Regulation Of TLR4and Inflammatory Cytokines IL-6, IL-8and MCP-1by Oleic Acid And1,25（OH）₂D₃in Human Vascular Smooth Muscle Cells

Objective:To investigate the regulation of TLR4and its downstream inflammatory cytokines by oleic acid in human aortic vascular smooth muscle cells.Methods:Human aortic vascular smooth muscle cells were treated with different doses of oleic acid for different time. The empty oleic acid was used to be control group, and then the LPS were used to be positive control. MMT methods were used to analyze the proliferation ability of human aortic vascular smooth muscle cells. TLR4, IL-6, IL-8and MCP-1mRNA expressions were detected by quantitative real-time PCR. TLR4, IL-6, IL-8and MCP-1protein levels were measured by western blot and ELISA.Results:Oleic acid induced the proliferation ability of human aortic vascular smooth muscle cells, but the proliferation ability of cells were inhibited with the rising of dose. The mRNA and protein expression of TLR4, IL-6, IL-8and MCP-1in HA-VSMC were upregulated by oleic acid with50μmol/L～200μmol/L in a time and dose dependent manner. The mRNA and protein expression levels of TLR4, IL-6, IL-8and MCP-1were all significantly enhanced after treated by200μmol/L oleic acid for24h. TLR4, IL-6, IL-8and MCP-1mRNA expression were respectively upregulated by6.5fold,7.4fold,7.4fold and7.1fold as compared with the control group (all P<0.05) by quantitative real-time PCR. Western blot analysis showed that the protein levels of TLR4(1.707±0.62) were markedly augmented by200μmol/L oleic acid than the control group (0.29±0.22, P<0.05), and the levels of IL-6, IL-8and MCP-1proteins were maximumly upregulated by2.16fold,1.93fold and2.06fold respectively in comparison with that of control group (all P<0.05) with studying by ELISA. Conclusion:Oleic acid regulated the proliferation ability of human aortic vascular smooth muscle cells in a time and dose dependent manner. Oleic acid induced the expression of TLR4mRNA and proteins in human aortic vascular smooth muscle cells. Oleic acid enhanced the levels of inflammatory cytokines IL-6, IL-8, MCP-1mRNA and proteins. Objective:To investigate the regulation of TLR4and inflammatory cytokines by1,25(OH)2D3in human aortic vascular smooth muscle cells.Methods:Human aortic vascular smooth muscle cells were cultured with various doses of1,25(OH)2D3, then the cells were harvested, total RNA were isolated for study of IL-6,IL-8,MCP-1mRNA expression by quantitative real-time PCR. Then HA-VSMC were treated with low dose1,25(OH)2D3to determine TLR4mRNA expression.Results:1,25(OH)2D3inhibited mRNA expression of IL-6,IL-8and MCP-1in HA-VSMC. The mRNA expression levels of IL-6, IL-8and MCP-1were all significantly downregulated after treated by1,25(OH)2D3. IL-6, IL-8and MCP-1mRNA expression were respectively downregulated by58%,69%and43%as compared with the control group (all P<0.05). The low dose1,25(OH)2D3markedly inhibited the expression of TLR4mRNA(P<0.05).Conclusion:1,25(OH)2D3inhibited inflammatory cytokines IL-6,IL-8,MCP-1mRNA expressions in human aortic vascular smooth muscle cells. The low dose1,25(OH)2D3markedly downregulated the expression of TLR4mRNA in human aortic vascular smooth muscle cells.