Cardiac collagen metabolism in murine viral heart diseases

AIM：To investigate the dynamic alteration of cardiac collagen metabolism in mice with acute,chronic myocarditis and dilated cardiomyopathy (DCM).METHODS：BALB/c mice infected with coxsackievirus B3 were used to establish animal models of acute,chronic myocarditis and dilated cardiomyopathy,while uninfected animals were also prepared and served as controls.After verification of models by histopathological methods and echocardiography,serum concentration of aminoterminal propeptide of type Ⅲ procollagen (PIIINP),aminoterminal propeptide of type Ⅰ procollagen (PINP) and carboxyterminal propeptide of type I procollagen (PICP) in each group of mice were detected by enzyme linked immunosorbent assay (ELISA).The expression of matrix metalloproteinase 1 (MMP-1) and its tissue inhibitor (TIMP-1) were determined by Western blotting analysis.The MMP-1 activity was also detected.RESULTS：Marked myocardial fibrosis was observed in all groups of CVB3-infected mice.Reparative fibrosis,promotion of synthesis and degradation of cardiac collagens were presented in heart tissue of acute myocarditis mice.Both reparative and reactive fibrosis,enhanced synthesis and lightened degradation of collagen were present in chronic myocarditis,while reactive fibrosis and excess collagen synthesis were confirmed in DCM.Expression and activity of MMP-1 was progressively decreased.TIMP-1 showed unchanged.The ratio of MMP-1/TIMP-1 was progressively descended.CONCLUSION：Collagen metabolism was special in different phase of viral heart diseases,which may play different roles in the progression and prognosis of these kinds of disease.     

Effect of hydrogen sulfide on myocardial fibrosis and PPARγ and NF-κB expression in rat model of diabetes

AIM: To explore the effects of hydrogen sulfide (H₂S) on the myocardial fibrosis in a rat model of diabetes and its mechanism.METHODS: Single intraperitoneal injection of streptozotocin (STZ) was utilized to establish a rat model of diabetes. Sodium hydrosulfide was used as an exogenous donor of hydrogen sulfide. Male SD rats were randomly divided into control group, STZ group, STZ+H₂S group and H₂S group. Eight weeks later, HE and VG staining methods were used to observe the collagen distribution and collagen volume fraction was measured by image analysis. The expression levels of type I collagen, PPARγ and NF-κB in the cardiac tissues were determined by Western blotting.RESULTS: Compared with control group, collagen distribution and the expression levels of type I collagen and NF-κB in the cardiac tissues were markedly increased (P<0.05), while PPARγ was significantly decreased in STZ group (P<0.05), but these indexes were reversed significantly in STZ+H₂S group (P<0.05). The expression levels of type I collagen, PPARγ and NF-κB had no significant difference between H₂S group and control group.CONCLUSION: Hydrogen sulfide attenuates cardiac fibrosis in diabetic rats, and its mechanism may be related to PPARγ-NF-κB signaling pathway.     

Effect of astragalus polysaccharides on expression of MMP2 and MMP9 in annulus fibrosus of cervical intervertebral discs from cervical spondylosis model rats

AIM: To investigate the effect of astragalus polysaccharides (AP) on the expression of matrix metalloproteinase 2 (MMP2) and MMP9 in the annulus fibrosus of cervical intervertebral discs from cervical spondylosis model rats. METHODS: The model rats were randomly divided into model group (M group), and low-dose and high-dose AP treatment groups (L-AP and H-AP groups). The rats in sham operation group were used as negative control group (NC group). In addition, all the annulus fibrosus tissues were used for primary cell culture. Histological analysis was performed using HE staining and Safranin O staining. The expression of MMP2, MMP9, tissue inhibitor of metalloproteinase 2 (TIMP2) and collagen Ⅳ at mRNA and protein levels was analyzed by immunohistochemistry, Western blot and RT-qPCR. Cell-collagen adhesion assay was used to detect annulus fibrosus cell-collagen adhesion. RESULTS: The intervertebral discs of M group were degenerated, while astragalus polysaccharide improved the degenerative disc disease in the rats with cervical spondylosis. Compared with NC group, the expression of MMP2 and MMP9 in the annulus fibrosus tissues of M group increased significantly, while the expression of TIMP2 and collagen Ⅳ was decreased significantly (P<0.05). Compared with M group, the expression of MMP2 and MMP9 in L-AP group and H-AP group was significantly decreased, while the expression of TIMP2 and collagen Ⅳ was significantly increased (P<0.05). The cell-collagen adhesion in M group was significantly lower than that in NC group (P<0.05). Compared with M group, the cell-collagen adhesion in L-AP group and H-AP group was increased significantly (P<0.05). Compared with NC group, the expression of MMP2 and MMP9 in annulus fibrosus cells of M group was increased significantly, while the expression of TIMP2 and collagen Ⅳ was decreased significantly (P<0.05). Compared with M group, the expression levels of MMP2 and MMP9 in L-AP group and H-AP group of fibrocytes were significantly decreased, while the expression of TIMP2 and collagen Ⅳ was significantly increased (P<0.05). CONCLUSION: Astragalus polysaccharides inhibit the expression of MMP2 and MMP9 in the annulus fibrosus of cervical intervertebral discs from cervical spondylosis model rats and regulate the dynamic balance of MMPs and TIMPs in the extracellular matrix, thus inhibiting the degradation of collagen in the intervertebral disc matrix and having the potential research value for the treatment of intervertebral disc degeneration.     

Effect of capsaicin on liver fibrogenesis in rats

AIM：To investigate the effects of capsaicin on rat hepatic stellate cells (HSCs) in vitro and on the liver fibrogenesis in vivo. METHODS：HSCs were cultured with different concentrations of capsaicin. The levels of reactive oxygen species (ROS) were tested with a DCFH-DA kit. The proliferation of HSCs was detected by CCK-8 assay. The expression of α-smooth muscle actin in HSCs was evaluated by Western blotting. The expression of fibrosis-related genes was detected by RT-PCR. The apoptosis of HSCs was measured by flow cytometry. The rat model of liver fibrosis was established by intraperitoneal injection of carbon tetrachloride. Capsaicin at different concentrations was given by gavage. The pathologic changes of the liver sections were observed under microscope with HE staining. Hydroxyproline content in the liver tissues and the levels of collagen Ⅲ and hyaluronic acid in the serum were also measured. RESULTS：Capsaicin inhibited the generation of ROS in a concentration-dependent manner. Compared with control, the proliferation and activation of HSCs were inhibited (P<0.05) and the apoptosis of HSCs was promoted by capsaicin (P<0.05). Capsaicin down-regulated the expression of tissue inhibitor of metalloproteinase 1 and transforming growth factor β 1 in activated HSCs (P<0.05). Capsaicin decreased the levels of hydroxyproline, collagen III and hyaluronic acid in the rats (P<0.05). CONCLUSION：Capsaicin inhibits the proliferation and activation, and promotes the apoptosis of hepatic stellate cells, thus down-regulating the fibrogenesis level of the liver in rats．     

Protective effect of aFGF delivery by ultrasound-targeted microbubble destruction on left ventricular function in diabetic cardiomyopathy rats

AIM：To observe the protective effect of delivery of acidic fibroblast growth factor (aFGF) to myocardium by ultrasound-targeted microbubble destruction (UTMD) on left ventricular function in diabetic cardiomyopathy (DCM) rats and to investigate the possible mechanisms. METHODS：Twenty-four rats were intraperitoneally injected with streptozocin to induce DCM and were randomly divided into DCM group and aFGF treatment group. Twelve healthy rats served as normal controls. The rats in aFGF treatment group were infused with SonoVue-aFGF mixed fluid through tail vein and UTMD was simultaneously performed. Four weeks after intervention, all rats underwent cardiac catheterization to mea-sure left ventricular end-systolic pressure (LVESP), left ventricular end-diastolic pressure (LVEDP) and the maximal increase/decrease rate of left ventricular pressure (LV±dp/dtmax). The microvessel density (MVD) of rat myocardial tissues was measured by immunohistochemical staining for CD31. The myocardial collagen volume fraction (CVF) was determined by improved Masson staining. The apoptotic index (AI) was detected by TUNEL method. RESULTS：Four weeks after intervention, the LVESP and LV±dp/dtmax in aFGF treatment group were significantly increased compared with DCM group (P<0.01), while the LVEDP in aFGF treatment group was significantly lower than that in DCM group (P<0.01). The MVD in aFGF treatment group was significantly increased compared with DCM group (P<0.01), but the CVF and AI in aFGF treatment group were significantly lower than those in DCM group (P<0.01). CONCLUSION: Delivery of aFGF to diabetic myocardium by UTMD could improve the left ventricular function of DCM rats and may be a new feasible therapeutic method for DCM.     

Effect of fat-specific protein 27 on activation of rat hepatic stellate cells in vitro

AIM：To investigate the effects of fat-specific protein 27 (Fsp27) on the proliferation and activation of hepatic stellate cells (HSCs) in vitro. METHODS：HSCs were isolated from the liver of SD rats. The mRNA and protein expression of Fsp27 in primary HSCs and activated HSCs was detected by real-time fluorescence quantitative PCR, immunofluorescence staining and Western blotting. After 72 h of transfection with Fsp27-carrying lentivirus (pLV-Fsp27), the proliferation of HSCs was tested by CCK-8 assay, the protein expression of α-smooth muscle actin (α-SMA) in HSCs was detected by Western blotting, and the mRNA expression of fibrosis-related proteins, including matrix metalloproteinase 2 (MMP-2), tissue inhibitor of metalloproteinase 1 (TIMP-1) and transforming growth factor beta 1 (TGF-β1), was determined by real-time fluorescence quantitative PCR. RESULTS：Rat HSCs were successfully isolated and cultured. The difference of Fsp27 expression between primary HSCs and activated HSCs was significant (P<0.01). The proliferation and activation of HSCs was inhibited 72 h after pLV-Fsp27 transfection (P<0.05). Fsp27 enhanced the mRNA expression of MMP-2 and down-regulate the mRNA expression of TIMP-1 and TGF-β1 in activated HSCs (P<0.05). CONCLUSION：Fsp27 inhibits the proliferation and activation of HSCs and regulates the expression of fibrosis-related proteins. Fsp27 may play an important role in maintenance of the quiescent phenotype of HSCs.     

Expression of matrix metalloproteinase-9, tissue inhibitor of metalloproteinase-1 and collagen type IV in lung of rats with multiple organ dysfunction syndrome

AIM: To determine the expression of matrix metalloproteinase-9 (MMP-9), tissue inhibitor of me-talloproteinase-1 (TIMP-1) and collagen type IV (IV-C) in the lung of rats with multiple organ dysfunction syndrome (MODS) and to investigate the mechanism of lung injury in MODS. METHODS: Adult male Sprague-Dawley (SD) rats (n=40) were randomly divided into sham control group and cecal ligation and puncture (CLP) model group. The rats in CLP group were divided into 4 subgroups as different intervals (6 h, 12 h, 24 h and 48 h), and there were 8 rats in each group. The rat model of MODS was established by CLP. All rats were sacrificed at various intervals. The functions of the liver, kidney and lung were determined by blood biochemical and blood gas analysis. The morphological changes of the lung tissues were observed with HE staining. The serum levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, MMP-9 and TIMP-1 were measured by ELISA. The expression of MMP-9 and TIMP-1 in the lung tissues was detected by RT-PCR and immunohistochemistry, and the expression of IV-C in the lung tissues was detected by immunofluorescence and Western blot. RESULTS: Compared with sham control group, the functions of the liver, kidney and lung were damaged at different degrees in model groups. No histopathological change in the lung tissues of sham control group was found, and the lung injury was serious in model groups. Compared with sham control group, the serum levels of TNF-α, IL-1β, MMP-9 and TIMP-1 in model groups increased significantly (P<0.05) and peaked at the interval of 12~24 h after modeling (P<0.01). The expression of MMP-9 and TIMP-1 in the lung tissues of model groups increased, and peaked at 12 and 24 h, respectively (P<0.01). The protein level of IV-C in MODS 6 h group was not changed as compared with control group, while that at the interval of 12~48 h after modeling was significantly decreased and dropped to the lowest at 24 h (P<0.01). CONCLUSION: MMP-9 and TIMP-1 play important roles in lung injury of MODS rats by regulating the synthesis and decomposition of IV-C which is the main component of extracellular matrix.     

Expression and significance of thrombospondin-1 in myocardium of type 2 diabetic cardiomyopathy rats

AIM: To investigate the expression and significance of thrombospondin-1 (TSP-1) in left ventricular myocardium of type 2 diabetic cardiomyopathy (DCM).METHODS: The rat model of DCM was established by eating a high-fat diet together with injection of low dose streptozocin (30 mg/kg) intrapertoneally.After 12 weeks,the content of collagen was quantified by Masson staining.The mRNA level of TSP-1 was determined by quantification real-time RT-PCR,while the protein level of TSP-1 was analyzed by Western blotting and immunohistochemistry.RESULTS: Compared with the control group,the content of collagen in the DCM group was increased greatly (11.01±3.05 vs 16.92±3.18,P<0.01).The mRNA and protein expressions of TSP-1 were significantly higher than those in control group (0.0089±0.0034 vs 0.0141±0.0037,P<0.05；96.38±16.80 vs 129.98±16.96,P<0.05).In DCM group,the mRNA and protein expressions of TSP-1 showed significantly positive correlations with the levels of fasting blood glucose and collagen (r=0.762,P<0.01; r=0.717,P<0.05; r=0.735,P<0.01; r=0.750,P<0.01).There was a significantly positive correlation of TSP-1 mRNA level with LVEDP (r=0.658,P<0.05).In contrast,there was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.605,P<0.05; r=-0.694,P<0.05).There was a significantly positive correlation of TSP-1 protein with LVEDP (r=0.716,P<0.05).There was a significantly negative correlation of TSP-1 protein with LVSP and -dp/dtmax (r=-0.633,P<0.05; r=-0.669,P<0.05).CONCLUSION: The increased expression of TSP-1 may play an important role in the development of myocardial interstitial fibrosis in DCM.     

Effects of chloroquine on autophagy and collagen metabolism in activated hepatic stellate cells in vitro

AIM: To explore the effects of chloroquine (CQ) on collagen Ⅰand collagen Ⅲ expression in activated rat hepatic stellate cell line HSC-T6 and the possible mechanism.METHODS: Transforming growth factor-β1 (TGF-β1) was used to activate HSC-T6 cells and 3 doses of CQ was administered for 24 h. The cells were divided into 5 groups as follows:control group, TGF-β1 group, TGF-β1+CQ (15 μmol/L) group, TGF-β1+CQ (30 μmol/L) group and TGF-β1 + CQ (60 μmol/L) group. Western blot was used to determine the expression of LC3-Ⅱ/LC3-I, P62 and α-SMA in activated HSC-T6 cells. The expression of collagen I and collagen Ⅲ was detected by immunocytochemical staining, Western blot and RT-qPCR. Western blot and RT-qPCR were also used to detect the expression of matrix metalloproteinase-13 (MMP-13), tissue inhibitor of metalloproteinase-1 (TIMP-1) and TIMP-2 at mRNA and protein levels.RESULTS: The ratio of LC3-Ⅱ/LC3-Ⅰ and P62 expression were increased after CQ intervention. Moreover, they were significantly higher in the TGF-β1+CQ groups than those in TGF-β1 group (P<0.01). The expression of collagen I and collagen Ⅲ at mRNA and protein levels was significantly increased in all TGF-β1+CQ groups as compared with TGF-β1 group (P<0.01), and it was markedly increased among TGF-β1+CQ groups in a dose-dependent manner. The expression of MMP-13 at mRNA and protein levels was significantly lowered and that of TIMP-1 and TIMP-2 was significantly increased in TGF-β1+CQ groups as compared with TGF-β1 group (P<0.05).CONCLUSION: Inhibition of autophagy by CQ in activated HSC-T6 cells up-regulates the expression of collagen I and collagen Ⅲ in a dose-dependent way, probably due to reduction of MMP-13 and enhancement of TIMP-1 and TIMP-2 expression.     

Expression of matrix metalloproteinase and tissue inhibitor of metalloproteinase in lung tissue of hypercapnia rat model

AIM: To study the expression of matrix metalloproteinase-9(MMP-9), matrix metalloproteinase-2(MMP-2) and the tissue inhibitor of metalloproteinase(TIMP-1) in the lung tissue of the hypercapnia rat.METHODS: Forty Wistar rats were randomly divided into a control group (group A, n=20) and hypercapnia group (group B, n=20). Group B received mix gas exposure (6% CO2, 21% O2, 72% N2) 7 h daily for 4 weeks. The parameters we would examine were as follow: arterial blood gas; the mean pulmonary artery pressure;MMP-2,MMP-9, TIMP-1, and NE activity in lung tissue. Masson pigmentation of elasticity fibre was analyzed by computer image analyzer. Histopathological changes of lung tissue were observed under light microscope. The protein expression of MMP (MMP-2, MMP-9) and TIMP (TIMP-1) in lung tissue were determined by immunocytochemistry.RESULTS: Decompensate respiratory acidosis (pH=7.20±0.04, PaCO2=7.84±0.15) developed in group B. The mean pulmonary artery pressure were similar between groups B and A (P>0.05). Tissue edema in the lung, endothelial cell damage of the small blood vessels, pulmonary micro thrombus formations and increased pulmonary capillary permeability were observed in group B. NE activity increased significantly (P<0.01). However, no significant change of MMP-2, MMP-9, TIMP-1 activity was found in group B and group A (P>0.05). There was significant decrease in the relative content of elasticity fibre in lung tissue in group B compared to group A (P<0.01). The expression of MMP-2 protein in the lung tissue of group B was lower than that in group A (P<0.01), but the expression of both MMP-9 and TIMP-1 proteins in the lung tissue in group B were higher than those in group A (P<0.01).CONCLUSION: Hypercapnia rat model is successfully reproduced by exposure of animals to the mix gas exposure (6% CO2, 21% O2, and 72% N2). The pulmonary artery pressure is not affected by hypercapnia. High concentration of CO2 causes increase of NE activity and decrease in the relative content of elasticity fibre. High concentration of CO2 causes the increase of MMP-2 protein expression and decrease in the MMP-9 and TIMP-1 protein expression.     

Effect of Kechuanning on airway remodeling and protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus

AIM:To investigate the effect of Kechuanning on airway remodeling and the protein level of p-ERK1/2 in lung tissues of asthmatic rats induced by virus. METHODS:The asthmatic rat model induced by respiratory syncytial virus was established. The experimental rats were divided into normal group, asthma model group, low dose (0.33 mL/kg), middle dose (3.0 mL/kg) and high dose (10 mL/kg) of Kechuanning groups, and PD98059 (3 mg/kg) group. The airway responsiveness of the rats was measured by animal ventilator. The pathological changes of the lung tissues were observed by HE staining. PAS staining and Masson staining were used to observe goblet epithelial cells metaplasia and airway collagen deposition. The expression of matrix metalloproteinases-9 (MMP-9) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) in the lung tissues of the rats was detected by immunohistochemical staining. The protein levels of ERK1/2 and p-ERK1/2 were determined by Western blot. RESULTS:Compared with model group, the airway responsiveness of the rats in middle dose and high dose of Kechuanning groups was significantly decreased (P<0.01), the injury of lung tissues was significantly decreased, the goblet epithelial cells metaplasia and airway collagen deposition were significantly reduced (P<0.01), and the expression of MMP-9 and TIMP-1 in the lung tissues was also significantly decreased (P<0.01). In addition, the protein level of p-ERK1/2 in high dose of Kechuanning group was significantly decreased compared with model group (P<0.01). CONCLUSION:Kechuanning may treat asthma by regulating the expression of p-ERK1/2 in the lung tissues and improving the airway remodeling symptoms of asthmatic rats induced by virus.     

Effects of spironolactone on atrial structural remodeling in rabbit model of chronic atrial fibrillation

AIM: To evaluate the effects and potential mechanism of spironolactone (SP) on atrial structural remodeling in rabbit model of chronic atrial fibrillation (AF). METHODS: The sternotomy was performed and the pacing electrodes were fixed to the left atria of New Zealand white rabbits. The animals were randomly divided into 3 groups. The rabbits were subjected to rapid atrial pacing (RAP) for 3 weeks in RAP group (intragastric administration with placebo) and RAP+SP group (intragastric administration with spironolactone at 20 mg·kg^-1·d^-1), respectively. The rabbits in sham group did not receive RAP and drugs. Before and after RAP, the structure and function of the atria were evaluated and AF inducibility was tested. After RAP, the atrial fibrosis was evaluated, and the expression levels of collagen I, collagen Ⅲ, matrix metalloproteinase (MMP)-2 and MMP-9 were determined. RESULTS: After 3 weeks of RAP, compared with sham group, obvious left atrial enlargement and dysfunction were observed in RAP group and RAP+SP group, but those had no significant differences in these 2 groups. Sustained AF was induced in 7, 5, and 0 rabbits in RAP group, RAP+SP group, and sham group, respectively. Compared with sham group, atrial interstitial fibrosis and the protein expression levels of collagen Ⅰ, collagen Ⅲ, MMP-2 and MMP-9 were all significantly increased in RAP group and RAP+SP group(P<0.05). Compared with RAP group, the the above indexes were all decreased in RAP+SP group(P<0.05). CONCLUSION: Spironolactone suppresses the atrial interstitial fibrosis and collagen expression, thus preventing atrial structural remodeling in rabbit model of chronic AF. The effect of spironolactone on reducing atrial MMP-2 and MMP-9 levels may be the potential mechanism.     

Relationship between matrix metalloproteinase-9 and tissue metalloproteinase inhibitor-1 and carotid atheromatous plaque stability

AIM: To investigate the correlation between matrix metalloproteinase-9 (MMP-9),tissue metalloproteinase inhibitor-1 (TIMP-1),MMP-9/TIMP-1 and carotid atheromatous plaque stability in cerebral infarction patients.METHODS: 80 patients with cerebral infarction were categorized as microemboli-negative group (n=70) and microemboli-positive group (n=10),20 normal human were served as control group.The MMP-9 and TIMP-1 levels in plasma were determined by mean of ELISA in 3 groups.RESULTS: The levels of MMP-9 and TIMP-1 in plasma were significantly higher in cerebral infarction patients than those in control group (P<0.01).The plasma MMP-9 content was positively correlated with TIMP-1 content (r=0.76，P<0.01).The ratio of MMP-9/TIMP-1 increased only in microemboli-positive patients (P<0.01).CONCLUSION: The results indicate that the plasma MMP-9 participates in pathophysiological process of cerebral infarction.The ratio of MMP-9/TIMP-1 shows a close relationship with carotid atheromatous plaque instability.     

Effects of nicotine on proliferation and migration of human umbilical cord mesenchymal stem cells

AIM: To explore the effects of nicotine on nitric oxide (NO), nitric oxide synthase (NOS), inducible nitric oxide synthase (iNOS), reactive oxygen species (ROS), mitochondrial membrane potential and cytokine secretion in human umbilical cord mesenchymal stem cells (MSCs). METHODS: MSCs were treated with different concentrations of nicotine. The content of NO was detected by nitrate reductase method. The activity of NOS and iNOS was mea-sured by ultraviolet spectrophotometry. ROS and mitochondrial membrane potential were detected by flow cytometry. The levels of intercellular adhesion molecule 1（ICAM-1）, stromal cell-derived factor 1 (SDF-1), matrix metalloproteinase 9 (MMP-9), tissue inhibitor of metalloproteinase 1(TIMP-1), transforming growth factor β1 (TGF-β1), insulin-like growth factor I (IGF-I) and basic fibroblast growth factor (bFGF) were determined by ELISA. RESULTS: At 24 h and 36 h after exposure to nicotine, the levels of NO were significantly increased in a dose-dependent manner. However, at 48 h, the levels of NO in 0.8 g/L group and 1.0 g/L group were lower than that in control group. The activity of NOS and iNOS were significantly increased in a dose-dependent manner. The level of ROS increased, while mitochondrial membrane potential decreased. After nicotine treatment, the secretions of SDF-1, TGF-β1, IGF-I and bFGF declined, while the levels of ICAM-1, MMP-9 and TIMP-1 increased. CONCLUSION: Nicotine may affect the proliferation, adhesion and migration of MSCs by increasing the levels of NO, NOS, iNOS and ROS and the production of ICAM-1, MMP-9 and TIMP-1, and decreasing the mitochondrial membrane potential and the secretion of SDF-1, TGF-β1, IGF-I and bFGF.     

Effect of insulin on early myocardial oxidative stress in severely burnt rats

AIM：To investigate the effect of insulin on early myocardial oxidative stress in severely burnt rats. METHODS：Twenty-four Sprague-Dawley rats were randomly divided into three groups (8 rats in each group): control group (sham scald group), scald injury group and scald injury + insulin group. The rats in the latter two groups were subject to third-degree burn with 30% total burn surface area (TBSA) on the back, and then received intraperitoneal injection of normal saline (40 mL/kg) immediately. The rats in scald injury + insulin group were subcutaneously injected with insulin (1 U/kg), while those in scald injury group received subcutaneous injection of the same volume of normal saline. All rats were sacrificed 24 h after scald, and blood samples from abdominal aorta and myocardial tissues were taken. Blood glucose (BG) content, blood lactate dehydrogenase (LDH) and creatine kinase (CK) activity, and myocardial oxidative and antioxidative indexes, including malondialdehyde (MDA), xanthine oxidase (XO), myeloperoxidase (MPO), superoxide dismutase (SOD), catalase (CAT) and glutathion peroxidase (GPx), were detected by spectrophotometry. RESULTS：(1) Compared with control group, BG levels in scald injury group and scald injury + insulin group were significantly elevated (P<0.05). But BG in scald injury + insulin group was significantly lower than that in scald injury group (P<0.05). (2) Compared with control group, the activity of LDH and CK in scald injury group was significantly increased (P<0.05), while that in scald injury + insulin group was significantly lower than that in scald injury group (P<0.05). (3) Compared with control group, the MDA content and the XOD and MPO activity in scald injury group were significantly increased (P<0.05), while the activity of SO, CAT and GPx was significantly decreased (P<0.05). Compared with scald injury group, the MDA content and the XO and MPO activity in scald injury + insulin group were significantly reduced (P<0.05), while the activity of SOD, CAT and GPx was significantly elevated (P<0.05). CONCLUSION：Insulin intervention attenuates early myocardial oxidative stress in burnt rats and decreases the rise in myocardial enzyme activity, thus exerting a cardioprotective effect.     

Hydrogen sulfide inhibits inflammatory responses induced by acute myocardial ischemia in isolated rat hearts

AIM：To investigate whether hydrogen sulfide (H2S) protects the hearts against inflammatory responses induced by acute myocardial ischemia in isolated rat hearts. METHODS：Rat acute myocardial ischemia injury was induced by ligation of the left anterior descending coronary artery for 4 h, and the normal perfusate was replaced with NaHS (5 μmol/L, 10 μmol/L and 20 μmol/L) perfusate accordingly in NaHS groups 2 h after ischemia. The changes of cardiac function in the myocardial ischemic injury rats were observed. The mRNA expression of TNF-α, IL-1β, IL-6, IL-10 and ICAM-1 was detected by real-time PCR. The protein level of nuclear factor-κB (NF-κB) in the myocardial tissues was detected by Western blotting. RESULTS：The cardiac function in ischemia group was lower than that in sham group (P<0.01). Compared with ischemia group, perfusion of NaHS resulted in the improvement of the cardiac function (P<0.05 or P<0.01). Compared with sham group, the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 in the cardiac tissues was significantly increased, and the mRNA expression of IL-10 in the cardiac tissues was significantly decreased in ischemia group (P<0.01). Compared with ischemia group, the perfusion of NaHS significantly decreased the mRNA expression of TNF-α, IL-1β, IL-6 and ICAM-1 (P<0.05 or P<0.01). The perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly increased the mRNA expression of IL-10 (P<0.01). The protein level of NF-κB in ischemia group was markedly higher than that in sham group (P<0.01). Compared with ischemia group, the perfusion of NaHS at concentrations of 10 μmol/L and 20 μmol/L significantly decreased the expression of NF-κB (P<0.05 or P<0.01). CONCLUSION：H2S protects the hearts against acute ischemia injury through inhibition of NF-κB activation and subsequent down-regulation of NF-κB-dependent inflammatory gene expression.     

Effect of idazoxan on permeability of blood-brain barrier and expression of MMP-9/TIMP-1 in mouse experimental autoimmune encephalomyelitis

AIM：To study the effect of idazoxan (IDA) on the permeability of blood-brain barrier (BBB) and the expression of matrix metalloproteinase 9 (MMP-9) and tissue inhibitor of metalloproteinase 1 (TIMP-1) in mouse experimental autoimmune encephalomyelitis (EAE).METHODS：Female C57BL/6 mice (n=36) were randomly divided into control group, EAE group and IDA group, with 12 mice in each group. EAE was induced by myelin oligodendrocyte glycoprotein 35-55 (MOG35-55). IDA (2 mg/kg, ip, bid) was administered for 15 d after immunization. The neurological defects of the mice were observed daily and scored. The pathological changes were observed under microscope with HE staining and LFB myelin staining. The BBB permeability was detected by Evans blue extravasation. The expression of MMP-9 and TIMP-1 in the brain of EAE mice was determined by Western blotting.RESULTS：Compared with EAE group, the score of neurological defects in IDA group was decreased, the inflammation was relieved, the BBB permeability was reduced, and the expression MMP-9 and the ratio of MMP-9/TIMP-1 were decreased (P<0.05).CONCLUSION：The neuroprotective effect of IDA on mouse EAE might be related to the down-regulation of MMP-9 and the ratio of MMP-9/TIMP-1, thus reducing the degradation of BBB and the permeability of BBB, and ameliorating the pathologic process of EAE.     

Relationship between 3-nitrotyrosine and myocardial cell apoptosis in rat diabetic cardiomyopathy

AIM: To explore the relationship between 3-nitrotyrosine (3-NT) level in hearts or blood and myocardial cell apoptosis in rat diabetic cardiomyopathy (DCM). METHODS: Sixty Sprague-Dawley (SD) rats (male, 8-week-old) were randomly divided into 4 groups: normal group, diabetic cardiomyopathy group (DCM group), diabetic rats treated with valsartan (40 mg·kg^-1·d^-1, D+V group) and DCM rats treated with valsartan (40 mg·kg^-1·d^-1, DCM+V group). Apoptotic index (AI) of rat cardiac myocytes was examined by TUNEL. The expression index (EI) of 3-NT in rat cardiac myocytes was examined by immunohistochemistry. The 3-NT concentration in rat serum was examined by ELISA. RESULTS: (1) Significant differences of the heart weight indexes among the 4 groups were observed (P<0.01). The heart weight indexes in DCM group and DCM+V group were higher than those in normal group and D+V group (P<0.01). (2) The EI of 3-NT in the cardiac myocytes was positively correlated with the AI of the cardiac myocytes in the same group (P<0.01), but the concentration of 3-NT in blood had no correlation with the AI of cardiac myocytes (P>0.05). (3) The difference of AI of cardiac myocytes among the 4 groups had statistical significance (P<0.01). The arrangement from high to low of AI was DCM group > D+V group and DCM+V group > N group (P<0.05). (4) The EI of 3-NT in DCM group was the highest as compared to other groups (P<0.05). (5) No statistical difference of 3-NT concentration in blood among the 4 groups was observed (P>0.05). CONCLUSION: (1) The expression of 3-NT in DCM myocardial tissues in SD rats is significantly increased and closely correlated with the apoptosis in myocardial cells. Valsartan inhibits 3-NT expression in DCM myocardial cells, thus inhibits the DCM myocardium apoptosis. (2) The 3-NT level in blood can not be true for reflection of 3-NT expression in DCM myocardial tissues and its effect on myocardial cell apoptosis.     

Long-term insulin treatment improves postischemic cardiac structure and function via increasing serum BNP levels

AIM：To investigate the influence of long-term insulin treatment on postischemic cardiac structural and functional changes, and to further explore the underlying mechanisms. METHODS：Adult male SD rats were randomly divided into 4 groups (8~10 rats per group): sham group, myocardial infarction (MI) + saline (1 mL·kg-1·d-1, hypodermic injection for 4 weeks) group, MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) group and MI + insulin (2 U·kg-1·d-1, hypodermic injection for 4 weeks) + wortmannin ［a phosphatidylinositol 3-kinase (PI3K) inhibitor; 15 μg·kg-1·d-1, intraperitoneal injection 15 min before each insulin treatment］ group. The rats in the latter 3 groups were subject to ligation of the left anterior descending coronary artery, while those in sham group underwent the same surgical procedures without tying the sutures. The cardiac structural and functional changes were observed by echocardiogram, heart catheterization and microscopy with HE and Masson trichrome staining. Blood glucose was determined by Roche blood glucose meter, and the serum levels of insulin and brain natriuretic peptide (BNP) were detected by ELISA. The protein expression and phosphorylation of PI3K, Akt, glycogen synthase kinase 3β (GSK3β) and p38 mitogen-activated protein kinase (p38 MAPK) in myocardial tissues were detected by Western blotting. The mRNA expression of BNP, β-myosin heavy chain (β-MHC) and atrial natriuretic peptide (ANP) in myocardial tissues was determined by real-time fluorescence quantitative PCR. RESULTS：At the end of the 4th week, MI rats receiving long-term insulin treatment showed decreased ratio of heart length/heart weight, smaller systolic left ventricle cavity, thicker systolic interventricular septum, and increased cardiac ejection fraction, left ventricular development pressure and instantaneous first derivate of left ventricle pressure (P<0.05 vs MI + saline group). Moreover, insulin treatment significantly increased the phosphorylation of PI3K and Akt and the serum level of BNP, and inhibited the phosphorylation of p38 MAPK (P<0.05 vs MI + saline group), but did not change the mRNA expression of BNP in myocardial tissues. The effects of insulin on BNP were not blocked by wortmannin (P>0.05 vs MI + insulin group). CONCLUSION：Insulin improves postischemic cardiac structure and function by increasing serum BNP levels possibly independent of PI3K-Akt signaling pathway.     

Effect of Yangyu Tuji on content of type Ⅰ, Ⅲ collagen and the expression of MMPs and TIMP-1 in wound caused by streptozotocin in rats

AIM: To study the effects of Yangyu Tuji (YYTJ) on delayed healing wound of diabetic rats caused by streptozotocin (STZ). METHODS: SD male rats were randomly divided into control group (control), model group (model); and 3 different dose groups of YYTJ. 55 mg/kg STZ were given by intraperitoneal injection except for control group. After 30 days, a round skin of 1.6 cm diametre was excised on all dorsal back of rats. The healing time and healing rate were observed according to re-epithelization. The content of collagenⅠ and Ⅲ was observed by Picric acid-Sirius red staining , Matrix metalloproteinase-1, 13 (MMP-1, -13), tissue inhibitor of metalloproteinases-1 (TIMP-1) by immuno-histochemistry assay. All data were analyzed by IPP software. RESULTS: The healing time in each group treated with YYTJ was shorter than that in model group (P<0.01), and the healing rate was increased (P<0.01, P<0.05). Content of type I collagen, ratio of type Ⅰ and Ⅲ collagen of high and mid dose group were significantly higher than that in model group (P<0.01) at 3rd, 7th, 11th day. The expression of MMP-1, -13 of each groups were higher than that in model group at 7th day (P<0.01, P<0.05), and MMP-1 trend to equal with model group at 11th day. MMP-13 was significantly lower than that in model group at 11th day (P<0.01, P<0.05). TIMP-1 of each group of wound was higher than that in model group at 3rd, 7th, 11th day (P<0.01, P<0.05). The ratio of type Ⅰ and Ⅲ collagens in each group was lower than that in model group at 11th day (P<0.01). Ratio of MMP-13 and TIMP-1 of high dose group and mid dose group were higher than that in model group at 3rd and 7th day (P<0.01). The ratio of each group was lower than that in model group at 11th day (P<0.01). Meanwhile, ratio of MMP-13 and TIMP-1 of high dose group and mid dose group were lower than that of lower dose group (P<0.05). CONCLUSION: It is possible that YYTJ accelerates wound healing by increasing collagen content of type Ⅰ and Ⅲ, especially type Ⅰ, as well as improves collagen deposition by regulating the balance of MMP and TIMP.