Over-expression of ACE2 gene alleviates Ang Ⅱ-induced oxidative stress in neural cells

AIM:To investigate the effect of over-expression of angiotensin-converting enzyme 2 (ACE2) gene on angiotensin Ⅱ (Ang Ⅱ)-induced oxidative stress and NADPH oxidase (NOX) expression in mouse neuroblastoma Neuro-2A cells. METHODS:The recombinant lentivirus encoding ACE2 gene was constructed and transfected into the Neuro-2A cells at a multiplicity of infection (MOI) of 10 for 72 h. The transfection efficiency of ACE2 gene and protein expression of ACE2 were detected, and the Neuro-2A cells were identified by detection of a neural cell marker. The Neuro-2A cells were divided into 7 groups:control group, eGFP group, ACE2-eGFP group, Ang Ⅱ treatment group, Ang Ⅱ-eGFP group, Ang Ⅱ-ACE2-eGFP group and Ang Ⅱ-ACE2-eGFP-A779 group. The Ang(1-7) level was determined by ELISA. The level of reactive oxygen species (ROS) in the cells was measured with a method of DHE staining. The protein expression of MAS receptor and NOX subunits (NOX2, NOX4, p47^phox and p67^phox) was detected by Western blot. RESULTS:Ang Ⅱ signi-ficantly increased ROS levels (P<0.01) and up-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox (P<0.01), but down-regulated MAS protein expression (P<0.01). Over-expression of ACE2 inhibited Ang Ⅱ-induced increase in ROS, down-regulated the protein expression of NOX2, NOX4, p47^phox and p67^phox,and still increased the Ang(1-7) level (P<0.01) and MAS receptor expression (P<0.01). An antagonist of the MAS receptor, A779, blocked the down-regulating effect of ACE2 on NOX expression (P<0.05). CONCLUSION:ACE2 over-expression antagonizes Ang Ⅱ-induced oxidative stress via MAS receptor in the neural cells.     

Angiotensin II regulates catalase expression and promotes fibroblast phenotype transformation through ERK1/2 pathway

AIM: To explore the effect of extracellular signal-regulated kinase 1/2 (ERK1/2) on the vascular adventitial remodeling in a hypertension rat model. METHODS: The rats were randomly divided into control group, mini-pump infusion of saline group and mini-pump infusion of angiotensin II (Ang II) group as the hypertension model. The systolic pressure and vascular morphology of the rats were examined. Adventitial fibroblasts were treated with Ang II, PD98059 (ERK1/2 inhibitor) and Ang II+PD98059. The catalase (CAT) expression in the cells was detected by Western blotting. RESULTS: Compared with control group and mini-pump infusion of saline group, the systolic pressure and carotid media thickness (stained by HE) in mini-pump infusion of Ang II group were significantly increased (P<0.01). Meanwhile, artery morphology in mini-pump infusion of Ang II group had obviously changed with a significant occurrence of pathological vascular remodeling. The result of Western blotting showed that the expression of CAT in the adventitial fibroblasts treated with Ang II+PD98059 was much higher than that in the cells treated with Ang II alone (P<0.05), indicating that down-regulation of CAT induced by Ang II was restored by ERK1/2 signaling pathway. CONCLUSION: Ang II down-regulates CAT through ERK1/2 pathway and promotes cell phenotype transformation, which lead to pathological vascular remodeling.     

Effects of short-chain acyl-CoA dehydrogenase on hypertensive vascular remodeling

AIM: To investigate the changes of short-chain acyl-CoA dehydrogenase (SCAD) in hypertensive vascular remodeling and to explore the relationship between SCAD and vascular remodeling in hypertension.METHODS: The spontaneously hypertensive rats (SHR; 24 weeks old) and Wistar rats (24 weeks old) were used as experimental control groups. The SHR and Wistar rats of 16 weeks old were trained by swimming as experimental groups. The systolic pressure was measured periodically. The thickness of vascular wall and the diameter of the vascular lumen were measured. The contents of ROS and ATP, the enzyme activity of SCAD, and the expression of SCAD at mRNA and protein levels in the aorta were determined. The free fatty acid in the serum and aorta was also measured.RESULTS: Compared with Wistar group, the diameter of vascular lumen decreased in SHR group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR group were increased significantly (P<0.05). Compared with SHR group, the diameter of vascular lumen increased in SHR+swim group. The thickness of vascular wall, the ratio of vascular wall and the diameter of vascular lumen, and the blood pressure in SHR+swim group were decreased significantly. Compared with control group, the expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, and the content of ATP were decreased in SHR group. However, the free fatty acid in the serum and aorta, and the content of ROS in the aorta were increased in SHR group. The expression of SCAD at mRNA and protein levels, the enzyme activity of SCAD, the content of ATP were increased in Wistar+swim group and SHR+swim group. However, the free fatty acid in serum and aorta, and the content of ROS in the aorta were decreased in Wistar+swim group and SHR+swim group.CONCLUSION: Decrease in SCAD expression may be associated with hypertensive vascular remodeling. Swimming training can reverse hypertensive vascular remodeling by increasing the expression of SCAD in the aorta.     

Up-regulation of miR-133a expression attenuates myocardial fibrosis in spontaneously hypertensive rats

AIM: To investigate the effect of up-regulated expression of microRNA-133a (miR-133a) on myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Wistar-Kyoto (WKY) rats with homologous normal blood pressure served as the normal control group. SHR were divided into SHR group, SHR+ adeno-associated virus (AAV) group and SHR+miR-133a-AAV group randomly. miR-133a carried by miR-133a-AAV was transfected into SHR heart by coronary perfusion. The rat tail artery pressure was monitored. The myocardial collagen deposition was observed by Masson staining. The expression of miR-133a in myocardial tissue was detected by real-time PCR. The protein levels of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) were determined by immunohistochemistry and Western blot. RESULTS: Compared with the WKY rats, the tail artery pressure of the SHR increased significantly. The expression of miR-133a in heart decreased, and the expression levels of TGF-β1 and CTGF increased (P<0.05), and myocardial fibrosis occurred. After up-regulating the expression level of miR-133a in the heart of SHR, the myocardial fibrosis was significantly reduced, and the expression levels of TGF-β1 and CTGF decreased (P<0.05). CONCLUSION: Up-regulation of the miR-133a expression improves myocardial fibrosis induced by hypertension, which may be related to inhibiting the protein expression of TGF-β1 and CTGF in myocardium.     

PP2A activation in mesenteric arteries of angiotensin Ⅱ-induced hypertensive rats

AIM: To investigate the mechanism of protein phosphatase 2A (PP2A) activation in mesenteric arteries of angiotensinⅡ(AngⅡ)-induced hypertensive rats. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to AngⅡinfusion (500 ng·kg^-1·min^-1) using osmotic minipump up to 14 d to established the hypertension model. The rats (n=40) were randomly divided into 4 groups:control group (n=10), AngⅡgroup (n=10), candesartan (CAN; AngⅡtype 1 receptor blocker)+AngⅡgroup (n=10) and CAN group (n=10). The rats in CAN+AngⅡgroup and CAN group were administered with candesartan ester at the dose of 10 mg·kg^-1·d^-1 by gavage on the first day after implantation of osmotic minipump. The rats were sacrificed on the 15th day after minipump implantation. Serum and mesenteric arteries were collected. Systolic blood pressure was measured by tail-cuff method. The serum levels of AngⅡ were measured by ELISA. The protein levels of endothelial nitric oxide synthase (eNOS), phosphorylated eNOS (Ser1177), PP2A catalytic subunit (PP2Ac), phosphorylated PP2Ac (Tyr307) and PP2A inhibitor 2 (I₂^PP2A) in the mesenteric arteries were determined by Western blot. The activity of PP2A in the arteries was detected using PP2A activity assay kit. RESULTS: Compared with control group, the systolic blood pressure in AngⅡgroup was significantly increased(P<0.05), while those in CAN+AngⅡgroup and CAN group were significantly decreased (P<0.05). The serum levels of AngⅡ in AngⅡ group and CAN+AngⅡ group were significantly higher than that in control group (P<0.05). Compared with control group, the phosphorylation levels of eNOS Ser1177 were decreased in AngⅡgroup (P<0.05), but the activity of PP2A was significantly increased (P<0.05), and Pearson correlation analyses showed a negative correlation between PP2A activity and eNOS S1177 phosphorylation (r=-0.842, P<0.05). Compared with AngⅡgroup, the phosphorylation levels of eNOS Ser1177 in CAN+AngⅡgroup were significantly increased (P<0.05), but the activity of PP2A was reduced (P<0.05). Compared with control group, the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries were decreased in AngⅡgroup (P<0.05), but increased in CAN+AngⅡgroup (P<0.05). No significant difference in all above-mentioned measures between control group and CAN group, nor in the levels of total eNOS and PP2Ac protein expression among all the groups was observed. CONCLUSION: AngⅡmay reduce the protein levels of phosphorylated PP2Ac (Tyr307) and I₂^PP2A in the mesenteric arteries of AngⅡ-induced hypertensive rats through AngⅡ/AngⅡ type 1 receptor-mediated signaling pathway, resulting in the activation of PP2A, then leading to down-regulation of eNOS S1177 phosphorylation, which ultimately mediates the occurrence of vascular endothelial dysfunction.     

Effects of sodium hydrosulfide on cardiac function and activity of renin-angiotensin-aldosterone system in rats with chronic heart failure

AIM: To investigate the effect of sodium hydrosulfide (NaHS) on cardiac function and activity of renin-angiotensin (Ang)-aldosterone (ALD) system (RAAS) in the rats with chronic heart failure (CHF).METHODS: The CHF rat model was established by abdominal aortic coarctation. SD rats were randomly divided into sham operation group, model group, low dose of NaHS group and high dose of NaHS group (n=6). The left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD) and left ventricular ejection fraction (LVEF) were measured before and after treatment by echocardiography in each group. The levels of renin, AngⅡ and ALD in the plasma were measured by ELISA. The expression of angiotensin-converting enzyme (ACE) and angiotensin Ⅱ type 1 receptor (AT1R) at mRNA and protein levels in the myocardium tissues was determined by qPCR and Western blot, respectively.RESULTS: After treatment with NaHS, compared with model group and before treatment, LVEDD and LVESD in low dose of NaHS group and high dose of NaHS group were decreased significantly, while LVEF was increased significantly (P<0.05). Compared with low dose of NaHS group, LVEDD and LVESD were decreased, while LVEF was increased in high dose of NaHS group (P<0.05). Compared with sham operation group, the levels of renin, AngⅡ and ALD in the plasma of model group were significantly increased (P<0.05), and the expression of ACE and AT1R at mRNA and protein levels in the myocardium tissues of model group were significantly increased (P<0.05). Compared with model group, the plasma levels of renin, AngⅡ and ALD in low dose of NaHS group and high dose of NaHS group were significantly decreased (P<0.05), and the myocardial expression of ACE and AT1R at mRNA and protein levels was significantly down-regulated (P<0.05). The plasma levels of renin, AngⅡ and ALD, and the myocardial expression of ACE and AT1R at mRNA and protein levels in high dose of NaHS group were significantly lower than those in low dose of NaHS group (P<0.05).CONCLUSION: NaHS inhibits the activation of RAAS, thus improving the cardiac function of CHF rats, and the effect of high-dose NaHS is better than that of low-dose NaHS.     

Preliminary study on relationship between renin-angiotensin-aldosterone system and hypertension in coal miners

AIM: To study the relationship between renin-angiotensin-aldosterone system and hypertension in coal miners. METHODS: The coal miners received questionnaire investigation and their blood pressure, height and weight were measured, plasma renin activity (PRA), plasma angiotensin Ⅱ(Ang Ⅱ) and aldosterone (ALD) were tested by means of radio immunoassay in coal miners with hypertension and nor-hypertension. RESULTS: It was found that levels of PRA, Ang Ⅱ and ALD were significantly higher in hypertensive group than in nor-hypertensive group (all P<0.01), average blood pressure had positive correlation with levels of PRA, Ang Ⅱ and ALD in all miners (r=0.371, r=0.412 and r=0.352). In coal miners with hypertension, levels of PRA, Ang Ⅱand ALD were significantly higher in underpit operation group than in ground operation group (all P<0.01), and install-repair group's were highest in underpit operation group; Levels of PRA, Ang Ⅱand ALD were significantly higher in high-salt diet group than in normal diet group (all P<0.01), but there was no significant change in age, length of service, family history, body mass index, smoking and alcoholism on levels of PRA, Ang Ⅱ and ALD. CONCLUSION: The renin-angiotensin-aldosterone system may potentially mediate underpit operation-and salt-induced hypertension pathogenesis in coal miners.     

Effect of rhynchophylline on TGF-β1/Smad pathway for processing ventricular remodeling in spontaneously hypertensive rats

AIM: To investigate the effect of rhynchophylline (Rhy) on blood pressure, cardiac hypertrophy and myocardial fibrosis in spontaneously hypertensive rats (SHR). METHODS: Spontaneously hypertensive rats were randomly divided into model group, high dose (10 mg·kg^-1·d^-1) and low dose (2.5 mg·kg^-1·d^-1) group of rhynchophylline, captopril group (17.5 mg·kg^-1·d^-1). Wistar-Kyoto rats were used as normal control. Respectively, systolic blood pressure was measured by tail cuff every 2 weeks. After 10 weeks, heart weight index and left ventricular weight index were calculated. The myocardial hydroxyproline and plasma angiotensin Ⅱ were detected. Moreover, basic myocardial histopathological changes and myocardial collagen fibres were observed by HE staining and Masson staining, respectively. The protein expression of TGF-β₁ and Smad3 in the myocardium was measured by the methods of immunohistochemistry and Western blot. RESULTS: Compared with SHR model group, Rhy significantly reduced blood pressure (P<0.05), the levels of HYP in the myocardium (P<0.05) and the levels of AngⅡ in the plasma (P<0.01). The pathological damages of the myocardial tissues and collagen deposition were attenuated. The protein expression of TGF-β₁ and Smad3 was significantly reduced by the treatment with Rhy (P<0.01). CONCLUSION: Rhynchophylline reduces blood pressure and adjusts to improve ventricular remodeling of SHR. The mechanism may be involved in the TGF-β₁/Smad pathway and reducing AngⅡ content.     

Effect of short-chain acyl-CoA dehydrogenase on cardiac hypertrophy induced by hypertension or exercise training

AIM: To investigate the differential expression of short-chain acyl-CoA dehydrogenase (SCAD) in cardiac hypertrophy induced by hypertension or exercise training. METHODS: Spontaneously hypertensive rats (SHR) were used as the model of pathological cardiac hypertrophy. The swim-trained rats were used as the model of physiological cardiac hypertrophy. The systolic pressure, cardiac hypertrophy parameters, echocardiogram parameters, free fatty acid in serum and cardiac muscle, and the expression and activity of SCAD in the left ventricle were measured. RESULTS: Compared with the control rats, trained rats developed an athletic heart, of which cardiac function was enhanced, whereas SHR developed hypertensive cardiac hypertrophy, of which cardiac function was deteriorated. Compared with the control rats, the ratios of left ventricular weight to body weight were both increased in trained rats and SHR, showing that the degrees of cardiac hypertrophy were similar in the 2 models. Compared with the control rats, the decrease of free fatty acid both in serum and myocardium indicated that the fatty acid utilization was increased in the left ventricle of trained rats. Meanwhile, the expression and activity of SCAD in the left ventricle of trained rats were increased. However, free fatty acid both in serum and myocardium were increased, indicating that the fatty acid utilization was decreased in the left ventricle of SHR. Furthermore, SHR had the decreased expression and activity of SCAD in the left ventricle. CONCLUSION: The changes of SCAD are different in cardiac hypertrophy induced by hypertension and exercise training, indicating that SCAD may be used as a molecular marker of physiological and pathological cardiac hypertrophy, and a potential therapeutic target of pathological cardiac hypertrophy.     

Effect of high-fructose diet on adipose tissue renin-angiotensin system in rats

AIM: To observe the effects of high-fructose diet on adipose tissue inflammation and renin-angiotensin system (RAS), and to reveal the role of Toll-like receptor 2 (TLR2) in this process.METHODS: Male SD rats (n=16) were randomly divided into control group, high fructose group, high fructose+siRNA negative control group, and high fructose+TLR2-siRNA group. The rats in control group were fed with a standard chow diet. The rats in high fructose group were fed with a diet with 60% fructose, and the rats in high fructose+TLR2-siRNA group and high fructose+siRNA negative control group were transfected with TLR2 siRNA and scrambled siRNA, respectively. Serum uric acid was measured and visceral adipose tissue was weighed at the 14th week. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), angiotensinogen (AGT), and angiotensin Ⅱ (Ang Ⅱ) were measured by ELISA. Infiltrating macrophages in the adipose tissues were measured with immunohistochemistry. The mRNA expression of IL-6, TNF-α, monocyte chemoattractant protein-1 (MCP-1), AGT, angiotensin-converting enzyme 1 (ACE1), angiotensin Ⅱ type 1 receptor (AT1R), and angiotensin Ⅱ type 2 receptor (AT2R) was detected by RT-qPCR. The protein level of TLR2 was determined by Western blot.RESULTS: High fructose-fed rats showed elevated serum uric acid, raising fat content, higher serum concentrations of IL-6, TNF-α, AGT and AngⅡ, and more infiltrating macrophages in the adipose tissues (P<0.05). Moreover, the mRNA levels of IL-6, TNF-α,MCP-1, AGT, ACE1, AT1R and AT2R in the adipose tissues were increased (P<0.05). When high fructose-fed rats were transfected with TLR2-siRNA, the dramatic decreases in TLR2 protein level and number of infiltrating macrophages in the adipose tissues were found. Both in serum and adipose tissues, the mRNA levels of inflammatory cytokines and RAS components were all significantly decreased (P<0.05).CONCLUSION: High-fructose diet up-regulates RAS in adipose tissues via activation of TLR2 inflammation signaling pathway.     

Effects of atorvastatin on expression of cytochrome P450 hydroaylase in spontaneously hypertensive rats

AIM: To investigate the effects of atorvastatin on blood pressure and expression of cytochrome P450 hydroaylase (CYP) 4A1 in spontaneously hypertensive rats (SHR). METHODS: SHRs (n=18) were randomly divided into three groups: SHR control group, 50 mg atorvastatin (HATV) group and 10 mg (LATV) group. Six male Wistar-Kyoto rats were selected as normal control group (WKY group). All rats were given vehicle once a day by gavage for 10 weeks. Systolic blood pressure (SBP) was measured before and after treatment every 2 weeks. The expressions of CYP 4A1 mRNA and protein in heart, liver, kidney, and aorta were detected by RT-PCR and Western blotting analysis, respectively. Plasma lipids were also measured.RESULTS: SBP in all SHR groups was much higher than that in WKY group before experiment (P<0.01). Compared with SHR control group, SBP significantly decreased in HATV group at 6, 8, 10 weeks and in LATV group merely at 10 weeks (P<0.01 or P<0.05). The expressions of CYP 4A1 mRNA and protein in heart, liver, kidney and aorta tissues of SHR control group were significantly higher than those in WKY group (P<0.01 or P<0.05). After 10 weeks, the levels of CYP 4A1 mRNA, protein and plasma lipids in HATV and LATV groups were markedly lower than those in SHR control group (P<0.01 or P<0.05).CONCLUSION: Atorvastatin down-regulates the expressions of CYP 4A1 mRNA and protein in SHR, which may be the mechanism of the favorable effects of statins on regulation of hypertension.     

Alterations in gene expression of sarcoplasmic reticulum Ca2+-ATPase and phospholamban detected by RNA array in spontaneously hypertensive rats

AIM: To explore the relationship between the alteration in gene expression of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) and phospholamban (PLB) in spontaneously hypertensive rats (SHR). METHODS: 294 samples of total RNA were obtained from the tissue of ventriculum , aortic smooth muscle, liver and kidney in SHR and normotensive rats (WKY). RNA array was used to determine the mRNA levels of SERCA and PLB. RESULTS: Compared with age-matched WKY rats, the systolic blood pressure increased higher in 6-week-old SHR (P<0.01). The cardiosomatic ratio was significantly higher in 10-week-old SHR (P<0.01), in cardiac sarcoplasmic reticulum, the mRNA levels of SERCA were significantly increased from 4 weeks (P<0.05 or P<0.01). In the aortic sarcoplasmic reticulum, the mRNA levels of SERCA were significantly increased from 4 weeks to 12 weeks (P<0.05 or P<0.01). There was no significant change in the expression of PLB between the two groups. The ratio of cardiac SERCA and PLB was significantly increased since 6-week-old (P<0.05 or P＜0.05)in SHR. The ratio of aortic SERCA and PLB in SHR was significantly increased since 4-week-old (P<0.05 or P<0.01) vs WKY. CONCLUSION: Our results provided the evidence that the abnormalities of intracellular Ca²⁺ hemostasis in SHR represent the progressive nature of essential hypertension.     

Effects of valsartan alone or in combination with benazepril on blood pressure and left ventricular hypertrophy in spontaneously hypertensive rats

AIM:To evaluate the effects of different doses of valsartan alone or with concomitant be-nazepril on blood pressure,left ventricular hypertrophy,RAASfunction and endoxi nlevel in spontaneously hy-pertensive rats(SHR).METHODS:Thirty SHR(fourteen-week-old,male)were divi ded into five groups(six rats in each group):SHR control group:fed with normal saline;benazepril group:fed with 1 mg·kg^-1·d^-1benazepril);low dose valsartan group:fed with 8 mg·kg^-1·d^-1valsartan;high dose valsartan group:fed with 24 mg·kg^-1·d^-1valsartan;combination drug therapy group:fed with valsartan(8 mg·kg^-1·d^-1)and benazepril(1 mg·kg^-1·d^-1),all for 8 weeks.WKY control group(n=6):fed with normal saline for 8 weeks.RESULTS:SBP,LVM/BW,TDMof SHR were remarkably lower than those of control after drug i n-tervene,and effect on SBP was most remarkable in high dose valsartan group and i nthe combi nation drug ther-apy group;effect on LVM/BW,TDM were most remarkable in combination drug therapy group.Renin activi-ties in plasma and myocardiumwere remarkably i ncreased in drug i ntervene groups.The levels of AngⅡi nplasma and myocardiumwere remarkably increased in two different dose of valsartan treati ng group,and thelarger dose of valsartan were,the higher levels of AngⅡin plasma and myocardium were;decreased in be-nazepril treati ng group and combination drug therapy group.Na⁺-K⁺-ATPase activities in myocardi umwere remarkably i ncreased and the level of endoxi n i n myocardium were remarkably decreased as SBP de-creased after drug intervene.CONCLUSION:Different dose of valsartan alone or combi ned with benazeprilcan decrease SBP of SHR,have the effect of inhibiti ng progression of ventricular hypertrophy.The effect ofcombination drug therapy group was most remarkable among five groups and can avoi d the si de effect of highAngⅡin plasma and myocardiumduri ng long-termuse of valsartan alone.     

Effects of dexamethasone on acute lung injury by inhibiting renin-angiotensin system in rats

AIM:To explore the relationship between renin-angiotensin system (RAS) and acute lung injury (ALI) in rats, and the effect of dexamethasone (DEX) on RAS was observed.METHODS: The rat model of ALI was induced by hemorrhagic shock and LPS administered intraperitoneally. The changes of MAP and the effects of DEX on MAP were observed. The mRNA expressions of ACE, AGT, AT1 and AT2 in lung tissue were assayed by RT-PCR. The changes of Ang I and Ang II in the serum and the effects of DEX on them were observed.RESULTS: The increasing of MAP was statistically obvious. MAP in hemorrhagic shock+LPS (HL) group recovered more slowly than that in HL+DEX(HLD) group. ACE, AGT, AT1 and AT2 mRNA expressions in HL group were increased, and higher than those in HLD group. The change of Ang II in serum in HL group was obviously higher than that in HLD group, while that of Ang I was not obvious.CONCLUSION:ALI activates RAS in rat lung, and promotes the production of Ang II then aggravates the injury of lung through increasing the expression of ACE and AGT. DEX decreases expression of Ang II.     

Renal expression of ACE/AngⅡ in ACE2 knockout mice after tourniquet shock and its relationship with renal injury

AIM: To investigate the effect of angiotensin-converting enzyme 2 (ACE2) on tourniquet shock (TS)-induced acute renal injury by observing the renal expression of angiotensin-converting enzyme/angiotensin Ⅱ (ACE/AngⅡ) and injury severity in ACE2 knockout (KO) mice. METHODS: The TS animal model was produced by bilateral tourniquet ligation in the inguinal region on both hind legs for 2 h to induce ischemia, and reperfusion was initiated by cutting latex rings for 4 h. Six-month-old male wild-type (WT) and ACE2 KO C57BL/6 mice were selected, and divided into 4 groups (6 mice in each group) including WT group, WT+TS group, KO group and KO+TS group. The expression of ACE and AngⅡ in the renal tissues was determined by Western blotting and ELISA, respectively. The renal content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD), blood urea nitrogen (BUN) and serum creatinine (Cr) were measured by chemical colorimetry. RESULTS: Compared with WT group, the expression of ACE/AngⅡ was obviously increased in WT+TS group, and significant renal oxidative stress injury was also observed. The expression of ACE/AngⅡ was elevated in KO mice, but no significant renal oxidative stress injury was found. Compared with WT+TS group, more highly increased expression of ACE/AngⅡ and more aggravated renal injury were exhibited in KO+TS group. CONCLUSION: Deletion of ACE2 gene exacerbates TS-induced renal oxidative stress injury by increasing local ACE/AngⅡ expression. The agonist targeting to ACE2 may be helpful for prevention and treatment of TS-induced acute renal injury.     

Expression of ERK and MKP-1 in vascular smooth muscle of spontaneously hypertensive rats with different ages

AIM: To investigate the expression of mitogen- activated protein kinase and mitogen-activated protein kinase phosphatase-1 in thoracic aorta smooth muscles of spontaneously hypertensive rats (SHR) and Wistar Kyoto rats (WKY) with different ages and the relationship between those and hypertension. METHODS: The caudal arterial pressure was measured by tail-cuff. Protein expression of p-ERK was detected by Western blotting, and MKP-1 mRNA in thoracic aorta smooth muscle was examined by RT-PCR. RESULTS: (1) The blood pressure of SHR was obviously higher than that of age-matched WKY (P<0.01), elevated with age (P<0.05) and became stable from 14-week-old. (2) The expression of p-ERK and MKP-1 in SHR was higher than that in WKY in 5-week-old rats, and the expression of p-ERK increased with age, while the expression of MKP-1 decreased with age (P<0.05). CONCLUSION: MKP-1 may play an important role in the development of hypertension in SHR. The decrease in the expression of MKP-1 that resulted in the activation of MAPK may induce vascular smooth muscle proliferation and hypertrophy.     

Role of benazepril on extracellular signal-regulated kinase activity and expression of B-type natriuretic peptide in spontaneously hypertensive rats

AIM：To investigate the role of benazepril on extracellular signal-regulated kinase (ERK) activity and expression of B-type natriuretic peptide in spontaneously hypertension rat (SHR).METHODS：Wistar Kyoto rats were used as control group.Twenty one 14-week-age SHR were randomized into 3 groups,7 rats each：benazepril group (10 mg·kg-1·d-1);hydralazine group (10 mg·kg-1·d-1) and sham group.In each group drugs or equal volume of vehicle (0.5% carboxymethyl cellulose) were administered respectively for 10 weeks by gavage.The ratio of left ventricle weight to body weight （LVW/BW） was measured to reflect myocardial hypertrophy.The caudal arterial pressure was measured by tail-cuff.Protein expression of p-ERK in myocardial tissue was detected by Western blotting,BNP mRNA in myocardial tissue was examined by RT-PCR,and protein expression of plasma BNP was detected by ELISA.RESULTS：1.Benazepril and hydralazine lowered the blood pressure after 10 weeks treatment (P<0.01).2.The ratio of LVW/BW in SHR benazepril group was significantly lower than that in SHR hydralazine group and SHR sham group (P<0.01),and similar to that in WKY group (P>0.05).3.The protein expression of p-ERK in myocardial tissue in SHR benazepril group was significantly lower than that in SHR hydralazine group and SHR sham group (P<0.01),and similar to that in WKY group (P>0.05).There was no significant difference of p-ERK expression between SHR hydralazine group and SHR sham group (P>0.05).4.The levels of plasma BNP and BNP mRNA in myocardial tissue in SHR benazepril group were significantly lower than that in SHR hydralazine group and SHR sham group (P<0.01),and similar to that in WKY group (P>0.05).There was no significant difference of plasma BNP and BNP mRNA in myocardial tissue between SHR hydralazine group and SHR sham group (P>0.05).CONCLUSIONS：Benazepril inhibited ERK activation,resulting in regression of myocardial hypertrophy and accompanied by the reduction of BNP level.However,in spite of the effect of lowering blood pressure,hydralazine did not prevent or regress cardiac hypertrophy and did not decrease the level of p-ERK and BNP in SHR.BNP level might serve as a therapeutic index for reversal of myocardial hypertrophy.     

Diuretic mechanism of U50 488H in spontaneously hypertensive rats

AIM: To investigate the diuretic effects of U50 488H, a selective κ-opioid receptor agonist, in spontaneously hypertensive rats (SHR) and explore their mechanism. METHODS: The physiological experimental technique was used to measure the blood pressure (BP), and the volume of urine output in normatensive WKY (Wistar-Kyoto) rats and SHR was measured. The plasma levels of some humoral factors were detected by radioimmunoassay. RESULTS: U50 488H decreased BP significantly in WKY rats and SHR, and the decrease of BP in SHR was more than that in WKY rats. U50 488H increased the urine volume dose-dependently in WKY rats and SHR. The urine volume of SHR increased by U50 488H was more than that of WKY rats. U50 488H (1 mg/kg) produced marked decrease in plasma concentration of ADH in WKY rats and SHR, and the decrease in SHR was significantly stronger than that in WKY. U50 488H also induced a decrease in plasma concentration of AngⅡin SHR, while it had no influence on the plasma concentration of AngⅡ in WKY rats. These effects of U50 488H were abolished by nor-BNI, a selective κ-opioid receptor antagonist. CONCLUSION: The significant decreases in plasma concentration of ADH and AngⅡmay be responsible for the effective diuresis of κ-opioid receptor stimulation by U50 488H in SHR.     

Effect of angiotensin Ⅱ on the remodeling of aorta in spontaneously hypertensive rats

AIM: To investigate the effects of a 10-weeks treatment with angiotensin Ⅱ (Ang Ⅱ) subtype I receptor antagonist losartan on vascular remodeling of thoracic aorta in male spontaneously hypertensive rats (SHR). METHODS: SHR were treated from 16 to 26 weeks of age with losartan at 15 mg/kg·d^-1 or 0.75 mg/kg·d^-1. RESULTS: Losartan (15 mg/kg·d^-1) treatment significantly decreased systolic blood pressure compared with the control group, while losartan (0.75 mg/kg·d^-1) had no the effect, losartan(15 mg) prevents the development of aortic hypertrophy by preventing hypertrophy of vascular smooth muscle cells (VSMC). In the losartan 0.75 group, these parameters were not changed. But in the losartan 15 and losartan 0.75 groups, the collagen content of the aortic media decreased significantly. CONCLUSION: It is inferred that the effect of Ang Ⅱ on stimulating VSMC growth of the aorta in SHR is dependent on arterial pressure, while the effect on collagen fibers is through pressure independent mechanism.