Study On The Effect Of Hypoxia And High Salt Co-Intervention On HA-VSMC And The Possible Mechanisms

Study objectives:（1）To investigate the effects of combined hypoxia and high salt intervention on the biological behavior of HA-VSMC and oxidative stress.（2）To investigate the effects of hypoxia and high salt interventions on HA-VSMC and the potential molecular mechanisms.Research methods:（1）Determination of salt and cobalt chloride concentrations,dosing amounts and duration of action.Primary cultured human aortic smooth muscle cells were used and passaged to the5th generation,and the high salt intervention experiment was divided into control group（132 mmol/L Na⁺）,medium concentration salt group（137 and 142 mmol/L Na⁺）,and high concentration salt group（147,152,157,162 and 167 mmol/L Na⁺）;hypoxia+high salt intervention experiment was divided into control group（132 mmol/L Na⁺）and the low oxygen+high salt group（152 mmol/L Na⁺+50,100,200,400 and 800μmol/L Co Cl₂）were incubated for 24h,48h,72h and 96h,respectively,and the HA-VSMC viability was measured by CCK-8 method and the proliferation rate（PR）was calculated.).（2）Effect of hypoxia and high salt intervention on the biological behavior of HA-VSMC and oxidative stress.The concentrations and doses of high salt and cobalt chloride were determined from（1）.HA-VSMC were de-synchronized for 24 h and divided into control（132mmol/L Na⁺）,hypoxic（200μmol/L Co Cl₂）,high salt（152 mmol/L Na⁺）,hypoxic+high salt（152 mmol/L Na⁺+200μmol/L Co Cl₂）groups.L Co Cl₂)were incubated for72 hours,and then the cells were collected for subsequent experiments:the scratch as-say and Transwell assay were selected to detect the migration ability of HA-VSMC;the colorimetric method of enzyme standard was selected to determine the content of SOD,MDA,ALP and calcium ions in HA-VSMC.3)Effects of hypoxia and high salt intervention on HA-VSMC and potential molecular mechanisms.The expression levels of OPN,Ang II and ET-1 proteins in HA-VSMC were de-tected using enzyme-linked immunosorbent assay;the m RNA levels of HIF-1α,Ang II,PCNA,ET-1,AT1R,OPN,SM22,α-SMA,type I collagen and type III collagen in HA-VSMC were analyzed using real-time fluorescence quantitative PCR technique for rel-ative expression;protein immunoblotting assay was used to detect the expression of HIF-1α,Ang II,OPN,SM22 and type I collagen in HA-VSMC.Results and analysis:（1）Determination of salt and cobalt chloride concentrations,dosing amounts and duration of action.The results of CCK-8 method showed that HA-VSMC proliferation was active at137-167 mmol/L Na⁺intervention for 72h compared to 132 mmol/L Na⁺,with the strongest pro-proliferative effect at 152 mmol/L Na⁺concentration.In the hypoxic group,compared with the control group,50-800μmol/L Co Cl₂ promoted HA-VSMC proliferation,with 200μmol/L In the low oxygen group,compared with the control group,50-800μmol/L Co Cl₂ could promote HA-VSMC proliferation,among which200μmol/L Co Cl₂ had the most significant pro-proliferation effect;compared with the control group,152 mmol/L Na⁺+200μmol/L Co Cl₂ also had the strongest pro-prolifer-ation effect in the low oxygen+high salt group.2)Effect of co-intervention of hypoxia and high salt on biological behavior of HA-VSMC and oxidative stress.According to the results of scratch assay and Transwell assay,the migration ability of HA-VSMC was significantly enhanced in the hypoxic group,high salt group and hypoxic+high salt group compared with the control group;in the colorimetric determi-nation of SOD,MDA,ALP and calcium ions in HA-VSMC by enzyme marker,com-pared with the control group,the expression of MDA,ALP and Calcium ion expression was upregulated and SOD activity was decreased in the hypoxic,high salt and hy-poxic+high salt groups compared to the control group.3)Effects of hypoxia and high salt interventions on HA-VSMC and potential molecular mechanisms.The expression of OPN,Ang II and ET-1 in HA-VSMC was measured by ELISA,and the expression of MDA,ALP and calcium ions was up-regulated and SOD activity was decreased in the hypoxia,high salt and hypoxia+high salt groups compared with the control group.1,AT1R,OPN,SM22,α-SMA,type I collagen and type III collagen m RNA levels were analyzed by real-time fluorescence PCR.Compared with the control group,the relative expression of HIF-1α,Ang II,PCNA,ET-1,AT1R,OPN,type I collagen and type III collagen m RNA levels were significantly increased in the hypoxic,high-salt and hypoxic+high-salt groups,while the relative expression of SM22 andα-SMA m RNA was decreased.The expression of HIF-1α,Ang II,OPN,SM22 and type I collagen in HA-VSMC was examined by protein immunoblotting assay.Compared with the control group,the expression of HIF-1α,Ang II,OPN and type I collagen were significantly increased and the expression of SM22 was decreased in the hypoxic,high-salt and hypoxic+high-salt groups,and the expression of each protein was basically consistent with the m RNA level.Conclusion:This study demonstrated that hypoxia,high salt,and combined hypoxia and high salt interventions could lead to HA-VSMC proliferation,migration,calcification,and phenotypic transformation.The co-intervention of hypoxia and high salt promoted up-regulation of HIF-1α,Ang II,and OPN protein expression,which may be related to HIF-1αsignaling pathway and RAAS activation.