Effects Of Yiqi Huoxue Recipe And Yiqi Huoxue Yangyin Recipe On Ang Ⅱ-Induced Senescence Of Human Umbilical Vein Endothelial Cells Based On PPAR γ/NF-κB Pathway

The occurrence and development of most cardiovascular diseases are accompanied by the phenomenon of vascular aging.Delaying vascular aging is one of the important strategies to prevent and treat cardiovascular diseases.Vascular endothelial cell senescence is the initial link of vascular aging and plays an important role in the occurrence and development of vascular aging.Therefore,starting from the molecular mechanism of endothelial cell aging,studying the changes of vascular aging has always been a research focus of the relationship between cardiovascular disease and aging,which is of great significance for the prevention and treatment of cardiovascular diseases.Hypertension is one of the common cardiovascular diseases,and its occurrence and development are accompanied by vascular aging and vascular remodeling.Therefore,hypertension can be used as a disease model of vascular aging for aging research.The activation of the Renin-Angiotensin-Aldosterone System(RAAS)is closely related to the occurrence of hypertension,and its main componentAngiotensin II(Ang II),can induce vascular endothelial cell senescence and increase inflammation factor expression,causing vascular damage.Peroxisome proliferatoractivated receptor gamma(PPAR γ)inhibits factors such as cytokines,chemokines,matrix metalloproteinases and adhesion molecules which can improve the inflammatory response of blood vessels and may affect the occurrence of vascular aging in the process of hypertension.Therefore,starting from the PPARγ/NF-κB inflammatory pathway,studying the changes of Ang II-induced vascular endothelial cell senescence in the process of vascular aging,and using traditional Chinese medicine to intervene and treat it,is of great significance for the prevention and treatment of hypertension.The previous study of the research group confirmed that Yiqi Huoxue Recipe could delay Ang II-induced endothelial cell aging and vascular aging in naturally aging rats by activating the PPAR γ pathway,and Yiqi Huoxue Yangyin Recipe could delay Dgal-induced vascular aging in mice.In this study,the Ang II-induced aging model of HUVECs was constructed,and the PPAR γ/NF-κB inflammatory pathway was used as the breakthrough point to explore the intervention effects of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe on Ang Ⅱ-induced vascular endothelial cell aging.The mechanism of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe in delaying vascular aging was elucidated from the cellular level.Study 1 Intervention of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe on HUVECs senescence induced by Ang ⅡObjective:To construct an aging model of HUVECs induced by Ang Ⅱ,and to observe the effects of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe on vascular endothelial cell senescence.Methods:By intervening HUVECs with different concentrations of Ang Ⅱ for 48 h,the experiments were divided into 8 groups:blank group,model group,10-4 mol/L,10-5 mol/L,10-6 mol/L,10-7 mol/L to determine the optimal concentration of Ang II to induce senescence.Senescence-associated-β-galactosidase(SA-β-gal)fluorescence staining was used to compare the fluorescence values,cell cycle analysis was used to observe the proportion of cells in each phase,and Western blot was used to detect the expression of p16 protein condition.After determining the optimal concentration for inducing senescence,different concentrations of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe were used to intervene the cells 12 h in advance,and then treated with Ang Ⅱ for 48 h at the same time,and the medium was changed every 12 h.β-gal fluorescence staining method compares the changes of fluorescence values to determine the appropriate concentration of the two parties to delay aging.Cell senescence was induced by 10-6 mol/L Ang Ⅱ,and 100 mg/L was used as the intervention concentration of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe,and divided into blank group,model group,RSC group,and only RSC group(without adding modeling drugs),RSCS group,RSCS only group(without modeling drugs),telmisartan group,DMSO group.The degree of cell senescence was detected by SA-β-gal chemical staining and fluorescent staining.The proportion of cells in each phase was determined by cell cycle analysis.The expression of p16 and p53 proteins was detected by immunofluorescence and Western blot.Results:(1)Compared with the blank group,the 10-6 mo/L Ang Ⅱ group showed senescent morphological changes under the light microscope,the fluorescence value of SA-β-gal staining increased(p<0.001),and the cell cycle was arrested at G0/G1 The proportion increased,the proportion of cells in S phase decreased,and the proportion of p16 protein increased.Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe group could reduce the fluorescence value of SA-β-gal staining(p<0.05,p<0.01);(2)In the formal experiment,compared with the blank group,the SA-β-gal blue staining rate and fluorescence value of cells in the model group increased,the proportion of cells in G0/G1 phase increased,and the proportion of cells in S phase decreased(p<0.001),p16 protein fluorescence expression increased(p<0.05),p53 protein fluorescence expression increased(p<0.01),p16 protein content expression increased,p53 protein content expression increased(p<0.001);Compared with the model group,the SA-β-gal blue staining rate and fluorescence intensity decreased in the RSC group,the proportion of cells in the G0/G1 phase of cell cycle decreased,and the proportion of cells in the S phase increased(p<0.001).The fluorescence values of p16 and p53 decreased(p<0.01).and the protein contents of p16 and p53 decreased.The fluorescence value of p53 decreased,the protein content of p16 decreased(p<0.001),and the protein content of p53 decreased(p<0.01).Conclusion:10-6 mol/L Ang Ⅱ stimulation for 48 h can accelerate the aging process of HUVECs,and 100 mg/L Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe can delay the aging of HUVECs.Study 2 Effects of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe on AngⅡ-induced HUVECs senescence via PPAR γ/NF-κB inflammatory pathwayObjective:To observe the effects of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe on PPAR γ/NF-κB inflammatory pathway in vascular endothelial cell senescence,to track the changes of SASP,and explore the possible mechanism of the two prescriptions in inhibiting cell senescence.Methods:10-6 mol/L Ang Ⅱ was used to induce senescence in cells for 48 hours.The experiment was divided into seven groups:blank group,model group,RSC group,RSC only group,RSCS group,RSCS only group and Telm group.Liquid-phase protein chip technology and Elisa method were used to determine the content of SASP factors,immunofluorescence method was used to detect the protein fluorescence expression of NF-κB p65,PPAR γ and AT1R,and Western blot method was used to detect the changes of NF-κB p65,p-NF-κB p65 and PPAR γ,AT1R protein content changes.Results:(1)Compared with the blank group,the contents of TGF-β1 and IL-6 in the model group increased(p<0.01),and the contents of CCL-2 and IL-8 increased(p<0.001);The expression of NF-κB p65 fluorescence increased(p<0.05),the fluorescence expression of PPARy decreased,and the fluorescence expression of AT1R increased(p<0.001);the protein contents of NF-κB p65,p-NF-κB p65 and AT1R increased(p<0.001),PPAR y protein content decreased(p<0.05);(2)Compared with the model group,the content of TGF-β1 in RSC group decreased(p<0.05),the content of IL-6 and CCL-2 decreased,and the content of MMP-3 and IL-8 increased(p<0.001),NF-κB p65 fluorescence decreased(p<0.01),PPARγfluorescence expression increased.AT1R fluorescence expression decreased(p<0.001).The expression of NF-κB p65,p-NF-κB p65,AT1R protein content decreased),and the content of PPARγ increased(p<0.001);the content of TGF-β1 decreased(p<0.05),the content of IL-6 and CCL-2 decreased,the content of IL-8 increased,the fluorescence expression of NF-κB p65 and AT1R decreased,and the fluorescence expression of PPARγ increased(p<0.001).Conclusion:100 mg/L Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe can delay vascular inflammatory endothelial cells by intervening PPAR γ/NF-κB pathway and inhibiting the expression of CCL-2,IL-6 and TGF-β1 factors.Experiment 3 Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe regulate NFκB inflammatory pathway and inhibit the senescence of HU VECs induced by AngⅡObjective:After silencing NF-κB p65 gene,to observe the changes of Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe in delaying vascular endothelial cell senescence,and to explore the mechanism of their delaying cell senescence.Methods:Adenovirus infection was used to reduce the expression of NF-κB p65 gene in cells.Fluorescence microscope photography and PCR technology were used to screen the optimal gene silencing time and MOI value.After successful gene silencing,10-6 mol/L Ang Ⅱ was used.Intervening cells for 48 hours to induce senescence,100 mg/L Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe intervened in cells.The experiment was divided into empty virus group,model group,model+NF-κB p65 silence group,RSC group,RSC+NF-κB p65 silence group,RSCS group,RSCS+NFκB p65 silence group,Telm group,Telm+NF-κB p65 silence group.The changes of p53 protein content were detected by Western blot.Results:(1)After adenovirus solution with different MOI intervened in HUVECs,the expression of fluorescence intensity was significantly enhanced at 48 h.When the MOI was 100,the fluorescence intensity of the shRNA2 and shRNA3 groups was significantly stronger than that of the empty virus group and shRNAl group at 48h,compared with the empty virus group,the expression of NF-κB p65 mRNA in the shRNA2 group decreased(p<0.001);(2)In the formal experiment,compared with the empty virus group,the protein content of NF-κB p65 in the model group increased(p<0.001);compared with the model group,the protein content of NF-κB p65 in the RSC group decreased(p<0.001),decreased in RSCS group(p<0.01);compared with RSC group,NF-κB p65 protein content in RSC+NF-κB p65 silence group was down-regulated(p<0.01),compared with RSCS group,the protein content of NF-κB p65 in the RSCS+NF-κB p65 silenced group was down-regulated(p<0.05),and compared with the Telm group,the protein content of NF-κB p65 in the Telm+NF-κB p65 silenced group was down-regulated(p<0.01).Compared with the empty virus group,the p53 protein content in the model group increased(p<0.05);compared with the model group,the p53 protein content in each group decreased,and the Telm group had a significant difference(p<0.01),and the other groups had significant differences(p<0.01).Compared with the RSC group,the protein content of p53 in the RSC+NF-κB p65 silence group was down-regulated(p<0.001),and compared with the RSCS group,the p53 in the RSCS+NF-κB p65 silence group was significantly different(p<0.001).Compared with the Telm group,the p5 3 protein content in the Telm+NF-κB p65 silence group was down-regulated(p<0.001).Conclusion:Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe can delay the senescence of HUVECs induced by Ang Ⅱ by inhibiting the NF-κB signaling pathway and down-regulating the expression of p53 protein.To sum up,this study used the Ang Ⅱ-induced aging model of HUVECs to prove that Yiqi Huoxue Recipe and Yiqi Huoxue Yangyin Recipe could delay endothelial cell aging through the PPAR y/NF-κB inflammatory signaling pathway.The mechanism of YiqiHuoxue Recipe and Yiqi Huoxue Yangyin Recipe in delaying vascular aging in the process of hypertension provides cytological evidence.