| objective The effect of IL-37 on human aortic vascular smooth muscle cells(HA-SMCs)was investigated by screening the optimal concentration of ox-LDL to create a calcification model and verifying the effect of IL-37 intervention on the calcification model.To provide some theoretical basis for the treatment and prognosis of vascular calcification(VC)and atherosclerosis(AS).methods1.Screening for optimal concentration to induce calcification in HA-SMCs: HA-SMCs were cultured in vitro and divided into blank group,(10,25,50,100)μg/m L treated ox-LDL group.cck8 method was used to detect cell proliferation activity.qpcr and ELISA,WB were used to detect smooth muscle actin(SMA),smooth muscle 22 α(SM22α),bone morphogenetic The expression of smooth muscle actin(SMA),smooth muscle 22 α(SM22 α),bone morphogenetic protein(BMP2),runx-associated transcription factor(RUNX2),OC,alkaline phosphatase(ALP),and IL-37 were detected by qpcr and ELISA and WB,respectively,and the model was validated by alizarin red calcification staining.2.HA-SMCs were divided into control group,ox-LDL-treated group,and IL-37(2h)+ox-LDL-treated group,and after 24 hours of starvation,the control group was changed to complete medium and the treated group was changed to calcified medium(10mmol/L β-glycerophosphate + 50 μ g/m L L-ascorbic acid + 100 nmol/L dexamethasone),and the cells were induced for 3 and 14 days.The expression of TLR4,NF-κB,osteogenesis-related factor MSX2,ALP,and RUNX2 were detected by qpcr and WB,the cell proliferation rate was detected by cck8,the apoptosis rate was detected by flow cytometry,and the degree of cell calcification was verified by alizarin red staining.3.HA-SMCs were divided into control group,ox-LDL treated group,IL-37(2h)+ox-LDL treated group,NF-κB inhibitor PDTC(2h)+ ox-LDL treated group,cells were starved for 24 h after passaging,control group was replaced with complete medium,treated group with calcified medium,(10mmol/L β-glycerophosphate + 50μg/m L Lascorbic acid + 100nmol/L dexamethasone),qpcr,western blot to detect the expression levels of TLR,NF-κ B,phosphorylated nuclear transcription factor(p-NF-κ B),RUNX2,MSX2,ALP,SMA,SM22α.results1.Gradient concentrations of ox-LDL(10,25,50,100 μ g/m L)were used to treat HA-SMCs for 3 days.WB results showed that the expression of IL-37 was highest in the 50 μg/m L ox-LDL-treated group.qpcr results showed that after 1,7 and 14 days of gradient concentrations of ox-LDL-treated cells,compared with the control group,the SMA,SM22α Expression was significantly decreased,with the lowest expression in the 50 μg/m L ox-LDL-treated group,and the difference was statistically significant.Therefore,50 μg/m L ox-LDL was selected as the modeling intervention concentration.qpcr results showed that the expression of BMP2 and RUNX2 was significantly higher in the supernatant after 3 days of treatment of cells with 50 μg/m L concentration of ox-LDL compared with the control group.elisa results showed that the expression of IL-37 was increased in the supernatant after 3 days of treatment of cells with 50 μg/m L concentration of ox-LDL.IL-37 expression was increased.ALP activity in the supernatant was detected by the ALP kit,suggesting that ALP activity in the supernatant of the ox-LDL-treated group was decreased,and the difference was statistically significant.2.HA-SMCs were divided into control and calcification modeling groups.After 24 hours of cell starvation,the control group was changed to complete medium and the modeling group was changed to calcification medium(10 mmol/L β-glycerophosphate+ 50 μg/m L L-ascorbic acid + 100 nmol/L dexamethasone)and 50 μg/m L ox-LDL was added to induce the cells for 14 days,and alizarin red staining was performed.The results showed that the calcification model was successfully established by the appearance of orange-red calcified nodules in the calcification model group compared with the control group.qpcr,ELISA and WB results suggested that the expression of IL-37 in both inside and outside cells was significantly higher in the calcification model group compared with the control group,and the expression of bone-related factors in the model group was also increased.3.HA-SMCs were divided into control group,ox-LDL-treated group,and IL-37(2h)+ox-LDL-treated group.After 24 hours of starvation,the control group was changed to complete medium,and the treated group was changed to calcified medium(10 mmol/Lβ-glycerophosphate + 50 μg/m L L-ascorbic acid + 100 nmol/L dexamethasone).3days after induction of cells,qpcr results showed that the expression of TLR4,NF-κB,RUNX2,MSX2,and ALP was significantly higher in the ox-LDL-treated group compared with the control group;compared with the ox-LDL-treated group,NF-κB,RUNX2,MSX2,and ALP were significantly lower in the IL-37(2h)+ ox-LDL-treated group,while lower than the control group.WB results showed that compared with the control group,the NF-κB expression was significantly higher in the ox-LDL-treated group,and TLR2 and RUNX2 were also increased at the same time,and the expression of the corresponding factors decreased after adding IL-37 treatment.4.Alizarin red staining showed a significant increase in calcified nodules in the ox-LDL-treated group compared with the control group,while the number of nodules in the IL-37(2h)+ox-LDL-treated group was significantly reduced after the addition of IL-37 treatment compared with the ox-LDL-treated group.The protective effect of IL-37 on HA-SMCs was clearly established,and increased pretreatment with IL-37 was able to significantly reduce the formation of calcified nodules.Compared with the blank group,(10,25,50,100)μg/m L ox-LDL treatment,SMA expression was the lowest in the 50 μg/m L concentration ox-LDL group,and BMP2 showed high expression,and the difference was statistically significant.Compared with the control group,the modeling group showed high expression of IL-37.(10,25,50,100)μg/m L ox-LDL treatment significantly increased cell proliferation activity in a concentration-and time-dependent manner,and cell proliferation activity was inhibited by adding 100ng/m L IL-37 pretreatment in the 50 μg/m L ox-LDL group.5.HA-SMCs were divided into control group,ox-LDL-treated group,NF-κB inhibitor PDTC(2h)+ ox-LDL-treated group,and cells were starved for 24 h after passaging,and the control group was replaced with complete medium and the treated group with calcified medium,(10mmol/L β-glycerophosphate + 50 μ g/m L L-ascorbic acid +100nmol/L dexamethasone).After 3 days of treatment,qpcr results showed that the bone transcription-related factors RUNX2,ALP,MSX2,and OC were significantly elevated in the ox-LDL-treated group compared with the control group.NF-κB was significantly inhibited and RUNX2 expression was decreased in the NF-κB inhibitor PDTC(2h)+ ox-LDL treated group compared to the ox-LDL treated group.WB results showed that the corresponding protein expression was consistent with the RNA expression trend of PCR results.It was confirmed that the expression of osteogenic-related factors was suppressed when the level of inflammatory factors was reduced,and the two were correlated.6.HA-SMCs were divided into control group,gradient concentration ox-LDL treatment group(10,25,50,100 μ g/m L),IL-37(2h)+ ox-LDL treatment group,and the expression of phosphorylated NF-κB was detected by Western blot,and the results showed that the phosphorylation level of NF-κ B was increased in a concentration-dependent manner after gradient concentration ox-LDL treatment,the phosphorylation level was strongest at 50 μg/m L,and the phosphorylation level was suppressed after increasing IL-37 pretreatment,and the difference was statistically significant.The expression of TLR4,NF-κB,and RUNX2 was significantly higher in the ox-LDL-treated group compared with the control group,and after adding IL-37 treatment,the expression of the corresponding factors was significantly decreased in the ox-LDL-treated group compared with the ox-LDL-treated group,the IL-37(2h)+ox-LDL-treated group,and the NF-κ B inhibitor PDTC(2h)+ox-LDL-treated group compared with the IL-37(2h)+ ox-LDL-treated group had the same trend,but the inhibitory effect of IL-37 was better than that of PDTC.qpcr results suggested that the expression of SMA,SM22α was significantly decreased in the ox-LDL-treated group compared with the control group,and after increasing IL-37,PDTC treatment,compared with the ox-LDL-treated group,the expression of SMA,SM22 α was significantly increased in the IL-37(2h)+ ox-LDL-treated group.SM22α expression was significantly increased,while SMA expression was increased while SM22 αexpression was decreased in the NF-κB inhibitor PDTC(2h)+ox-LDL-treated group compared with the ox-LDL-treated group.Therefore,we suggest that IL-37 inhibits inflammatory pathways while suppressing osteogenic factor expression,which affects cell phenotypic transformation and exerts a protective effect against calcification.conclusionIn conclusion,we suggest that IL-37 can inhibit HA-SMCs osteogenic transformation by inhibiting TLR expression,inhibiting NF-κB phosphorylation,inhibiting the expression of downstream osteogenic transcription-related factors,and thus inhibiting HA-SMCs calcification,and for the protective effect of vascular calcification,IL-37 may serve as a novel target,providing some theoretical basis for vascular calcification and AS treatment and prognosis. |
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