dose groups of CSDRW were set up in the experiment.ELISA kits were used to detect biochemical indexes such as blood lipids,liver and inflammatory factors in serum,oil red O staining was used to observe the area of lipid plaques in the inner wall of mouse aorta,and HE staining was used to observe the aortic tissue and liver tissue lesions in mice.The results showed that:(1)Effects on body weight and blood lipids:compared with the negative group,mice in the model group had significantly increased body weight(P<0.05),highly significant increase in TC,TG,LDL-C levels(P<0.01),highly significant decrease in HDL-C levels(P<0.01),and highly significant increase in AI levels(P<0.01);compared with the model group,mice in the low-dose and high-dose groups had significantly increased body weight(P<0.05),significantly decreased TC,TG,LDL-C levels(P<0.05),significantly increased HDL-C levels(P<0.01),and significantly increased AI levels(P<0.01).Compared with the model group,the mice in the low and high dose groups had significantly lower body weight(P<0.05),significantly lower TC,TG and LDL-C levels(P<0.05),significantly higher HDL-C levels(P<0.05),and significantly lower AI levels(P<0.01).It indicates that CSDRW can improve obesity and lipid metabolism disorders caused by high-fat diet.(2)Effects on aortic intimal lipid accumulation:The aortic intimal lipid accumulation was analyzed by oil red O staining.The results showed that the percentage of lipid plaque area in the inner wall of aorta was significantly increased in the model group mice compared with the negative group(P<0.01),while the mice in the low and high dose groups of CSDRW decreased by 0.85%and 41.81%,respectively,compared with the model group,and the difference was significant in the high dose group(P<0.05).It indicates that CSDRW can reduce aortic plaque area and improve atherosclerosis.(3)Effects on inflammatory factors:The detection of serum and liver inflammatory factors revealed that compared with the negative group,the levels of pro-inflammatory factors IL-1β,IL-6,iNOS and TNF-α were significantly increased(P<0.05),and the levels of anti-inflammatory factors IL-10 and eNOS were significantly decreased(P<0.05)in the model group;compared with the model group,the levels of IL-1β,IL-6,TNF-α and eNOS were significantly decreased(P<0.05)in the wine intervention group.and TNF-α and iNOS were significantly lower(P<0.05),and the anti-inflammatory factors IL-10 and eNOS were significantly higher(P<0.05).It indicates that CSDRW can reduce the inflammatory state of the organism and is beneficial in fighting against atherosclerosis.(4)Effects on liver oxidative stress:compared with the negative group,GSH-PX and SOD levels were significantly lower(P<0.05)and MDA levels were significantly higher(P<0.05)in the model group;compared with the model group,GSH-PX and SOD levels were significantly higher(P<0.05)and MDA levels were significantly lower(P<0.05)in the high-dose group.It indicates that the liver oxidative stress status due to high-fat diet can be significantly improved after wine intervention.(5)Effects on the histological structure of the aorta and liver:through the pathological histological study of the aorta and liver,it was found that the endothelial cells of the aortic vessels were intact in the negative group,and there were no other abnormalities in the orderly arrangement of the smooth muscle of the vascular intima;in the model group,the shed endothelial cells were seen,the intima was significantly thickened,and the lumen was significantly narrowed;through the CSDRW intervention,the intima of the aorta was significantly smoother compared with the model group,and the official lumen The endothelium was significantly thickened and the lumen was significantly narrowed.In addition,in the model group,the arrangement of liver cell cords was disturbed,the hepatic sinusoids were significantly narrowed,the cell morphology was abnormal and steatosis occurred,and the lipid droplet area of liver tissue was increased,but the high-dose group could significantly improve the related damage.It indicates that high dose of Cabernet Sauvignon dry red wine can improve the liver inflammation and steatosis caused by high-fat diet.(6)Effects on liver function:by analyzing AST and ALT in serum and liver,it was found that compared with the negative group,serum AST vitality was significantly increased(P<0.05),ALT was extremely significantly increased(P<0.01),and liver ALT and AST levels were significantly decreased(P<0.05)in the model group;compared with the model group,serum AST and ALT levels were extremely significantly Compared with the model group,the serum AST and ALT levels in the high-dose group were significantly lower(P<0.01),and the liver ALT and AST levels in the high-dose group were significantly higher(P<0.05).This indicates that the liver function of the atherosclerosis model mice was significantly damaged.CSDRW could reduce the serum AST and ALT levels and increase the liver ALT and AST levels,indicating that there was no damage to liver function in the high dose group of CSDRW.3.Mechanism of atherosclerosis intervention by Cabernet Sauvignon dry red wine.We used ApoE-/-mice as experimental animals to establish atherosclerosis model by high-fat diet,and used RT-qPCR,Western-Blot and immunofluorescence techniques to study the mRNA and protein expression of key genes in inflammation and lipid metabolism-related pathways in aorta and liver,to elucidate the molecular signaling pathways of atherosclerosis intervention by Cabernet Sauvignon dry red wine and reveal its The molecular mechanisms of these effects were revealed.Using 16S rDNA high-throughput sequencing technology,we analyzed the changes of intestinal microbiota in atherosclerosis model mice to decipher the relationship between intestinal flora and atherosclerosis after Cabernet Sauvignon dry red wine intervention.The results showed that:(1)Immunofluorescence technique was used to analyze key proteins MyD88,NF-κB,and iNOS on the aortic NF-κB pathway.results showed that.The immunofluorescence of MyD88 and NF-κB was highly significant(P<0.01)and that of iNOS was significantly higher(P<0.05)in the model group compared with the negative group;the immunofluorescence of MyD88,NF-κB,and iNOS was highly significant lower(P<0.01)in the high-dose group compared with the model group.It indicates that CSDRW exerts anti-inflammatory effects in vivo by regulating the regulation of NF-κB pathway and its upstream and downstream proteins on the aorta.(2)Western-Blot technique was used to study the protein expression of TLR4,PI3K,AKT,NF-κB,iNOS,TNF-α,and eNOS key genes on the liver NF-κB pathway.The results showed that compared with the negative group,the protein expression levels of TLR4 and PI3K were significantly higher(P<0.05),AKT,NF-κB,iNOS,TNF-α were highly significantly higher(P<0.01),and eNOS were highly significantly lower(P<0.01)in the liver of mice in the model group;compared with the model group,the high-dose group Compared with the model group,the protein expression levels of TLR4,PI3K,NF-κB,AKT,iNOS,and TNF-α were highly significantly decreased(P<0.01),and the protein expression levels of eNOS were highly significantly increased(P<0.01)in the high-dose group.It indicates that CSDRW can slow down the atherosclerotic process by reducing the inflammatory response in the liver.(3)RT-qPCR,Western-Blot and immunofluorescence techniques were used to study the mRNA and protein expression of key genes on the PPARγ-ABCA1-LXR-α pathway of hepatic lipid metabolism in CSDRW intervention in atherosclerosis.The results showed that the mRNA levels of PPARγ,ABCA1,and LXR-α were significantly lower(P<0.05)and the mRNA expression of SERBP-1c was significantly higher(P<0.05)in the liver of mice in the model group compared with the negative group;the mRNA levels of PPARγ,ABCA1,and LXR-α were significantly higher in the liver of mice in the high-dose group compared with the model group(P<0.05),and the mRNA expression of SERBP-1c was highly significantly decreased(P<0.01);WB analysis revealed that compared with the negative group,the liver protein expression of AMPK,PPARγ,ABCA1 was highly significantly decreased(P<0.01),the protein expression of LXR-α was significantly decreased(P<0.05),and the protein expression of SERBP-1c was significantly In the high-dose group,the protein expression of AMPK and ABCA1 was significantly higher(P<0.01),PPARγ was significantly lower(P<0.05)and LXR-α was significantly higher(P<0.05);immunofluorescence assay showed that compared with the negative group,the fluorescence expression of PPARγ,ABCA1 and LXR-α in the liver of the model mice was significantly lower(P<0.01).The fluorescence expression of PPARγ,ABCA1 and LXR-αin the liver of mice in the model group was highly significantly reduced compared with the negative group(P<0.01);the fluorescence expression of PPARγ,ABCA1 and LXR-α in the high-dose group was highly significantly increased compared with the model group(P<0.01).It indicates that CSDRW can regulate upstream and downstream proteins through regulating PPARγ-ABCA1-LXR-α pathway in the liver to alleviate liver lipid metabolism-related functions and histopathological damage.(4)Intestinal flora 16S rDNA sequencing revealed no significant difference inα-diversity and showed significant separation in β-diversity among the groups of animals(P<0.05).In terms of community composition at the phylum level,CSDRW could down-regulate the abundance of the phylum Bifidobacterium and Akkermania,significantly up-regulate Bifidobacterium(P<0.05),down-regulate Akkermansia(P<0.05),and significantly down-regulate the abundance of the phylum Wartybacter,Thick-walled Bacteria,Actinomycetes,Bifidobacterium and Akkermansia spp.compared to the model group(P<0.05,P<0.01).In terms of community composition at the genus level,Ackermannia spp.and unclassificd_f_Lachnospiraceae,were highly significantly lower(P<0.01),Bifidobacterium spp.were significantly higher(P<0.05),and Enterorhabdus was highly significantly higher(P<0.01)in the model group compared to the negative group.After CSDRW intervention,Ackermania spp.and unclassificd_f_Lachnospiraceae were significantly higher(P<0.05)and Bifidobacterium spp.and Enterorhabdus were significantly lower(P<0.05).Mathematical models were developed using the Spearman correlation coefficient method,and analysis revealed that three genera of Ackerman,Christensenellaceae_R-7 and Eubacterium_fissicatena were significantly negatively correlated(P<0.01)with the percentage of aortic intimal plaque area,TC,TG,LDL-C,and significantly correlated with HDL-C positive correlation(P<0.01).It is suggested that CSDRW may intervene in the development process of atherosclerosis by remodeling the intestinal flora.Conclusion:CSDRW can effectively improve blood lipids and aortic intimal lipid accumulation and alleviate hepatic steatosis in animals with atherosclerosis model.The mechanism of action may be through regulating the expression of key genes such as TLR4,NF-κB and TNF-α in the NF-κB pathway,and PPARγ,ABCA1 and LXR-α in the PPARγ-ABCAl-LXR-α signaling pathway to remodel the intestinal flora of model mice,especially upregulating the expression of Akkermansia,Christensenellaceae_R-7 and Eubacterium_fissicatena genera to control atherosclerosis formation. |
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