The Study On The Role And Mechanism Of MKP5 In The Palmitic Acid-induced Vascular Endothelial Cell Injury

Background:Vascular endothelial cell injury is closely related to the development of cardiovascular diseases such as atherosclerosis,diabetic macroangiopathy,hypertension and coronary heart disease,and the disorder of lipid metabolism characterized by hyperlipidemia is the main risk factor of such cardiovascular diseases.In hyperlipidemia,lipolysis is accelerated and the level of free fatty acids in the blood increases significantly,which reduces the diastolic factor of vascular endothelial cells and also triggers oxidative stress and inflammatory response,inducing apoptosis and causing vascular dysfunction.Mitogen-activated protein kinase phosphatases5(MKP5)is a negatively regulated phosphatase of Mitogen-activated protein kinase(MAPK).which plays a regulatory role in obesity-related diseases such as diabetes,pulmonary fibrosis and fatty liver.It has been shown that MKP5 has an inhibitory effect on macrophage inflammatory response and islet cell apoptosis induced by obesity and fat accumulation,but the role and mechanism of MKP5 in vascular endothelial cell injury have not been reported.Palmitic acid(PA),a major component of saturated free fatty acids,is commonly used to construct models of lipotoxic injury,Therefore,studying the regulatory role of MKP5 on palmitic acid-mediated vascular endothelial cell injury may provide a new theoretical basis and potential molecular targets for the treatment of hyperlipidemiarelated cardiovascular diseases.Aims:To regulate MKP5 expression in human umbilical vein endothelial cells HUVEC and to explore the regulatory role of MKP5 in palmitic acid(PA)-induced vascular endothelial cell injury and its molecular mechanism.Methods:1.HUVEC were treated with different concentrations of PA(100,200,300,400,500,600 μM).The proliferation activity of HUVEC was detected by CCK8 method.HUVEC were treated with PA(400 μM)for different time(0,3,6,9,12 h).Western blot was used to detect the expression of MKP5 protein,MKP5 protein,apoptosisassociated protein(Bax,Bcl-2,cleaved Caspase 3)and phosphorylation of ER stress kinase IRE-1α.2.HUVEC were transfected with pc DNA3.1 and pc DNA3.1-MKP5 plasmids respectively to construct a stable MKP5 overexpression cell line(HUVEC-MKP5)and a control cell line(HUVEC-PC).After HUVEC-MKP5 and HUVEC-PC were stimulated with 400 μM PA for 9 h,the proliferation activity of HUVEC-MKP5 and HUVEC-PC were detected by CCK8 method;the apoptosis protein(Bax,Bcl-2,cleaved Caspase 3,cleaved Caspase 9)and the expression of endoplasmic reticulum stress related proteins(GRP78,p IRE-1α,p EIF-2α,CHOP,cleaved caspase 12)were detected by Western blot,and the gene transcript level of transcription factor XBP-1s was detected by q PCR.The DCFH-DA reactive oxygen detection kit was used to detect the changes in intracellular ROS content,The contents of NO,GSH,MDA,SOD,CAT and T-AOC were detected by corresponding kits.The q PCR method was used to detect the expression of oxidative stress molecules(p22phox,HIF-1α)and inflammatory cytokines(IL-6,IL-1β,TNF-α,VCAM-1 and ICAM-1).The phosphorylated protein levels of P38,JNK and ERK in the MAPK pathway were detected by Western blot.3.HUVEC were further infected with lentivirus that inhibits MKP5 expression and the control virus Lenti-SC for 24 h,respectively,replaced with DMEM complete culture medium and continue to incubate for 12 h.After treatment with 400 μM PA for9 h,the expression of apoptosis proteins(Bax,Bcl-2,cleaved Caspase9,cleaved Caspase3)and endoplasmic reticulum stress related proteins(GRP78,p IRE-1α,cleaved Caspase12)were detected by Western blot.The changes of intracellular ROS content were detected by DCFH-DA reactive oxygen species assay kit,and the expression of oxidative stress molecules(p22phox,HIF-1α)and inflammatory cytokines(IL-6,IL-1β,TNF-α)were detected by q PCR method.The phosphorylated protein levels of P38,JNK and ERK in MAPK pathway were detected by Western blot.Results:1.PA inhibited the proliferation activity of HUVEC,induced endoplasmic reticulum stress and mitochondrial apoptosis.CCK8 assay showed that the proliferation activity of HUVEC decreased in a dosedependent manner after PA treatment.Under 400 μM PA treatment,the cell proliferation activity decreased by about 52%.Western blot showed the expression level of MKP5 protein showed a trend of decreasing first and then increasing,and the inhibitory effect on MKP5 expression was most obvious when treated for 9 h.Further detection of mitochondrial apoptosis and endoplasmic reticulum stress showed that PA could increase the expression of Pro apoptotic protein Bax in Bcl-2 family,decrease the expression of anti apoptotic protein Bcl-2,increase the activation of cleaved caspase3,and up regulate the phosphorylation level of IRE-1 α,indicating that PA stimulated HUVEC to induce mitochondrial apoptosis and endoplasmic reticulum stress.PA(400μM,9 h)was selected for subsequent experiment.2.MKP5 inhibited PA induced mitochondrial apoptosis in HUVEC.The results of cell proliferation assay showed that the overexpression of MKP5 significantly inhibited the decrease in cell proliferation activity induced by PA.Western blot results showed that MKP5 significantly down regulated PA induced cleaved Caspase 3 and cleaved Caspase 9 protein expression,and up regulated Bcl-2/Bax ratio.Furthermore,the inhibition of MKP5 expression further promoted the cleavage activation of Caspase 3 and Caspase 9 induced by PA,and the ratio of Bcl-2/Bax further decreased,indicating that MKP5 can inhibit PA induced mitochondrial apoptosis.3.MKP5 alleviated the endoplasmic reticulum stress induced by PA.Western blot results showed that PA could significantly increase the protein levels of GRP78,p IRE-1α and p EIF-2α,while the overexpression of MKP5 could inhibit the high expression of these proteins induced by PA.q PCR results showed that MKP5 significantly inhibited the increase of XBP-1s gene expression induced by PA.At the same time,PA induced the expression of endoplasmic reticulum stress apoptosis related proteins CHOP and cleaved Caspase 12,while the overexpression of MKP5 could significantly down regulate the above apoptotic indexes induced by PA.MKP5 knockdown can further promote GRP78 expression and Caspase12 splicing activation,activate IRE-1α,and aggravate PA induced endoplasmic reticulum stress injury.These results indicate that MKP5 can inhibit endoplasmic reticulum stress induced by PA.4.The overexpression of MKP5 inhibited oxidative stress and inflammation induced by PA.The results of oxidative stress analysis showed that PA can induce an increase in the production of ROS,down regulate the level of NO,increase the production of peroxidation product MDA,inhibit the levels of antioxidant enzymes SOD,CAT and non-enzymatic antioxidant GSH,and reduce the content of cell T-AOC.However,After the overexpression of MKP5,could significantly decreased ROS production,increased NO bioavailability,down regulated MDA level and up-regulated antioxidant capacity of HUVEC.At the same time,q PCR results showed that PA could increase the m RNA levels of p22 phox and HIF-1α,while the overexpression of MKP5 significantly inhibited the stimulation of PA on the above-mentioned oxidative stress factors.These results indicate that MKP5 can inhibit the oxidative stress response of cells.q PCR further confirmed that PA up-regulated the m RNA levels of proinflammatory factors IL-6,IL-1β and TNF-α and vascular adhesion molecules VCAM-1 and ICAM-1,while MKP5 overexpression significantly inhibited the expression levels of pro-inflammatory factors and adhesion molecules,inhibit the inflammatory response induced by PA.MKP5 knockdown further promoted ROS production and caused upregulation of m RNA levels of p22 phox and HIF-1α.It also exacerbated PAinduced increase in expression of pro-inflammatory factors IL-6,IL-1β and TNF-α,which exacerbated cellular oxidative stress and inflammatory damage.It indicates that MKP5 has the ability to inhibit PA-induced oxidative stress and inflammatory response.5.MKP5 regulated the signal transduction of MAPK pathway induced by PA.Western blot results showed that with the extension of PA treatment time,p P38 in the MAPK pathway reached a peak at 3 h,and did not change significantly when the time was extended to 12 h.p JNK and p ERK reached their peak at 3 h,and then gradually decreased.After the overexpression of MKP5,have a different degrees of inhibition of p P38,p JNK and p ERK expression(P<0.05).Further interference with the expression MKP5 can intensify the activation of PA on the MAPK pathway.Therefore,MKP5 may regulate PA induced HUVEC injury through P38,JNK and ERK pathways.Conclusion:1.MKP5 can alleviate PA induced proliferation inhibition,oxidative stress,inflammatory response,endoplasmic reticulum stress and mitochondrial apoptosis of vascular endothelial cells.2.MKP5 may inhibit lipotoxicity-related vascular endothelial cell injury through negative regulation of P38,JNK and ERK pathways...