The Action And Mechanism Of The Slow Release Hydrogen Sulfide Donor SMP-12 On Atherosclerosis

Objective:Atherosclerosis(AS)is a serious disease endangering human health.The disorder of lipid metabolism is a major cause of AS,and the formation of foam cells is the key step.These suggest that abnormal expression of HMG-CoA,LDLR,NF-κB and COX-2 influence the development of AS.H2S isrecognized as a novel gaseous signal moleculer.It plays important roles in anti-oxidant,anti-apoptosis,anti-inflammatory,anti-fibrosis,the protection of cells and blood vessels,myocardial protection,anti-AS.In our study,we used SMP-12 as an H2S slow release donor,which was carried by SMP-10 and the chemically is stable.It overcomes the shortcomings of NaHS oxidation and toxicity of preparing the solution of 42S.And the slow releasing H2S by SMP-12 is important to simulate the endogenous of H2S.This study is investigated the effect of the SMP-12 on AS compared with the NaHS,and then explore its mechanism of anti-AS.Methods:Animal experiments:the clean grade of healthy male SD rats randomly divided into five groups(230 ± 10)g,and each group has 5 rats for a three-month modeling.①Normal group:basal diet;②Model diet groups:high-fat diet + saline gavage;③NaHS group:high-fat diet + NaHS gavage;④SMP-12 group:high-fat diet+ SMP-12 gavage;⑤SMP-10 group:high-fat diet+SMP-10 gavage.Each group rats inject the vitamin D3 except the normal group for accumulation of 80 million units of vitamin D3.Plasma was measured for TC,TG,LDL-C,HDL-C before and after modeling levels.The HE staining ofaortic and liver,and the plasma content of H2S were determinated.Then we determinated the total cholesterol of liver by the total cholesterol kit.Meanwhile the mRNA and protein of HMG-CoA reductase and LDLR were quantitated by qRT-PCR and Western-blot.Cell experiments:The cells of RAW264.7 were cultured and passaged and then seed into 6-well plates in logarithmic phase.Normal group:serum-free medium for 48 hours;Model group:serum-free DMEM+60mg/L Ox-LDL cultured 48h;NaHS,SMP-12,SMP-10 group:serum-free DMEM was added to a final concentration of 60mg/L Ox-LDL for 24h culture + NaHS(250μmol),SMP-12(25μmol),SMP-10(50μmol)for another 24h.To detect the accumulation of the lipid in macrophages by enzymatic assay and Oil Red O staining,while collecting six-well plates for each group of cells to extract RNA and proteins to detect the mRNA and protein of NF-κB and COX-2 by qRT-PCR and Western-Blot.Results:The results of animal experiments:The serum lipids were normal and no differences between groups before modeling;The TC,LDL-C of the model group are increased compared with the normal group after modeling,The TC,LDL-C of NaHS and SMP-12 group are reduced compared with model group,especially the SMP-12 group.The HDL-C of the SMP-12 group isincreasedly,all of them are statistically significant.The normal group of HE staining of aorta have uniform aortic wall,and the intimal smooth muscle cells arranged in neat rows in the film,having no AS plaque;While the model group showed obvious atherosclerotic plaques and shedding,SMP-12 group,NaHS group are significantly reduced atherosclerotic lesions,especially the SMP-12 group of the vessel wall integrity,only part of the thickening,and have no plaque.After the HE staining of liver,thenormal liver cells were polygonal,larger,arranged in neat rows.There are large,round central core,no intracellular lipid droplets,and lobular orderliness.The liver cell of the model and SMP-10 group were swelling,and have many lipid droplets,and nuclear home on-side mainly.The lipid droplets in the SMP-12 and NaHS were significantly reduced,especially the SMP-12 group.The HE staining of liver is consistent with the result of total cholesterol.So the model of atherosclerosis proved to be successful.The content of H2S of plasma of model group were lower than normal;while the NaHS and SMP-12 groups were significantly increased,especially the SMP-12 group.The mRNA and protein of NF-κB and COX-2 are deteceed by qRT-PCR and Western-Blot,which showed that the SMP-12 and NaHS groups were significantly reduced HMG-CoA reductase mRNA and protein levels and increased LDLR mRNA and protein expression.The results of cells experiments:Ox-LDL leads to deposition of intracellular lipid,which suggested that the foam cells are formationed.The SMP-12,NaHS,and SMP-10 groups can inhibit the accumulation of lipid in macrophages,and inhibit the formation of foam cells,especially the SMP-12 group.The NaHS and SMP-12 groups can inhibit the mRNA and protein of NF-κB and COX-2.The effect of SMP-12 is better.Conclusion:SMP-12 and NaHS have the effect of anti-atherosclerotics and the SMP-12 was better.The mechanism may be related to the inhibition of NF-κB and COX-2,lessen the expression of HMG-CoA reductase and theaugment expression of LDLR,SMP-12 is better for thetreatment of AS.