| BackgroundObesity is becoming a global health issue,especially in most industrialized countries.The epidemic of obesity is closely related with the increased occurrence of cardiovascular and metabolic diseases 1.The accumulated adipose tissue is also a main source of inflammation and this explains the endurance of sub-health status in obesity.The level of free fatty acids(FFAs)released from adipose tissue is a great marker of obesity in individuals.The elevated level of FFAs in the serum has been shown to be detrimental to various target organs,such as liver,pancreas,and skeletal muscle.The mechanism may be closely related to the inflammatory response of human umbilical vein endothelial cells,but its mechanism is not completely clear.Objective1.To investigate the effects of FFAs on human umbilical vein endothelial cells proliferation and apoptosis.2.To study the function of FFAs on the release of inflammatory factors in human umbilical vein endothelial cells.3.To clarify the role of FFAs on human umbilical vein endothelial cells by regulating TLR4 signaling pathway to promote the expression of inflammatory factors.Methods1.The effects of FFAs on human umbilical vein endothelial cells proliferation and apoptosis.(1)Human umbilical vein endothelial cells proliferation was detected by CCK-8 after human umbilical vein endothelial cells treated with 100 ?M,200 ?M and 300?M FFAs for 24 h or 48 h.(2)Human umbilical vein endothelial cells apoptosis was detected by CCK-8 after human umbilical vein endothelial cells treated with 100?M,200?M and 300?M FFAs for 24 h or 48 h.(3)Human umbilical vein endothelial cells proliferation was detected by CCK-8 after human umbilical vein endothelial cells treated with 100 ?M,200 ?M and 300?FFAs for 24 h or 48 h and infected with TLR4 interference.(4)Human umbilical vein endothelial cells apoptosis was detected by CCK-8 after human umbilical vein endothelial cells treated with 100 ?M,200?M and 300?M FFAs for 24 h or 48 h and infected with TLR4 interference.2.The effects of FFAs on the release of inflammatory cytokines in human umbilical vein endothelial cells(1).The expression level of IL-6,IL-8,CCL5 and CXCL10 were detected by ELISA after human umbilical vein endothelial cells treated with 100 ?M,200 ?M and 300?M FFAs for 24 h.(2).The expression level of IL-6,IL-8,CCL5 and CXCL10 mRNA were detected by qRT-PCR after human umbilical vein endothelial cells treated with 100?M,200 ?M and 300 ?M FFAs for 24 h.(3).The expression level of IL-6,IL-8,CCL5 and CXCL10 were detected by ELISA after human umbilical vein endothelial cells treated with 100 ?M,200 ?M and 300 ?M FFAs for 24 h and infected with TLR4 interference.(4).The expression level of IL-6,IL-8,CCL5 and CXCL10 mRNA were detected by qRT-PCR after human umbilical vein endothelial cells treated with 100?M,200 ?M and 300 ?M FFAs for 24 h and infected with TLR4 interference.3.The role of FFAs on human umbilical vein endothelial cells by regulating TLR4 signaling pathway to promote the expression of inflammatory factors(1).The expression level of MyD88,NF-?B and IKK-? protein were detected by Western blot after human umbilical vein endothelial cells treated with 100 ?M and 200 pM FFAs for 24 h.(2).The expression level of MyD88,NF-?B and IKK-(3 mRNA were detected by qRT-PCR after human umbilical vein endothelial cells treated with 100 ?M and 200?M FFAs for 24 h.(3).The expression level of MyD88,NF-?B and IKK-? protein were detected by Western blot after human umbilical vein endothelial cells treated with 100?M and 200 ?M FFAs for 24 h and infected with TLR4 interference.(4).The expression level of MyD88,NF-?B and IKK-P mRNA were detected by qRT-PCR after human umbilical vein endothelial cells treated with 100 ?M and 200?M FFAs for 24 h and infected with TLR4 interference.Results1.FFAs treatment could inhibit Human umbilical vein endothelial cells proliferation and promote cell apoptosis and TLR4 interference reversed the effects of FFAs on Human umbilical vein endothelial cellsCompared with control group,the cell proliferation of Human umbilical vein endothelial cells treated with 300 ?M FAAs was decreased at 24 h(P<0.05).The cell proliferation of Human umbilical vein endothelial cells treated with 100 pM,200 ?M and 300 ?M FAAs were significantly lower than that in control group at 48h,and the difference between the two groups was statistically significant(P<0.05).The cell apoptosis rate of Human umbilical vein endothelial cells treated with 100 ?M FAAs were significantly higher than that in control group at 24 h and 48h,and the difference between the two groups was statistically significant(P<0.05).After Human umbilical vein endothelial cells treated with different dosages of FFAs for 24 h or 48 h and infected with TLR4 interference,the cell proliferation activity was not statistically significant compared with the control group(P>0.05).Meanwhile,After Human umbilical vein endothelial cells treated with 100 ?M FFAs for 24 h or 48 h and infected with TLR4 interference,the cell apoptosis was not statistically significant compared with the control group(P>0.05)2.FFAs treatment could promote the release of various inflammatory factors and TLR4 interference reversed the effects of FFAs on Human umbilical vein endothelial cellsIn order to further analyze the effects of FFAs on the release of inflammatory cytokines in human umbilical vein endothelial cells,the expression level of IL-6,IL-8,CCL5 and CXCL10 were detected by ELISA and qRT-PCR after human umbilical vein endothelial cells treated with 100?M,200?M and 300?M FFAs for 24 h.The results of ELISA showed the expression level of IL-6,IL-8 and CCL5 in Human umbilical vein endothelial cells treated with 100pM,200?M and 300?M FAAs were significantly higher than that in control group at 24 h(P<0.05)and the expression level of CXCL10 in Human umbilical vein endothelial cells treated with 300?M FAAs were significantly higher than that in control group at 24 h(P<0.05).The results of qRT-PCR showed the expression level of IL-6 and CCL5 mRNA in Human umbilical vein endothelial cells treated with 100?M,200?M and 300?M FAAs were significantly higher than that in control group at 24 h(P<0.05)and the expression level of CXCL10 and IL-8 mRNA in Human umbilical vein endothelial cells treated with 200?M and 300?M FAAs were significantly higher than that in control group at 24 h(P<0.05).The expression level of IL-6,IL-8,CCL5 and CXCL10 in Human umbilical vein endothelial cells treated with FAAs and infected with TLR4 interference was not statistically significant compared with the control group(P>0.05).It suggested that TLR4 interference reversed the release of various inflammatory factors by FFAs on Human umbilical vein endothelial cells.3.FFAs promoted the expression of inflammatory cytokines by regulating TLR4 signaling pathway in Human umbilical vein endothelial cellsTo investigate the underlying molecular mechanisms of the release of various inflammatory cytokines,we measured the level of TLR4 downstream signaling molecules by Western blot and qRT-PCR.The results of Western blot showed the expression level of MyD88,NF-?B and IKK-(3 protein in Human umbilical vein endothelial cells treated with 100?M and 200pM FAAs were significantly higher than that in control group at 24 h(P<0.05)and the expression level of MyD88,NF-?B and IKK-? protein in Human umbilical vein endothelial cells treated with 200?M FAAs and infected with TLR4 interference was significantly decreased(P>0.05).The results of qRT-PCR showed the expression level of MyD88,NF-?B and IKK-? mRNA in Human umbilical vein endothelial cells treated with 100?M and 200?M FAAs were significantly higher than that in control group at 24 h(P<0.05)and the expression level of MyD88,NF-?B and IKK-? mRNA in Human umbilical vein endothelial cells treated with 200?M FAAs and infected with TLR4 interference was significantly decreased(P>0.05).ConclusionsFFAs could inhibit Human umbilical vein endothelial cells proliferation,promote cell apoptosis,and promote the release of inflammatory cytokines IL-6,IL-8,CCL5 and CXCL10 by regulating TLR4 signaling pathway.TLR4 might be a potential target in the treatment of FFAs induced inflammation and damage. |
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