Studies On The Differential Expression Of NAD(P)H Oxidases In The Development Of Cardiac Hypertrophy And The Effect Of Atorvastin

OBJECTIVE To detect the expression changes of NAD(P)H oxidaseregulation isoform P47phox and catalytic subunit NOX homologues in thedevelopment of the cardiac hypertrophy induced by two kidney two clip andinvestigate the effects of atorvastin on cardiac hypertrophy and the mechanismrelgulated by NAD(P)H oxidase. METHODS The two kidney two clip method(two-kidney two-clip,2K2C) was used to build renovascular hypertension rat model,the rats which the blood pressure was higher than160mmHg was selected for theexperiment at the fourth week after surgery. The experiment was divided into two parts.In the first one, rats were divided into6groups,10rats in each group：sham-operatedrats for4weeks group (S4);2K2C rats for4weeks group (K4); S8: sham-operated ratsfor8weeks group;K8:2K2C rats for8weeks group;S12:sham-operated rats for12weeks group;K12:2K2C rats for12weeks group, investigate differential expression ofNAD(P)H oxidase in different period. In the second part, rats were divided into5groups,10rats in each group:(1) Sham: rats received sham operation and were giventhe same amount of normal saline everyday；(2)2K2C: rats received two-kidney two-clip operation and were given the same amount of normal saline everyday；(3)AtoH:2K2C rats were given atorvastatin (10mg·㎏-1per day)；(4) AtoL:2K2C rats weregiven atorvastatin (5mg·㎏-1per day)；(5) Can:2K2C rats were given candesartan(10mg·㎏-1per day). All groups were intragastric administered medicine for4weeksafter surgery. Blood pressure and hemodynamic parameters were measured. Leftventricular weight to body weight (LVW/BW) ratio was calculated. Paraffin-embeddedhearts were cut into5μm slices, which were stained with hematoxylin-eosin (HE) andMASSON for morphological analysis. The oxidative fluorescence dihydroethidiumdye determined by laser confocal fluorescent microscopy was used to evaluate in situO2–generation in the LV. Western-blot analysis and PCR was performed to investigate ANF, BNP, β-MHC and the mRNA and protein expression of P47phox and NOX2，NOX4. RESULTS (1) Compared with sham-operated group, the AoSP、AoDP、LVESP、LVEDP and LVW/BW ratios were markedly elevated in2K2C groups at thefourth week after surgery while±dp/dtmaxwas significantly decreased. The expressionof ANF, BNP and β-MHC increased significantly in a time-dependent manner.(2) ThemRNA transcription and protein expression of NOX2, NOX4and p47phox in2K2Crats increased significantly at4,8,12week after operation.(3) The drugs wereadministered for4weeks,±dp/dtmaxincreased significantly in atorvastin or candesartangroups. However, atorvastin had no significant blood-lowing effects compared withcandesartan.(4) HE staining showed that the hypertrophy increased significantly inleft ventricle in2K2C rats. MASSON staining showed that the collagen content inmyocardium increased significantly. Both atorvastin and candesartan partly reversedthese effects，the high dose atorvastin have better anti-hypertrophy effects.(5)Generation of ROS in LV increased greatly in2K2C rats while ROS in myocardiumdecreased sharply in the rats treated by atorvastin and candesartan.(6) The mRNA andprotein expression of NOX2, NOX4and p47phox increased significantly in2K2C ratsbut they were down-regulated after treament with atorvastin and candesartan.CONLUSIONS (1) The expression levels of P47phox, NOX2and NOX4increasedevelopment of cardiac hypertrophy in a time-dependent manner, which indicates thatNAD(P)H oxidase contribute to the deteriorate of cardiac hypertrophy.(2) Atorvastinand candesartan had a remarkable inhibitory effect on the expression of NOX2, NOX4and p47phox, which may be the one of their anti-hypertrophy mechanisms.