EMMPRIN Induces MMP-9 Expression In Macrophages Via JAK2/STAT3 Signaling

[Objective]Atherosclerosis(athersclerosis,AS)is a chronic inflammation disease.Multiple risk factors affected the speed and ponderancy of the disease.For the past few years,a large number of related inflammatory factors had been extensively researched to indicate that athersclerosis is not only closely related to multiple risk factors,but also influenced by the microscopic regulation of inflammatory factors[1].Extracellular matrix metalloproteinase inducer(EMMPRIN)had been shown to upregulate in the circulation of patients with stable angina pectoris and acute coronary syndromes,and also matrix metalloproteinases,which degradated the fibrous cap of plaque and affected the stability of the plaques,the formation of thrombus,and the progress of coronary artery disease[2],playing an important role in development and incidence of acute coronary syndrome(ACS).EMMPRIN expression incereased in the plaque and platelet surface,and that closely related to thrombus formation.Foreign researchers have used MMP-9 as a target factor for exploring vulnerable marker markers,but which signaling pathway regulated the progressof EMMPRIN-MMPs was not entirely clear,and study found the relationship bettween JAK2/STAT3 pathway and atherosclerosis process is inseparable.JAK2/STAT3 signaling pathway is closely related to the protective effect of cardiomyocytes.Whatever the model of ischemia-reperfusion injury or hypoxia/reoxygenation model and direct oxidative stress model,studies realized that JAK2/STAT3 signaling was significantly up-regulated after cardiomyocyte recovery and blood supply.JAK2/STAT3 has a dual function on cardioprotection and atherogenic atherosclerosis.In the atherosclerosis model,JAK2/STAT3 directly regulated the expression of MMP-9 protein and promoted the leukocytes to adhere to subcutaneous and the expression of tissue factor,which subsequently activated the coagulation system and promoted the formation of thrombosis.In the model of myocardial ischemia,this pathway also showed some positive effects on reducing myocardial ischemia,improving left ventricular function,reducing myocardial oxidative stress and inhibiting the production of inflammatory waterfalls.Yang Y,H and his colleagues[3]found that JAK2/STAT3 directly mediates the expression of MMP-9 in macrophages under the stimulation of LPS,and after inhibiting this signaling,the secretion of MMP-9 and the rate of macrophages migrate to subcutaneous were reduced,also potently proved that JAK2 and STAT3 protein play an important role in the early stage of atherosclerosis.Therefore,EMMPRIN-induced expression of MMP-9 is an important factortor of accelerating progression of atherosclerosis.Looking for a signaling of regulation of macrophage secretion of MMP,9 is essential.JAK2/STAT3 signaling has a very close relationship with cardiovascular disease,promoting the role of atherosclerosis,,so we hypothesis that EMMPRIN-induced MMP-9 secretion is through the channel to achieve.We established the THP-1 macrophage model to verify the relationship of this pathway with atherosclerosis and EMMPRIN/MMP-9,which offer theoretical evidence and experimental basis for the clinical treatment of atherosclerosis.[Methods]1.Culture and induction of THP-1 cells THP-1 cells were cultured in a complete culture medium at 37 ℃ in an incubator containing 95%air and 5%CO2.The cells were adjusted to 1 x 106 cells/ml and transferred to a six-well plate,each inoculated with 2 ml.According to the literature,the optimum conditions were induced to ultimately concentration with 5 ng/ml PMA.After incubation for 3 h in serum-free medium,add 1 ng/ml concentration EMMPRIN to stimulate.The corresponding time period THP-1 cells were collected for the next experiment.2.The expression of JAK2,STAT3 and MMP-9 mRNA in macrophages were detected by RT-PCR at Oh,6h,12h,18h and 24h after 0h,respectively.The expression of JAK2,STAT3 and MMP-9 in THP-1 cells were detected by RT-PCR.The levels of JAK2,STAT3,p-STAT3 and MMP-9 were measured by Western blot.3.In order to investigate the role of EMMPRIN in the regulation of MMP-9 expression in macrophages,the experimental group was divided into three groups:control group(RPMI1640 medium group),EMMPRIN stimulation group,EMMPRIN+JAK2 inhibitory group(AG490),EMMPRIN+ AG490 + siRNA-STAT3 intervention group,EMMPRIN stimulation 6 hours later,cells in the six-well plate was collected.[Results]1.THP-1 macrophage model was established successfully.THP-1 cells after the application of PMA stimulation 48h,90%of the successful differentiation of cells,protruding pseudopodia,mononuclear cells from the suspended state into adherent growth of macrophages,we could think that the experimental model was established successfully.2.JAK2,STAT3,MMP-9 expression in macrophages increased after EMMPRIN stimulated2×10~6 THP-1 macrophage cells were successfully induced,and RT-PCR and Western blot were used to detect the expression of the corresponding gene and protein at lng/ml EMMPRIN stimulation.Compared with the control group,the expression of JAK2,STAT3 and MMP-9 in EMMPRIN group reached the peak at 6 hours and decreased at 24 hours,and the three trends were consistent.3 JAK2/STAT3 regulates the expression of MMP-9 in macrophagesCompared with the control group,JAK2,STAT3 and MMP-9 were significantly increased in the EMMPRIN group at 6h after stimulation,which was consistent with the results of the previous trial,while the EMMPRIN + AG490 group compared to the control group,MMP-9 gene and protein were 0.3 bold that of the control group,the pathway inhibited nearly 67%.(P<0.05).The expression of Ng-siRNA-STAT3 in the siRNA-STAT3-group was significantly lower than that in the EMMPRIN group(the results were not listed).The siRNA-STAT3 and AG490 double The STAT3 gene and protein level were almost completely inhibited,and the expression of MMP-9 was decreased by 80.4%compared with EMMPRIN group.In addition,the content of p-STAT3(Tyr705)protein in this experiment was found to be significantly up-regulated in EMMPRIN alone group,and the protein content of the three groups was decreased by 64.4%in AG490 inhibitor group.When STAT3 protein of siRNA-STAT3 group was almost completely inhibited,the expression of MMP-9 was comparable to that of AG490 group.[Conclusions]1.EMMPRIN promotes secretion of MMP-9 in macrophages.2.EMMPRIN up-regulates JAK2 and STAT3 expression.3.After inhibiting JAK2 or(and)STAT3 protein,EMMPRIN induced down-regulation of MMP-9 expression in macrophages,and MMP-9 expression was significantly decreased after both of them were inhibited simultaneously4.After inhibiting p-STAT3(STAT3-siRNA)protein,EMMPRIN-induced MMP-9 expression was down-regulated,and EMMPRIN-induced secretion of MMP-9 might play a role through p-STAT3(Tyr705).[Limitations]Because of no more time,this experiment proves that the possibility of EMMPRIN/JAK2/STAT3/MMP-9 is existent,buy not unique.Experimental results show that the regulation of JAK2、STAT3、MMP-9 in macrophages are consistent,which initiative proved the role of JAK2/STAT3 signaling in the regulationof EMMPRIN-MMP-9 in macrophages that needed co-positioning experiments can be confirmed.