| Objective: Arterial calcification（AC）,a common pathological process in a variety of diseases,is a type of ectopic calcification,and the regulation is active,driven by vascular smooth muscle cells（VSMCs）,to the transdifferentiation of osteoblast-like cell phenotype.Previous studies have shown that L-carnitine（LC）is a key drug for antioxidant damage,which can protect cells from oxidative damage.Whether L-carnitine,an antioxidant,can inhibit the osteoblast-like differentiation of VSMCs byβ-glycerophosphate（β-GP）requires further investigation.Methods:（1）α-smooth muscle actin（α-SMA）is a characteristic protein of VSMCs,and α-SMA was identified by immunofluorescence,and then the purchased cells were verified to be VSMCs.（2）10 mmol/L of β-GP was used to induce osteoblast-like differentiation of cells,so as to construct a mouse calcified cell model,different concentrations（100,200,400,600,800μmol/L）of L-carnitine were applied to mouse VSMCs cultured in vitro,the viability of the cells was measured by CCK8 assay,and appropriate low,medium,and high concentrations（200,400,800μmol/L）of L-carnitine were selected as subsequent experimental treatment concentrations.（3）The subsequent experiments were divided into five groups: control group,calcification（β-GP）group,and low,medium,and high concentration L-carnitine（LC+β-GP）groups.The success of calcification was verified by alizarin red staining,and the degree of cell calcification was assessed by the area of calcified nodules.（4）Reactive oxygen species kits were used to detect five groups of cells and verify reactive oxygen species（ROS）levels by microscopic mean fluorescence intensity.（5）The m RNA expression of Run related transcription factor 2（RUNX2）,bone morphology protein-2（BMP-2）,nicotin adenine dinucleotide phosphate oxidases（NOX4）and catalase（CAT）were determined by q PCR.Results:（1）Green fluorescent muscle filaments arranged in the cytoplasm were observed by immunofluorescence（IF）,which was verified as α-SMA and further demonstrated as VSMCs.（2）Treatment of mice VSMCs with different concentrations（100,200,400,600 and 800μmol/L）of L-carnitine and 10mmol/L β-GP for 24 h,48h,72 h had no significant effect on cell viability.（3）Alizarin red S staining results showed that: 5 mineralized nodules could be observed,and the nodules were most obvious in the calcified group,and orange mineralized nodules were significantly reduced in the L-carnitine all drug-treated groups compared with the calcified group,and the difference was statistically significant（P < 0.05）.（4）The results of ROS detection showed that ROS generation was significantly enhanced in the calcification group compared with the control group,and the difference was statistically significant（P < 0.05）.ROS generation was significantly reduced in the L-carnitine all drug-treated groups compared with the calcification group,and the difference was statistically significant（P < 0.05）.ROS generation was significantly reduced in the L-carnitine all drug-treated groups compared with the calcification group,and the difference was statistically significant（P < 0.05）.（5）The results of q PCR showed that the m RNA expression of all indicators in the calcification group was significantly enhanced compared with the control group,and the difference was statistically significant（P < 0.05）.With the increase of L-carnitine drug treatment concentration,the contents of the measured m RNAs of the four indicators decreased.Compared with the calcification group,the m RNA expressions of RUNX2,BMP-2 and NOX4,CAT were significantly reduced in the medium and high concentrations of L-carnitine,and the differences were statistically significant（P < 0.05）.Conclusion: L-carnitine significantly inhibited the osteoblast-like differentiation of mouse VSMCs,and the mechanism may be related to the oxidative stress pathway.And within a certain range,the degree of osteoblast-like differentiation of mouse VSMCs decreased with increasing L-carnitine concentration. |
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