| BackgroundIt has been verified that the incidence of renal vascular hypertension is closely related to the renin-angiotensin-aldosterone system(RAAS).Meanwhile a higher level of serum matrix metalloproteinase-9(MMP-9)has also been observed in renovascular hypertensive rats,however,the role of MMP-9 in the pathogenesis of renal vascular hypertension remains unclear.In this study,JNJ0966,a specifically inhibitor of the MMP-9 expression,were administrated in the rat models of renovascular hypertension,to observe the effect of the inhibition of MMP-9 expression on the blood pressure level and cardiovascular remodeling in the models,and to explore the role of MMP-9 in the pathogenesis of renal hypertension.Method1.Model establishment:44 8-weeks old SPF male Sprague Dawley rats were randomly divided into sham group(n=8)and two-kidney two-clip renovascular hypertension surgical model group(n=36).After fasting for 12 hours(only water provided),all rats weight and non-invasive tail artery blood pressure were performed before surgical proceeding,10%chloral hydrate anesthesia laparotomy were performed.In the model group,bilateral renal arteries were narrowed by 0.20mm silver clip,while in the sham group,bilateral renal arteries were not narrowed.Postoperative intramuscular injection of benzylpenicillin was performed for 3 days to prevent infection.On the 14th day after operation,non-invasive tail artery blood pressure was measured again to determine whether the model building was successful.2.Experimental grouping:successful rats were randomly divided into three groups:model group,low-dose group and high-dose group,and the same number of sham operation rats were selected as the sham operation group.From the failed model rats,rats with renal pyknosis were selected as the failed model group.3.Method:in the sham group and the model group,1 ml of the solvent(20%β-hydroxypropyl-cyclodextrin)was given to the rats daily,once a day.In the low dose group and the high dose group,10 mg/Kg and 30 mg/kg of JNJ0966 were dissolved in a solvent and 1ml were gavaged to rats daily,once a day respectively.The blood pressure was remeasured on the 8th day of the intervention,then the rats were sacrificed.The left ventricular mass index(LVWI=left ventricular weight/body weight)was calculated.The changes of myocardial interstitial and myocardial cells were observed by HE staining in left ventricular myocardial tissue sections.HE staining of the superior mesenteric artery and abdominal aorta was used to observe and calculate the ratio of artery wall to lumen.The area ratio of collagen fibers in the intima media(i.e.,collagen volume fraction,CVF)was observed by Masson staining.Western blotting was used to detect the MMP-9 level of myocardial tissue;serum MMP-9 and AngⅡ levels were detected by ELISA;urease-glutamate dehydrogenase assay serum urea nitrogen,creatine oxidase assay serum creatinine and enzyme cycle assay serum cysteine.Results1.Modeling and grouping:At the second weekend after surgery,the blood pressure of 8 rats in the sham operation group[(112.094±8.189)mmHg],and blood pressure of 18 rats in the two-kidney two-clip model group increased[(149.45±8.340)mmHg],reaching the diagnostic criteria of hypertension,and the modeling was successful.The blood pressure of the other 11 models failed to achieve the diagnostic criteria of hypertension[(109.369±9.227)mmHgl,among these rats,the blood pressure of 6 rats with renal pyknosis(model-failure group)was[(108.536±6.412)mmHg].7 rats died during and after operation.The results of the grouping were:sham operation group,model group,low-dose group,high-dose group,and model-failure group(n=6,respectively).2.MMP-9 specific inhibitor significantly reduced the blood pressure levels of renovascular hypertensive ratsCompared with the model group[(171.107±15.61)mmHg],the blood pressure of the low dose group[(138.961±2.741)mmHg]and the high dose group[(133.882±10.54)mmHg]decreased significantly(P<0.05),but it was still significantly higher than that of the sham group[(114.189±8.495)mmHg](P<0.05).There was no significant difference between the high dose group and the low dose group(P>0.05).3.MMP-9 specific inhibitor significantly improved ventricular remodeling in renovascular hypertensive ratsMyocardial fiber rupture was observed in the model group.LVWI((2.7678±0.401)mg/g)and myocardial cell cross-sectional area(3294.17±520.53)was significantly higher in the model group than that in the sham operation group((1.844±0.129)mg/g,(1653.50±261.37),respectively)(P<0.05).Compared with the model group,these two indicators of the low dose group((2.278±0.243)mg/g,(1848.83±293.83),respectively),and the high dose group((2.219±0.233 mg/g,(1656.17±324.61),respectively)were decreased significantly(P<0.05),and the myocardial fiber destruction was significantly improved.However,there was no significant difference between the low-dose group and the high-dose group(P>0.05).There was no significant difference in LVMI((2.086 ±0.206)mg/g)between the model-failure group and the sham operation group(P>0.05)4.MMP-9 specific inhibitor significantly improved vascular remodeling in renovascular hypertensive ratsCompared with the sham operation group,the ratio of superior mesenteric artery wall to lumen of the model group was significantly increased((0.876±0.197)vs(1.127±0.275)),and the abdominal aortic CVF was significantly decreased((55.103±13.663)vs(41.491±7.603).)%)(P<0.05).Compared with the model group,the ratio of superior mesenteric artery wall to lumen of the low-dose group(0.825±0.215)and high-dose group(0.853±0.053)was significantly decreased(P<0.05),and the CVF of abdominal aorta was significantly increased((54.083±5.154)%,(60.072±11.731)%,respectively),but the difference between the low dose group and the high dose group was not significant(P>0.05).There was no significant difference in the CVF of superior mesenteric artery and ratio of abdominal aortic wall to lumen between every groups(P>0.05).5.The expression level of MMP-9 protein in myocardium of rats with renovascular hypertension was increased,MMP-9 specific inhibitor significantly reduced the expression level of MMP-9Compared with the sham operation group,the MMP-9 protein level in the myocardium of the model group was significantly increased[(0.120±0.230)vs(3.941±1.904)](P<0.05).After intervention,compared with the model group,the MMP-9 protein levels in the low-dose group(0.153±0.127)and the high-dose group(0.003±0.005)were significantly decreased(P<0.05),which was similar to the sham group(P>0.05),but the high dose group was significantly lower than the low dose group(P<0.05).6.Serum MMP-9 and AngⅡ levels were increased in the renovascular hypertensive rats,MMP-9 specific inhibitor significantly reduced their levelsCompared with the sham operation group,the serum MMP-9 and Angll levels of the model group were significantly increased((20.036±5.989)vs(37.831±16.703)ng/ml,(21.575±4.746)vs(27.567).±4.716)pg/ml)(P<0.05).After intervention,compared with the model group,the serum levels of MMP-9((21.715±4.660),(21.890±9.029)ng/ml),and Angll((22.032±5.180),(19.151±3.347)pg/ml,respectively)of the low-dose group and the high-dose group were significantly decreased(P<0.05),which similar to the sham group(P>0.05),while there was no significant difference between the low dose group and the high dose group(P>0.05).7.There was no abnormality in serum creatinine,urea nitrogen and homocysteine in the renovascular hypertensive ratsCompared with the sham operation group,there was no significant difference in serum creatinine,urea nitrogen and homocysteine between the model group and the sham operation group,however serum urea nitrogen and creatinine levels of low-dose and high-dose group were significantly increased((6.843±1.820);(6.634±1.100)vs(4.587±0.808)mmol/L,(38.333±6.218);(39.00±3.00)vs(28.667±6.408)umol/L,respectively)(P<0.05).The level of surum urea nitrogen,creatinine and homocysteine were significantly higher in the model-failure group(with renal pyknosis)than those in the sham operation group and the model group((7.670±0.963)vs(4.587±0.808)and(5.595±1.222)mmol/L;(40.33±4.590)vs(28.667±6.408)and(30.333±6.831)umol/L;(10.275±1.142)vs(8.010±1.602)and(8.022±0.713)μmol/L,respectively.](P<0.05).ConclusionsSpecific inhibition of MMP-9 expression can significantly reduce the blood pressure and improve cardiovascular remodeling in renovascular hypertensive rats.The increase of MMP-9 may be one of the pathogenesis of renal vascular hypertension. |
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