Tetramethylpyrazine Reg?lates Lipopolysaccharide-induced Cytoskeletal Remodeling Of Human Umbilical Vein Endothelial Cells Via MiR-143-3p And MiR-194-5p

Objective:To investigate the effect of tetramethylpyrazine on the permeability of human umbilical vein endothelial cells(HUVECs)induced by Lipopolysaccharide(LPS)and the expression of cytoskeleton-associated proteins and micrornas(mi RNAs)Methods:HUVECs were digested with 0.25% trypsin and isolated for primary culture.The endothelial cells was identified by immunohistochemistry with factor VIII antibody endothelial cells.The safe concentration range of tetramethylpyrazine in HUVECs was determined by CCK8 method.From the safe range of tetramethylpyrazine,three groups of drug concentration with gradient increase were selected as the Treatment Group.Cells were randomly divided into 7 groups: Normal control group,LPS group(1?g/ml),low-dose TMP group(60 ?g/ml + LPS 1 ?g/ml),medium-dose TMP group(120 ?g/ml + LPS 1 ?g/ml),high-dose TMP group(240 ?g/ml + LPS 1 ?g/ml),pure TMP group(240 ?g/ml),and fasudil group(3.25 ?g/ml + LPS 1 ?g/ml).Establishment of endothelial cell permeability model in the Transwell Cell,the electrical resistance of each group was measured by voltage resistance meter,the permeability of cells in each group was detected by Dextran method,and cytoskeleton of each group was detected by immunofluorescence technique?The Rac1-GTPase active protein was pulled down by Rac1 Pull-Down Assay.The protein expression of Rac1-GTPase and Rac1,LIMK1 and p-LIMK1 in HUVECs were detected by Western blot.High-throughput sequencing technique was used to detect the differential expression of mi RNAs in normal control group,LPS injury group and high-dose TMP group.Real-time PCR was used to detect the expression of m RNA of Rac1,LIMK1 and the expression of mi R-143-3p and mi R-194-5p in each group.Results:1.When the concentration of TMP was in the range of 0-240 ?g/ml the decrease of the activity of HUVECs induced by LPS was significantly improved.When the concentration of TMP exceeded 480 ?g/ml,the activity of HUVECs was inhibited by TMP itself.2.Lps could induce morphological changes of endothelial cells,increase and coarsening of stress fibers,marked staining of microfilaments,destruction of intercellular compactness and enlargement of intercellular space With the increase of TMP concentration,the intracellular microfilament staining decreased,the stress fibers decreased and became thinner,and the tight junction recovered gradually.Morphological observation showed that TMP concentration below 30 ?g/ml had no protective effect on LPS-induced HUVECs cytoskeleton remodeling.3.TMP has protective effect on LPS-induced disruption of HUVECs barrier function.Using transendothelial resistance(TEER)and permeability coefficient(PA)measurements,the results showed that TMP(60 ?g/ml,120 ?g/ml,240 ?g/ml)could inhibit the decrease of TEER and the increase of endothelial permeability induced by LPS(P<0.05).4.Western blot showed that TMP could inhibit the up-regulation of Rac1-gtpase,p-LIMK1,Rac1,LIMK1 in HUVECs induced by LPS(P<0.05).5.High throughput sequencing results: There were 59 mi RNAs in LPS group compared with normal control group and TMP High Dose Protection Group(P<0.05).There were29 down-regulated mi RNAs in LPS group and 30 up-regulated mi RNAs in TMP group,and 30 down-regulated mi RNAs in LPS group and TMP group.6.QPCR results showed that TMP inhibited the down-regulation of RAC1,LIMK1 m RNA(P<0.05)and the up-regulation of mi R-143-3p and mi R-194-5p(P<0.05)in LPS-induced HUVECs.In combination with its protein level,the regulation of RAC1 /LIMK1 signaling pathway by TMP may come from the post-transcriptional level.Conclusions:(1)Tetramethylpyrazine can effectively inhibit the increase of HUVECs permeability induced by lipopolysaccharide;(2)The mechanism of lipopolysaccharide-induced cytoskeleton injury by TMP may be related to the down-regulation of Rac1-GTPase active protein and LIMK1 phosphorylated protein expression in RAC1 / LIMK1 signaling pathway;(3)LPS could decrease the expression of mi R-143-3p and mi R-194-5p in endothelial cells(4)The expression changes of RAC1 and LIMK1 induced by tetramethylpyrazine in LPS-injured endothelial cells may be regulated by the post-transcriptional level of mi R-194-5p and mi R-143-3p.