| 1.BackgroundAcute cardiovascular events caused by atherosclerosis and its complications are the leading killer of human beings.Acute coronary syndrome(ACS)is the key cause of death in coronary atherosclerotic disease.ACS is mainly caused by the rupture of plaque which leads to acute luminal stenosis or occlusion of the coronary artery.The most important cause of ACS is unstable plaque.Growing evidences has shown that the macrophages apoptosis is an important cause of the vulnerable plaque formation.The macrophage apoptosis occurs through every stage of atherosclerosis.In the early stage of atherosclerosis,the macrophages apoptosis can reduce the cellular composition.In the late stage of atherosclerosis,the macrophages apoptosis cause plaque instability.The mechanism of unstable plaque resulted by macrophages apoptosis has two aspects as follows:1.The macrophage apoptosis leads to the reduction of the number of cells,macrophage can not effectively phagocytize the apoptosis smooth muscle cells and macrophages,and secondary necrosis occurs,a large number of cell plasma contents is released,leading to the formation of a plaque lipid core;2.The macrophages apoptosis can lead to the release of intracellular cholesterol,leading to the significant increase of cholesterol crystals in the lipid core of plaque,and can also promote macrophages to secrete a large number of MMPs to damage the fibrous cap,leading to the instability of plaque.The formation of foam cells derived from macrophages is the result of dysregulation of cholesterol intake,storage and efflux.Macrophages phagocyte ox-LDL into cells by scavenger receptors CD36 and SR-A1 on the surface of cells.The phagocytic ox-LDL is converted to free cholesterol under the action of late endosome in macrophages.Macrophages have advanced enzymatic mechanism,and free cholesterol can be converted into cholesterol ester by ACAT1.Cholesterol ester can be converted to free cholesterol by Nceh1.Free cholesterol in macrophages can be transported to the extracellular by ABCA1 and ABCG1,transported to the liver by HDL and then excreted from the body.Ox-LDL contains a large number of active molecules which can cause macrophage apoptosis,such as 25-HC and 7-KC.25-HC and 7-KC in macrophages can lead to ER stress,and long-term ER stress can lead to apoptosis of macrophages.Growing studies have shown that knockdown of ABCA1 or ABCG1 can significantly increase the apoptosis rate of macrophages.Therefore,promoting the efflux of cholesterol from macrophages can reduce the apoptosis of macrophages.CTRP9(C1q-TNF-related protein-9)is highly homologous of adiponectin.CTRP9 has a variety of biological activities,such as improving insulin resistance,anti-atherosclerosis and anti-inflammation.The mechanisms of CTRP9 anti-atherosclerosis as follows:CTRP9 can reduce the injury of endothelial cells,improve the function of endothelial cells,inhibit the phenotypic transformation and proliferation of vascular smooth muscle cells,reduce the release of inflammatory factors in macrophages and inhibit the formation of foam cells.CTRP9 also alleviates myocardial ischemia-reperfusion injury.Currently,the effect of CTRP9 on the macrophages apoptosis in atherosclerotic plaques is not investigated.Therefore,we proposed the following hypothesis:CTRP9 may alleviate the macrophages apoptosis by enhancing cholesterol efflux through AdipoR1/AMPK pathway.2.Objectives(1)The effect of CTRP9 on lipid deposition in macrophages;(2)The effect of CTRP9 on the expressions of cholesterol efflux related proteins in macrophages and the mechanisms involved;(3)Whether CTRP9 alleviates macrophage apoptosis by enhancing cholesterol efflux;(4)The effect of CTRP9 on the macrophages apoptosis and its mechanisms;3.MethodsIn this study,mouse peritoneal macrophages were used as the research object.Experimental methods include Western blot,Oil red "O",CCK8,TUNEL,cellular immunofluorescence and so on.(1)Different concentrations of ox-LDL(0,50,100,150,200?g/mL)and different concentrations of CTRP9(0,0.1,0.3,1,5?g/mL)were used to stimulate the cells for 24h and 12h,respectively.We detected the effects of ox-LDL and CTRP9 on the cell viability.(2)The cells were pretreated with different concentrations of CTRP9(0,0.1,0.3,1?g/mL)for 12h,and then stimulated with 50?g/mL ox-LDL for 24h.We investigated the effects of different concentrations of CTRP9 on lipid deposition in macrophages.(3)The cells were stimulated with 1?g/mL CTRP9 for different times(0h,6h,12h,24h).And macrophges were pretreated with different concentrations of CTRP9(0,0.1,0.3,1 ?g/mL)for 12h,then stimulated with 50?g/mL ox-LDL for 24h.The effects of CTRP9 on the expressions of LXR?,ABCA1,ABCG1 and CYP27A1 were observed.(4)The cells were pretreated with 1?g/mL CTRP9 for 12h,and then stimulated with 50?g/mL ox-LDL for 24h.The effects of CTRP9 on the expressions of ACC,FAS,HMGCR,LDLR,SR-A1,CD36,LRP1,LIPE,Nceh1,ACAT1,ApoE and SR-B1 were investigated.(5)The cells were pretreated with LXR? inhibitor GSK2033(10?M)for 1h,then stimulated with 1?g/mL CTRP9 for 12h,and stimulated with 50?g/mL ox-LDL for another 24h.We explored whether LXRa was involved in the effects of CTRP9 on the expression of ABC A1 and cholesterol efflux.(6)The cells were stimulated with 1?g/mL CTRP9 for different times(0h,6h,12h,24h),and the effect of CTRP9 on PPARy expression was observed.The cells were pretreated with PPARy inhibitor T0070907(1?M)for 1h,then stimulated with 1?g/mL CTRP9 for 12h,and then stimulated with 50 ?g/mL ox-LDL for another 24h to observe whether PPARy was involved in the effects of CTRP9 on the expressions of LXR?,ABCA1 and cholesterol efflux.(7)The cells were pretreated with LXRa inhibitor GSK2033(10?M)and PPARy inhibitor T0070907(1?M)for 1h,then stimulated with 1?g/mL CTRP9 for 12h,and then stimulated with 50?g/mL ox-LDL for another 24h to observe whether CTRP9 alleviated macrophage apoptosis by PPARy/LXRa pathway.(8)The cells were stimulated with 1?g/mL CTRP9 for different times(0h,6h,12h,24h)to observe the effects of CTRP9 on the expressions of Wnt3a and ?-catenin.The cells were pretreated with 1?g/mL CTRP9 for 12h and then stimulated with 50?g/mL ox-LDL for 24h to observe the effect of CTRP9 on the nuclear translocation of ?-catenin.The macrophages were transfected with Ad-Wnt3a for 48h,then pretreated with 1 ?g/mL CTRP9 for 12h and stimulated with 50?g/mL ox-LDL for another 24h to observe whether CTRP9 regulated the expressions of PPARy,LXR?,ABCA1 and cleaved-PARP1 and decreased the macrophages apoptosis through Wnt/?-catenin pathway.(9)The cells were pretreated with AMPK inhibitor Compound C(1.5 ?M)for 1h,and then stimulated with 1?g/mL CTRP9 for 12h and 50 ?g/mL ox-LDL for another 24h to observe whether CTRP9 regulated the expressions of Wnt3a,PPAR?,LXR?,ABCA1 and cleaved-PARP1 through AMPK activation.(10)After transfected with SiAdipoR1 for 48h,CTRP9 was stimulated with 1?g/mL CTRP9 for 12h and 50?g/mL ox-LDL for another 24h to observe whether CTRP9 regulated the phosphorylation of AMPK and the expressions of Wnt3a,PPAR?,LXR?,ABCA1 and cleaved-PARP1 and alleviated the macrophages apoptosis mediated by AdipoR1.(11)Statistical analysis.4.Results(1)ox-LDL decreased cell viability in a dose-dependent manner and CTRP9 did not affect cell viability.Different concentrations of ox-LDL(0,50,100,150,200?g/mL)and different concentrations of CTRP9(0,0.1,0.3,1,5?g/mL)stimulated macrophages for 24h and 12h,respectively.Ox-LDL decreased cell viabilty in a dose-dependent manner(p<0.05).0.1?g/mL,0.3?g/mL,1?g/mL CTRP9 did not affect the cell viability.5?g/mL CTRP9 decreased the cell viability,but there was no statistical significance difference(p>0.05).(2)CTRP9 decreased lipid deposition in macrophages in a dose dependent manner.Different concentrations of CTRP9(0,0.1,0.3,1 ?g/mL)were used to stimulate cells for 12h and 50?g/mL ox-LDL for another 24h.CTRP9 reduced lipid deposition in macrophages in a dose-dependent manner.(3)CTRP9 increased the expressions of cholesterol efflux related proteins.1?g/mL CTRP9 were used to stimulate macrophages for different times(0h,6h,12h,24h)and cells were pretreated for 12h with different concentrations of CTRP9(0,0.1,0.3,1?g/mL)and 50?g/mL ox-LDL for another 24h.CTRP9 increased the expressions of LXR?,ABCA1,ABCG1 and CY27A1 in a time-dependent and concentration-dependent manner.(4)CTRP9 promoted the expressions of PPARy downstream proteins which were cholesterol efflux related proteins by inhibiting Wnt/?-catenin pathway through AdipoR1/AMPK.Compared with ox-LDL group,the expressions of LXR?,ABCA1,ABCG1 and CYP27A1 were significantly increased(p<0.05)in ox-LDL+CTRP9 group,ox-LDL+T0901317(LXRa agonist)group and ox-LDL+Rosiglitazone(PPARy agonist)group.Compared with ox-LDL+CTRP9 group,the expression of ABCA1 in ox-LDL+CTRP9+GSK2033(LXRa inhibitor)group was significantly decreased(p<0.05).Compared with ox-LDL+CTRP9 group,the expressions of LXRa and ABCA1 in ox-LDL+CTRP9+T0070907(PPARy inhibitor)group were significantly decreased(p<0.05).Compared with ox-LDL+CTRP9+Ad-GFP group and ox-LDL+CTRP9 group,the expressions of ABCA1,LXR? and PPAR? in ox-LDL+CTRP9+Ad-Wnt3a group were significantly decreased(p<0.05).Compared with ox-LDL+CTRP9 group,the expression of ABCA1,LXR? and PPAR? in ox-LDL+CTRP9+Compound C group were significantly decreased(p<0.05),and the expression of Wnt3a were significantly increased(p<0.05).Compared with ox-LDL+CTRP9+SiNC group and ox-LDL+CTRP9 group,the expressions of ABC A1,LXR?,PPAR? and the phosphorylation of AMPK in ox-LDL+CTRP9+SiAdipoR1 group were significantly decreased(p<0.05),and the expression of Wnt3a was significantly increased(p<0.05).(5)CTRP9 inhibited the Wnt/?-catenin pathway through AdipoR1/AMPK to promote the expression of PPAR? and reduce the apoptosis of macrophages.Compared with ox-LDL group,the expression of cleaved-PARP1 was significantly decreased in ox-LDL+CTRP9 group(p<0.05).Compared with ox-LDL+CTRP9 group,cleaved-PARP1 expression was significantly increased in ox-LDL+CTRP9+GSK2033 group and LDL+CTRP9+T0070907 group(p<0.05).TUNEL results showed that the apoptosis rate of macrophages in ox-LDL+CTRP9+T0070907 group was higher than ox-LDL+CTRP9 group(p<0.05).Compared with ox-LDL+CTRP9+Ad-GFP group and ox-LDL+CTRP9 group,cleaved-PARP1 expression was significantly increased in ox-LDL+CTRP9+Ad-Wnt3a group(p<0.05).TUNEL results showed that the apoptotic rate of macrophages in ox-LDL+CTRP9+Ad-Wnt3 a group was significantly increased compared with ox-LDL+CTRP9+Ad-GFP group and ox-LDL+CTRP9 group(p<0.05).Compared with ox-LDL+CTRP9 group,cleaved-PARP 1 expression was significantly increased in ox-LDL+CTRP9+Compound C group(p<0.05).Compared with ox-LDL+CTRP9 groups and ox-LDL+CTRP9+SiNC group,cleaved-PARP1 expression and the apoptotic rate of macrophages were significantly increased in ox-LDL+CTRP9+SiAdipoR1 group(p<0.05).5.Conclusion(1)CTRP9 decreased lipid deposition in macrophages;(2)CTRP9 promoted the expressions of cholesterol efflux related proteins;(3)CTRP9 alleviated the macrophages apoptosis by enhancing cholesterol efflux;(4)CTRP9 inhibited Wnt/?-catenin through AdipoR1/AMPK pathway;(5)CTRP9 attenuated the apoptosis of macrophage through the AdipoR1/AMPK pathway.1.BackgroundAtherosclerosis is the common pathological basis of coronary atherosclerotic heart disease,stroke and peripheral vascular diseases.In the early stages of atherosclerosis,monocytes pass through the endothelium space to subintima and differentiate into macrophages.Macrophages phagocyte ox-LDL resulting in intracellular lipid deposition,eventually becoming foam cells.Foam cells formation is an important marker of atherosclerosis and the inhibition of the formation of foam cells is an important target of atherosclerosis prevention and treatment.Reverse cholesterol transport(RCT)is the process that the excess cholesterol in peripheral cells was transferred by the proteins on the cell membrane from intracellular to extracellular to form mature HDL.HDL is transported to liver,where it is selective taken up by SR-B1 on the surface of hepatocytes,converted to bile acids and excreted from the body.RCT is the only way to ger rid of excess cholesterol in the body and is an important mechanism of anti-atherosclerosis.The excess cholesterol in cells was transported by ABCA1 and ABCG1 to ApoA1 and HDL,respectively,to form mature HDL particles which is the initional step of RCT.The initial step of RCT is the rate-limiting step.The expression of ABCA1 and ABCG1 is reduced in atherosclerosis,and overexpression of ABCA1 and ABCG1 can reduce the formation of atherosclerosis.HDL is selective uptaken by SR-B1 on the surface of hepatocytes and cholesterol in liver is excreted from the liver by ABCG5/G8 which are also important steps in RCT.ABCG5/G8 mainly secretes cholesterol in liver cells into bile in the form of bile salts.Study have shown that the formation of atherosclerosis can be reduced by promoting the cholesterol clearance through SR-B1 and ABCG5/G8-dependent manner.Therefore,every step of RCT is closely related to the formation of atherosclerosis and it is also an important target for the prevention and treatment of atherosclerosis.CTRP9(Clq-TNF-related protein-9)is a novel adipocytokine.It is highly homologous to adiponectin and has similar biological functions.CTRP9 plays an important role in the regulation of glucose and lipid metabolism and cardiovascular diseases.Growing studies has demonstrated the protective rol of CTRP9 in atherosclerosis.CTRP9 plays an anti-atherosclerosis role mainly through the following mechanisms:CTRP9 can reduce the damage of endothelial cells and improve the diastolic function of endothelial cells;CTRP9 can inhibit the phenotypic transformation and proliferation of vascular smooth muscle cells,reduce the release of inflammatory factors in macrophages and inhibit the formation of foam cells.CTRP9 also improved myocardial ischemia-reperfusion injury.At present,there is no study about the effect of CTRP9 on reverse cholesterol transport.Therefore,the present study proposed the following hypothesis:reverse cholesterol transport is decreased.Because the expression of reverse cholesterol transport releated proteins was decreased in CTRP9-/-mice.2.Objectives(1)The serum lipid levels of WT mice and CTRP9-/-mice were measured;(2)The cholesterol efflux rate of WT mice and CTRP9-/-mice was detected;(3)The expressions of reverse cholesterol transport related proteins in livers of WTmice and CTRP9-/-mice were detected.3.MethodsIn this study,CTRP9-/-and WT(C57BL/6J)mice were used as research objects.After 8 weeks of high-fat feeding,the serum TC,LDL-C,HDL-C,TG levels of WT and CTRP9-/-mice were detected.WT mice and CTRP9-/-mice were intraperitoneally injected with macrophages loaded by 3?Ci/mL 3H-cholesterol and 50?g/mL ox-LDL.Then,we collected serum,liver and feces.The liquid scintillation counting method was used to determine the cholesterol efflux rate in mice.Immunohistochemical staining was used to detect the expression of SR-B1 and ABCG5/G8 in livers of CTRP9-/-and WT mice.4.Results(1)The lipid levels in serum.After 8 weeks of high-fat feeding of WT and CTRP9-/-mice,serum concentrations of TC,LDL-C,TG,and HDL-C were measured.There were no significant differences in TC,LDL-C,HDL-C and TG between WT ND group and CTRP9-/-ND group(p>0.05).TC,LDL-C,HDL-C and TG in WT HFD group and CTRP9-/-HFD group were significantly increased(p<0.05).LDL-C and TG in CTRP9-/-HFD group were significantly higher than WT HFD group(p<0.05).However,HDL-C in CTRP9-/-HFD group was significantly lower than WT HFD group(p<0.05).(2)The cholesterol efflux rate of CTRP9-/-HFD group was reduced.At 24h,counts in serum of CTRP9-/-ND group and CTRP9-/-HFD group were significantly lower than WT ND group and WT HFD group(p<0.05).At 48h,counts in serum of CTRP9-/-HFD group were significantly lower than WT HFD group(p<0.05),and there was no significant difference between CTRP9-/-ND group and WT ND group(p>0.05).Counts in liver and feces of CTRP9-/-ND group were no significantly different from WT ND group(p>0.05).There was no significant difference between CTRP9-/-HFD group and WT HFD group(p>0.05).Counts in feces of CTRP9-/-HFD group was significantly lower than WT HFD group(p<0.05).(3)The expressions of SR-B1 and ABCG5/G8 in liver of CTRP9-/-HFD group were decreased.Compared with WT ND group,the expression of SR-B1 in liver was no significant difference in CTRP9-/-ND group(p>0.05).However,the expression of SR-B1 in liver of WT HFD group was significantly higher than CTRP9-/-HFD group(p<0.05).There was no significant difference of the ABCG5/G8 expression in liver of CTRP9-/-ND group and WT ND group(p>0.05).Compared with CTRP9-/-HFD group,ABCG5/G8 expression in liver of WT HFD group was significantly increased(p>0.05).5.Conclusions(1)TC,LDL-C,TG were increased and HDL-C was decreased in serum of CTRP9-/-HFD group;(2)The cholesterol efflux rate of CTRP9-/-HFD group was significantly reduced;(3)The expression of reverse cholesterol transport-related protein in liver were significantly decreased in CTRP9-/-HFD group. |
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