| Background:Atherosclerosis(AS)is a chronic inflammatory disease caused by the accumulation of lipids in the arteries.Macrophages are the main immune cells involved in its progression.Macrophages take up oxidized low-density lipoprotein(ox-LDL)through scavenger receptors,convert ox-LDL to free cholesterol(FC)through cholesterol esterification,and excrete FC through cholesterol efflux.However,when lipid metabolism is disturbed in macrophages,excess lipids accumulate in the cells and form large numbers of lipid droplets,leading to the transformation of macrophages into foam cells.Foam cell accumulation and mass death promote plaque formation,which is a key factor in disease progression.In contrast,Pyrotosis is a form of programmed pro-inflammatory cell death induced by inflammasomes.In macrophages,ox-LDL was found to activate NLRP3(Nucleotide-binding oligomerization domain-like receptor protein 3)inflammasomes and its downstream scorch death pathway to induce macrophage pyrotosis.In the early stages of AS,macrophage pyrotosis in the aorta may further promote the formation of macrophage-derived foam cells,which in turn promote the formation of atherosclerotic plaques.As the disease progresses,macrophage pyrotosis may accelerate necrotic core formation and cause plaque destabilisation in the late stages of AS.Mitogen-activated protein kinase phosphatases 7(MKP7)belongs to the MKPs family and plays an important regulatory role in the proliferation,differentiation and cytokine response of macrophages,dendritic cells and T cells.Recent studies have reported that overexpression of MKP7 in endothelial cells can promote the progression of AS.However,the role of MKP7 in macrophages in the pathogenesis of AS has rarely been reported.Aims:The study aims to explore the role of MKP7 in macrophage foaming and pyrotosis through up-regulating or inhibiting the expression of MKP7 in mouse macrophage cell line RAW264.7 cells and mouse primary peritoneal macrophages Methods:1.In order to determine the optimal concentration of ox-LDL for treatment of RAW264.7 cells,RAW264.7 cells were treated with different concentrations(0,25,50,75 and 100 μg/m L)of ox-LDL for 24 hours.The viability of RAW264.7 cells was measured by CCK8 assay and Western blot was used to detect the expression of MKP7 protein.According to the results,The concentration of ox-LDL at the highest MKP7 protein expression was selected to treat RAW264.7 cells for different times(12,24 and 48 h)and the expression of MKP7 protein was detected by Western Blot to determine the treatment time.2.RAW264.7 cell were transfected with pRM02 and pRM02-MKP7 plasmids respectively to construct a stable MKP7 overexpression cell line named RAW-MKP7,and its control cells were named RAW-PR;or RAW264.7 cells were infected with a lentivirus expressing MKP7 interference sequence(Lenti-sh MKP7,its control virus was Lenti-SC)for 24 h.Macrophages were induced with 50 μg/m L ox-LDL for 24 hours.The intracellular lipid droplet content was observed using Oil Red O staining;the total cholesterol(TC)and free cholesterol(FC)assay kits were used to analyse intracellular TC,FC,cholesteryl ester(CE)concentrations,thus assessing intracellular lipid metabolism.The transcript expression levels of cholesterol esterification key enzyme ACAT1,hydrolysis key enzyme n CEH,cholesterol efflux transport proteins ABCA1 and ABCG1,and scavenger receptor CD36,SR-AI,LOX-1were detected by q PCR method,and the protein expression of scavenger receptor CD36,SR-AI,SR-B1 was detected by Western Blot;Macrophage phagocytosis was detected using Dil-ox-LDL when MKP7 expression was inhibited to analyze the regulatory role of MKP7 in ox-LDL-induced macrophage foaming.The migration ability of macrophages was measured using Transwell assay.At the same time,Lenti-SC and Lenti-sh MKP7 were used to infect primary peritoneal macrophages and treated with ox-LDL for 24 hours.The expression of CD36 and SR-AI was detected by Western Blot and the phagocytic function was detected by Dil-ox-LDL.3.In the primary peritoneal macrophages of mice overexpressing(infected with Retro-MKP7 expressing MKP7 retrovirus,whose control virus was Retro-PLN)or inhibiting the expression(infected with Lenti-SC and Lenti-sh MKP7)of MKP7,after24 h of induction with ox-LDL,LDH detection kit was used to detect the release of LDH.The changes in intracellular ROS levels were detected by the ROS Assay Kit.The expression and activation levels of proteins related to the pyrolysis pathway were detected by Western Blot assay.Results:1.Ox-LDL inhibited the viability of RAW264.7 cells and induced the expression of MKP7The CCK8 assay showed that the viability of RAW264.7 cells gradually decreased with increasing concentrations of ox-LDL treatment compared to the control group(P<0.01).Meanwhile,Western Blot results showed that the expression of MKP7 increased with increasing ox-LDL concentration,reached a peak at 50μg/m L ox-LDL treatment and then decreased.After treating RAW264.7 cells with 50μg/m L ox-LDL,the expression of MKP7 gradually increased with increasing treatment time,peaked at 24 h and then decreased.The expression of MKP7 increased gradually with the treatment time,peaking at 24 h and then decreasing.Based on the above results,we selected 50 μg/m L ox-LDL to induce macrophages for 24 h for subsequent experiments2.MKP7 promotes the formation of macrophage-derived foam cellsOil Red O staining showed that ox-LDL induced a significant increase in intracellular lipid droplet content in RAW264.7 cells(P<0.01).Inhibition of MKP7 expression reduced ox-LDL-induced lipid deposition,while MKP7 overexpression could further promote ox-LDL-induced intracellular lipid deposition.Detection of intracellular TC,FC and CE content showed that interference with MKP7 expression reversed ox-LDL-induced increases in intracellular TC and CE concentrations(P<0.01)and decreases in FC concentrations(P<0.01),while MKP7 overexpression further promoted ox-LDL-induced changes in TC,CE and FC concentrations.Subsequently,we explored the role of MKP7 in lipid deposition in foam cells at the molecular level.The q PCR results showed that ACAT1 expression was significantly increased(P<0.01)and n CEH,ABCA1 and ABCG1 expression was significantly decreased in the ox-LDL-treated group(P<0.05),while disturbance of MKP7 expression reversed the expression changes of the above molecules(P<0.05),and overexpression of MKP7 further promoted the upregulation of ACAT1 expression(P<0.05)and decreased the expression of n CEH,ABCA1 and ABCG1(P<0.01).The above results indicate that MKP7 may participate in the formation of foam cells by regulating the esterification,hydrolysis and efflux of cholesterol in macrophages.Next,we further investigated whether MKP7 is involved in the uptake of ox-LDL by macrophages.q PCR results showed that the expression of scavenger receptors CD36,SR-AI,and LOX-1 was significantly upregulated in the ox-LDL-induced group(P<0.05).,while interference with the expression of MKP7 suppressed the changes in the expression of the above molecules(P<0.05).MKP7 overexpression further contributed to the increase in the expression of scavenger receptor expression(P<0.05).Western Blot showed that the expression of CD36,SR-AI and SR-B1 was significantly reduced after inhibition of MKP7 expression(P<0.05),while overexpression of MKP7 further promoted the increase of ox-LDL-induced scavenger receptor expression(P<0.05).Macrophages can also take up ox-LDL through phagocytosis,and we subsequently examined changes in macrophage phagocytosis.After treatment of RAW264.7 cells with 50 μg/m L Dil-ox-LDL for 6 h,the number of red fluorescent cells increased significantly in the Dil-ox-LDL-treated group compared to the control group,and their number decreased significantly after interfering with the expression of MKP7.The results of Transwell showed that the migration ability of RAW264.7 cells was diminished by ox-LDL treatment,while the interference of MKP7 expression improved the ox-LDL-induced impairment of macrophage migration function,while macrophage migration function was further impaired by MKP7 overexpression..In this experiment,the expression of CD36,SR-AI and Dil-ox-LDL uptake were detected after silencing MKP7 expression in primary mouse peritoneal macrophages,and the results were consistent with those in RAW264.7 cells.3.MKP7 promotes pyroptosis of primary peritoneal macrophagesThe results of LDH activity assay showed that LDH release was markedly increased in the ox-LDL induced group(P<0.01),and interfering with the expression of MKP7 significantly inhibited the release of LDH(P<0.01),while MKP7 overexpression further promoted the release of LDH(P<0.01).ROS results showed that ROS production of RAW264.7 cells was significantly increased in the ox-LDL treated group(P<0.01),and intracellular ROS levels were significantly reduced after inhibition of MKP7 expression(P<0.01),while MKP7 overexpression further promoted ox-LDL-induced increase in ROS production(P<0.01).In this study,we further explore the role of MKP7 in the pyroprosis of primary macrophages at the molecular level.Compared with the control group,the expression of NLRP3,ASC,cleaved-Caspase1,mature IL-1β and cleaved-GSDMD in ox-LDL group was significantly up-regulated(P<0.05).After inhibiting the expression of MKP7,the expression levels of NLRP3,ASC,cleaved-Caspase1,mature IL-1β and cleaved-GSDMD dramatically decreased in ox-LDL group(P<0.05).The activation of NLRP3-mediated inflammatory bodies and its downstream pyroptosis pathway was further promoted after MKP7 overexpression(P<0.05),indicating that MKP7 promotes the pyroptosis of macrophages.Conclusion:1.MKP7 may promote macrophage foaming by promoting scavenger receptor expression,cholesterol esterification,diminished macrophage migratory capacity and inhibition of cholesterol hydrolysis and cholesterol efflux.2.MKP7 can promote the pyroptosis of macrophages induced by ox-LDL. |
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