Sestrinl Inhibits Ox-LDL-induced Activation Of NLRP3 Inflammasome In Atherosclerosis

Cardiovascular and cerebrovascular diseases caused by atherosclerosis are one of the main causes of disability and death in China.Atherosclerosis can affect all large and medium-sized arteries,including the coronary,carotid and cerebral arteries,the aorta and its branches,and the major arteries of the limbs.To study the cellular and molecular mechanism of atherosclerosis is of great significance for the development of new methods for prevention and treatment of atherosclerosis.Currently,the treatment of atherosclerosis mainly focuses on improving lipid metabolism,adjusting lifestyle,anti-platelet,improving vascular status,and controlling other basic diseases.However,studies have shown that inflammatory reaction is an important pathological change in atherosclerotic lesions.Inflammatory reaction participates in the whole process of atherosclerotic lesions and plays an important regulatory role in the occurrence,progression and rupture of plaques.Macrophages,lymphocytes and cytokines play important roles in the above reaction process.Atherosclerosis features the imbalance between lipid metabolism and a macrophage-mediated dysfunctional immune reaction.Low-density lipoprotein(LDL),particularly its modified type(ox-LDL),acts as the major source of lipid accumulation in atherosclerotic lesions.Atherosclerosis begins with the penetration and retention of circulating lipoproteins into the arterial wall.Circulating monocytes are recruited to and populate in the atherosclerotic lesions where they turn into macrophages and uptake lipoproteins,excessive cholesterol accumulation in the cytosol induces the formation of cholesterol crystals,macrophages turn into foam cells eventually.The above pathological changes can activate NLRP3 inflammasome,induce endoplasmic reticulum stress response,activate inflammatory response signaling pathway and trigger inflammatory response.It is therefore important to thoroughly understand the molecular mechanisms underlying the modulation of macrophage activities in atherosclerotic arteries.Current studies have shown that regulating the effects of macrophages on activation of inflammasomes and other inflammatory signaling pathways is a potentially promising strategy for the treatment of atherosclerosis.Sestrins(SESNs)belongs to a group of stress-inducible proteins which modulate metabolic homeostasis of multiple cell types.A growing body of evidence suggests that deficiencies in endogenous SESNs provoke an array of metabolic disorders including insulin resistance,fat accumulation,mitochondrial dysfunction,and oxidative damage.Among SESNs,the function of SESN2 has been intensively studied.SESN2 has been shown to act as an LKB1-AMPK scaffold to activate AMPK signal pathway in cardiomyocytes.In macrophage cell line RAW264.7,SESN2 profoundly restrains the inflammatory reaction including nitric oxide release,the expression of inducible nitric oxide synthase,as well as the production classical pro inflammatory cytokines.However,the expression pattern and functional characteristics of SESN1 remain elusive.Similar to SESN2,SESN1 is a p53 target gene and its product is an antioxidant which activates AMPK signal pathway while inhibiting the mechanistic target of rapamycin in complex 1(mTORC1)signal pathway.Therefore,SESN1 is considered a tumor suppressor in follicular lymphoma that controls mTORC1 activity.Furthermore,SESN1 acts as a leucine sensor and is required for leucine-dependent activation of mTORC1 in skeletal muscle.Human senescent CD4+T cells express Sesnl,Sesn2 and Sesn3.Inhibition of Sesnl,Sesn2 and Sesn3 in senescent T cells induced functional reversal of senescence.To our knowledge,the role of SESN1 in atherosclerosis or macrophages has not been elucidated.Objective:To investigate the regulatory effect of sestrinl protein on ox-ldl-induced NLRP3 inflammasome activation in macrophages of atherosclerotic mice.To clarify the expression of sestrinl in macrophages and the mechanism of its inhibition of NLRP3.To illuminate the regulatory effect of sestrinl on atherosclerotic lesions and the potential molecular mechanism,and to provide a theoretical strategy for the treatment of atherosclerosis based on the control of inflammatory response.Methods:ApoE-/-mice were used to establish the model of atherosclerosis.Wild-type C57BL/6J mice were fed normally.1:Expression level of sestrinl protein in aortic macrophages and monocytes of atherosclerotic mice.Monocytes and macrophages were extracted and separated from the aorta of the two types of mice.Flow cytometry was used to measure the contents of monocytes and macrophages of the two types of mice and and rt-pcr was used to measure the mRNA expression levels of sestrinl,sestrin2 and sestrin3 proteins;2:Lentivirus transfected macrophages.Macrophages of C57BL/6J mice were transfected independently with two lentiviral vectors of sesnl-GFP-encoding and GFP-encoding.After transfection,the levels of NLRP3,NLRC4,sesreinl,IL-1β,and apoptotic protease caspase-1 in the transfected macrophages were detected and compared with macrophages not transfected.Sestrinl protein subtypes in macrophages transfected with sesnl-GFP-encoding were detected;3:To detect the effect of sestrinl protein on the activation of NLRP3 inflammasome.C57BL/6J mice macrophages were treated with LPS and ox-LDL,and protein levels of sestrinl and sestrin2 were detected.The macrophages were then treated independently by the above two lentiviruses,and the binding levels of ACS and NLRP3 inflammasomes,as well as the expression levels of caspase-1 p20,IL-1β,pro-IL-1β,and IL-18 after the treatment of the two lentiviruses were detected,as well as the content of NLRP3 inflammasomes in macrophages without ox-LDL treatment;4:Study on the effect of sestrinl protein on NF-κB signaling pathway.Macrophages in C57BL/6J mice were treated independently by the above two lentiviruses and divided into two groups.The macrophages in the two groups were partially ox-LDL treated and partially untreated and then divided into four groups,Levels of IL-1α,IL-6,IL-1βexpression,IKKβ phosphorylation,NF-κB p50 and p65,IκBα were measured;5:Effect of sestrinl protein on NLRP3 inflammasome activation induced by cholesterol crystallization.The macrophages of C57BL/6J mice were treated independently by two lentiviruses and divided into two groups.After CHC and LPS treatment,the levels of IL-1β,caspase-1p20,and pro-caspase-1,as well as the binding level between ACS and NLRP3 were detected,and the expression levels of the receptors related to lipid uptake by macrophages were detected;6:Study on the effect of sestrinl overexpression on macrophage-mediated inflammatory response.The intrinsic monocytes and macrophages of ApoE-/-mice were removed,and macrophages treated independently by the two lentiviruses were adoptively injected into mice.After 2 weeks,the levels of aortic macrophages in mice were compared,and the levels of inflammatory factors IL-1β,IL-6,TNF-α and CXCL1 were detected;7:Effect of knocking down sestrinl protein on macrophage-mediated inflammatory response.Using shS1 lentivirus and shC lentivirus,as well as the same procedure in method 6,expression levels of IL-1β,IL-6 and TNF-α expression in the aorta of mice treated with two lentiviruses were detected and compared.Results:1.SESN1 is up-regulated in aorta monocytes and macrophages in atherosclerotic mice1.1 Flow cytometry revealed the existence of myeloid cells in the aorta of C57BL/6J mice and ApoE-/-atherosclerosis mice.1.2 Flow cytometry showed a significant increase in the content of monocytes in the aorta of ApoE-/-atherosclerotic mice compared with C57BL/6J mice.1.3 PCR analysis showed that compared with C57BL/6J mice,the mRNA expression level of sesreinl protein was significantly increased in the aortic macrophages of ApoE-/-mice with a statistical difference(P<0.001),and moderately increased in monocytes with a statistical difference(P<0.05).1.4 PCR analysis showed that compared with C57BL/6J mice,mRNA expression level of sesrein2 protein was significantly increased in aortic macrophages of ApoE-/-mice,with statistical difference(P<0.01),but no significant difference in mononuclear cells(P>0.05).1.5 PCR analysis showed that compared with C57BL/6J mice,the mRNA expression level of sesrein3 protein showed no significant difference in the aortic macrophages and monocytes of ApoE-/-mice(P>0.05).2.Lentiviral infection induces the overexpression of exogenous SESN12.1 Immunoblot analysis showed that sestrinl protein level was significantly increased in macrophages transfected with sesnl-gfp-encoding lentivirus compared with macrophages transfected with gfp-encoding lentivirus,while sestrin2 and sestrin3 levels were unchanged,indicating successful transfection.2.2 Immunoblot analysis showed that compared with macrophages of C57BL/6J mice,the expression levels of NLRP3 and NLRC4 in macrophages transfected with gfp-encoding and sesnl-gfp-encoding were both increased,with statistically significant differences compared with macrophages untransfected(P<0.05).There was no significant difference between the two transfected macrophages(P>0.05).2.3IL-1β expression was not detected in the two lentiviral transfected macrophages.2.4 Immunoblot analysis showed that the expression of caspase-1 p20 was not significantly detected in the two lentiviral transfected macrophages,and there was no significant difference in the pro-caspase-1 protein between the three types of macrophages.3.SESN1 inhibits oxLDL-induced activation of NLRP3 inflammasome3.1 After LPS and ox-ldl treatment,immunoblot analysis showed no significant change in sestrinl expression level in treated macrophages compared with untreated macrophages.3.2 Analysis showed that sestrin2 expression was up-regulated in treated macrophages.3.3 Immunocoprecipitation-assay showed that ASC binding to NLRP3 was significantly weaker in macrophages transfected with sesnl-GFP-encoding lentivirus than in macrophages transfected with GFP-encoding lentivirus,with statistical difference(P<0.01).3.4 Immunoblot analysis showed no significant difference in the expression of pro-caspase-1 in macrophages transfected with the two lentiviruses(p>0.05),while the content of caspase-1 p20 in macrophages transfected with sesn1-GFP-encoding was significantly lower than that of macrophages transfected with GFP-encoding,showing statistical difference(P<0.05).3.5 The level of mature IL-1β in macrophages transfected with sesnl-GFP-encoding was significantly lower than that in macrophages transfected with GFP-encoding(P<0.05),but there was no significant difference in IL-1β mRNA level between the twoes(P>0.05).3.6 There was no significant difference in the levels of pro-IL-1β in macrophages treated with different lentiviruses(P>0.05),but there was significant difference in the expression levels of IL-1β,which was statistically significant(P<0.05).3.7 There was no significant difference in IL-18 mRNA level between macrophages treated with two lentiviruses(P>0.05).4.SESN1 impedes oxLDL-induced NF-κB signaling4.1 mRNA levels of IL-1β,IL-6 and IL-1α were significantly increased after treatment with ox-LDL alone,with statistically significant differences compared with untreated macrophages(IL-1β:P<0.001,IL-6:P<0.05,IL-1α:P<0.01).mRNA levels of IL-6 and IL-1β mRNA in the ox-LDL-treated macrophages transfected with sesn1-GFP-encoding lentivirus were lower than those in the macrophages transfected with GFP-encoding lentivirus(IL-1β:P<0.01,IL-6:P<0.05).4.2 IKKβ activation was induced by ox-LDL,and IKKβ activation was significantly decreased after transfection with sesn1-GFP-encoding lentivirus,which was statistically different from that of GFP-encoding lentivirus transfected macrophages(P<0.05).4.3 ox-LDL inhibited IκBα activation and increased the expression levels of NF-κB p50 and p65.IκBα activation was enhanced and the expression levels of NF-κB p50 and p65 were decreased in those macrophages transfected with sesn1-GFP-encoding lentivirus(IκBα:P<0.05,p50:P<0.01,p65:P<0.05).5.SESN1 suppresses cholesterol crystal-induced activation of NLRP3 inflammasome5.1 Levels of IL-1β in macrophages transfected with GFP-encoding were higher than those transfected with sesn1-GDP-encoding lentivirus,with statistically significant differences(P<0.001).5.2 The binding level of ACS and NLRP3 and the level of caspase-1 p20 were significantly lower in macrophages transfected with sesn1-GFP-encoding than those transfected with GFP-encoding lentivirus(P<0.05).5.3 Oil red O staining indicated less lipid accumulation and foam cell formation in LS1-infected macrophages as compared with LC-infected macrophages.5.4 Compared with macrophages transfected with GFP-encoding lentivirus,the expression of lipid receptors in macrophages transfected with sesn1-GFP-encoding lentivirus was not significantly different(P>0.05).6.SESN1 overexpression diminishes macrophage-mediated aorta inflammationCompared with mice transfected with GFP-encoding lentivirus,the expression levels of IL-1β,IL-1βmRNA,IL-6,TNF-α and CXCL1 were decreased in the aorta of mice transfected with lentivirus sesn1-GFP-encoding,showing statistically significant differences(IL-6:P<0.01,TNF-α:P<0.001,IL-1β:P<0.001,IL-1βmRNA:P<0.05,CXCL1:P<0.01).7.SESN1 knockdown enhances macrophage-mediated aorta inflammation7.1 The activation level of caspase-1 in shS1-transfected macrophages was significantly higher than that in shC-transfected macrophages(P<0.01).7.2 Compared with mice transfected with shC,the expression levels of IL-1β,IL-6 and TNF-α in macrophages were significantly higher in mice transfected with shS1,with statistically significant differences(IL-6:P<0.001,TNF-α:P<0.001,IL-1β:P<0.05).7.3 Compared with mice transfected with shC macrophages,the expression level of TNF-α in the local aorta of mice transfected with shSl was increased,with statistically significant difference(P<0.05).Conclusions:1.The content of local monocytes in the aorta of atherosclerotic mice increased,indicating that the recruitment and subintimal infiltration of monocytes in the lesion were enhanced,which was consistent with the basic pathological process of atherosclerotic lesions.Both Sestrinl and sestrin2 levels were elevated in aortic macrophages,suggesting that both proteins play a role in regulating inflammatory responses in atherosclerosis.However,sestrinl level in C57BL/6J mice was significantly lower than that in atherosclerotic mice,indicating that sestrinl protein expression level was up-regulated in atherosclerotic inflammatory response.This study has not yet clarified the regulatory mechanism.2.Sestrinl protein level was elevated in macrophages of C57BL/6J mice transfected with LS1,indicating successful transfection.However,the expression levels of NLRP3 and NRRC4 inflammarosomes in the macrophages after transfection of LC and LS1 were both increased,indicating that lentivirus itself would cause the increased expression levels of inflammarosomes,which was the basis for the subsequent test of sestrinl’s regulation of the expression levels of atherosclerotic inflammarosomes.However,IL-1β,Caspase-1 p20 and factors directly related to inflammatory response,were not detected in the two transfected macrophages,and there was no difference in the expression level of pro-caspase-1 p20 in the two groups of macrophages,indicating that the inflammatory pathway was not activated in the two lentiviral transfected macrophages.Sestrinl typing showed that type 1 was dominant in regulating macrophage-mediated inflammatory responses.3.Sestrinl protein inhibited the activation of NLRP3 inflammasome in macrophages of C57BL/6J mice induced by ox-LDL.After LPS and ox-LDL treatment,sestrinl protein level did not change significantly,but sestrin2 protein level increased,which was consistent with previous research results.As an antioxidant,sestrin2 expression was up-regulated in the case of oxidative damage or inflammation.In the case of sestrinl protein expression,the binding level of ACS and NLRP3 was significantly inhibited,and the expression level of pro-inflammatory cytokines IL-1βand caspase-1 p20 was decreased,indicating that sestrinl had a significant inhibitory effect on ox-LDL-induced NLRP3 activation.However,sestrinl protein also showed inhibition of NLRP3 inflammasome in LPS-treated macrophages,suggesting that sestrinl inhibition of NLRP3 is not ox-LDL specific dependent.4.Sestrinl inhibits ox-LDL and induces activation of the NF-κB inflammatory signaling pathway.After treatment of macrophages with ox-LDL alone,the mRNA levels of inflammatory cytokines IL-1α,IL-6 and IL-1β expression were significantly increased,and the expression levels of NF-κB p50 and p65 were increased.Combined with the phosphorylation of IKKβ and the inhibitory effect of IκBα,it was suggested that ox-LDL could definitely induce inflammatory pathway activation.The above pro-inflammatory effects were improved in the presence of sestrinl protein.This indicates that sestrinl protein can inhibit ox-LDL-induced activation of NF-κB mice increased,indicating that the recruitment and subintimal infiltration of monocytes in the lesion were enhanced,which was consistent with the basic pathological process of atherosclerotic lesions.Both Sestrinl and sestrin2 levels were elevated in aortic macrophages,suggesting that both proteins play a role in regulating inflammatory responses in atherosclerosis.However,sestrinl level in C57BL/6J mice was significantly lower than that in atherosclerotic mice,indicating that sestrinl protein expression level was up-regulated in atherosclerotic inflammatory response.This study has not yet clarified the regulatory mechanism.2.Sestrinl protein level was elevated in macrophages of C57BL/6J mice transfected with LS1,indicating successful transfection.However,the expression levels of NLRP3 and NRRC4 inflammarosomes in the macrophages after transfection of LC and LS1 were both increased,indicating that lentivirus itself would cause the increased expression levels of inflammarosomes,which was the basis for the subsequent test of sestrinl’s regulation of the expression levels of atherosclerotic inflammarosomes.However,IL-1β,Caspase-1 p20 and factors directly related to inflammatory response,were not detected in the two transfected macrophages,and there was no difference in the expression level of pro-caspase-1 p20 in the two groups of macrophages,indicating that the inflammatory pathway was not activated in the two lentiviral transfected macrophages.Sestrinl typing showed that type 1 was dominant in regulating macrophage-mediated inflammatory responses.3.Sestrinl protein inhibited the activation of NLRP3 inflammasome in macrophages of C57BL/6J mice induced by ox-LDL.After LPS and ox-LDL treatment,sestrinl protein level did not change significantly,but sestrin2 protein level increased,which was consistent with previous research results.As an antioxidant,sestrin2 expression was up-regulated in the case of oxidative damage or inflammation.In the case of sestrinl protein expression,the binding level of ACS and NLRP3 was significantly inhibited,and the expression level of pro-inflammatory cytokines IL-1βand caspase-1 p20 was decreased,indicating that sestrinl had a significant inhibitory effect on ox-LDL-induced NLRP3 activation.However,sestrinl protein also showed inhibition of NLRP3 inflammasome in LPS-treated macrophages,suggesting that sestrinl inhibition of NLRP3 is not ox-LDL specific dependent.4.Sestrinl inhibits ox-LDL and induces activation of the NF-κB inflammatory signaling pathway.After treatment of macrophages with ox-LDL alone,the mRNA levels of inflammatory cytokines IL-lα,IL-6 and IL-1β expression were significantly increased,and the expression levels of NF-κB p50 and p65 were increased.Combined with the phosphorylation of IKKβ and the inhibitory effect of IκBα,it was suggested that ox-LDL could definitely induce inflammatory pathway activation.The above pro-inflammatory effects were improved in the presence of sestrinl protein.This indicates that sestrinl protein can inhibit ox-LDL-induced activation of NF-κB inflammatory signaling pathway.5.Sestrinl inhibits NLRP3 inflammasome activation induced by cholesterol crystals.The levels of ACS binding to NLRP3,IL-1β and caspase-1 p20 in macrophages after cholesterol crystallization treatment were significantly increased,while the expression of sestrinl protein significantly inhibited the above pathological process.Moreover,oil red O staining showed that in the presence of sestrinl protein,foam cells in the lesion were reduced and lipid deposition in macrophages was improved.As an important pathological change of atherosclerosis,sestrinl protein played a good role in prevention,but macrophages showed that lipid receptors were not significantly changed.Further studies are needed to determine the cause of the decrease in the number of foam cells.6.Changing the expression level of sestrinl protein in mice can regulate the inflammatory response of atherosclerotic lesions.As shown in results 6 and 7,the expression levels of IL-1β,IL-1β mRNA,IL-6,TNF-α and CXCL1 in the aorta of mice with high expression of sestrinl protein were significantly lower than those of mice transfected with LC lentivirus.After the knockout of sestrinl protein expression,the expression level of inflammatory factors in mice increased significantly.Through these in vivo experiments,we proved that sestrin1 protein can inhibit the inflammatory response associated with atherosclerosis,and in the absence of sestrinl,it can aggravate local inflammation and promote the development of atherosclerotic lesions.In summary,sestrinl protein expression is up-regulated in atherosclerosis.It inhibited the activation of ox-LDL and CHC-induced NLRP3 inflammasome,inhibited the activation of NF-κB inflammatory signaling pathway by regulating the inhibition of IKKβ phosphorylation and enhanced the activation of IκBα,inhibited the secretion of related inflammatory factors,and finally improved the local inflammatory response of atherosclerotic lesions.In addition,sestrinl protein can reduce the lipid deposition under intima and reduce the formation of foam cells,which plays a certain preventive role in relieving local inflammatory reactions.In vivo studies revealed more clearly that sestrinl improved the inflammatory response and tissue damage in the aortic region of atherosclerotic mice.