Role of nucleolin in Ang II-induced phenotypic transformation of vascular smooth muscle cells

AIM: To investigate the effect of nucleolin on angiotensin II (Ang II)-induced phenotypic transformation of vascular smooth muscle cells (VSMCs). METHODS: Ang II was used to induce the phenotypic transformation of VSMCs. The spatial and temporal expression patterns of nucleolin, and the effects of Ang II on the expression of VSMC phenotypic transformation markers α-smooth muscle actin (α-SMA), calponin, smooth muscle protein 22 α (SM22α) and osteopontin (OPN) were investigated. The techniques of gene over-expression and RNA interference were used to assess the effect of nucleolin on the expression of Ang II-mediated VSMC phenotypic transformation markers. RESULTS: The expression of α-SMA, SM22α and calponin at the mRNA and protein levels was gradually decreased by Ang II stimulation, while the expression of OPN at mRNA and protein levels was gradually increased. The expression of nucleolin was gradually up-regulated in the VSMCs treated with Ang II at different concentrations for various duration (P<0.05). Ang II induced nucleolin translocation from the nucleus to cytoplasm. Over-expression of nucleolin promoted the VSMC phenotypic transformation induced by Ang II. Down-regulation of nucleolin suppressed the promotion of phenotypic transformation. CONCLUSION: Nucleolin promotes Ang II-induced phenotypic transformation of VSMCs, and its mechanism may be related to its function of cytoplasmic translocation.     

Effects of SM22α on migration and invasion abilities of colorectal cancer cells

AIM:To explore the effects of smooth muscle 22α protein (SM22α) on the migration and invasion abilities of colorectal cancer cells, and to investigate its molecular mechanism. METHODS:The SM22α-over-expressing cells were constructed by lentivirus transfection. The cell migration ability was detected by wound healing assay. The changes of cell migration and invasion abilities were measured by Transwell assay. RT-qPCR was used to detect the changes of SM22α mRNA level. The protein levels of extracellular signal-regulated kinase (ERK), p-ERK, matrix metalloproteinase-9 (MMP-9) and SM22α were determined by Western blot. RESULTS:HCT116 cells with SM22α over-expression were constructed successfully. SM22α inhibited the migration and invasion abilities of colorectal cancer cells. SM22α over-expression decreased the protein levels of p-ERK and MMP-9 (P<0.05).CONCLUSION:SM22α inhibits the migration and invasion abilities of colorectal cancer cells by inhibiting ERK/MMP-9 signaling pathways.     

Retrovirus-mediated cellular repressor of E1A-stimulated genes inhibits neointima formation in the rat carotid artery after balloon injury

AIM: To evaluate the effects of over-expression of cellular repressor of E1A-stimulated genes (CREG) mediated by retrovirus on neointima formation in injured rat carotid. METHODS: The pluronic F127 containing pLNCX/CREG or pLNCX/GFP retroviral vectors was placed around the injured rat carotid.The neointima,media areas and the intima to media ratio were calculated.Expressions of CREG,SM α-actin and Ki-67 were detected. RESULTS: The GFP expression was observed at day 2 in pLNCX/GFP groups.The expression of exogenous CREG was also significantly increased in arteries at day 2 after pLNCX-CREG infection.Over-expression of CREG significantly suppressed neointima formation,attenuated the expression of Ki-67 and up-regulated SM α-actin expression. CONCLUSION: Over-expression of CREG inhibits VSMCs proliferation and promotes VSMCs differentiation after vascular injury.It suggests that modulation of CREG expression or activity may be a viable approach to treat neointimal restenosis after percutaneous coronary intervention.     

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 Xijiao Dihuang and Yinqiao San decoction on ICAM-1 and VCAM-1 expression in mouse lung tissues and rat pulmonary microvascular endothelial cells infected with influenza virus

AIM：To investigate the effects of Xijiao Dihuang and Yinqiao San decoction (XDY) on the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in mouse lung tissues and rat pulmonary microvascular endothelial cells (RPMVECs) infected with influenza virus, and to explore its mechanism for treatment of viral pneumonia. METHODS：Fifty-four male BALB/c mice were randomly divided into normal group, model group and XDY group (n=18 in each group). The viral pneumonia model was established by intranasally dripping influenza A (H1N1) virus into the mice. The mice in XDY group were treated with XDY 1 h after dripping the virus. The expression of ICAM-1 and VCAM-1 in lung tissues was examined by immunohistochemical staining 2, 4 and 6 d after infection. On the other hand, RPMVECs were obtained from male Wistar rats and primarily cultured. The cells were randomly divided into control group, virus group, virus+XDY group, tumor necrosis factor α (TNF-α) group and TNF-α+XDY group. The mRNA and protein expression of ICAM-1 and VCAM-1 was evaluated by real-time PCR and flow cytometry 24 h after infection. RESULTS：Virus exposure increased ICAM-1 and VCAM-1 expression in mouse lung tissues (P<0.01), and XDY treatment attenuated this effect (P<0.01). Virus and TNF-α both led to the increases in mRNA and protein expression of ICAM-1 and VCAM-1 in RPMVECs (P<0.01), which were also reduced by treatment with XDY (P<0.01). CONCLUSION：Treatment with XDY decreases virus-induced ICAM-1 and VCAM-1 expression, suggesting an important role of XDY in treatment of viral pneumonia.     

Placental growth factor is involved in angiotensin II-induced cardiac fibroblast activation

AIM：To explore the role of placental growth factor (PLGF) in the process of angiotensin II (Ang II)-induced activation of cardiac fibroblasts (CFs). METHODS：Primary culture and identification of CFs from neonatal Sprague-Dawley rats were performed. The method of fluorescence immunocytochemistry was employed to observe the expression of alpha-smooth muscle actin (α-SMA). Real-time PCR and Western blotting were used to determine mRNA and protein levels. The cell proliferation was observed by WST-1 assay. RESULTS：Compared with control group, the PLGF expression at mRNA and protein levels in Ang II-treated CFs was significantly increased, whereas the mRNA expression of PLGF was decreased in the CFs treated with telmisartan and Ang II. Treatment with PLGF induced the proliferation of CFs and increased the protein expression of α-SMA. Treatment with PLGF for 60 min significantly increased the protein levels of p-ERK1/2 in the CFs. Compared with Ang II group, the proliferation of CFs was depressed and the protein expression of α-SMA was attenuated in Ang II+anti-PLGF group.The mRNA expression levels of type I and type III collagens were also down-regulated. CONCLUSION：PLGF might be involved in the process of Ang II-induced proliferation of CFs and fibrosis.     

Effect of 27nt-miRNA on regulation of SM22α expression in vascular smooth muscle cells and its effect on cell viability,migration and phenotypic changes

AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs). METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs. Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion. The cell viability was measured by MTT assay. The migration ability was detected by scratch assay. The mRNA and protein expression of SM22α was determined by RT-PCR, immunocytochemical staining and Western blot. RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05), the migration ability was increased (P<0.05) and the expression of SM22α at mRNA and protein level was decreased (P<0.05). Compared with negative control lentiviral group, the cell viability in 27nt-miRNA over-expression group was decreased (P<0.05), the migration ability was decreased (P<0.05), and the mRNA and protein expression of SM22α was increased (P<0.05). While in anti-27nt-miRNA group, the cell viability was increased(P<0.05), the migration ability was increased (P<0.05), and the mRNA and protein expression of SM22α was decreased (P<0.05). CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.     

he effects of constant magnetic field on apoptosis,adhesion molecule expression in endothelial cells and their adhesion rates with monocytes

AIM: To investigate the effects of constant magnetic field on apoptosis, secretion and expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in human umbilical vein endothelial cells (HUVEC), and their adhesion rates with THP-1 monocytes induced by angiotensin Ⅱ (AngⅡ).METHODS: The third passage of cultured HUVEC was used.There were six groups: control group, Ang Ⅱ (10^-6 mol/L) group, Ang Ⅱ with 1, 5, 10 or 20 gausses of constant magnetic field group.Samples were collected 24 h after incubation with or without magnetic field.Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and propidinm iodide staining with flow cytometry.Secretion and expression of ICAM-1 and VCAM-1 were detected by ELISA and immunocytochemistry, respectively.Adhesion rate between HUVEC and THP-1 was measured by counting method.RESULTS: Ang Ⅱ at concentration of 10^-6mol/L induced apoptosis in HUVECs (P<0.05 vs control), whereas in 1, 5, 10 and 20 gausses group, apoptosis of HUVECs was significantly lower than that in Ang Ⅱ group (P<0.05).Ang Ⅱ at concentration of 10-6 mol/L significantly increased secretion and expression of ICAM-1 and VCAM-1 (P<0.05 vs control), whereas secretion and expression of ICAM-1 and VCAM-1 in 1, 5, 10 and 20 gausses group significantly decreased, compared with Ang Ⅱ group (P<0.05).The adhesion rates between HUVEC and THP-1 significantly increased 24 h after incubation of HUVEC with Ang Ⅱ (P<0.05 vs control), in contrast, the adhesion rates between HUVEC and THP-1 in 1, 5, 10 and 20 gausses group significantly decreased, compaed with Ang Ⅱ group (P<0.05).CONCLUSIONS: One gauss to 20 gausses of constant magnetic field antagonizes the effects of Ang Ⅱ on HUVEC, decreases apoptosis and expression of ICAM-1 and VCAM-1 in HUVEC, and also decreases the adhesion rates between HUVEC and monocytes induced by Ang Ⅱ.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.     

MEF2A/2D regulates depolarization induced differentiation via Rho signaling pathway in VSMCs

AIM：To investigate the role of myocyte enhance factor 2 (MEF2) in the regulation of depolarization-induced differentiation via Rho signaling pathway in vascular smooth muscle cells (VSMCs). METHODS： In primarily cultured mouse portal vein VSMCs, the techniques of immunofluorescence, Western blotting, real-time RT-PCR and siRNA transfection were applied to determine the membrane translocation of RhoA, the phosphorylation of LIM kinase (LIMK) and cofilin-2, and the mRNA expression of 4 MEF2 isoforms, myocardin and SM22α under high KCl-induced depolarization. RESULTS：RhoA membrane translocation occurred 30 s after depolarization, while phosphorylation of LIMK and cofilin-2 peaked at 10 min and 30 min, with increment of 42.20% and 32.75%， respectively. The mRNA expression of MEF2A and 2D was increased by 47.63% and 48.15%, respectively. In MEF2A/2D knockdown VSMCs, the mRNA expression of myocardin was not sensitive to depolarization, while SM22α expression was not affected. CONCLUSION：MEF2A/2D, acting on myocardin, is involved in the regulation of depolarization-induced differentiation via Rho signaling pathway in VSMCs     

Therapeutic effect of neuregulin-1β on pressure overload-induced myocardial hypertrophy in rats

AIM：To investigate the therapeutic effect and the mechanism of neuregulin-1β (NRG-1β) on the rat model of myocardial hypertrophy induced by pressure overload．METHODS：Eight weeks after coarctation of abdominal aorta, the Wistar rats were randomly divided into 4 groups: myocardial hypertrophy (model) group, sham operation (sham) group, NRG-1β treatment group (intravenous injection of NRG-1β at dose of 10 μg/kg daily for 7 d) and NRG-1β+Herceptin (HERCE) treatment group ［intravenous injection of NRG-1β (10 μg/kg) plus HERCE (10 μg/kg) daily for 7 d］. The characteristics of heart functions were evaluated by the methods of hemodynamics and echocardiography. Masson staining was employed to observe the pathological changes of myocardial tissues. The concentration of angiotensin II (Ang II) in myocardial tissues was measured by radioimmunoassay. The level of tumor necrosis factor α (TNF-α) in myocardial tissues was detected by ELISA. The mRNA expression of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2-associated X protein (bax) in the myocardium was determined by RT-PCR. RESULTS：The left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were higher, while the left ventricular end-systolic diameter (LVESD) and left ventricular end-diastolic diameter (LVEDD) were smaller in NRG-1β group than those in model group. The left ventricular end-systolic pressure (LVESP) and maximal rate of increase/decrease in left ventricular pressure (±dp/dtmax) were higher, and left ventricular end-diastolic pressure (LVEDP) was significantly lower in NRG-1β group than those in model group. Compared with model group, treatment with NRG-1β decreased collagen volume fraction (CVF), reduced the Ang II and TNF-α, increased bcl-2 mRNA expression, and decreased bax mRNA expression in myocardial tissues. No difference of the above parameters between model group and NRG-1β+HERCE treatment group was observed. CONCLUSION：NRG-1 reduces the expression of Ang II and TNF-α in myocardial tissues in pressure-overload rats, thus reducing Ang II and TNF-α mediated myocardial interstitial remodeling. Increase in the mRNA expression of bcl-2 and decrease in the mRNA expression of bax by NRG-1 inhibit myocardial cell apoptosis, which is responsible for its role of improving cardiac function of myocardial hypertrophy induced by pressure overload.     

Effects of xijiao dihuang decoction on the expression of adhesion molecules in vascular endothelial cells of adrenine and hypothermia-treated rats

AIM: To study the effects of xijiao dihuang decoction, a Chinese medicine, on the expression of adhesion molecules in vascular endothelial cells (VECs) of adrenine and hypothermia-treated rats. METHODS: The expression of VCAM-1, ICAM-1, PECAM-1, iNOS and their corresponding mRNA in VECs was assayed by immunohistochemistry analysis and RT-PCR, respectively. RESULTS: Immunohistochemistry analysis showed the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS are higher in adrenine and hypothermia-treated rats than that in controls, xijiao dihuang decoction decreased the expression of VCAM-1, ICAM-1, PECAM-1 and iNOS in a dose-dependent manner in adrenine and hypothermia-treated rats. CONCLUSION: Xijiao dihuang decoction can down-regulate the expression of adhesion molecules in vascular endothelial cells of adrenine and hypothermia-treated rats.     

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.     

Effect of doxycycline on matrix metalloproteinase activity and vascular remodeling in balloon-injured rat carotid arteries

AIM:To examine the inhibition of matrix metalloproteinase (MMP) activity by doxycycline (Doxy) and its effect on vascular smooth muscle cells (SMCs) proliferation,neointimal hyperplasia and vascular remodeling.METHODS: The model of rat common carotid artery injury was established by balloon-dilatation.Doxy was administered to the animals of treatment group at dose of 30 mg·kg^-1·d^-1.The activity of MMPs in the tissue of injured carotid arteries was measured by gelatin zymography.The thickness and area of neointimal,lumen area and the proliferation of SMCs were measured by histological and morphometric analysis.RESULTS:1.After Doxy treatment,the activity of MMP-9 in the carotid arteries was reduced by 26.3% and 34.5% compared to that in rats without Doxy treatment at 24 hours and 3 days after balloon injury,respectively (P<0.01).The activity of MMP-2 was also reduced by 40.0% at 7 days after injury (P<0.01).2.The thickness of neointimal were significantly decreased by 32.0% and 38.8% (P<0.01) and the lumen area was increased by 58.0% and 90.4 % at 14 and 28 days after injury in the Doxy-treated rats compared to those in control rats,respectively (P<0.01).Doxy treatment significantly reduced intimal SMCs proliferation from 62.76%±1.02 % in the controls to 43.23%±1.06% at 7 days after injury (P<0.01).CONCLUSION: Doxy treatment inhibits the activity of MMPs,the SMCs proliferation of intimal,neointimal hyperplasia and vascular remodeling,suggesting that Doxy treatment is useful in preventing restenosis after PTCA.     

Effect of angiotensin-(1-7) on angiotensin II-induced rat renal interstitial fibroblast activation

AIM: To explore the influence of angiotensin-(1-7) on angiotension II (Ang II)-induced activation and extracellular matrix secretion in rat renal interstitial fibroblasts (NRK-49F cells). METHODS: The NRK-49F cells were maintained and sub-cultured, then the cells were divided into control group, Ang II group, Ang-(1-7) group and Ang II+Ang-(1-7) group. The expression of α-smooth muscle actin(α-SMA),transforming growth factor β₁ (TGF-β₁) and insulin-like growth factor I(IGF-I) was detected by the method of immunocytochemistry when the cells were cultured for 72 h. The content of TGF-β₁, IGF-I and collagen type I(Col I) in the cultured supernatants were measured by ELISA. RESULTS: In control group and Ang-(1-7) group, only basic expression of α-SMA and almost no expression of TGF-β₁, IGF-I and Col I were observed. Compared with control group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was increased in Ang II group. Compared with Ang II group, the expression of α-SMA, TGF-β₁, IGF-I and Col I was significantly decreased in Ang II+Ang-(1-7) group.CONCLUSION: Ang-(1-7) inhibits the activation of renal interstitial fibroblasts and decreases the Ang II induced secretion of Col I by suppressing TGF-β₁ and IGF-I expression.     

Relationship between renal hypertension and cardiac α1-adrenoceptor autoantibodies in rats

AIM: To examine the autoantibody against α₁-adrenoceptor and its biologic activities during the development of renal hypertension. METHODS: Renal hypertension of rat was achieved by clipped renal artery, the titre of autoantibody to α₁-adrenoceptor was detected using ELISA immunoassay. Furthermore, the biological offects of these autoantibodies on cultured cardiomyocytes were also examined. RESULTS: After two weeks of clipping renal arteries, both the frequency of occurrence and the titre of autoantibodies to cardiac α₁-adrenergic receptor were significantly increased as compared with the control of pre-treatment. The increased autoantibodies lasted for several weeks and then automatically decreased gradually to the pre-clipping level at 12 weeks. The biological effects of these autoantibodies displayed an "agonistic-like" activities on the beating frequency of cultured neonatal cardiomyocytes. CONCLUSION: Autoantibodies against α₁-adrenoceptor may play a role in the elevation of peripheral vascular resistance and in the development of cardiac hypertrophy in rats with renal hypertension.     

Regulatory effect of miR-199a-5p on cardiomyocyte hypertrophy in rat

AIM：To investigate the expression of miR-199a-5p in rat cardiomyocyte hypertrophy models. METHODS：The in vivo cardiomyocyte hypertrophy model was established by transverse abdominal aortic constriction (TAAC) and the in vitro model was induced by angiotensin II. The content of miR-199a-5p was detected by qRT-PCR in the plasma of the TAAC rats and in the cardiomyocytes (CM) of the newborn rats. The CM was isolated and transfected with miR-199a-5p mimic or inhibitor at concentration of 100 nmol/L by Lipofectamine RNAiMAX. The mRNA levels of atrial natriuretic factor (ANF) and β-myosin heavy chain (β-MHC) were detected by qRT-PCR. Tritium-labeled leucine incorporation was employed to determine the protein synthesis rate in the CM. The method of cyto-fluorescent staining was applied to measure the changes of the CM surface area. RESULTS：Compared with control group, the content of miR-199a-5p significantly increased in the TAAC rats and in the CM induced by angiotensin II. In addition, over-expression of miR-199a-5p in the CM up-regulated the mRNA expression of ANF and β-MHC, accelerated the protein synthesis rate and enlarged the CM surface area. In the CM transfected with miR-199a-5p inhibitor following induced by angiotensin II, the hypertrophy effect receded inversely (P<0.05). CONCLUSION:Over-expression of miR-199a-5p may promote cardiomyocyte hypertrophy, and repression of miR-199a-5p may inhibit cardiomyocyte hypertrophy in the CM.     

Effects of Huaiyu decoctum on serum concentrations of TNF-α, IL-6 and IL-10 in rats after anorectal operation

ATM: To investigate the effects of Huaiyu decoctum on the serum concentrations of TNF-α, IL-6 and IL-10 in rats after anorectal operation. METHODS: Sprague-Dawley rats (n=40) were randomly divided into 4 groups:normal group, model group, low-dose Huaiyu decoctum group and high-dose Huaiyu decoctum group. The concentrations of TNF-α, IL-6 and IL-10 in the rat serum were measured by ELISA. The pathologic changes of the anorectal tissues were observed under microscope with HE staining. The protein expression of ICAM-1, VCAM-1 and NF-κB was determined by Western blotting. RESULTS: After Huaiyu decoctum administration, TNF-α and IL-6 concentrations in the serum were significantly decreased, and IL-10 concentration was increased as compared with model group. Moreover, Huaiyu decoctum markedly attenuated edema and hyperemia in the rats after anorectal operation. The protein expression of intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and NF-κB in the anorectal tissues was obviously inhibited by Huaiyu decoctum treatment. CONCLUSION: Huaiyu decoctum improves the recovery of anorectal tissues after operation by decreasing the serum concentrations of TNF-α, IL-6 and IL-10, and reducing the protein expression of ICAM-1, VCAM-1 and NF-κB in the anorectal tissues.