Mechanism of carbon monoxide protects rat intestine against LPS

AIM: To study the effect of exogenous carbon monoxide on rat intestine attacked by LPS. METHODS: The experimental rats were divided into 6 groups randomly: normal group, lipopolysaccharide (LPS, 5 mg/kg) group, CO inhalation (250 mL/M3) group, CO intraperitoneal injection (2 mL/kg) group, LPS (LPS 5 mg/kg) with CO inhalation (250 mL/M3) group and LPS (LPS 5 mg/kg) with CO intraperitoneal injection (2 mL/kg) group. The PaO₂, PaCO₂, SO₂ and COHb were monitored. Rat intestine malondialdehyde (MDA) with thiobarbitric acid (TBA) and superoxide dismutase (SOD) with hydroxylamine were detected and heme oxygenase-1 (HO-1) mRNA expression were checked by RT-PCR. RESULTS: Low concentration of CO inhalation (250 mL/M3) and CO intraperitoneal injection (2 mL/kg) did not cause the rat hypoxia. Compared to control group, exposure exogenous CO, the MDA content in LPS attacked rat intestine decreased, the SOD activity and the expression of HO-1 mRNA increased. CONCLUSIONS: Low concentration of CO inhalation (250 mL/M3) and low dose CO intraperitoneal injection (2 mL/kg) are safe to rat. Exposure exogenous CO protects rat intestine against LPS attack, induces the HO-1 mRNA expression and exerts anti-inflammation via endogenous CO.     

Heme oxygenase decreases the generation of ROS in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells

AIM:To investigate the role of heme oxygenase (HO) in AngⅡ induced proliferation and hypertrophy of cultured vascular smooth muscle cells.METHODS:(1) Western blotting analysis was carried out to detect protein level of HO-1 in the tissues.(2) ［3H］-TdR, ［3H］-leucine incorporation was measured in cultured vascular smooth muscle cells.(3) 2,7-dichlorofluorescin diacetate (DCFH-DA) as an index was used to determine the cellular reactive oxygen species (ROS) level.RESULTS:(1) No significant difference in HO-1 protein expression level between AngⅡ-stimulated and control groups was observed, but HO-1 protein level in Hemin-induced group was higher than that in other two groups (P<0.01).No significant increase in HO-1 protein expression was found in zinc-protoporphyrin IX (ZnPPIX) group.(2) After AngⅡ stimulation, ［3H］-TdR and ［3H］-leucine incorporations of vascular smooth muscle cells (VSMCs) were increased.Hemin inhibited this increase.The higher concentration of Hemin, the more significant was the inhibitory effect.On the contrary, ZnPPIX promoted the increase in the effect of AngⅡ by inhibiting HO.(3) Fluorescence intensity in AngⅡ group was obviously higher than that in control groups (P<0.01).Compared with AngⅡ group, Hemin group decreased 62.7%, but ZnPPIX group increased 39.5%.CONCLUSION:Hemin induces HO-1 expression and inhibits the effect of AngⅡ to stimulate proliferation and hypertrophy of VSMCs.The mechanism may be related to its inhibition of ROS production.     

Relationship between expression of heme oxygenase-1 mRNA and pulmonary hypertention induced by hypoxic hypercapnia

AIM: To study the effect of chronic hypoxic hypercapnia on expression of heme oxygenase-1 (HO-1). METHODS: Sprague-Dawley rats were randomly divided into three groups: control group(A),hypoxic hypercapnic group(B), hypoxic hypercapnia+hemin group(C). HO-1 and HO-1 mRNA were observed in pulmonary arterioles by the technique of immunohistochemistry and in situ hybridization. RESULTS: ① mPAP and weight ratio of right ventricle (RV) to left ventricle plus septum (LV+S) were significantly higher in rats of B group than those of A and C group (P<0.01). Differences of mCAP were not significant in three groups(P>0.05). ② Blood CO concentration was significantly higher in rats of B group than that of A group (P<0.01), it was much higher in C group than that of B group(P<0.01). ③ Light microscopy showed that vessel well area/total area (WA/TA), density of medial smooth muscle cell (SMC) and media thickness of pulmonary arterioles were much higher in rats of B group than those of A and C group (P<0.01). ④ The observation by electron microscopy showed proliferation of medial smooth muscle cells and collageous fibers of pulmonary arterioles in rats of B group, hemin could reverse the changes mentioned above. ⑤ HO-1 and HO-1 mRNA in pulmonary arterioles was significantly higher in rats of B group than those of A group(P<0.01), and they were significantly higher in rats of C group than those of B group (P<0.01). CONCLUSION: Expression of HO-1 mRNA and HO-1 in pulmonary arterioles was enhanced by hypoxic hypercapnia. Hemin partly inhibited pulmonary hypertension and pulmonary vessel remodeling by enhancing the expression of HO-1 mRNA and HO-1.     

Effects of p204 expression on expression of p21 and proliferation of vascular smooth muscle cells in rats

AIM: To observe the effect of interferon-inducible protein 204 (p204) on the expression of p21 and proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Interferon alpha (IFN-α) and small interference RNA (siRNA) targeting p204 gene ( Ifi204 ) was used to intervene cultured VSMCs in vitro instantaneously, then the cell vitality was determined by MTT assay to reflect the cell proliferation. The cell cycle was analyzed by flow cytometry. The expression of p204 and p21 at mRNA and protein levels was determined by semi-quantitative RT-PCR and Western blotting. RESULTS: In rat VSMCs, IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality and the G₁/S phase transition, and up-regulated the expression of p21 at mRNA and protein levels. Transfection of Ifi204 siRNA restrained the expression of p204 and p21, increased the cell vitality and promoted the G₁/S phase transition. CONCLUSION: The expression of p204 restrains the proliferation of rat VSMCs, probably by activating the expression of p21.     

Effect of curcumin on heme oxygenase-1 expression in the lung of rats treated with LPS

AIM: To study the regulatory effect of curcumin on expression of heme oxygenase-1 (HO-1) in the lung of rat treated with LPS. METHODS: Eighteen rats were divided into three groups injected with different agents via lingua vein: control group (animals received equivalent saline), LPS group (animals received a bolus dose of LPS 5 mg·0.5 mL-1·kg-1) and LPS+curcumin group (animals received AP-1 inhibitor curcumin 20 mg·0.5 mL-1·kg-1 20 min before the injection of LPS 5 mg·0.5 mL-1·kg-1). The expression of HO-1 mRNA and protein in the lung were examined 7 h after LPS administration by reverse transcribed polymerase chain reaction (RT-PCR) and Western blotting, respectively. Carboxyhemoglobin (HbCO) formation within pulmonary tissue was measured to represent CO content. RESULTS: The results showed that HO-1 mRNA and protein expression as well as CO content in the lung of rats in LPS group were significantly higher than those in control group (P<0.01), while the parameters mentioned above in LPS+curcumin group were markedly lower than that in LPS group. CONCLUSION: The increased HO-1 expression in the lung of rat induced by LPS may be regulated by activating AP-1.     

Effects of platelet-derived growth factor and angiotensinⅡ on the expression of cell cycle related genes in vascular smooth muscle cells

AIM: To investigate the different expressions of cell cycle related genes in hyperplastic and hypertrophic vascular smooth muscle cells caused by platelet-derived growth factor (PDGF-BB) and angiotensinⅡ(AngⅡ). METHODS: Rat aorta media smooth muscle cells were cultured. PDGF-BB and AngⅡ were added into serum-free medium at a concentration of 20 μg/L and 10^-6 mol/L , respectively. Vascular smooth muscle cells (VSMCs) were harvested after stimulated for 24 hours. The expression of cell cycle related genes was measured by DNA chips(Atlas cDNA Expression Arrays, Clontech Laboratories, Inc.). RESULTS: The expression of cyclin D3 mRNA ,cyclin G1 mRNA,p57 mRNA,p16 mRNA,E2F-3 mRNA and DP2 mRNA were higher in PDGF-BB than those in AngⅡstimulated VSMCs. p15 mRNA,p19 mRNA,E2F-1 mRNA, E2F-5mRNA,and N-myc mRNA were only detected in PDGF-BB stimulated group. But the expression of p53-associated protein mRNA were higher in AngⅡstimulation group. The expression of PCNA mRNA, c-myc binding protein mRNA,p53-dependent cell growth regulater mRNA,cyclinC mRNA, cyclinB1 mRNA, E2F-3mRNA were similar in the two groups. CONCLUSION: The procession of cell cycle relys on the coordination of many regulater molecules expressed in different phases. Our study preliminarily definite the genes that express during PDGF-stimulated VSMC's hyperplasia and Ang II-stimulated VSMC's hypertrophy.     

The expression of heme oxygenase-1 in vascular smooth muscle cells induced by lipopolysaccharide

AIM:To study the alterations of heme oxygenase-1 mRNA in vascular smooth muscle cells(VSMC) induced by lipopolysaccharide(LPS) and the role of heme oxygenase(HO)/carbon monoxide(CO)pathway in the disorders of regulation of cardiovascular system by LPS. METHODS: LPS (final concentrations 10 mg/L,30 mg/L and 50 mg/L) was added in cultured VSMCs for 6 h respectively or 10 mg/L LPS for 9 h and 18 h. MDA content, LDH release and the rate of trypan blue uptake of VSMC were measured. HO-1 mRNA expression was examined by Northern Blot. RESULTS:VSMC HO-1 mRNA expression was increased gradually with the increasing of LPS concentration. When final concentration of LPS was 50 mg/L, the HO-1 mRNA expression of VSMC was increased by 176.7% compared with control. When LPS final concentration was 10 mg/L, the HO-1 mRNA expression increased gradually along with the culture time. When cultured for 18 h, the HO-1 mRNA expression of VSMC was increased by 195.6% compared with control. Only at LPS 50 mg/L for 6 h and 10 mg/L for 18 h, the rate of trypan blue uptake,MDA content and LDH release were significantly increased. CONCLUSION: LPS can induce the HO-1 mRNA expression of VSMC and that were dose-dependent and time-dependent. The inducible HO may play an important role in the pathogenesis of vascular system under LPS.     

Role of heme oxygenase-1 in cardioprotection against anoxia/re oxygenation induced injury and itsmechanisms

AIM:To investigate the role of HO-1 in pro tection of rat hearts against anoxia/reoxygenation-induced injury and its under lying mechanism.METHODS:Cardiac contractility,lactate dehydrogenase (LDH) and infarct area were analyzed by the Langendorff method in isolated rat hearts.RESULTS:After intraperitoneal injection of HO-1 inducer hemin,CO concentration in rat blood enhanced (P<0.01 vs control group).Pretreatm ent with hemin prevented the increase in LVEDP and decrease in LVDP,±dp/d tmax during the anoxia and reoxygenation period in hearts.Hemin had n o effect on changes of coronary flow,but it really inhibited the release of LDH from anoxia/reoxygenation hearts.Hemin also reduced the infarct area in anoxia heart after 2 h reoxygenation (P<0.01).CO concentration in rat blood redu ced after intraperitoneal injection of HO-1 inhibitor ZnPP (P<0.01 vs contr ol group).ZnPP aggravated the decrease in LVDP and ±dp/dtmax.Co mpared with anoxia/reoxygenation heart,pretreatment of ZnPP enhanced the LDH re lease and enlarged the infarct area (P<0.05).GC inhibitor methylene blue a nd cyclooxygenase-2 (COX-2) inhibitor celecoxib both partly abolished the protec tion effect of hemin on LVEDP,LVDP and ±dp/dtmax.Pretreatment o f methylene blue or celecoxib also cancelled the inhibition of LDH release and r eduction of infarct area caused by hemin (P<0.05).CONCLUSION:HO-1 inducer hemin protects heart from anoxia/reoxy genation-induced injury.The cardiac protection of HO/CO might be through GC pathway,and the activation of COX-2 might be also involved in this process.     

Role of heme oxygenase-1/carbon monoxide system in pulmonary ischemia-reperfusion injury

AIM: To investigate the effect of heme oxygenase-1 (HO-1)/carbon monoxide (CO) system on pulmonary ischemia-reperfusion injury (PIRI) in rabbits. METHODS: Single lung ischemia and reperfusion animal model was used in vivo. The rabbits were randomly divided into three groups (n=10 in each), control group (C), PIR group (I-R), PIR+ hemin group (H) and PIR+zinc protoporphyrin IX (ZnPP) group (Z). Changes of several parameters which included plasma carboxyhemoglobin (COHb) at different time points, wet to dry ratio of lung tissue weight (W/D), the injured alveoli rate (IAR) and the HO-1 enzymatic activity were measured at 180 min after reperfusion in lung tissue. The tissue slides were also stained by immunohistochemistry (IHC) and in situ hybridization (ISH) for HO-1 to detect the expression of HO-1 in lung and to analyze the optical density. The lung tissue was prepared for electron microscopic observation at 180 min after reperfusion. RESULTS: The plasma content of COHb in I-R, H, and Z group increased in a time-dependent manner after I-R. But the increment of H group was higher than that of I-R group, while that of Z group was lower. The HO-1 activity in lung tissue was highest in H group, followed by IR group, Z group, and C group (P<0.05 and P<0.01). Except C group, HO-1 was upregulated in all other groups in the pulmonary endothelial cells, part of pulmonary vascular smooth muscle cells, extima of vessels and epithelial cells of airway. H group had the highest average optical density value, then the IR group, Z group and C group (P<0.05 and P<0.01). The value of W/D and IAR was highest in Z group, the second was in IR group, then the H group and C group (P<0.05 and P<0.01). The abnormal changes of the lung tissue in morphology in I-R group, Hemin treatment mitigated the injury of I-R in H group and ZnPP exacerbated the impairment of ultrastructure in Z group were also observed. CONCLUSION: HO-1/CO system possesses notable protective effects on lung during pulmonary ischemia-reperfusion injury in rabbits.     

Effects of caveolin-1 scaffolding domain peptide on LPS-induced acute lung injury in mice

AIM: To investigate the effects of caveolin-1 (Cav-1) scaffolding domain peptide, cavtratin, on lipopolysaccharide (LPS)-induced mouse acute lung injury and heme oxygenase-1 (HO-1) activity. METHODS: Adult male BALB/c mice were randomly divided into 6 groups (n=8 to 10):control, Antennapedia internalization sequence (AP), LPS, LPS+hemin, LPS+ hemin+cavtratin and LPS+hemin+cavtratin+zinc protoporphyrin IX (ZnPP) groups. After LPS administration for 24 h, the lung pathological changes, the wet/dry weight (W/D) ratio of lung tissues, total cell number in bronchoalveolar lavage fluid and serum lactate dehydrogenase activity were measured. The co-localization of HO-1 and Cav-1 was displayed by immunofluorescence, and the HO-1 activity were detected. The mRNA expression of pro-inflammatory cytokines IL-1β, IL-6, TNF-α, MCP-1 and iNOS was detected by real-time PCR. RESULTS: The mice in LPS+hemin+cavtratin group had the decreased interaction between HO-1 and Cav-1, and the increased HO-1 activity compare with LPS group (P<0.05). Compared with LPS group, the pulmonary damage was attenuated in LPS+hemin+cavtratin group, and the injury indexes, including W/D ratio, total cell number in bronchoalveolar lavage fluid and lactate dehydrogenase activity in the serum, and the mRNA expression of inflammatory cytokines all decreased (P<0.05). HO-1 activity inhibitor ZnPP abolished the above protective effect of cavtratin on the lung tissues with LPS-induced acute lung injury. CONCLUSION: Cavtratin has beneficial effects on the lung with LPS-induced acute injury by restoring the HO-1 activity.     

Effect of heme oxygenase-1/endogenous carbon monoxide system on restenosis of rabbit carotid artery after balloon injury

AIM: To investigate the effect of heme oxygenase-1 (HO-1)/carbon monoxide (CO) system on restenosis of carotid artery after balloon angioplasty.METHODS: Fifty rabbits were randomly divided into 5 groups: control group given normal chow (C group), sham group(Sh group), 1.5% cholesterol diet group (Ch group), 1.5% cholesterol diet plus hemin group(Hm group)and zinc protoporphyrin IX group(Zn group).The experiment lasted for 10 weeks. At the beginning of the 3rd week, the animals in Ch group, Hm group and Zn group underwent balloon injury at one side of common carotid artery.RESULTS: Compared with those in C group, the production of arterial nitric oxide (NO) and activity of constructive nitric oxide synthase (cNOS) were significantly decreased, while CO production and HO-1 expression were significantly increased (all P<0.01) in Ch group. Compared with those in Ch group, the arterial CO production and HO-1 expression in Hm group were markedly increased, while the expression of endothelin-1 (ET-1), the intimal area and the ratio of intimal area/medial area(I/M) were distinctly reduced. Compared with those in Ch group, the arterial CO production and HO-1 expression in Zn group were obviously decreased, while the expression of ET-1, the intima area and the ratio of I/M were significantly increased.CONCLUSION: The HO-1/CO system improves the endothelium function and restrains neointimal proliferation by compensating and regulating NOS/NO system and lowering ET-1 expression so as to inhibit the restenosis.     

Effects of lentivirus-mediated transfection of shRNA targeting Adra1d gene on calcium and calmodulin in rat aortic vascular smooth muscle cells

AIM:To investigate the effects of lentivirus-mediated transfection of shRNA targeting α_1D-adrenergic receptor (Adra1d) gene on calcium ion (Ca²⁺) and calmodulin (CaM) in vascular smooth muscle cells (VSMCs) of rat aorta. METHODS:Single oligonucleotide sequences of shRNA targeting rat Adra1d gene were design and synthesized, and then the shRNA was constructed and cloned into GV248 vector. The U6-shRNA carrier and expression vector were transfected into 293T cells together and packed with lentivirus, and the supernatant was collected and concentrated by overspeed centrifugation. The VSMCs of rat aorta were transfected with recombinant lentivirus vector. The interference effects were identified by RT-qPCR and Western blot. The concentration of Ca²⁺ in VSMCs was detected by laser confocal inspection, and the expression of CaM at mRNA and protein levels in the VSMCs was determined by RT-qPCR and Western blot. RESULTS:The lentiviral shRNA expression vector was successfully constructed. The titer of the concentrated virus was 3×10¹¹ TU/L. The mRNA and protein expression levels of Adra1d in the rat aortic VSMCs were significantly reduced after transfection. The interference efficiency of Lv-shRNA4-Adr to Adrald gene was greater than 85%. After target silencing of Adra1d gene, compared with scrambled group, the Ca²⁺ fluorescence intensity of rat aortic VSMCs was significantly increased. Moreover, the mRNA and protein expression levels of CaM were also increased significantly. CONCLUSION:A lentiviral shRNA expression vector targeting rat Adra1d gene was successfully constructed, which significantly increased Ca²⁺ concentration and CaM expression in rat aortic VSMCs.     

Expression of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3

AIM: To observe the dynamic variation of oxygenase-1 protein and mRNA on mouse acute viral myocarditis caused by coxsackie viruses B3.METHODS: A total 72 inbred male BALB/c mice of 4-6 weeks were divided randomly into 2 groups as follows: 32 mice were inoculated intraperitoneally (ip) with virus free 1640 culture solution 0.1 mL on day 0 as blank group (C); 40 mice were ip 0.1 mL tissue culture infectious dose 50 (TCID50 is 10-4.36/mL) coxsackie viruses B3 (CVB3) on day 0 as VMC group (V), then each mouse in both groups was ip 0.1 mL NS every day. 8 mice in each of C group and V group were sacrificed on 4, 8, 15, and 21 d respectively after infections. The blood specimens gathered by taking out the eyeballs of mice were tested for the content of carboxyhemoglobin (COHb) using spectrophotometer method.The heart tissue slides were also stained by immunohistochemistry (IHC) for HO-1 and in situ hybridization (ISH) for HO-1 mRNA. The histological and ultrastructure changes were observed under light microscope and electron microscope.RESULTS: (1)The histopathological changes of myocardial cells: many inflammatory cells were found in the heart and large area myocardial cells necrosis was observed under light microscope. The inflammatory area was reduced at late stage in the heart of mouse in group V, while the myocardium in group C was normal. (2) The myocardial observation by electron microscopy: the myofibril in group V was dissolved and mitochondrial membrane disappeared, mononuclear cell infiltration was also observed under electron microscopy, which contained many lysosomes. The myocardial cells in group C were normal. (3) The changes of blood COHb level: compared with group C, the group V COHb level showed significantly higher on the day 8 and day 15 after CVB3 innoculation (0.047±0.005 vs 0.031±0.004; 0.076±0.006 vs 0.030±0.005, P<0.01). No obvious change in group C was observed. (4) The result of HO-1 IHC staining: myocardial cells had positive expression in group V, group C was negative. The absorbance (A) values in group V was significantly higher than that in group C (P<0.01) at different time points. (5) The result of HO-1 ISH was similar to HO-1 IHC. The A values in group V was all higher than that in group C (P<0.01).CONCLUSION: Viral myocarditis caused by coxsackie viruses B3 induces the expression of HO-1 mRNA and protein, and these expressions may play self-protection in inflammation injury in myocardial cells.     

Effect of interferon-inducible protein p204 on proliferation of vascular smooth muscle cells in rats

AIM: To study the effect of interferon-inducible protein p204 on the proliferation of vascular smooth muscle cells (VSMCs) in rats. METHODS: Cultured VSMCs were treated with interferon alpha (IFN-α) and p204 gene (Ifi204) small interfering RNA (siRNA) in vitro instantaneously. The cell vitality was detected by MTT me-thod,and the cell cycle was analyzed by flow cytometry. The expression of mRNA and proteins was determined by real-time qRT-PCR and Western blotting,respectively.RESULTS: IFN-α induced the increase in the expression of p204 at mRNA and protein levels, reduced the cell vitality, inhibited the cell cycle of G₁/S transition, and down-regulated the expression of Ras protein in VSMCs. Meanwhile, the phosphorylation levels of Raf and ERK were decreased. Transfection of Ifi204 siRNA restrained the expression of p204, increased the cell vitality and promoted the cell cycle of G₁/S transition in VSMCs. The up-regulation of Ras protein expression and the increased phosphorylation levels of Raf and ERK were also observed.CONCLUSION: The expression of p204 restrains the proliferation of VSMCs in rats by inhibiting the activation of Ras/Raf/MEK/ERK signal pathway.     

Eukaryotic expression of human Arresten gene and its effect on the proliferation and migration of vascular smooth muscle cells

AIM: To express human Arresten gene in eukaryotic cell,and to investigate its effect on the proliferation and migration in vitro of rat primary cultured thoracic aortic vascular smooth cells (VSMCs).METHODS: COS-7 cells were transfected with recombinant eukaryotic expression plasmid pSecTag2-AT or control plasmid pSecTag2 mediated by liposome.48 hours after transfection,polymerase chain reaction (RT-PCR) was used to detect the expression of Arresten mRNA in the cells,while Western blotting assay was applied to detect expressed Arresten protein in concentrated supernatants.VSMCs were then co-cultured with the concentrated supernatants;and its proliferation was detected using cell counting kit-8 (CCK-8) in vitro.Migration of VSMCs was assayed by a microchemotaxis chamber and a polycarbonate filter (Transwell's chamber) with pores of 8 μm in diameter.RESULTS: RT-PCR revealed that the genome of Arresten-transferred cells contained a 449bp specific fragment of Arresten gene.Successful protein expression in supernatants was confirmed by Western blotting.CCK-8 assay showed that the proliferation of VSMCs was inhibited significantly by Arresten protein as compared with control group (P<0.01).Transwell's chamber showed that the number of control group,pSecTag2 transfected group and pSecTag2-AT transfected group were 28.70±3.97,26.10±4.53 and 14.00±3.33 (P<0.01).CONCLUSION: Arresten protein expressed in eukaryotic cells inhibits the proliferation and migration of VSMCs effectively in vitro.     

Phosphorothioate-modified antisense TGF-β1 oligodeoxynucleotide inhibits neointimal hyperplasia after vascular balloon injury in rats

AIM: To evaluate the effects of antisense TGF-β1 oligodeoxynucleotide (AS TGF-β1) on the expression of TGF-β1, deposition of extracellular matrix (ECM) and the neointima formation in the arteries after balloon injury. METHODS: The unmodified and phosphorothioate-modified AS TGF-β1 which containing 15 bases and surrounding the initiation codon region (ATG) of rat TGF-β1 complementary DNA (cDNA) were designed. At the same time, sense TGF-β1 oligodeoxynucleotide (S TGF-β1) with the base sequence complement to AS TGF-β1 was synthesized as a control. The oligodeoxynucleotides were introduced into in vivo and in vitro experiments, respectively. RESULTS: The AS TGF-β1 significantly inhibited the protein expression of TGF-β1 in a concentration-dependent manner, and S TGF-β1 did not have the same effect. Furthermore, no effect of the AS TGF-β1 on the mRNA expression of TGF-β1 in injured VSMCs was observed. Moreover, for the injured VSMCs, AS TGF-β1 significantly and concentration-dependently inhibited the basal DNA synthesis. Both AS TGF-β1 and S TGF-β1 did not exhibit dose-dependent effects on DNA synthesis in uninjured VSMCs. Fibronectin (FN) mRNA expression in injured VSMCs was significantly decreased by AS TGF-β1 in a concentration (001~1 μmol/L)-dependent manner. AS TGF-β1 significantly increased the mRNA expression of contractile marker SM22α, and decreased the mRNA expression of synthetic markers osteopontin and matrix Gla, especially at the concentration of 001 μmol/L and 01 μmol/L. After treatment with AS TGF-β1 (90 μg·kg-1·d-1) for 28 d, the neointima formation was significantly inhibited, and the area ratio of intima/media was markedly decreased by 68% compared with untreated group, but S TGF-β1 had no effect on neointimal formation. CONCLUSION：The AS TGF-β1 specifically inhibits the protein expression of TGF-β1 in the VSMCs derived from injured arteries. Moreover, it significantly inhibits DNA synthesis and cell proliferation, and decreases the expression of FN. Therefore, AS TGF-β1 dramatically attenuates neointima formation after balloon njury. The effects of AS TGF-β1 on the injured VSMCs may be associated with its reverse effects on the alteration of VSMC phenotype after balloon injury.