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@#Objective To investigate the optimal administration combination of β-aminopropionitrile (BAPN) and Angiotensin Ⅱ (Ang-Ⅱ) in the establishment of SD rat aortic dissection (AD) model and the related complications. Methods Forty-two three-week-old male SD rats were randomly divided into 7 groups: a group A (0.25% BAPN), a group B (0.40% BAPN), a group C (0.80% BAPN), a group D [1 g/(kg·d) BAPN], a group E [1 g/(kg·d) BAPN+ 1 μg/(kg·min) saline], a group F [1 g/(kg·d) BAPN+1 μg/(kg·min) Ang-Ⅱ] and a group G (control group). There were 6 rats in each group. The intervention period was 4 weeks (groups E and F were 4 weeks+5 days). Rats were dissected immediately if they died during the experiment. After the intervention, the surviving rats were sacrificed by pentobarbital sodium, and the whole aorta was separated and retained. Hematoxylin-eosin staining was used to observe the changes of aorta from the pathological morphology. Results There was no statistical difference in the survival rate among the groups after 4 weeks of BAPN intervention (P>0.05). After 5 days of mini-osmotic pumps implantation, the survival rate of rats was higher in the group E than that in the group F (P=0.008), and the incidence of AD in the group E was lower than that in the group F (P=0.001). BAPN could affect the food and water intake of rats. After BAPN intervention for 4 weeks, the body weight of rats in the group G was higher than those in the intervention groups (P<0.05). BAPN combined with Ang-Ⅱ could make the aortic intima thick, elastic fiber breakage, arrangement disorder, and inflammatory cell infiltration in rats, which conformed to the pathological and morphological changes of AD. BAPN could also affect mental state and gastrointestinal tract. Conclusion The combination of BAPN [1 g/(kg·d)] and Ang-Ⅱ [1 μg/(kg·min)] can stably establish AD model in rats, which will provide a stable carrier for further study of the pathogenesis and therapeutic targets of AD. However, the complications in this process are an unstable factor. How to balance the influence of BAPN on other tissues and organs in the process of AD model establishment remains to be further studied.
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BACKGROUND:Cardiac hypertrophy is an adaptive response of the heart to physiological and pathological stimuli such as pressure overload.It is of compensatory significance in the early stage,but if the stimulation continues,it can cause cardiomyopathy leading to heart failure.MicroRNAs are involved in the regulation of cardiac hypertrophy.However,the role of miR-20a in pressure overload-induced cardiac hypertrophy has not been reported. OBJECTIVE:To investigate the role of miR-20a in pressure overload-induced cardiac hypertrophy and the underlying mechanisms. METHODS:Transverse aortic constriction was used to induce cardiac hypertrophy in vivo and angiotensin Ⅱ was used to induce H9c2 cell models of cardiac hypertrophy in vitro.MiR-20a was overexpressed in vivo by intramyocardial injection of miR-20a overexpressing adenovirus and in vitro by transfecting miR-20a mimic into H9c2 cells.Cardiac hypertrophy was assessed by measuring heart weight/body weight ratio,cell surface area,and myocardial fibrosis.The expression levels of atrial natriuretic peptide,brain natriuretic peptide,β-myosin heavy chain and miR-20a were detected by real-time fluorescence quantitative PCR.Mitochondrial fission was detected by MitoTracker.The downstream target genes of miR-20a were predicted by RNAhybrid software. RESULTS AND CONCLUSION:(1)The expression level of miR-20a was significantly decreased in both hypertrophic cardiomyocytes and hearts(P<0.05).(2)At the animal level,overexpression of miR-20a significantly inhibited transverse aortic constriction-induced cardiac hypertrophy,including decreasing the upregulated expression level of hypertrophic marker genes(P<0.05),reduced the enlarged heart volume,reducing the increased heart weight/body weight ratio(P<0.01),reducing the increased myocardial cross-sectional area(P<0.05),and attenuating fibrosis(P<0.01).(3)At the cellular level,overexpression of miR-20a significantly inhibited angiotensin Ⅱ-induced cardiomyocyte hypertrophy,including decreasing the upregulated expression levels of atrial natriuretic peptide(P<0.05),brain natriuretic peptide(P<0.01)and β-myosin heavy chain(P<0.05),reducing the increased protein/DNA ratio(P<0.01),and suppressing the increased cell surface area(P<0.05).(4)Overexpression of miR-20a significantly inhibited angiotensin Ⅱ-induced mitochondrial fission(P<0.05).(5)The results of RNAhybrid software analysis showed that miR-20a and the mRNA 3'untranslated region of cAMP-dependent protein kinase inhibitor alpha were well complementary and the predicted binding sites were highly conserved.(6)In conclusion,miR-20a is significantly down-regulated in pressure overload-induced cardiac hypertrophy.Overexpression of miR-20a inhibits cardiac hypertrophy at both the cellular level and animal level and attenuates angiotensin Ⅱ-induced mitochondrial fission.
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BACKGROUND:Previous studies have shown that salidroside has an ameliorative effect on multi-organ fibrosis.However,the protective effect of salidroside on angiotensin ⅱ-induced fibrosis in cardiac fibroblasts is unclear. OBJECTIVE:To investigate the protective effects of salidroside on angiotensin ⅱ-induced oxidative stress and extracellular matrix deposition in cardiac fibroblasts of Sprague-Dawley rats and its mechanism of action. METHODS:Angiotensin Ⅱ was used to induce fibrosis in cardiac fibroblasts,and there were five experimental groups:normal control group,model group(final concentration of angiotensin Ⅱ in culture medium was 1 μmol/L),salidroside low and high dose groups(treatment with salidroside 50,100 μmol/L for 2 hours,followed by co-incubation with angiotensin Ⅱ for 48 hours),SIRT1 inhibitor group(treatment with SIRT1 inhibitor EX527 10 μmol/L for 2 hours,followed by high dose of salidroside for 2 hours and then co-incubation with angiotensin Ⅱ for 48 hours).The cell viability was detected using the cell counting kit-8 method,the cell migration rate was detected by Transwell,the intracellular reactive oxygen species level was detected by DCFH-DA fluorescent probe,and the intracellular malondialdehyde content,superoxide dismutase and catalase activities were detected by relevant kits.The protein and mRNA expression levels of SIRT1,LOXL2,α-SMA,type I collagen and type Ⅲ collagen were detected by western blot and qRT-PCR,respectively. RESULTS AND CONCLUSION:The cells were identified as cardiac fibroblasts by Vimentin fluorescence.Compared with the normal control group,cell viability,cell migration rate,reactive oxygen species level,and malondialdehyde content were significantly increased,superoxide dismutase and catalase activities were significantly decreased,LOXL2,α-SMA,type I collagen,type Ⅲ collagen mRNA and protein expression were significantly increased,and SIRT1 protein expression level was significantly decreased in the model group(all P<0.01).Compared with the model group,the above indexes showed opposite changes in the salidroside low and high dose groups(all P<0.05).Moreover,salidroside showed dose-dependent regulation.Compared with salidroside groups,cell migration rate and α-SMA protein expression level were significantly increased in the SIRT1 inhibitor group(both P<0.001).To conclude,salidroside has a protective effect on angiotensin Ⅱ-induced cardiac fibroblasts and can dose-dependently inhibit oxidative stress and extracellular matrix deposition.
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Objective To investigate the effect of YTH domain family protein 2(YTHDF2)on an-giotensin Ⅱ(Ang Ⅱ)-induced hypertrophy and apoptosis of primary neonatal rat cardiomyocytes.Methods The expression level of YTHDF2 was detected in the primary neonatal rat cardiomyo-cytes with or without Ang Ⅱ stimulation(AngⅡ group and normal group).The cells were divid-ed into blank group(transfected with siRNA+PBS),siYTHDF2 group(transfected with siYTHDF2+PBS),model group(siRNA+Ang Ⅱ)and experimental group(siYTHDF2+Ang Ⅱ)to investi-gate the effects of silencing YTHDF2 on the hypertrophy and apoptosis of cardiomyocytes.West-ern blotting and RT-qPCR were used to detect the expression of YTHDF2 at protein and mRNA levels,and RT-qPCR was employed to measure the mRNA levels of myocardial hypertrophic related genes atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP)and beta-myosin heavy chain(β-MHC),and cardiomyocyte apoptosis related genes Bax and B lymphocytoma 2 gene(Bcl-2).The surface area of cardiomyocytes was observed by α-actin immunofluorescence staining.Cardiomyocyte apoptosis was observed by TUNEL staining,and the binding relationship between YTHDF2 and Bcl-2 was verified by immunoprecipitation.Results The expression of YTHDF2 at protein and mRNA levels were significantly higher in the AngⅡ group than the nor-mal group(1.49±0.03 vs 0.97±0.09,1.50±0.08 vs 1.00±0.07,P<0.05).Compared with the blank group,the surface area of cardiomyocytes was notably enlarged,apoptotic rate was obvi-ously increased,the mRNA levels of ANP,BNP,β-MHC and Bax were significantly increased,and that of Bcl-2 was remarkably decreased in the model group(P<0.05).The experimental group obtained decreased surface area and apoptotic rate of cardiomyocytes,lower mRNA levels of ANP,BNP,β-MHC and Bax,and increased mRNA expression of Bcl-2(P<0.05).Conclusion Silencing YTHDF2 can alleviate Ang Ⅱ-induced hypertrophy and apoptosis in primary neonatal rat cardiomyocyte,and YTHDF2 inhibits the expression of Bcl-2 by binding to it.
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ObjectiveTo explore the mechanism of modified Liuwei Dihuangtang in preventing and treating renal injury in diabetic kidney disease (DKD) via the angiotensin-converting enzyme 1 (ACE1)/angiotensin Ⅱ (AngⅡ)/angiotensin Ⅱ type 1 receptor (AT1R) axis. MethodFifty male SD rats were randomized into a normal group (n=8) and a modeling group (n=42). The rats in the modeling group were fed with a high-sugar and high-fat diet for 6 weeks and intraperitoneally injected with 35 mg·kg-1 streptozotocin (STZ) to establish the model of DKD. After successful modeling, the rats were randomized into model, traditional Chinese medicine (modified Liuwei Dihuangtang granules 21 g·kg-1), western medicine (losartan potassium, 33 mg·kg-1), and integrated Chinese and western medicine (losartan potassium 33 mg·kg-1 combined with modified Liuwei Dihuangtang granules 21 g·kg-1) groups. The levels of fasting blood glucose (FBG), urinary protein (Up), blood urea nitrogen (Bun), and serum creatinine (SCr) were measured in each group after 8 consecutive weeks of drug intervention. Enzyme-linked immunosorbent assay was employed to determine the serum levels of ACE1, AngⅡ, and AT1R. Western blot was employed to measure the protein levels of ACE1, AngⅡ, and AT1R in the renal tissue. The pathological and morphological changes of the renal tissue were observed after hematoxylin-eosin (HE) staining, Masson staining, and periodic acid Schiff 's (PAS) staining. The fecal samples of rats in each group were collected for 16S rDNA high-throughput sequencing. ResultCompared with the normal group, the model group showed elevated levels of Up, FBG, Bun, SCr, ACE1, AngⅡ, and AT1R (P<0.01), serious lesions in the renal tissue, up-regulated protein levels of ACE1, AngⅡ, and AT1R (P<0.01), increased Firmicutes/Bacteroidetes (F/B) ratio, decreased relative abundance of Lactobacillus, and increased relative abundance of Moralella and Bifidobacteria. Compared with the model group, drug intervention lowered the levels of Bun, SCr, ACE1, AngⅡ, and AT1R (P<0.01) and alleviated the pathological changes in the renal tissue. Chinese medicine and integrated Chinese and western medicine lowered the levels of Up and FBG (P<0.01), and western medicine and integrated Chinese and western medicine down-regulated the protein levels of ACE1, AngⅡ, and AT1R. In addition, Chinese medicine down-regulated the protein levels of AngⅡ (P<0.01) as well as ACE1 and AT1R (P<0.05). Chinese medicine and integrated Chinese and western medicine decreased the F/B ratio, and western medicine and Chinese medicine increased the relative abundance of Blautia. Chinese medicine and integrated Chinese and western medicine increased the relative abundance of Lactobacillus, Ruminococcus undetermined genera, and Bifidobacteria, decreased the relative abundance of Moralella, and increased the Chao 1 and Ace indexes (P<0.05). Compared with the western medicine group, the integrated Chinese and western medicine group showed lowered levels of Up (P<0.01), Bun (P<0.05), and ACE1 and AT1R (P<0.01), down-regulated protein levels of ACE1, AngⅡ, and AT1R (P<0.05), alleviated pathological changes in the renal tissue, increased relative abundance of Bifidobacteria, and increased Chao 1 and Ace indexes (P<0.05). ConclusionModified Liuwei Dihuangtang combined with losartan potassium can mitigate renal fibrosis by regulating the ACE1/AngⅡ/AT1R axis, increasing the relative abundance of Lactobacillus and Bifidobacterium, reducing the relative abundance of Moralella, improving the richness and evenness of intestinal flora, and alleviating pathological damage in the renal tissue.
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OBJECTIVE To investigate the inhibitory effect and the possible mechanism of hydrogen sulfide (H2S) on the proliferation of cardiac fibroblasts. METHODS The heart of neonatal SD rats was collected, and cardiac fibroblasts were separated with differential centrifugation. Using sodium hydrosulfide as the donor of H2S, the effects of H2S on the proliferation of cardiac fibroblasts induced by angiotensin Ⅱ (Ang Ⅱ), hydroxyproline content and the expression of sirtuin 3 (SIRT3) protein were detected. After SIRT3 knockdown with siRNA technology, the effects of H2S on the proliferation of cardiac fibroblasts induced by Ang Ⅱ, hydroxyproline content, the expressions of collagen Ⅰ (Col Ⅰ), collagen Ⅲ (Col Ⅲ ) and optic atrophy protein 1 (OPA1) were detected. RESULTS H2S could inhibit the proliferation of Ang Ⅱ-induced cardiac fibroblasts, reduce the content of hydroxyproline and increase the expression of SIRT3 (P<0.05). After down-regulating the expression of SIRT3 with siRNA technology, the inhibition of H2S on the proliferation of Ang Ⅱ-induced cardiac fibroblasts and the reduction of hydroxyproline content were both inhibited, and the effect of H2S on reducing the expression of Col Ⅰ and Col Ⅲ and enhancing the expression of OPA1 was also significantly weakened. CONCLUSIONS H2S inhibits the proliferation of Ang Ⅱ -induced cardiac fibroblasts through increasing the expression of SIRT3.
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ObjectiveTo explore the underlying mechanism of modified Zhenwutang in delaying renal interstitial fibrosis in chronic renal failure (CRF) by observing the effects of modified Zhenwutang on the expression of angiotensin Ⅱ (Ang Ⅱ), angiotensin Ⅱ type 1 receptor (AT1R), nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4), transforming growth factor-β1 (TGF-β1), type I collagen (COL1A1), and type Ⅲ collagen (COL3A1) in the serum and renal tissues of adenine-induced CRF rats. MethodFifty male SPF-grade SD rats were randomly divided into a normal group (n=10) and an experimental group (n=40) using a random number table. After one week of adaptive feeding, the experimental CRF model was established in rats by administering adenine at 150 mg·kg-1·d-1 orally. Three rats from each group were randomly selected to evaluate the model induction. After successful modeling, rats in the experimental group were randomly divided into a model group, low-, medium, and high-dose modified Zhenwutang groups, and a benazepril hydrochloride group, with six rats in each group. The rats were orally administered the corresponding drugs once daily for four weeks. At the end of the first week, 13th week, and 17th week of the experiment, 24 hour urinary protein quantification (24 h-UTP) was measured. At the end of the 17th week, the rats were euthanized, and blood samples were collected from the abdominal aorta for the measurement of total protein (TP), albumin (ALB), creatinine (Cr), and blood urea nitrogen (BUN) in the serum. Enzyme-linked immunosorbent assay (ELISA) was used to measure the expression levels of serum Ang Ⅱ. Hematoxylin-eosin (HE) staining and Masson's trichrome staining were performed to observe the pathological changes in renal tissues. Immunohistochemistry (IHC) was performed to observe the expression of AT1R, NOX4, TGF-β1, COL1A1, and COL3A1. Real-time fluorescence-based quantitative polymerase chain reaction (Real-time PCR) was used to observe the mRNA expression levels of AT1R, NOX4, and TGF-β1. Western blot was conducted to measure the protein expression levels of AT1R, NOX4, and TGF-β1. Result① Compared with the normal group, the model group showed a significant increase in 24 h-UTP (P<0.01). The levels of Cr and BUN in the model group were significantly higher (P<0.01), while the levels of TP and ALB were significantly lower (P<0.01). The serum Ang Ⅱ level in the model group was significantly elevated (P<0.01). The model group exhibited widening of the renal glomerular mesangial space, necrotic glomeruli, increased interstitial width with extensive inflammatory cell infiltration, brownish precipitates blocking the renal tubular lumens, irregular renal tubules, and significant deposition of collagen fibers in the renal interstitium. Additionally, the collagen fibers around the renal vessels, outside the parietal layer of the renal sacs, glomerular basement membrane, and tubular basement membrane increased significantly. The expression of AT1R and NOX4 in the glomeruli and renal tubules of the model group was significantly enhanced, and TGF-β1 expression also significantly increased in the renal tubules. The expression of COL1A1 and COL3A1 in the renal interstitium significantly increased. The mRNA expression of AT1R and TGF-β1 in the model group significantly increased (P<0.01), while NOX4 mRNA expression significantly decreased (P<0.01). The protein expression of AT1R, NOX4, and TGF-β1 was significantly enhanced (P<0.01). ② Compared with the model group, modified Zhenwutang significantly reduced 24h-UTP (P<0.01), decreased levels of Cr and BUN (P<0.01), increased levels of TP and ALB (P<0.01), reduced serum Ang Ⅱ level (P<0.01), alleviated renal pathological damage, reduced expression of AT1R, NOX4, TGF-β1, COL1A1, and COL3A1 in the glomeruli, renal tubules, and renal interstitium, reduced mRNA expression of AT1R and TGF-β1 (P<0.01), increased NOX4 mRNA expression (P<0.01), and weakened protein expression of AT1R, NOX4, and TGF-β1 (P<0.01). The modified Zhenwutang groups showed a significant dose-effect trend. ConclusionModified Zhenwutang may delay renal interstitial fibrosis in CRF rats by reducing the expression of Ang Ⅱ, AT1R, NOX4, and TGF-β1 in the serum and renal tissues, thereby alleviating renal pathological damage, reducing proteinuria, protecting renal function, and delaying the progression of CRF. The modified Zhenwutang group exhibited a dose-effect trend.
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Objective To investigate the relationship between serum levels of microRNA-130a(miR-130a),angiotensin Ⅱ(AngⅡ)and the degree of coronary artery lesions in patients with acute coronary syn-drome(ACS).Methods A total of 160 ACS patients admitted to the hospital from January 2021 to February 2023 were selected as the ACS group.According to total Gensini score,ACS patients were divided into mild group(57 cases),moderate group(54 cases)and severe group(49 cases).At the same time,160 healthy peo-ple were selected as the control group.The clinical data of all subjects were collected.The serum levels of miR-130a and AngⅡ were detected by real-time fluorescence quantitative PCR and enzyme-linked immunosor-bent assay,respectively.The clinical data and serum levels of miR-130a and AngⅡ were compared between control group and ACS group.The serum levels of miR-130a and AngⅡ were compared between ACS patients with different degrees of disease.Pearson correlation analysis was used to analyze the correlation between ser-um miR-130a level and AngⅡ in ACS patients.Multivariate Logistic regression was used to analyze the risk factors of ACS.Receiver operating characteristic curve was used to analyze the diagnostic value of serum miR-130a and AngⅡ levels for moderate and severe ACS.Results Compared with the control group,the ACS group had significantly higher proportions of patients with a history of hypertension and diabetes and signifi-cantly higher serum levels of miR-130a and AngⅡ(P<0.05).The serum levels of miR-130a and AngⅡ were increased sequentially in the mild group,moderate group and severe group(P<0.05).Serum miR-130a level was positively correlated with AngⅡ level in ACS patients(P<0.05).Hypertension,diabetes history and ele-vated serum levels of miR-130a and AngⅡ were independent risk factors for ACS(P<0.05).The area under the curve(AUC)of serum miR-130a,AngⅡ and their combination in the diagnosis of moderate ACS were 0.728,0.823 and 0.885,respectively,and the AUC of the combination of miR-130a and AngⅡ was higher than that of miR-130a,AngⅡ(P<0.05).The AUC of serum miR-130a,AngⅡ and their combination in the diagnosis of severe ACS were 0.731,0.730 and 0.825,respectively.The AUC of the combination of miR-130a and AngⅡ was higher than that of miR-130a and AngⅡ(P<0.05).Conclusion ACS patients serum miR-130-a,AngⅡ level is higher,and the serum miR-130a,AngⅡ levels are associated with the ACS degree of cor-onary artery lesions,the combination of the both degree of coronary artery lesions with high diagnostic value.
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Objective To explore the effect of circ_0038467 on angiotensin Ⅱ(Ang Ⅱ)-induced cardiomyocyte damage and its possible mechanism.Methods Ang Ⅱ was used to induce rat cardiomyocyte H9C2 to establish a cell injury model.si-NC,si-circ_0038467,miR-NC,miR-495 mimics were transfected into H9C2 cells and then treated with 1 μmol/L Ang Ⅱ for 24 h.si-circ_0038467 and anti-miR-NC,si-circ_0038467 and anti-miR-495 were co-transfected into H9C2 cells and treated with 1 μmol/L Ang Ⅱ for 24 h.qRT-PCR method was used to detect the expression levels of circ_0038467 and miR-495.A kit was used to detect the level of MDA and the activity of LDH and SOD.Flow cytometry was used to detect the rate of apoptosis.The dual lu-ciferase reporter experiment was used to detect the targeting relationship between circ_0038467 and miR-495.Western blot was used to detect the protein expression of cleaved Caspase-3 and cleaved Caspase-9,Bcl-2,p-P65 and p-IKBa.Results The expres-sion of circ_0038467 in H9C2 cells induced by Ang Ⅱ was increased(P<0.05),while the expression of miR-495 was decreased(P<0.05).After transfection of si-circ_0038467 or miR-495 mimics,the activity of LDH and the level of MDA were decreased(all P<0.05),the rate of apoptosis and the protein levels of cleaved Caspase-3,cleaved Caspase-9 were decreased(all P<0.05),while the activity of SOD was increased(P<0.05).Circ_0038467 could target miR-495.Co-transfection of si-circ_0038467 and anti-miR-495 could antagonize the effect of si-circ_0038467 on Ang Ⅱ-induced oxidative stress and apoptosis of H9C2 cells.In addition,circ_0038467 could activate the NF-κB pathway by targeting miR-495.Conclusion circ_0038467 regu-lates oxidative stress and apoptosis of cardiomyocytes by targeting miR-495/NF-KB pathway.
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Objective The purpose of this study was to investigate the protective effect of dexmedetomidine(DEX)on pathological car-diomyocyte hypertrophy.Methods An in vitro cell population was established in neonatal rats.The rats were divided into six groups:control group(C)without serum for 24 h,model group(A)with angiotensin Ⅱ(Ang Ⅱ)for 24 h,dexmedetomidine group(AD)with Ang Ⅱ+DEX(5μmol/L)for 24 h,C'group with serum-free culture for 48 h,A'group with Ang Ⅱfor 24 h,and AD'group with DEX+Ang Ⅱfor 24 h.The morphological changes of cells were observed by immunofluorescence.The protein expressions of atrial natriuretic peptide(ANP),brain natriuretic peptide(BNP),and myosin heavy chain(β-MHC)were detected by western blot,and the cell activity was detected by CCK-8.Results Compared with group C,the size of cells in group A was larger,and that in group AD was even more significant.Simi-lar observations were found for hypertrophy related proteins.Compared with group C,the expression of ANP,BNP,and βMHC increased in group A,although the increase in AD group was more obvious.CCK-8 detection showed that compared with group C,the activity of group A decreased and that of group AD increased significantly.Compared with the C'group,the expression of hypertrophy-related pro-tein in the A'group was significantly increased,but the expression of ANP and BNP protein in the AD'group was significantly lower than that in the A'group.The differences were statistically significant(P<0.05).Conclusion Dexmedetomidine can alleviate the occur-rence of pathological hypertrophy through compensatory mechanisms similar to physiological myocardial hypertrophy,and may play a role in myocardial protection.
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Objective @# To explore the method of using high-fat diet combined with angiotensin-Ⅱ ( Ang-Ⅱ) and β-aminopropionitrile (BAPN) to establish the model of aortic dissection in mice.@*Methods @#24 C57BL /6J mice (4 weeks old,male) were randomly divided into control group[intraperitoneal injection of 0.9% sodium chloride solu- tion 10 ml / (kg · d) ]and experimental groups[Ang-Ⅱ 4 mg / ( kg · d) group,Ang-Ⅱ 4 mg / ( kg · d) + BAPN 0. 33 g / (kg · d) group],each group with 8 mice ; all mice were given a high-fat diet and the mice weights were measured at the same time point and administered according to the weight standard.The dead mice were dissected immediately and the aorta was taken out for pathological section,then observed under the microscope.The morphology of aorta was detected by small animal ultrasound and the mice with obvious dissection were killed and dissected directly. @*Results @#After administration,the activity and appetite of mice in the high-fat diet combined with Ang-Ⅱ + BAPN group decreased most significantly,and the mortality rate of aortic dissection rupture and the success rate of modeling in this group were higher than those in the high-fat diet combined with Ang-Ⅱ group,while there was no significant change in the control group.Under the ultrasound of small animals,compared with the other two groups,the mice in the high-fat diet combined with Ang-Ⅱ + BAPN group showed the formation of abdominal aortic vascular false lumen and vascular enlargement.The mice that died during the administration were dissected immediately,and a large number of blood clots in the abdominal cavity and around the blood vessels could be seen.The mice with aortic dissection or aortic aneurysm could be seen under ultrasound in small animals,and severe adhesion between the vascular wall and the surrounding tissues could be found when dissected,while no obvious abnormalities were found in the blood vessels of the control group.The results of the staining showed that the false lumen of blood vessel wall was formed and the arrangement of elastin and collagen was disordered in the mice of high fat diet com- bined with Ang-Ⅱ + BAPN group.The thickness of blood vessel wall in each group was statistically analyzed,and it was found that the blood vessels in the two experimental groups were thicker than those in the control group,which was statistically significant (P<0. 001) .The vascular wall of Ang-Ⅱ + BAPN + high-fat diet group showed severe elastin degradation.@*Conclusion @# High-fat diet combined with Ang-Ⅱ 4 mg / (kg · d) and BAPN 0. 33 g / (kg · d) can establish an efficient model of aortic dissection in mice.
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Objective @# Angiotensin Ⅱ Type 2 receptor (AT2R) is a major receptor of angiotensin Ⅱ , which has a protective effect on damage to various tissues and organs.In this study,an overexpression plasmid of AT2R was con- structed to explore the effect of overexpression of AT2R on lipopolysaccharide ( LPS ) -induced inflammatory re- sponse in mouse hepatocytes (AML12 cells) .@*Methods @#Mice tissues and organs were used as samples to amplify the gene of interest (AT2R) fragments containing EcoR Ⅰ and Hind Ⅲ restriction sites,and then the final product was obtained by digestion and ligation.The final positive clones were sequenced and identified.The pCMV-Flag-N- AT2R plasmid was transfected into HEK 293T cells,and the expression of Flag protein was detected by the Western blot method after 24 h.AT2R expression on AML12 cells was observed by Western blot and laser confocal microsco- py.AML12 cells were treated differently and divided into control group,LPS treatment group,pCMV-Flag-N-AT2R group and pCMV-Flag-N-AT2R + LPS group,and cell viability was detected by CCK8.Western blot detected PCNA proteins and observed cell proliferation ; Cytoinflammatory factor levels were detected by qPCR ; Western blot detec- ted the expression level of nuclear transcription factor NF-кB (p65) in cells. @*Results @#The identification and se- quencing results of EcoR I and Hind III double restriction showed that pCMV-Flag-N-AT2R plasmid was successful- ly constructed,and the detection results of the Western blot method showed successful expression of AT2R protein. Laser confocal observed that there were AT2R receptors on AML12 cells,and AT2R recombinant plasmids could be expressed on AML12; Compared with the control group,the viability and proliferation ability of LPS-treated AML12 cells were weakened,while the levels of IL-6 and TNF-α increased,and the expression level of nuclear transcription factor NF-кB (p65) increased.Compared with the LPS group,the viability and proliferation of cells in AML12 cells treated with pCMV-Flag-N-AT2R and LPS were enhanced,while the levels of IL-6 and TNF-α decreased.The ex- pression of the nuclear transcription factor NF-кB ( p65 ) decreased.@*Conclusion @# AT2R overexpression plasmids were successfully constructed and successfully expressed on AML12 cells,and AT2R could inhibit the inflammatory response of LPS-induced AML12 cells.
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OBJECTIVE@#To investigate the effect of inhibitor of growth protein-2 (Ing2) silencing on angiotensin Ⅱ (AngⅡ)-induced cardiac remodeling in mice and explore the underlying mechanism.@*METHODS@#An adenoviral vector carrying Ing2 shRNA or empty adenoviral vector was injected into the tail vein of mice, followed 48 h later by infusion of 1000 ng · kg-1 · min-1 Ang Ⅱ or saline using a mini-osmotic pump for 42 consecutive days. Transthoracic echocardiography was used to assess cardiac geometry and function and the level of cardiac hypertrophy in the mice. Masson and WGA staining were used to detect myocardial fibrosis and cross-sectional area of cardiomyocytes, and myocardial cell apoptosis was detected with TUNEL assay. Western blotting was performed to detect myocardial expressions of cleaved caspase 3, ING2, collagen Ⅰ, Ac-p53(Lys382) and p-p53 (Ser15); Ing2 mRNA expression was detected using real-time PCR. Mitochondrial biogenesis, as measured by mitochondrial ROS content, ATP content, citrate synthase activity and calcium storage, was determined using commercial assay kits.@*RESULTS@#The expression levels of Ing2 mRNA and protein were significantly higher in the mice with chronic Ang Ⅱ infusion than in saline-infused mice. Chronic infusion of AngⅡ significantly increased the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) and reduced left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) in the mice. Ing2 silencing obviously alleviated AngⅡ-induced cardiac function decline, as shown by decreased LVEDD and LVESD and increased LVEF and LVFS, improved myocardial mitochondrial damage and myocardial hypertrophy and fibrosis, and inhibited cardiomyocyte apoptosis. Chronic AngⅡ infusion significantly increased myocardial expression levels of Ac-p53(Lys382) and p-p53(Ser15) in the mice, and Ing2 silencing prior to AngⅡ infusion lessened AngⅡ- induced increase of Ac-p53(Lys382) without affecting p53 (ser15) expression.@*CONCLUSION@#Ing2 silencing can inhibit AngⅡ-induced cardiac remodeling and dysfunction in mice by reducing p53 acetylation.
Assuntos
Animais , Camundongos , Angiotensina II , Proteína Supressora de Tumor p53 , Acetilação , Volume Sistólico , Remodelação Ventricular , Função Ventricular Esquerda , Miócitos CardíacosRESUMO
Objective:To explore the molecular mechanism of the effect of the histone methylase zeste gene enhancer homolog 2 (EZH2) on the proliferation and apoptosis of human hypertrophic cardiomyocytes AC16.Methods:The AC16 hypertrophic cardiomyocyte model was constructed by adding angiotensin Ⅱ to the AC16 cell culture medium. The cells were divided into four groups, including the blank control group, the angiotensin Ⅱ group, the empty vector + angiotensin Ⅱ group, and the EZH2 overexpression + angiotensin Ⅱ group. The expression levels of EZH2 and brain natriuretic peptide ( BNP) genes were measured using fluorescent quantitative PCR. The EZH2, trimethylation of lysine at position 27 of histone H3 (H3K27me3), and BNP proteins expression were detected by Western Blot. The MTS method was used to detect the proliferation of AC16 cell. The Annexin V-FITC/PI double staining method was used to detect the apoptosis of AC16 cell. Results:Compared with the blank control group, the expression levels of EZH2 and H3K27me3 in the angiotensin Ⅱ group were decreased, the expression level of BNP was increased, cell proliferation was decreased, and apoptosis was increased (all P < 0.001). Compared with the empty vector + angiotensin Ⅱ group, the expression levels of EZH2 and H3K27me3 in the EZH2 overexpression + angiotensin Ⅱ group were increased, the expression level of BNP was decreased, the cell proliferation level was increased, and the apoptosis level was decreased (all P < 0.001). There was no significant difference between the angiotensin Ⅱ group and the empty vector + angiotensin Ⅱ group (all P > 0.05). Conclusions:Histone methylase EZH2 has an effect on the proliferation and apoptosis of AC16 cell, providing a reference for the treatment of myocardial hypertrophy and revealing the exact pathogenesis of myocardial hypertrophy.
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Objective:To establish a candidate reference method for the determination of angiotensin Ⅱ in human plasma by isotope dilution liquid chromatography tandem mass spectrometry (ID-LC-MS/MS) and to evaluate its performance.Methods:Using [ 13C 6- 15N]-angiotensin Ⅱ as the internal standard, the plasma was accurately weighed by gravimetric method and mixed with a certain amount of internal standard. At the same time, enzyme inhibitor was added. After zinc sulfate solution protein precipitation and reversed-phase solid-phase extraction plate treatment, it was analyzed by liquid chromatography tandem mass spectrometry. The multi reaction ion monitoring mode(MRM)was selected by mass spectrometry to detect specific ion fragments of angiotensin Ⅱ and internal standard. The linearity, sensitivity, precision, recovery rate and uncertainty of the performance of the established method were evaluated according to ISO15193. Results:Angiotensin Ⅱ had good linearity in the range of 10-1 000 pg/g ( r=0.999 5), the lower limit of quantification was 7.68 pg/g, the analytical recoveries were 97.14% to 102.85%, intra-batch imprecision≤3.21%, inter-batch imprecision≤2.96%, and total imprecision≤3.67%. Conclusion:A method for the determination of plasma angiotensin Ⅱ was established by ID-LC-MS/MS. The method is accurate and reliable, and is expected to be a reference method for the determination of plasma angiotensin Ⅱ.
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Objective:To observe the morphological and hemodynamics changes of aortic segments in mice with angiotensinogen Ⅱ(Ang II) combined with β-aminopropionitrile(BAPN) induced-aortic dissection by color Doppler ultrasound(CDUS).Methods:Twenty male mice of 6-8 weeks old C57BL/6 were randomly divided into two groups: the model group( n=10) was induced by intraperitoneal injection of Ang Ⅱ combined with BAPN to establish mice model with aortic dissection; the control group( n=10) was intraperitoneally injected with normal saline.The body weight, systolic and diastolic blood pressure of the mice were routinely recorded. On the 42th day, CDUS was used to measure the indexes of ascending aorta(AoA), descending thoracic aorta(DAo) and suprarenal aorta(SAo) in both groups, including the inner diameter of the cross section, peak systolic velocity(PSV), the end diastolic velocity(EDV), the resistance index(RI), the pulsatility index(PI), time average mean velocity(TAMV), the heart rate(HR) and the maximal shear rate(SR). Then, the aortas were harvested from the root to the bifurcation of the renal artery. The pathological changes of the aortic wall were observed using hematoxylin-eosin(HE) staining. Results:①There were statistically significant differences in body weight, systolic blood pressure, diastolic blood pressure and heart rate between the model group and the control group(all P<0.05). Compared with the control group(0/10), the incidence of the AoA dissection(8/10) in the model group was obviously higher, the difference was statistically significant( P<0.05); while the incidence of the DAo dissection(4/10) and the SAo dissection(3/10) in the model group was slightly higher, the differences were not statistically significant (all P>0.05). ②Compared with the ascending aorta of the control group, the inner diameter, PSV, EDV, TAMV, PI and SR in the model group were significantly higher(all P<0.05), while RI showed no significant difference between the two groups ( P>0.05). For the descending thoracic aorta, PSV, EDV, TAMV, PI and SR in model group were higher than those of the control group(all P<0.05), however the inner diameter and RI were not significantly different between the two groups (all P>0.05). And for the superior renal aorta, PSV, TAMV, RI, PI and SR in the model group were obviously higher than the control group(all P<0.05), whereas the inner diameter and EDV were not significantly different between the two groups (all P>0.05). ③The HE of the tissue section in the model group showed, the aortas were obviously dilated, irregular, with inhomogeneously thickening wall; the endothelial cell nuclei were slightly stained, and some intima and middle layer ruptured and protruded outward to form dissecting aneurysms. The adventitias were markedly infiltrated with inflammatory cells. Conclusions:Ultrasonography could primarily evaluate the hemodynamic changes of aorta in hypertension with aortic dissection, and the PSV, TAMV, PI and SR of aorta may be important indicators for early predicting the occurrence of aortic dissection in hypertension.
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【Objective】 To explore B lymphocytes’ role and mechanisms in angiotensinⅡ/phenylephrine (AngⅡ/PE) induced cardiomyopathy so as to understand the role of inflammatory cells in myocardial injury. 【Methods】 AngⅡ/PE was administered to wild-type (WT) and B cells deficiency (μMT) mice for 14 days or 28 days. The mice were analyzed by blood pressure measurement, echocardiography imaging, flow cytometry, and histology. Cardiac fibrosis was evaluated by Masson staining. 【Results】 Compared with the control group, the left ventricular mass (P<0.01), heart mass/tibia length ratio (P<0.01) and cross-sectional area of cardiomyocytes in AngⅡ/PE group were significantly increased (P<0.01). After 2 weeks of AngⅡ/PE treatment, B lymphocytes (P<0.05), CD45+ leukocytes (P<0.05), CD64-Ly6C+ monocytes (P<0.05), CD64+Ly6C-macrophages (P<0.05) and Ly6g+ neutrophils (P<0.05) were recruited in myocardial tissue. Compared with WT_AngⅡ/PE group, the heart weight/tibia length ratio (P<0.05), left ventricular weight (P<0.05) and myocardial cell cross-sectional area (P<0.05) of μMT_AngⅡ/PE mice were significantly improved. CD45+Ly6C+CD64- monocytes (P<0.05) and CD45+Ly6C-CD64+ macrophages (P<0.05) were significantly decreased. 【Conclusion】 B lymphocytes deficiency improves AngⅡ/PE induced cardiac hypertrophy by reducing the infiltration of CD45+Ly6C+CD64- monocytes and CD45+Ly6C- CD64+ macrophages.
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[Abstract]Objective:To evaluate the efficacy and safety of fermented cordyceps powder combined with angiotensin-converting enzyme inhibitors (ACEI)/angiotensin Ⅱ receptor blocker (ARB) in the treatment of diabetic kidney disease (DKD). Method:The randomized controlled trials (RCTs) concerning the treatment of DKD with fermented cordyceps powder plus ACEI/ARB were retrieved from Pubmed, Embase, Cochrane library, China National Knowledge Infrastructure (CNKI), Chinese BioMedical Literature Database on disc (CBMdisc), Wanfang Data Knowledge Service Platform, and Chongqing Weipu Database for Chinese Technical Periodicals (VIP). The quality of the included articles was evaluated by the Cochrane Collaboration's tool, followed by data analysis using RevMan 5.3. Result:A total of 48 RCTs were included, involving 4 562 cases. As revealed by Meta-analysis, the effective rate of fermented cordyceps powder combined with ACEI/ARB was higher than that of ACEI/ARB [risk ratio (RR)=1.20, 95% confidence interval (CI) (1.15,1.24), <italic>P</italic><0.000 01]. Moreover, such combination effectively reduced urinary albumin excretion rate [standardized mean difference (SMD)=-2.61,95%CI (-3.17,-2.05),<italic>P</italic><0.000 01],24-h proteinuria[SMD=-1.75,95%CI (-2.15,-1.35),<italic>P</italic><0.000 01], serum creatinine(Scr)[mean difference (MD)=-14.57,95%CI (-17.94,-11.21),<italic>P</italic><0.000 01], blood urea nitrogen(BUN)[MD=-1.05,95%CI (-1.29,-0.81),<italic>P</italic><0.000 01], cystatin C (Cys-C) [MD=-0.52,95%CI (-0.68,-0.36),<italic>P</italic><0.000 01], fasting blood glucose(FBG)[MD=-0.59,95%CI (-0.93,-0.25),<italic>P</italic>=0.000 6], hemoglobin A1c(HbA1c)[MD=-0.50,95%CI(-0.75,-0.24),<italic>P</italic>=0.000 1], tumor necrosis factor-<italic>α</italic>(TNF)-<italic>α </italic>[SMD=-1.68,95%CI (-2.21,-1.15),<italic>P</italic><0.000 01], C-reactive protein(CRP) [SMD=-1.35,95%CI (-1.77,-0.93),<italic>P</italic><0.000 01], and interleukin-6 (IL-6) [SMD=-1.52,95%CI (-1.98,-1.07),<italic>P</italic><0.000 01]. There was no significant difference in the incidence of adverse events between the two groups [RR=0.77,95%CI (0.49,1.21),<italic>P</italic>=0.25]. Conclusion:Fermented cordyceps powder combined with ACEI/ARB is more effective than ACEI/ARB in the treatment of DKD, which is worthy of clinical promotion and use. More multi-center RCTs with a large sample size are needed for verification.
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Objective:To explore the protective effect and the mechanism of Danggui Shaoyaosan(DSS) on angiotensin Ⅱ (AngⅡ)/transient receptor potential cation channel 6 (TRPC6) pathway in nephrotic syndrome (NS) rats. Method:In animal experiments, doxorubicin (4 mg·kg<sup>-1</sup> for the 1<sup>st</sup> week and 2 mg·kg<sup>-1</sup> for the 2<sup>nd</sup> week) was injected twice to the tail vein of rats to induce NS model in 160 rats, which were then randomly divided into model group (normal saline), losartan group (30 mg·kg<sup>-1</sup>·d<sup>-1</sup>), and low-(4.3 g·kg<sup>-1</sup>·d<sup>-1</sup>), medium-(8.6 g·kg<sup>-1</sup>·d<sup>-1</sup>), and high-dose (17.2 g·kg<sup>-1</sup>·d<sup>-1</sup>) DSS groups. Besides, a normal group was also set. After intervention for four weeks, ultrastructure changes of the kidney were identified by transmission electron microscopy (TEM). The 24-hour urine protein was detected by kits. Radioimmunoassay was used to detect the content of AngⅡ and Calcineurin (CaN) in plasma. Western blot was used to detect the protein expression of TRPC6, angiotensin Ⅱ type 1 receptor (AT1R), podocyte slit diaphragm-specific protein (Nephrin), and cysteine-aspartic acid protease-3 (Caspase-3) in the renal cortex. Immunohistochemistry was used to detect the expression of TRPC6 and AT1R in the slit diaphragm. In cell experiments, AngⅡ stimulated MPC5 podocytes. The cells were randomly divided into a normal group, an AngⅡ group, an AngⅡ+SAR7334 (TRPC6-specific inhibitor) group, an AngⅡ+5%DSS group, an AngⅡ+10%DSS group, and an AngⅡ+15%DSS group. Western blot was used to detect the protein expression of TRPC6, AT1R, Nephrin, and Caspase-3 in podocytes. Result:Compared with the normal group, the model group showed increased 24-hour urine protein content (<italic>P</italic><0.01) and AngⅡ and CaN in plasma (<italic>P</italic><0.01), incomplete glomerular structure, the extensive fusion of podocyte process with elevated fusion rate (<italic>P</italic><0.01), increased expression distribution of AT1R and TRPC6 in the renal cortex, and up-regulated protein expression of AT1R, TRPC6, and Caspase-3 in renal tissues (<italic>P</italic><0.01), and reduced Nephrin protein expression (<italic>P</italic><0.01). Compared with model group, the losartan group and the high-dose DSS group exhibited decreased 24-hour urine protein content (<italic>P</italic><0.01) and the content of AngⅡ and CaN in plasma (<italic>P</italic><0.01), improved glomerular structure, reduced fusion rate of podocyte process (<italic>P</italic><0.01), diminished expression distribution of TRPC6 and AT1R in the renal cortex, declining protein expression of AT1R, TRPC6 and Caspase-3 in renal tissues (<italic>P</italic><0.01), and elevated Nephrin protein expression (<italic>P</italic><0.01). Additionally, compared with the normal podocytes, AngⅡ-stimulated podocytes showed increased protein expression of AT1R, TRPC6 and Caspase-3 (<italic>P</italic><0.01), and decreased expression of Nephrin (<italic>P</italic><0.01). Compared with the AngⅡ group, the AngⅡ+SAR7334 group displayed reduced protein expression of AT1R, TRPC6, and Caspase-3 (<italic>P</italic><0.01) and increased protein expression of Nephrin (<italic>P</italic><0.01). Conclusion:DSS can improve the pathological characteristics of NS presumedly by inhibiting the interaction between AngⅡ and TRPC6 in podocytes and improving the structural integrity of podocytes to repair the damage of glomerular molecular barrier and slow down the progression of NS-induced proteinuria.
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Objective:To study the effect of Fushengong prescreption on the regulation-antagonism effect of angiotensin converting enzyme-angiotensin Ⅱ-angiotensin Ⅱ 1 receptor (ACE-AngⅡ-AT1R) axis and angiotensin converting enzyme 2-angiotensin (1-7)-Mas receptor[ACE2-Ang(1-7)-MASR] axis of rats with chronic renal failure(CRF), and to explore its mechanism of delaying the development of CRF. Method:The 65 male SD rats were randomly divided into normal group (<italic>n</italic>=10) and modeling group (<italic>n</italic>=55). The normal group was routinely reared, while the modeling group were administered by gavage with 0.25 g·kg<sup>-1</sup>d<sup>-1 </sup>adenine suspension for 28 days. After the model was successfully established, the survival model rats were randomly divided into model group, benazepril group(0.01 g·kg<sup>-1</sup>·d<sup>-1</sup>)and low,medium and high dose of Fushengong prescreption groups (4,8,16 g·kg<sup>-1</sup>·d<sup>-1</sup>). The normal group and model group were administered the same volume of normal saline by gavage, lasted for 28 days. After the experiment, systolic blood pressure (SBP) and diastolic blood pressure (DBP) of caudal artery were measured, and 24-hour urine was collected to determine 24-hour urine protein (24 h U-pro). The content of serum creatinine(SCr) and blood urea nitrogen (BUN) in the serum were measured, the histological morphology was observed by hematoxylin eosin(HE)staining, and the degree of renal interstitial fibrosis was observed by Masson staining. Enzyme linked immunosorbent assay (ELISA) was used to determine the contents of AngⅡ, Ang (1-7) and Cystatin C (CysC) in serum and renal homogenate. The protein level of ACE, ACE2, AT1R and MASR were detected by Western blot. The expression of ACE and ACE2 protein in renal tissues were detected by immunohistochemistry. Result:Compared with normal group, the expression levels of SCr, BUN and CysC in model group were significantly increased(<italic>P</italic><0.05), the content of AngⅡ in serum and kidney tissues were significantly increased, the content of Ang (1-7) were significantly decreased(<italic>P</italic><0.05), the expression of ACE and AT1R protein in renal tissues were significantly increased(<italic>P</italic><0.05), and the expression of ACE2 and MASR protein were significantly decreased(<italic>P</italic><0.05). Compared with model group and benazepril group, after the intervention with Fushengong prescreption, the serum SCr,BUN and CysC decreased(<italic>P</italic><0.05),the content of AngⅡ in serum and kidney tissues decreased significantly,Ang(1-7) increased significantly(<italic>P</italic><0.05), the expression of ACE and AT1R protein in renal tissues decreased significantly(<italic>P</italic><0.05), ACE2 and MASR protein increased significantly(<italic>P</italic><0.05). The high-dose Fushengong prescreption has the best effect. The high, medium and low-dose effects of Fushengong prescreption were dose-dependent. Conclusion:Fushengong prescreption improved renal function and pathological change of kidney in adenine-induced rats with chronic renal failure. The mechanism may be related to the inhibition of ACE-AngⅡ-AT1R axis and promotion of ACE2-Ang(1-7)-MASR axis ,which leads to the delaying of the progression of chronic renal failure.