Effect Of Tea Polyphenols On The Expression Of Inflammatory Factors In Endothelial Cells Induced By Lipopolysaccharide

Objective: To observe the effect of tea polyphenols on the expression of inflammatory factors in human umbilical vein endothelial cells induced by porphyromonas gingivalis lipopolysaccharide and investigate the mechanism of its action.Methods: 1.Human umbilical vein endothelial cells were cultured in vitro by enzyme digestion.Factor ? related antigen in endothelial cells was identificated by immunofluorescence.The normal passage culture was carried out and the cells in good growth state were taken for experiment.2.CCK-8 was used to detect cell growth activity of HUVEC treated with TP(5,10,20,40,80,160 mg/L)for 12,24,48 h or Pg-LPS(0.001,0.01,0.1,1,10 ?g/m L)for 4,8,24 h.TP and Pg-LPS action concentration and time were screened respectively,then the experiment was divided into four groups: negative control group(Control),positive control group(LPS),LPS+TP1 group and LPS+TP2 group.3.The morphological changes of cells in the four experimental groups were observed by the ordinary optical inversion microscope.4.The cytokines MCP-1 and IL-6 of the four experimental groups in cell culture fluid were detected by ELISA.5.The protein expression of MCP-1,IL-6 and the protein expression of NF-?B p65 in nucleus and cytoplasm in the four experimental groups was detected by Western blot.Results: 1.The primary cultured cells had typical morphological characteristics of endothelial cells,and they were identified as HUVEC by immunofluorescence of factor ? related antigens.2.The results of CCK-8 showed that the cell proliferation rate increased with the increase of TP concentration(P<0.05)compared with negative control group when the HUVECs were treated with 5,10,20,and 40 mg/L TP for 12 h.When the concentration of TP increased to80 mg/L,the cell proliferation rate decreased(P<0.05),and the proliferation rate decreased to negative at 160 mg/L.Under the same concentration of TP,the cell proliferation rate decreased with time increased(P<0.05).Therefore,two concentrations of 5 mg/L and 40 mg/L TP acting on cells for 12 h were used to interfere with the inflammatory response of HUVEC induced by Pg-LPS.Under the same time,the cell inhibition rate increased with the increase of concentration(P<0.05)when the HUVECs were treated with Pg-LPS(0.1,1,10 ?g/m L)for 4,8,24 h.Under the same concentration,the cell inhibition rate: 24 h>8 h>4 h.The 10 ?g/m L Pg-LPS acting on cells for 24 h was selected as the experimental condition for the subsequent inflammatory response model.3.Observation of four groups of cells with inverted phase contrast microscope: The most of the cells adhered to the wall and the cells were fusiform or irregular triangles in the negative control group.The part of the cells became longer,the density of cells decreased,the gap became larger and there were more dewall cells in LPS group.The cell density was slightly higher than that in the LPS group and the dewall cells were also less in the TP intervention groups.4.The results of ELISA showed that the secretion of IL-6 and MCP-1 increased significantly(P<0.05)in LPS group compared with the negative control group.TP pretreatment could significantly reduce the secretion of IL-6 and MCP-1(P<0.05),and the secretion in 40 mg/L TP pretreatment group(LPS+TP2)was lower than that in 5 mg/L TP pretreatment group(LPS+TP1)(P<0.05).5.The results of western blot showed that the protein expression of IL-6,MCP-1 increased significantly(P<0.05)in LPS group compared with the negative control group.The protein expression of IL-6 and MCP-1 in TP intervention groups was lower than that in LPS group(P<0.05),and 40 mg/L TP pretreatment group(LPS+TP2)was more significant than that in 5 mg/L TP pretreatment group(P<0.05).The protein expression of NF-?B p65 increased in nucleus but decreased in cytoplasm in the LPS group compared with the negative control group(P<0.05).The protein expression of NF-?B p65 decreased in nucleus but increased in cytoplasm in the TP intervention groups compared with the LPS group(P<0.05).TP inhibited the translocation of NF-?B p65 from the cytosol to the nuclear fraction.Conclusion: The present study suggests that TP can inhibit the up-regulation of MCP-1and IL-6 expression in human umbilical vein endothelial cells induced by Pg-LPS,which may be related to the inhibition of NF-?B p65 activation.