Effect Of Probucol And Irbesartan, Alone And In Combination, On Insulin PI3K/Akt Signaling And Other Related Factors Of Hypertension

Objective:(1) To investigate the effect of probucol and irbesartan on blood pressure,insulinresistance,endothelial function,oxidative stress and kidney interstitial fibrosis ofSHR.(2) To investigate the effect of probucol and irbesartan on insulin PI3K/Aktsignaling pathway of SHR.(3) To investigate the effect of probucol and irbesartan on oxidative stressimpairment,endothelial dysfunction and other related factors of HUVEC.Methods(1) In the first part of animal experiments,hypertension model rats were treatedwith probucol and irbesartan.Then,the change of hypertension,heart rates weremeasured.The glycometabolism and serum eNOS,NO,SOD,MDA were measuredwith biochemistry methods.The level of serum insulin,plasma Angâ…¡and kidneyAngâ…¡were measured with radio-immunity methods.The change of serumadiponectin and PAI-1 were measured by ELISA.The pathological change of kidneywas investigated after stained by HE.The CVF of kidney interstitial was calculatedafter stained by trinitrophenol-sirius red.(2) The change of the key protein of the insulin PI3K/Akt signaling pathway ofSHR after treated with probucol and irbesartan was measured by the method ofwestern blotting.(3) HUVECs were cultured byâ… type collagenase,andâ…§factor was identifiedby immunohistochemisty.The endothelium cell model of oxidative stress was madeby H₂O₂,the level of NO,NOS,SOD,MDA,PAI-1 of the cell culture fluid aftertreated with probucol and irbesartan were measured.Results(1) Part 1:1) Compared with SHR-C group,the blood pressure of SHR-Irb groupand SHR-Pro+Irb group were lower.2) The result of OGTT showed there wereglucose utilization dysfunction in SHR-C group and SHR-Pro group.3) The level ofplasma Angâ…¡and kidney Angâ…¡of SHR-Irb group and SHR-Pro+Irb group were lower than those of SHR-C group.4) The level of serum NO,SOD and PAI-lofSHR-Irb,SHR-Pro+Irb groups were higher than those of SHR-C group.5) The levelof serum eNOS and adiponectin of SHR-C group was lower than those of SHR-Pro,SHR-Irb,SHR-Pro+Irb group.6) The level of serum MDA and kidney interstitialCVF of SHR-C group was higher than those of the other three SHR groups.(2) Part 2:1)There had no difference of the level of liver Akt relative protein inSHR groups and WKY group.2) Compared to SHR-C group,the level of liverp-Akt(Thr308) relative protein of the other three SHR groups were higher,among thethree groups,SHR-Pro+Irb group was the most highest.3) The level of liverp-Akt(Ser473) relative protein of the SHR-Irb and SHR-Pro+Irb group were higherthan that of SHR-C group.(3) Part 3:1) Theâ…§factor of HUVEC was identified by the method ofimmunohistochemisty,and a lot of brown and finely ground particle can be seen.2)The effect of different concentration of probucol and irbesartan on HUVEC under theoxidative stress state,the OD of C,Pro2(5μM),Pro3(10μM),Irb3(10μM),Pro3(10μM)+Irb3(10μM) group were higher than that of H group.3) The level ofNO in culture fluid of C,Prol(2.5μM),Pro2 (5μM) and Pro3(10μM) group werelower than that of H group.4) The level of iNOS in culture fluid of C,Pro2 (5μM)and Pro3(10μM) group were lower than that of H group.5) The level of eNOS inculture fluid of C,Pro2 (5μM) and Pro3(10μM) group were higher than that of Hgroup.6) The level of SOD in culture fluid of C group was higher than that of Hgroup,and there had no difference in those of Pro 1 (2.5μM),Pro2 (5μM),Pro3(10μM)and H group.7) The level of MDA in culture fluid of C,Prol(2.5μM),Pro2 (5μM)and Pro3(10μM) group were lower than that of H group.8) The level of PAI-â… inculture fluid of C and Pro3(10μM) group were lower than that of H group.9) Thelevel of NO in culture fluid of C,Irb2(5μM) and Irb3(10μM) group were lower thanthat of H group.10) The level of iNOS in culture fluid of C,Irb3(10μM) group werelower than that of H group.11) The level of eNOS in culture fluid of C,Irb3(10μM)group were higher than that of H group.12) The level of SOD,MDA and PAI-1 inculture fluid of C,Irb 1 (2.5μM),Irb2(5μM) and Irb3(10μM) group were higher thanthat of H group.13) Compared to Pro+ Irb group,the level of NO in culture fluid of C group was lower,however,those of H and Irb (10μM) group were higher.14)Compared to Pro + Irb group,the level of iNOS in culture fluid of C group was lower,however,those of H,Pro (10μM) and Irb (10μM) group were higher.15) Comparedto Pro+Irb group,the level of eNOS in culture fluid of C group was higher,however,those of H and Pro (10μM) group were lower.16) Compared to Pro+ Irb group,thelevel of SOD in culture fluid of C group was higher,however,those of H and Pro(10μM) group were lower.17) Compared to Pro+Irb group,the level of MDA inculture fluid of H and Pro (10μM) group were higher.18) Compared to Pro+Irbgroup,the level of PAI-1 in culture fluid of H and Pro (10μM) group were higher.Conclusion(1) Oxidative stress had the closely relationship with insulin resistance andendothelial dysfunction.Probucol and irbesartan could improve insulin sensitivityand endothelial function of SHR.The mechanism of irbesartan to improve oxidativestress maybe concerned with SOD,besides irbesartan itself could inhibit NADPHoxidase.In this experiment,the antioxidant probucol did not decreased the bloodpressure of SHR,however,it played an antihypertension effect in combination withirbesartan.Irbesartan could decrease the blood pressure and the level of serum PAI-1of SHR.(2) The progress of kidney impairment according to hypertension was complex.The change of RAAS and oxidative stress in kidney of SHR related to the kidneyimpairment of SHR.And probucol and irbesartan could postpone the kidney fibrosisof SHR and had a certain degree of protection to kidney.(3) In the glycometabolism target tissue,the PI3K/Akt signaling mediated theimportant progress of glycometabolism such as glycogen synthesis and glucosetransport;in the endothelium,the same signaling pathway mediated the NOproduction and maintain the normal function of vascular endothelium.Theimpairment of the phosphorylation of Akt Thr308 of insulin PI3K/Akt signalingpathway may be a cause for insulin resistance of SHR.Procucol and irbesartan couldimprove the insulin sensitivity by increasing the phosphorylation ofAkt Thr308.(4) Oxidative stress could injury PI3K/Akt/eNOS signaling pathway in HUVEC.However,probucol and irbesartan could increase the clearance of oxygen radicals in endothelium,and then protected the normal function of endothelium.They maybehave some kind of synergistic reaction according to improve the increased level ofPAI-1 caused by oxidative stress.