| Objective: To observe the correlation between the expression level of mi R-148a-3p and the concentration of interleukin 1β(IL-1β)in chondrocytes(CC),and to further explore the effects of mi R-148a-3p on the secretion of inflammatory cytokines,apoptosis and chondrocyte phenotype in chondrocytes induced by IL-1β.Method:1.Rabbit chondrocytes isolated in vitro were observed by fluorescence inverted microscope and identified by toluidine blue staining.2.0ng/ml,5ng/ml,10ng/ml,and 20ng/ml IL-1β cytokines were used to intervene the P2 generation chondrocytes,respectively,and intracellular RNA was extracted 24 hours later for q RT-PCR detection to explore the relationship between the expression level of mi R-148a-3p and IL-1β concentration.3.P2-generation chondrocytes were treated with 10ng/ml IL-1β cytokine for 24 h.The m RNA and protein expressions of matrix metalloproteinase-13(MMP-13),type II collagen(COL-II)and proteoglycan(ACAN)were detected by extracting RNA and protein from chondrocytes.The inflammatory changes were induced in chondrocytes.4.P2-generation chondrocytes in good condition with a growth density of 30-50% were treated with mi R-148a-3p mimic,inhibitor and negative control(NC)respectively using Lipofectamine 2000.(All mi R-148a-3p related products were transfected with red fluorescent modifier).After setting different transfection concentration gradients and time gradients,cell RNA was extracted for q RT-PCR detection to explore the optimal transfection conditions for subsequent mi R-148a-3p.5.P2-generation chondrocytes were implanted with mi R-148a-3p mimic,inhibitor and NC after the cell density reached 70% and were treated with 10ng/ml IL-1β for 24 h.The concentration of interleukin 6(IL-6)and interleukin 10(IL-10)in the supernatant was detected by ELISA.The expressions of Bcl-2,Bax,Caspase-3,MMP-13,ACAN,Col-II and SOX9 were detected by Western-blot.Results:1.Primary chondrocytes isolated and cultured began to spread at 48 h,showing spindle,triangle,or polygonal shape;4-5 days like "paving stones" shape;The surrounding cell matrix showed obvious refraction.Toluidine blue staining shows a blue-purple cytoplasm stain.2.Compared with the control group,the expression level of mi R-148a-3p in chondrocytes in 5,10 and 20ng/ml intervention groups was significantly increased(P<0.01).3.Compared with the control group,m RNA relative expression level of MMP-13 was significantly increased(P<0.01),COL-Ⅱ was significantly decreased(P<0.01),and ACAN expression was significantly decreased(P<0.05)in 10ng/ml IL-1β intervention group.The relative protein quantification showed that the expression of MMP-13 was increased(P<0.05),while the expressions of COL-Ⅱ and ACAN were significantly decreased(P< 0.01).4.With the same transfection concentration,the expression of mi R-148a-3p increased with the extension of transfection time;At the same transfection time,the expression level of mi R-148a-3p reached the plateau at the transfection concentration of 50 n M.The optimal transfection concentration of mi R-148a-3p mimics was 50 n M,the optimal transfection concentration of inhibitors was 100 n M,and the optimal transfection time was 72 hours.5.Compared with NC control group,IL-6 level of IL-1β+mimic group was significantly increased(P<0.01),while IL-6 level of IL-1β+inhibitor group was decreased(P<0.05).IL-10 levels of IL-1β+mimic group were significantly decreased(P<0.01),but IL-10 levels of IL-1β+inhibitor group were not different.The expressions of IL-6 and IL-10 were significantly different between IL-1β+mimic group and IL-1β+inhibitor group(P<0.01).6.Compared with NC control group,Bax and Caspase-3 protein expressions of IL-1β+mimic group were increased(P<0.05),while Bax and Caspase-3 protein expressions of IL-1β+inhibitor group were not different.Bcl-2 protein expression of IL-1β+mimic group was decreased(P<0.05),while Bcl-2 protein expression of IL-1β+inhibitor group was increased(P<0.05).The protein expressions of Bax,Caspase-3 and Bcl-2 were significantly different between IL-1β+mimic group and IL-1β+inhibitor group(P<0.01).7.Compared with the NC control group,m RNA relative expression levels showed that MMP-13 expression of IL-1β+mimic group was significantly increased(P<0.01),while COLII,ACAN and SOX9 expression were significantly decreased(P<0.01).The expression of MMP-13 was decreased in IL-1β+inhibitor group(P<0.05),but the expressions of COL-II,ACAN and SOX9 were not different.The expressions of MMP-13,COL-II,ACAN and SOX9 were significantly different between IL-1β+mimic group and IL-1β+inhibitor group(P<0.01).8.Compared with the NC control group,the relative protein quantitative showed that the expression of MMP-13 in IL-1β+mimic group was significantly increased(P<0.01),and the expressions of COL-II,ACAN and SOX9 were decreased(P<0.05).The expression of MMP-13 was decreased in IL-1β+inhibitor group(P<0.05),the expression of COL-II was increased(P<0.05),the expressions of ACAN and SOX9 were significantly increased(P<0.01).The expressions of MMP-13,COL-II,ACAN and SOX9 were significantly different between IL-1β+mimic group and IL-1β+inhibitor group(P<0.01).Conclusions: 1.The expression of mi R-148a-3p in chondrocytes has a dose-dependent relationship with the concentration of IL-1β,and may be regulated by IL-1β.2.Inhibition of mi R-148a-3p in chondrocytes in vitro alleviates inflammatory changes caused by IL-1β,partially inhibits apoptosis,maintains chondrocyte phenotype,and alleviates extracellular matrix(ECM)degradation. |
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