EMMPRIN Regulates Autophagy Levels Via The TLR-4 Signaling Pathway In Macrophages

Objective:Atherosclerosis(AS)is a complex chronic vascular inflammatory disease.The inflammatory response runs through the lesion.The main mechanisms include lipid accumulation and chronic inflammation of the arterial wall.The aggregation of macrophages in the inflammatory response is a sign of AS.EMMPRIN can up-regulates the expression of matrix metalloproteinases(MMPs)and activates multiple downstream pathways to induce inflammatory responses.TLR-4/NF-x:B signaling pathway is a classical inflammatory pathway,it has been found that EMMPRIN can activate NF-κB signaling pathway in the downstream of TLR4,so that up-regulate inflammatory response and accelerate atherosclerosis.Autophagy is a lysosomal degradation process that maintains cellular homeostasis and is closely related to AS.Activation of autophagy within a certain range can reduce inflammation,reduce accumulation of lipid droplets in cells,eliminate dysfunctional mitochondria and reduce intracellular oxygen free radicals(ROS)levels that inhibit apoptosis.Excessive autophagy leads to excessive cell death,thinning of the plaque fibrous cap,high expression of inflammatory factors,and aggravation of plaque rupture、Both TLR4 signaling pathway and EMMPRIN in AS are associated with autophagy.Activing the TLR4 pathway can up-regulates autophagy,and EMMPRIN also up-regulates autophagy levels,and activation of autophagy within a certain range slows plaque rapture.Whether the TLR-4 signaling pathway is involved in EMMPRIN regulation of autophagy is still unclear.This study was designed to investigate whether EMMPRIN regulates autophagy in human THP-1 macrophages via the TLR4 signaling pathway.Methods:1.Culture and differentiation of human THP-1 monocytes:Human THP-1 monocytes were cultured in a complete medium at 37 ℃ in an incubator containing 95%air and 5%carbon dioxide.After growing to a certain amount,the number of cells was adjusted to 1×10^6 cells/ml and transferred to a six-well plate,and 2 ml of the cell suspension was inoculated into each well.The phorbol ester was adjusted to a final concentration of 5 ng according to the relevant literature.After induction of culture for 48 hours,the cells were washed 3 times with PBS,and cultured for 3 hours in serum-free medium,and the obtained human THP-1 macrophages were treated correspondingly according to different groups.2.Determine the stimulation concentration and stimulation time of EMMPRIN:The toxicity of EMMPRIN to human THP-1 macrophages at different time points was detected by CCK8.Combined with the previous study of our group,the optimal stimulation concentration and stimulation time of EMMPRIN were determined.3.Experimental grouping and detection of related protein expression and autophagosomes in each experimental group:To better illustrate EMMPRIN regulating autophagy by TLR4 signaling pathway in human THP-1 macrophage,the experiments were divided into the following three groups:The blank control group,EMMPRIN stimulation group and TAK-242 group were detected by Westerninblot for the expression level of TLR-4,NF-κB,LC3,Beclinl and P62 protein;immunofluorescence was used to detect the fluorescence expression of LC3,Beclin1 and P62;electron microscopy for autophagosomes.Results:1.Successful establishment of human THP-1 macrophage model:after 48 hours of stimulation with PMA(phorbol ester)at a final concentration of 5 ng/ml,90%of human THP-1 monocytes changed from a suspended state to an adherent growth,and the morphology changed from a circular shape to an elliptical shape,a spinous shape or a long shape.The fusiform shape,partially protruding from the pseudopod,increased in volume,indicating that human THP-1 monocytes have successfully differentiated into macrophages.2.Determination of EMMPRIN optimal stimulation concentration and optimal stimulation time:Combined with the previous study of our group and CCK-8 toxicity test,it was found that 1 ng/ml of EMMPRIN stimulated human THP-1 macrophages for 6 hours,EMMPRIN activity was relatively higher and the cytotoxicity is relatively low,so the optimal stimulation concentration of EMMPRIN is 1 ng/ml,and the optimal stimulation time is 6h.3.The expression of TLR-4 and NF-κB protein was significantly increased after EMMPRIN stimulated human THP-1 macrophages:1 ng/ml of EMMPRIN stimulated human THP-1 macrophages for 6 h,compared with the blank control group,The expression level of TLR-4 and NF-κB protein was significantly increased(P<0.05).4.EMMPRIN stimulated human THP-1 macrophages and increased the expression of LC3-Ⅱ and Beclinl protein,and decreased the expression of P62 protein:1 ng/ml of EMMPRIN stimulated human THP-1 macrophages for 6 h,Western Blot found:Compared with the blank control group,the expression level of LC3-Ⅱ protein in EMMPRIN group was significantly increased(P<0.05).The expression level of Beclinl protein was not statistically significant,but it was higher than that in the blank control group;the expression level of P62 protein was decreased(P<0.05).Immunofluorescence assay showed that the expression levels of LC3-II and Beclinl protein in EMMPRIN group were significantly higher than those in the blank control group(P<0.05).5.After inhibiting TLR4 signaling pathway,NF-κB protein expression level decreased,LC3-Ⅱ and Beclinl protein expression level also decreased,P62 protein expression level increased:after 5umol/L TAK-242 stimulated human THP-1 macrophage for 4 hours,the cells were washed 3 times with PBS and then stimulated with 1 ng/ml of EMMPRIN for 6 hours.Western Blot showed that the expression of TLR4 and NF-kB protein in TAK-242 group was significantly lower than that in EMMPRIN group(P<0.01).At the same time,the levels of LC3-Ⅱ and Beclinl protein were also decreased(P<0.05),and the expression level of P62 protein was increased(P<0,01).Immunofluorescence assay showed that the expression levels of LC3-Ⅱ and Beclinl protein in TAK-242 group were lower than those in EMMPRIN group(P<0.05).The fluorescence expression level of P62 protein was not statistically significant,but still higher than that in EMMPRIN group..6.More autophagic bodies were found after EMMPRIN stimulation:5umol/L TAK-242 stimulated human THP-1 macrophages for 4 hours,washed with PBS 3 times,and then stimulated with Ing/ml EMMPRIN for 6 hours.Compared with the EMMPRIN group and the blank control group,autophagosomes were not found in the TAK-242 group;more autophagic bodies were found in the EMMPRIN group compared with the blank control group.Conclusions:1.EMMPRIN can promote the expression of TLR-4 and NF-κB in human THP-1 macrophages,suggesting thatRIN may positively regulate TLR4 signaling ’pathway and promote inflammatory response.2.EMMPRIN can promote the expression of LC3-Ⅱ and Beclinl in human THP-1 macrophages,down-regulate the expression of P62 protein,and find more autophagosomes,indicating that EMMPRIN may lead to exeessive autophagy of macrophages.3.After EMMPRIN activates TLR-4/NF-κB signaling pathway,the expression levels of autophagy markers LC3-Ⅱ and Beclinl are up-regulated,P62 protein expression is decreased,and autophagosomes can be found;TLR4 signaling pathway is inhibited,When TLR-4 and NF-κB protein was down-regulated,the expression of autophagy markers LC3-Ⅱ and Beclinl was also down-regulated,while the expression of P62 protein was elevated,and no autophagosomes were found,suggesting that EMMPRIN may regulate macrophages excessive autophagy through TLR4 signaling pathway,is expected to provide a new experimental basis for clinical treatment targeting.