The Effects Of Let-7b On Oxidative Stress In Atherosclero-Sis And The Contribution Of Rosuvastatin And/or Probucol

Background Oxidative stress has played critical role in atherosclerosis.Recently,the pleiotropic effects apart from lowering serum cholesterol levels of rosuvastatin and probucol are widespread studied.The combined effects of rosuvastatin and probucol on oxidative stress have never been covered.Studies have demonstrated that microRNA play a significant role in atherosclerosis,miRNA lethal-7b(let-7b),which first found in human,is found highly expressed in endothelial cells and taken part in the oxidative stress in hepatocytes.But it is unknown whether let-7b play an important role in oxidative stress during atherosclerosis.The impact of rosuvastatin and/or probucol on let-7b has never been clarified.Objective:To investigate the effects and mechanism of rosuvastatin and/or pro-bucol as well as let-7b on oxidative stress in HUVEC from molecular bio-log y level and protein level in vitro.And to elucidate the underlying mechanisms of rosuvastatin and/or probucol as well as let-7b on oxidative stress in AS b y pat-hological and molecular processes in vivo.Methods:The experiment was divided into three parts.First,H2O2(50μmol/L)was used to stimulat cultured HUVEC for 24h to construct oxidative stress cellular model,the content of MDA and SOD was deceted,then let-7b mimics/inhibitor was transiently transfected into HUVECs,let-7b and the mRNA levels and protein levels of Bach 1,the competitive Nrf2 and HO-1 were detected with real-time RT-PCR and western blotting,to confirm the let-7b on oxidative stress,In the second part,rosuvastatin,probucol and and combination of the two drugs were added into the HUVEC stimulated by H2O2.Then let-7b inhibitor was transiently transfected into HUVECs,above-mentioned indexes were detected as before.In the third part,C57BL/J6 mice were administered with high fat diet as control group(C,n=8).30 apoE-/-mice(divided into 4 groups)were fed with the same diet for 12 weeks to construct AS models(M,n=8),among these groups,probucol group(P,n=7),rosuvastatin group(R,n=7)and combination group(PR,n=7)were feeded with probucol,rosuvastatin and two drugs separately.Animals were sacrificed after 12 weeks,the serum lipid profiles and SOD/MDA were measured,HE stain,Masson stain and Sudan black stain were carried out.let-7b was detected with real-time RT-PCR,protein levels of Bach1,the competitive Nrf2 and HO-1 were decected by immunohistochemistry and western blotting.Results:First,the celluar model of HUVEC was successfully structed,The expression of let-7b was lowered by H2O2(50μmol/L)in HUVEC than control group(P<0.05),well the Bachl was improved,Nrf2 and HO-1 were decreased(P<0.05).The level of Bachl protein was decreased well Nrf2 and HO-1 were increased(P<0.05)after overexpression of let-7b.In the second part,The levels of SOD were increased well the MDA were descended;The levels of Bachl descended and the levels of Nrf2 and HO-1 protein improved after administered with probucol,rosuvastatin and combination separately(P<0.05),while the levels changed after transfectd with let-7b inhibitor.In the last part,the AS animal model was succeed;the levels of TC,LDL and apoB obviously increased in the M group,while HDL decreased.The levels of TC,LDL and apoB could be down regulated by both probucol and rosuvastatin and the combination could get lower result(P<0.05).HE stain,Masson Stain and Sudan black stain showed prominent atheromatous plaque in M group,rosuvastatin and probucol both could hamper the progress of plaque well combination of the two drugs could prevent AS.The high levels of let-7b,Nrf2 and HO-1 protein were detected in aorta of M group,and the much higher expression of let-7b,Nrf2 and HO-1 were detected after drugs were used,while Bachl protein was decreased(P<0.05).Conclusion:1.The oxidative stress model of HUVEC was successfully structed by stimulated by H2O2 50umol/L for 24h.2.Overexpression of let-7b in HUVEC could suppress the Bachl,then improve the Nrf2 and HO-1 level,which indicates let-7b as an protector for HUVEC under oxidation through the passageway of Bachl/Nrf2-HO-1.3.Probucol and rosuvastatin could improve the oxidative stress probably by the anti-oxidation effects,the mechanism might including improving the level of HO-1 by up-regulating the expression of let-7b,which could inhibit the Bachl.The combination of probucol and vrosuvastatin have the synergism effect.4.The animal model of AS could be constructed by apoE-/-mice fed with high fat diet for 12 weeks.Probucol and rosuvastatin could hamper the atheromatous plaque through decreasing cholesterol and by the anti-oxidation effects.The underlying mechanism might include improving the level of HO-1 by up-regulating the expression of let-7b,which could inhibit the Bachl.The combination of probucol and rosuvastatin have the synergism effect.