Vascular damages in rats immunized by alpha1-adrenoceptor peptides

Autoantibodies against the α1-adrenoceptor which had agonist activity as norepinephrine might play roles in the progression of hypertension, but whether the autoantibodies could induce vascular remodeling as norepinephrine is not clear. In this paper, the models with antibodies against the α1-adrenoceptor were made by immunizing Wistar rats with the synthesized the second extracellular loop of α1-adrenoceptor peptides. The homo-age male Wistar rats received BSA in the same immunizing manner and male spontaneous hypertensive rats （SHR） were used as control. All the rats were raised for one year. The blood pressure and morphological changes of arteries were measured. In the end, despite the systolic blood pressure of immunized rats had no difference with normal control, the media thickness of aortas and ratio of media to lumen in the third-order arteries of mesenteric vasculature were increased in immunized rats. The observation with electron microscope showed that the mitochondria of vascular smooth muscle cells （VSMCs） had notable hyperplasia, and the interstitial collagen fibril was increased too. The effects of purified antibodies against α1-adrenoceptor on the proliferation of cultured VSMCs, and the expressions of c-jun, c-fos and α1-adrenoceptor were detected. The results showed that the antibodies could promote the proliferation of cultured VSMCs, and enhance the expression of c-jun both in vitro and in vivo. So we concluded that antibodies against the α1-adrenoceptor could contribute to vascular damages in rats by stimulating the growth of VSMCs which might be caused by the increased c-jun expression, and might play particular roles in the pathological changes of hypertension.     

Vascular Damages in Rats Immunized by α_1-Adrenoceptor Peptides

Autoantibodies against the α1-adrenoceptor which had agonist activity as norepinephrine might play roles in the progression of hypertension, but whether the autoantibodies could induce vascular remodeling as norepinephrine is not clear. In this paper, the models with antibodies against the α1-adrenoceptor were made by immunizing Wistar rats with the synthesized the second extracellular loop of α1-adrenoceptor peptides. The homo-age male Wistar rats received BSA in the same immunizing manner and male spontan...     

Endoplasmic reticulum stress induced by Thapsigargin in vascular smooth muscle cells of rat coronary artery

AIM: To establish the endoplasmic reticulum stress( ERS) cell model in vascular smooth muscle cells( VSMCs) of SpragueDawley( SD) rats. METHODS: Under sterile condition,the coronary arteries were isolated from SD rats. The primary VSMCs were cultured by tissue-sticking method,and observed the basic morphological characteristics under optical microscope. The marker proteins of VSMCs including α-smooth muscle actin( α-SMA) and smooth muscle myosin heavy chain( SM-MHC) were identified by immunofluorescence technique. VSMCs were treated with thapsigargin(0. 5,1 and 2 μmol / L) for 24 h,and the expression levels of binding immunoglobulin protein( Bi P) and C / EBP homologus protein( CHOP),the marker molecules of ERS,were detected using Western blotting. RESULTS: VSMCs climbed out from coronary artery tissues after about six days,and the cells had a nice state and formed the VSMC-like typical " peak valley". The results of immunofluorescence technique show that the marker proteins of VSMCs,α-SMA and SM-MHC were expressed significantly. The results of Western blotting show that the protein expression levels of Bi P and CHOP were increased by thapsigargin in a dose-dependent manner. CONCLUSION: VSMCs can be successfully cultured by tissue-sticking method and built the ERS model induced by thapsigargin.     

Autoantibodies against G-protein-coupled receptors modulate heart mast cells

Mast cells are believed to be involved in myocardial tissue remodelling under pathophysiological conditions. We examined the effects of autoantibodies against G-protein-coupled receptors in sera of patients with heart diseases on myocardial mast cells in the cultured neonatal Sprague-Dawley rat heart cells. Cells collected at day 3 and 10 of the culture were preincubated with autoantibodies against α-adrenoceptor and angiotensin Ⅱ ATl-receptor, agonist phenylephrine and angiotensin Ⅱ, and control IgG. The pretreated cultured cells were stained for selected mast cell markers tryptase, chymase and TNF-α The cultured cells were also processed for observation with electron microscopy. The autoantibodies-treatment of the 3-day cultured cells caused both increased intensity of immunofluorescence （p 〈 0.05） and their enlarged diameters of the mast cells when compared to age-matched ones. In contrast, the fluorescence of preincubated 10-day-old mast cells was decreased compared with controls （p 〈 0.01）. In control samples, the fluorescence of 10-day-old mast cells was significantly higher than that of 3-day-old ones （p 〈 0.001）. Results of electron microscopy examination demonstrated there was an increased granulation of treated 3-day-old mast cells, while a degranulation of mast cells at day 10 of application. The results suggest the modulation effect of the autoantibodies against G-protein-coupled receptors on mast cells, indicating a potential functional link between the autoantibodies against G-protein-coupled receptors and the mast cells in progression of heart disease.     

Preparation and application of polyclonal antibody against a recombinant laccase

A laccase gene from Trametes sp. 420 was recombinantly expressed in Pichia pastoris, producing the enzyme rLacD. Six mutant enzymes were produced by site-directed mutation at six potential glycosylation sites in the enzyme rLacD respectively. To probe the mutants with lower activities sensitively and specifically, the antiserum containing specific polyclonal antibodies were prepared by immunizing healthy male rabbits, about 4-month-old and 2 kilogram weight, using pure rLacD as an immunogen. Antibodies were collected after the fifth immunization injection. The antiserum had titres of 1：32 in double immunodiffusion test and of 1：128,000 in enzyme-linked immunosorbent assay （ELISA）. The results obtained by Western blot analysis showed that the antiserum could react with rLacD and its mutants with highly specific and sensitive affinities.     

Expression and purification of Ctomp2aa167-aa434 and preparation of its mAb

Chlamydia trachomatis outer membrane protein 2 (Ctomp2) is a major immunogen in chlamydial infections and a highly genus-conserved structural protein of all Chlamydia species . To purify the protein and to prepare monoclonal antibodies (mAbs) against it, the recombinant protein was induced by IPTG, which was confirmed by SDS-PAGE and purified by means of a Ni2 -charged resin column. The denatured protein was refolded in the GSH-GSSH buffer gradually and identified by Western blotting. Then the BALB/c mice were immunized with the recombinant protein to prepare the mAb against Ctomp2. The obtained mAbs were characterized. Genital specimens were tested with indirect ELISA mostly made of the mAb and cell culture in 84 patients with genital symptoms. The results showed that high-level expression of the recombinant protein was achieved, which existed as inclusion body and amounted to 38 % of total bacterium protein. A mAb against Ctomp2 was obtained. It belongs to IgG 2b. The titers were as high as 1:40 000. The Western blotting showed that the mAb only reacted with the recombinant protein. It had no crossing reactions against E. coli, N. gonorhoea, M. hominis, U. urealyticum and M. penetrans . It had high specifity. In comparison with gold standard test-cell culture, the sensitivities, specificities, positive predictive values and negative predictive values of indirect ELISA were 95.24%, 100%, 100% and 98.44%, respectively. The above-mentioned research work contributed not only to the further study of the structure and function of this protein , but also to the establishment of the method for its clinical application, for it had not been reported before.     

Agonistic AT1 Receptor Autoantibody Increases in Serum of Patients with Refractory Hypertension and Improves Ca^2＋ Mobilization in Cultured Rat Vascular Smooth Muscle Cells

Agonistic AT1 receptor autoantibodies （AT1-AAs） have been described in the patients with malignant hypertension or preeclampia. Furthermore, AT1-AAs were highly associated with refractory hypertension. Function of vascular smooth muscle cells （VSMCs） is important in the regulation of blood pressure. We investigated and compared the ability of angiotensin II （Ang II） and AT1-AAs to stimulate the intracellular calcium mobilization and cellular proliferation of rat VSMCs. Twenty-two patients with refractory hypertension, 24 patients with non-refractory hypertension and 37 normotensives were recruited. The serum of each patient was detected for the presence of AT1-AAs by ELISA. Ang II and the AT1-AAs from the sera of patients were used to stimulate rat VSMCs in vitro. AT1-AAs were detected in 10/22, 3/24 and 3/37 of patients with refractory hypertension, non-refractory hypertension and normotensives, respectively. AT1-AAs led the increase intracellular calcium mobilization in a dose-dependent manner and cellular proliferation of VSMCs just as Ang II. Both of these effects caused by AT1-AAs were blocked with losartan or a peptide corresponding to a part of the second extracellular loop of AT1 receptor. Since AT1-AAs exhibited pharmacological activity in rat VSMCs just as Ang II, they might play a role in the elevation of peripheral vascular resistance and in vascular remodeling. And AT1-AAs were suggested to involve in resistance to antihypertensive therapy.     

Protection of guinea pigs against Leptospira interrogans serovar Lai by LipL21 DNA vaccine

In this study, the full lipL21 gene fragment encoding outer membrane protein LipL21 was cloned from L. interrogans serovar Lai and inserted into eukaryotic expression vector pcDNA3.1（＋）. The guinea pigs were immunized with pcDNA3.1（＋）-lipL21, pcDNA3.1（＋） or PBS. Six weeks after the second immunization, the splenocytes were isolated to detect their proliferative ability by lymphocyte transformation experiments. In addition, microscopic agglutination test was used for quantitative detection of specific antibodies. The rest guinea pigs were challenged intraperitoneally with L. interogans sorevar Lai. Then, protective effect was evaluated on the basis of survival and histopathological lesions in the kidneys, lungs, and liver. The lipL21 gene was successfully expressed in COS-7 cells through recombinant pcDNA3.1（＋）-lipL21. The titer of specific antibodies substantially increased, and the stimulation index of splenocytes increased significantly. Hence, the pcDNA3.1（＋）-lipL21 could protect the immunized guinea pigs from homotypic Leptospira infection. Furthermore, no obvious pathologic changes were observed in the pcDNA3.1（＋）-lipL21 immunized guinea pigs. The results showed that the protective effect with pathogenic strains of Leptospira was shared by LipL21 mediated through a plasmid vector. Consequently, these results indicated that the lipL21 DNA vaccine was a promising candidate for the prevention of leptospirosis.     

Construction and expression of a single chain antibody mimicing human ovarian cancer antigen CA125

One concept for immune therapy of cancer involves induction of antigen mimic antibodies to trigger the immune response against tumor cells. Anti-idiotypic antibodies directed against the antigen-binding site of antibodies specific for tumor antigen may functionally and even structurally mimic antigen and induce anti-anti-idiotypic immune response. Monoclonal antibody WJ02 is one of such anti-idiotypic antibodies, which contains internal image of CA125. In order to improve the immunospecificity of mAb WJ02, we constructed a single chain of mAb WJ02 in Vl-linker-Vh orientation. The scFv-WJ02 could be expressed and secreted in the recombinant Pichia pastoris system. The secreted scFv protein with a molecular weight of 30 kD retained the biological activity of mAb WJ02, which was proved by a direct binding assay and inhibition experiment. Our results indicated that the scFv-WJ02 could be used as a possible tool for idiotypic therapy against ovarian cancer, which might enhance the possibility of eliminating nonspecific responses induced by mAb WJ02.     

Response to hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 from elemene combo tumor cell vaccine

To analyze immune response to murine hepatocarcinoma Hca-F of mice immunized with heat shock protein 70 （HSP70） derived from elemene combo tumor cell vaccine （EC-TCV） of Hca-F, HSP70 was isolated from EC-TCV by ADP affinity chromatography. Mice were immunized with HSP70 intraperitoneally three times and spleen calls were sampled. For cells, their proliferation and cytotoxicity against Hca-F were measured with MTT assay and their phenotypes were analyzed with flow cytometry. Spleen cells of immunized mice with HSP70 exhibited more potent cytotoxicity against Hca-F and proliferation than that of normal control mice, but less potent than that of mice immunized with EC-TCV. Among three groups, the percent of T6 T lymphocytes in the mice immunized with HSP70 （35.5%） was the highest compared with 6.25% in normal mice, and 28.4% in the mice immunized with EC-TCV. Immunization of HSP70 derived from EC-TCV could elicit potent immune response to Hca-F. HSP70 is one of elements inducing anti-tumor immune responses against Hca-F.     

Immunogenicity and protective efficacy of DNA vaccine against visceral leishmaniasis in BALB/c mice

The current study was designed to examine the protective efficacy of DNA vaccines based on gp63 and Hsp70 against murine visceral leishmaniasis.Inbred BALB/c mice were immunized subcutaneously twice at an interval of three weeks with pcDNA3.1(+) encoding T cell epitopes of gp63 and Hsp70 individually and in combination.Animals were challenged intracardially with 10~7 promastigotes of Leishmania donovani 10 days post immunization and sacrificed 1,2 and 3 months post challenge.The immunized animals revealed a significant reduction(P < 0.05)in splenic and hepatic parasite burden as compared to the infected controls.Maximum reduction in parasite load(P < 0.05) was observed in animals treated with a combination of pcDNA/gp63 and pcDNA/Hsp70.These animals also showed heightened DTH response,increased IgG2a,elevated Th1 cytokines(IFN-γ and IL-2) and reduced IgG1 and IL-10 levels.Thus,mice immunized with the cocktail vaccine exhibited significantly greater protection in comparison to those immunized with individual antigens.     

Preparation and determination of immunological activities of anti-HBV egg yolk extraction

To prepare an effective immune preparation to treat hepatitis B, hens were immunized with hepatitis B vaccines, and then anti-HBV egg yolk extraction （anti-HBV EYE） was refined from egg yolk by a dialyzable method. Its chemical characteristics were identified by ultraviolet spectrum, HPLC, Lowry analysis and pharmacopocia-raleted methods. The specific immunological activity was examined by leukocyte adherence inhibition （LAI） in vitro and delayed type hypersensitivity （DTH） in vivo. Anti-HBV EYE was a small dialyzable substance with molecular weight less than 12 kD containing 18 kinds of amino acids. The preparation could obviously inhibit LAI and DTH which was similar to hepatitis B virus-specific transfer factor of pig spleen. However, there were no similar effects observed in the nonspecific transfer factor （NTF） group, control egg yolk extraction （CEYE） group and hepatitis A virus （HAV） group. The results suggested that anti-HBV EYE contained hepatitis B virus-specific transfer factor （STF） and had the antigen-specific cell immune activity similar to PSHBV-TF. The STF obtained from egg yolk of the hens immunized with specific antigen, might be a potential candidate for immunoregulation in hepatitis B prevention and treatment.     

Autophagy inhibits PDGF-BB-induced calcification in vascular smooth muscle cells

AIM: To investigate the relationship between autophagy and calcification in vascular smooth muscle cells( VSMCs) after plateletderived growth factor( PDGF)-BB stimulation. METHODS: Cultured VSMCs were stimulated with PDGF-BB for different time,the expression of vascular calcification-related proteins and autophagy-related proteins were detected by Western blot. The interaction between Beclin1 and PI3KC3 was detected by co-immunoprecipitation. RESULTS: The expression of BMP2 and ALP showed a trend from decline to rise. ALP slumped at 12 h,and BMP2 slumped at 6 h. Moreover,the expression of Beclin-1 showed a trend from rise to decline,and peaked at 12 h. The conversion of LC3-Ⅰto Ⅱ increased in a time-dependent manner,and peaked at 24 h. The expression of BMP2 and ALP was increased in VSMCs incubated with PDGF-BB and autophagy inhibitor 3-MA,compared with PDGFBB-stimulated VSMCs. Furthermore,the interaction between Beclin1 and PI3KC3 was enhanced at 6 h after PDGF-BB stimulated,peaked at 12 h,and kept in high level at 24 h. Moreover,the phosphorylation level of Beclin1 was enhanced by PDGF-BB stimulation,and peaked at 6 h. CONCLUSION: Our findings demonstrate that PDGF-BB-induced autophagy inhibits VSMC calcification by enhancing Beclin1 phosphorylation and interaction between Beclin1 and PI3KC3.     

Target Organ Protection from a Novel Angiotensin Ⅱ Receptor （AT1） Vaccine ATR12181 in Spontaneously Hypertensive Rats

Hypertension produces pathophysiological changes that are often responsible for the mortality associated with the disease. It is evident that overactive renin-angiotensin systems play a central role in the development of hypertension and target organ damage associated with hypertension. We have previously found that a novel angiotensin H receptor （AT1） vaccine-ATR12181 attenuated the development of high blood pressure （BP） in spontaneously hypertensive rat （SHR） model of human essential hypertension. Our objective was to determine whether this attenuation of high BP is associated with prevention of target organ damage induced by hypertensive state. SHRs were immunized against a peptide （coded ATR12181） from the extracelluar portion of the ATIA receptor by repeated subcutaneous injections of peptide-tetanus-toxoid complex in combination with Freund＇s adjuvant. A 64 weeks long-term observation was performed. Repeated vaccinations resulted in the induction of anti-ATR12181 antibodies. At the end of observation, vaccinated SHRs manifested lower BP, decreased cardiac hypertrophy and attenuation of kidney injuries, mRNA levels of c-los and c-jun in heart and kidneys were decreased in vaccinated SHRs. Since a self antigen was used, safety of vaccine was concerned. However, the signs of autoimmune diseases were not observed in the sections of heart and kidney. These data demonstrated that repeated immunization against a domain of the extracellular portion of the AT1 receptor was able to cause a target organ protection against hypertension. Active immunization against the AT1 receptor may be considered as a promising new strategy in the treatment of hypertension.     

Elementary study on the antigenicity of SARS-CoV Mc region

To test the antigenic activity of M protein (Mc protein) in the inner membrane of SARS-CoV, SARS-CoV Mc protein's bases locating inside the membrane were cloned, the His-fusion protein was expressed in E. coli and analyzed for its antigenic activity. Among those 7 clinically diagnosed patients' sera , there were 5 positive and 2 negative in reaction with His-fusion protein. All of the 20 healthy persons' sera and rabbit anti-OC43 and 229E were of negative reaction with His-fusion protein. The animals immunized with His-fusion protein have produced multi-clonal antibody. The His-fusion protein could specially react with clinically diagnosed SARS patients' sera and the animals immunized with His-fusion protein could produce specifically multi-clonal antibody, but it could not react with the sera of healthy persons and the rabbit anti-OC43 and 229E.     

The Experimental Study on Constructing the Tissue Engineered Myocardium-like Tissue in vitro with Bone Mesenchymal Stem Cells

Objective:Unlike other tissues,myocardium has not substitute whick can be used to repair damaged cardiac tissue.This paper proposes engineering 3-D myocardium-like tissue constructs in vitro with bone mesenchymal stem cells(BMSCs) of infant and poly-lactic-co-glycolic acid(PLGA)in vitro.Methods:Bone marrow was obtained from the sternal marrow cavum outflow of infant with congenital heart disease (CHD)undergoing cardiac operation.BMSCs were obtained by density gradient centrifugation.The cells in passages two were induced in DMED with 10 umol/L 5- Azacytidine(5-Aza)for 24 h.When the induced BMSCS were cultured nearly into filled,the cells were planted in the scaffold of PLGA in 5.5×106 cells/cm2.The cell- scaffold complex has been cultured in the shake cultivation for 1 week,then the complex has been planted in the dorse of the nude mouse.When the experiment had been finished,the histology,immunology,real time PCR and so on were done.Results: The BMSCs of infant with congenital heart disease have the properties of the stable growth and the rapid proliferation.The immunohistochemistry showed that tissue engineered myocardium constructed in vitro expressed some cardiac related proteins such asα-actin,Cx-43,Desmine,cTNI and so on.The transparent myofilaments,gap junctions and intercalated disk-like structure formation could be observed in the 3D tissue-like constructs by transmission electron microscope(TEM).The engineered myocardium-like tissue had the auto-myocardial property as assessed by real time- PCR and so on.Conclusion:The engineered myocardial tissue-like constructs could be built with infant BMSCs and PLGA in vitro.Our results may provide the first step on the long road toward engineering myocardial material for repairing the defect or augmenting the tract in CHD,such as ventricular septal defect,tetralogy of Fallot and so on.     

Immunogenicity of the recombinant adenovirus type 5 vector with type 35 fiber containing HIV-1 gag gene

The immune efficiency of a recombinant adenovirus type 5 with type 35 fiber containing HIV-1 gag gene (rAd5/F35-mod.gag) was investigated in BALB/c mice, in which the rAd5/F35-mod.gag was firstly identified with PCR, then transfected to 293 cells and the in vitro expression level of Gag protein was determined by Western blotting and indirect immuno-fluorescent assay. Mice were immunized with intramuscular injections of rAd5/F35-mod.gag, rAd5-mod.gag or DNA and were boosted after 3 weeks. To test the effect of pre-existing anti-viral immunity on immunization, mice were also injected with Ad5-GFP vector and then immunized 4 and 7 weeks later with Ad5/F35-mod. gag vector. The P24-specific IgG antibody in sera of immunized mice was determined by ELISA and the specific cytotoxic T lymphocyte (CTL) response was assayed by intracellular cytokine staining. It was demonstrated that the rAd5/F35-mod. gag vector could express efficiently the HIV Gag protein in 293 cells in vitro and induce strong HIV-specific immune responses in vivo. The strongest CTL and serum IgG response occurred when mice were immunized twice with injection of rAd5/F35 alone, but the anti-Ad5 antibody after primary infection with adenovirus could inhibit the specific immune responses induced by rAd5/F35 vector. It is concluded that single immunization with recombinant adenovirus rAd5/F35-mod. gag can induce specific CTL and serum IgG antibody responses in mice, but the immunogenicity of rAd5/F35 is comparably weaker than that of rAd5.     

一种新生SD大鼠皮质源性神经元的培养方法

BACKGROUND: Primary culture in vitro of neurons plays an important role in the development, regeneration, signal transduction mechanisms, neuropharmacology and gene expressions of the nervous system. OBJECTIVE: To establish a simple method for primary culture of high-purity cortical neurons in neonatal Sprague-Dawley rats. METHODS: Cortical tissues were acquired from neonatal Sprague-Dawley rats born 1 day. In traditional experimental group, the whole cortex was removed; in improved experimental group, the cortical tissues, 2-3 mm thick on the brain surface were removed. Single cell suspensions were prepared after papain digestion and centrifugation and were then seeded onto 24-well culture plates containing neuron solutions for primary culture (1×105 per well). Cells were identified by neuronal specific markers MAP-2 and Tuj1 after 3-day culture. The number of neurons and neurite length were observed under inverted phase contrast microscope and recorded at 6, 24, 48 and 72 hours, 5 and 7 days of culture, resprctively. RESULTS AND CONCLUSION:The cultured cells expressing MAP-2 and Tuj1 were neurons that could be used in the following experiments. The purity of neurons in the improved experimental group was 92% at 3 days, while only 51% in the traditional experimental group. Cells in both two groups had attached to the wall presenting with small processes at 6 hours, and a simple neural network formed at the 3rd day until dense neural networks could be found at the 5th day. To conclude, our culture method herein is simple and convenient, and can be used to produce neurons with high purity, which will be helpful for the experimental studies on cortical neurons from Sprague-Dawley rats. 中国组织工程研究杂志出版内容重点：组织构建;骨细胞;软骨细胞;细胞培养;成纤维细胞;血管内皮细胞;骨质疏松;组织工程     

Protective Effects of Overexpression TCR Vβ5.2-HSP70 and TCR Vβ8.2-HSP70 against Collagen-Induced Arthritis in Rats

Collagen-induced arthritis （CIA） is an animal model, which closely resembles human rheumatoid arthritis （RA） in pathogenesis and pathology. Evidence suggests that the inhibition of T lymphocytes or their functions can alleviate the progression of arthritis. So the administration of arthritogenic T cell receptor （TCR） variable region peptide or DNA vaccines encoding pathogenic TCR Vβ variable region may provide useful information for designing specific immunotherapies against autoimmune diseases. Heat shock proteins （HSPs） have the function of raising antigenic immunogenicity and HSP70 has a protective effect against arthritis. We previously demonstrated the presence of pathogenic predominant T cell receptor Vβ5.2 and Vβ8.2 clonotypes in the joints of CIA rats. In this study, we constructed the recombinant eukaryotic expression vectors pTARGET-TCR Vβ5.2/8.2-HSP70, and evaluated their protective effects on CIA rats. Protective effects were observed in CIA rats by injecting these recombinant DNA vaccines, which could alleviate arthritis index, decrease the levels of IFN-~ and anti-CII antibody in serum, and increase the levels of IL-4. Pathological changes were not as serious as those observed in control CIA rats. The rat injected with two combined vaccines showed better protective effects than CIA rats administered with individual vaccine. These results showed that recombinant DNA vaccines pTARGET-TCR Vβ5.2-HSP70 and pTARGET-TCR Vβ8.2-HSP70 could significantly alleviate the arthritic symptoms of CIA rats, and better protective effects could be achieved if these two vaccines were used in combination. Cellular ＆ Molecular Immunology.     

Resveratrol inhibits angiotensin II-induced ERK1/2 activation by downregulating quinone reductase 2 in rat vascular smooth muscle cells

Our previous studies showed that resveratrol could inhibit the proliferation of vascular smooth muscle cells (VSMCs) and repress mRNA and protein expression of quinone reductase 2 (NQO2).This study further explored the potential mechanisms whereby resveratrol inhibits the proliferation of rat VSMCs.Lentiviral vectors that incorporated NQO2 small interfering RNA (siRNA) were constructed and transduced into rat VSMCs.The cell proliferation was detected using the bromodeoxyuridine (BrdU) assay.Cultured rat VSMCs were stimulated with angiotensin II and the level of reactive oxygen species (ROS) was measured using a ROS assay kit.A realtime quantitative PCR was used to detect NQO2 mRNA levels.Extracellular signal-regulated kinase (ERK1/2) and NQO2 protein expression were determined by Western blotting analysis.The inhibitory effect of resveratrol (10 and 50 μmol/L) on the proliferation of rat VSMCs in the NQO2 siRNA group was significantly weaker than that in the normal and scrambled siRNA group (P < 0.01).The ROS level in the NQO2 siRNA and resveratrol (50 μmol/L) treatment groups were lower than that in the normal and scrambled siRNA groups (P < 0.01 in both).Compared with the normal and scrambled siRNA group,the phosphorylation of ERK1/2 was significantly decreased in the NQO2 siRNA and resveratrol (50 μmol/L) treatment group (P < 0.01 in both).In conclusion,high concentration of resveratrol inhibits angiotensin II-induced ERK1/2 phosphorylation and subsequent proliferation by down-regulation of NQO2 in cultured rat VSMCs.