Metallothionein inhibits rat vascular fibroblasts activation induced by homocysteine

AIM:To study the effects of exogenous metallothionein (MT) and ZnCl₂-induced MT production on biological action of homocysteine(HCY)in vascular fibroblasts.METHODS:[³H]-TdR, [³H]-Pro incorporation and LDH leakage were measured, the cellular viabilities were calculated by trypan blue exclusion test and the intracellular contents of MT were assayed by [¹⁰⁹Cd]-hemoglobin saturation method in cultured rat vascular fibroblasts.RESULTS:Proliferation, collagen production of vascular fibroblasts in HCY-treated group were significantly increased compared with control group in a concentration-depedant manner. HCY (500 μmol/L) increased LDH leakage and decreased the cellular viabilities (P＜0.05 or P＜0.01). [³H]-TdR incorporation, [³H]-Pro incorporation, collagen secretion and LDH leakage were all decreased in MT (1×10^-5 mol/L, 1×10^-4mol/L) plus HCY(500 μmol/L) incubated group, compared with HCY alone group, respectively (P＜0.05 or P＜0.01). MT content in ZnCl₂ pretreatment group was increased compared with control group. Proliferation, collagen production and LDH leakage in HCY group pretreated with ZnCl₂ were decreased whereas the cellular viabilities were increased compared with HCY alone group.CONCLUSIONS:The results shows that HCY induces proliferation and collagen production of vascular fibroblasts. Both exogenous MT and endogenous MT induced by ZnCl₂ inhibite the above-mentioned effects of HCY on vascular fibroblasts. MT inhibites vascular fibroblast activation induced by HCY, which may be related to its vascular protection.     

Effects of chemotherapy on free radicals in the rat glioma

AIM: To observe the role of free radicals in the inhibitory effect of chemotherapy on glioma cells. METHODS: C6 glioma cells were cultured in vitro, and treated with carmustine (B), teniposide (V), or/and nimodipine (N). Furthermore, the glioma-bearing rats were treated with B plus N, B+V+lisplatin (D)+N, or B+V+D+N+angiotensin Ⅱ. The MDA content and superoxide dismutase (SOD) activity in the culture supernatant and cortical brain tissue were assayed. RESULTS: B, V and N significantly decreased MDA content and SOD activity in the supernatant of glioma cell culture and C6 glioma cells. Chemotherapy reduced MDA content and increased SOD activity in the cortical brain tissue of tumor-bearing rats, with highest efficiency in B+V+D+N+angiotensin Ⅱ group. The survival time of tumor-bearing rats in B+V+D+N+angiotensin Ⅱ group was longer than that in other chemotherapy group. CONCLUSION: The antitumor effects of combined chemotherapy may be involved in the free radical metabolism.     

Ischemic preconditioning protects against hepatic ischemia-reperfusion injury by attenuating leukotriene C4 production

AIM：To investigate the effect of ischemic preconditioning (IP) on leukotriene C4 (LTC4) generation during hepatic ischemia-reperfusion(I/R) injury.METHODS：Adult male Sprague-Dawley rats were randomly divided into sham (control) group, I/R group and IP+I/R group. The rat liver was subject to 60 min of partial hepatic ischemia followed by 5 h of reperfusion. LTC4 content was measured by reversed-phase high-performance liquid chromatography (RP-HPLC). The activity of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was determined, and the changes of histological structures were observed to assess the tissue injury. The reduced glutathione (GSH) level in the liver tissues was also examined by biochemical methods.RESULTS：IP markedly decreased LTC4 content in rat livers compared with I/R group. The activity of serum ALT and AST in I/R group and IP+I/R group was higher than that in control group. Compared with I/R group, the levels of serum ALT and AST were markedly decreased, and the level of GSH in hepatic tissues was elevated in IP+I/R group. In addition, the structural damage of the rat liver tissues in IP+I/R group displayed slighter than that in I/R group.CONCLUSION：IP treatment reduces LTC4 production accompanied with improving the liver function and histological structure during I/R injury, suggesting that the beneficial effects of IP may be involved in its repressing LTC4 generation during hepatic I/R injury.     

The protective effect of preconditioning on rabbit liver during ischemia and reperfusion and its potential mechanism

AIM:To investigate the protective effect of preconditioning on rabbit liver during total ischemia and reperfusion and its mechanism. METHODS: Using hepatic ischemia and reperfusion injury (HIRI) model in rabbits, animals were randomly divided into three groups: control group (A), non-preconditioning group(B) and preconditioning group(C), different effects of preconditioning on several parameters including alanine aminotransferase (ALT) activity and levels of nitric oxide (NO) and malondialdehyde (MDA) in plasma or liver tissue as well as hepatocellular morphological changes were measured and observed during HIRI. RESULTS:In C group NO levels of plasma and liver tissue were higher than those in B group (P＜0.05);While MDA levels and ALT value in plasma were lower than those in B group (P＜0.05 and P＜0.01); and there were not significant differences between C and A group (P＞0.05); abnormal morphological chages of liver cells in A group were ameliorated remarkably too during HIRI. CONCLUSION:Preconditioning can attenuate HIRI by improving NO level and reducing oxygen free radicals level.     

Levels of histone modifications in activated primarily cultured rat hepatic stellate cells

AIM: To investigate the changes of histone modifications during the activation of primarily cultured rat hepatic stellate cells (HSCs) and the relationship between histone modification patterns and α-smooth muscle actin (α-SMA) expression, and to explore the roles of histone modifications in the activation of HSCs. METHODS: The rat HSCs were isolated by in situ perfusion of collagenase combined with density gradient centrifugation, cultured in vitro and identified by immunofluorescence staining. The morphological features of the cells were observed under inverted microscope. The changes of desmin and α-SMA during the activation of HSCs were detected by immunofluorescence staining and Western blotting. The levels of histone 3 lysine 4 dimethylation (H3K4me2), histone 3 lysine 9 dimethylation (H3K9me2), histone 3 lysine 9 acetylation (acH3K9) and histone 4 lysine 12 acetylation (acH4K12) in quiescent HSCs and activated HSCs were determined by Western blotting.RESULTS: The morphology of HSCs shifted from a quiescent phenotype to highly activated myofibroblast during the culture. Immunofluorescence staining and Western blotting showed that the expression levels of α-SMA and desmin were increased over time and reached maximum at 15 d. According to the results of cell morphology and immunofluorescence staining, the cells cultured for 24 h and 15 d were quiescent and activated HSCs, respectively. Compared with quiescent HSCs, there were higher H3K4me2 and lower H3K9me2, acH3K9 and acH4K12 modification levels in activated HSCs (P<0.01). CONCLUSION: Histone modifications show anomalous expression during the activation of primarily cultured rat HSCs. Histone modifications may contribute to the transdifferentiation of HSCs and the development of hepatic fibrosis.     

Ulinastatin protects rat pulmonary tissues from paraquat-induced acute injury

AIM: To investigate the protective effects of ulinastatin on the rats with paraquat-induced acute lung injury and its mechanisms. METHODS: The Wistar rats (n=108) were randomly divided into control group, paraquat group and ulinastatin group. The rats in paraquat group and ulinastatin group were given paraquat by gavage, while the rats in control group were given sterile saline by gavage. The rats in ulinastatin group were also given ulinastatin treatment. The serum levels of MDA, SOD, IL-6, IL-10 and TNF-α were measured after 1 d, 3 d, 7 d, 14 d, 21 d and 28 d. The expression levels of p38 MAPK, MMP-2 and TIMP-1 in the lung were also measured. RESULTS: The levels of SOD in 1 d, 3 d and 7 d in paraquat group and ulinastatin group were significantly lower than those in control group (P<0.01). The level of SOD in ulinastatin group was significantly higher than that in paraquat group (P<0.05). The levels of MDA, IL-6, IL-10 and TNF-α in 1 d, 3 d and 7 d in paraquat group and ulinastatin group increased compared with control group (P<0.01), and those in ulinastatin group were significantly lower than those in paraquat group (P<0.05). The levels of p38 MAPK and TIMP-1 in 1 d, 3 d, 7 d, 14 d, 21 d and 28 d in paraquat group and ulinastatin group were higher than those in control group (P<0.01), and those in ulinastatin group was significantly lower than those in paraquat group (P<0.05). The level of MMP-2 in 1 d, 3 d, 7 d, 14 d and 21 d in paraquat group and ulinastatin group increased compared with control group (P<0.01), and that in ulinastatin group was significantly lower than that in paraquat group (P<0.05).CONCLUSION: Ulinastatin protects the lung tissues of rats from paraquat-induced acute lung injury by inhibiting p38 MAPK signaling pathway and ameliorating inflammatory and oxidative responses.     

Effects of angiotensin II and losartan on collagen synthesis in rat hepatic stellate cells

AIM: To investigate the effects of angiotensin II (Ang II) and AT_1a receptor antagonist (losartan) collagen synthesis in rat hepatic stellate cells (HSCs). METHODS: ① Rat HSCs were isolated, cultured and identified. ② Rat HSCs were incubated in the medium with different concentrations of AngII or losartan, then the quantity of collagen was examined by -proline release assay. RESULTS: ① The yield of HSCs was 2×10⁷-3×10⁷/per rat, their viability and purity was more than 95% and 90%, respectively. ② The yield of collagen in HSCs significantly got a rise in a concentration-dependent manner when HSCs were incubated with AngII (10^－6mol/L-10^－10 mol/L) (P<0.05). While HSCs were influenced by the antagonist of AT_1a (10^－6 mol/L-10^－9 mol/L), the quantity of collagen dropped greatly (P<0.05). CONCLUSIONS: Ang II stimulates HSCs to produce more collagen. Losartan inhibits the cell to synthesize collagen via AT_1a receptor (P<0.05). The results indicate that Ang II may play an important role in the development of hepatic fibrosis, and using AT_1a antagonist may offer a new strategy to prevent hepatic fibrosis.     

siRNA targeting fas protects donor liver against cold preservation and ischemic reperfusion injury in rat liver transplantation

AIM:To investigate the silencing effect of fas siRNA to alleviate ischemic-reperfusion (I/R) injury in liver transplantation. METHODS:Three pairs of 21-nt synthesized fas siRNAs were transfected into BRL cells respectively for evaluation of silence efficacy, and the most effective fas siRNA was chosen in vivo for experiment. In cold preservation experiment, siRNA was transfected in vivo by hydrodynamics method. After 48 h, livers of fas siRNA group and control group were harvested and cold preserved, and cell apoptosis and fas expression was evaluated at 2, 4 and 6 h. Orthotopic liver transplantation was performed in fas siRNA group and blank control group. At 1, 3, 6, 12 and 24 h after transplantation, blood and liver samples were collected for evaluation of serum ALT levels, Fas protein and mRNA expression, and apoptosis by TUNEL staining. RESULTS:fas siRNA2, which began at nt 315, inhibited fas gene expression much more than other siRNAs. As to cold preservation, apoptosis index (AI) and fas expression in fas siRNA group was lower than that in control group at each checked point (P<0.01). At 1, 3, 6, 12, and 24 h after blood reperfusion of liver transplantation, the serum ALT level in fas siRNA group was much less than that in control group. The cell apoptosis in fas siRNA group was substantially decreased, and the expressions of fas mRNA and protein were dramatically reduced. CONCLUSION:fas-mediated apoptosis plays an important role in I/R injury of rat liver transplantation. Silencing fas by siRNA holds therapeutic promise to limit I/R injury.     

Shenmai injection protects rat cardiomyocytes from angiotensin Ⅱ-induced apoptosis in vitro

AIM: To study the protective effects of Shenmai injection, a Chinese medicine, on angiotensin Ⅱ (AngⅡ)-induced rat cardiomyocyte apoptosis in vitro and the probable mechanism. METHODS: Cultured cardiomyocytes from neonatal rats were stimulated with AngⅡ. Cell viability were measured by MTT, and apoptosis was 'evaluated suing Hoethst33258 fluorescent dye staining and flow cytometry. Fluo-3/AM was used to test the change in intracellular free calcium. RESULTS: It was found that incubation with AngⅡ (10^-7mol/L) for 48 h increased cardiomyocyte apoptosis, Shenmai injection (0.5 g/L, 1.0 g/L) inceased myocyte viability (P＜0.05). Shenmai injection (1.0 g/L) significantly decreased the AngⅡ-induced rat cardiomyocyte apoptosis (P＜0.05) and decreased fulorescent intensity of intracellular calcium. CONCLUSION: Shenmai injection has a significant inhibitory effect on AngⅡ-induced rat cardiomycoyte apoptosis in vitro by alleviating intracellular calcium overload.     

Valsartan protects a rat model of adriamycin-induced dilated cardiomyopathy from left ventricular remodeling and failure

AIM: To investigate whether and how AT1 receptor blocker， valsartan， attenuates left ventricular remodeling and failure in a rat model of adriamycin（ADR）-induced dilated cardiomyopathy. METHODS: Weight-matched adult male Wistar rats were randomly divided into 3 groups as follows: 1) the ADR group, in which 2.5 mg/kg of ADR was weekly injected via a tail vein for 10 weeks (n=25); 2) concomitant AT1 receptor blocker valsartan and ADR, in which valsartan was administered by daily gavage at a dose of 30 mg·kg-1·d-1 (n=10); 3) control group (n=10). Hemodynamics and echocardiographic measurements were obtained at 12 weeks after treatment. Finally, left ventricle (LV) samples were collected at 12 weeks. The hydroxyproline content was determined by the methods of chloramines T. The expression of MMP-2, MMP-9 and tissue inhibitors of metalloproteinase-1 (TIMP-1) were measured by Western blotting. MMP-2 and -9 gelatinolytic activities were measured by gelatin zymography. RESULTS: Mortality was significantly lower in valsartan -treated rats than that in ADR rats (20% versus 40%, P<0.01). The dilatation of LV cavity was significantly attenuated in ADR-induced dilated cardiomyopathy rats given valsartan. Valsartan partially normalized LV contractile function, which was significantly reduced in ADR rats. The hydroxyproline content was increased in ADR-DCM group and significantly reduced by valsartan treatment (P<0.01). The protein levels of LV MMP-2 and MMP-9 were increased in ADR rats and attenuated by valsartan treatment (both P<0.01). However, no change in TIMP-1 was observed (P>0.05). The activities of LV myocardial MMP-2 and -9 gelatinolytic were increased significantly in ADR rats (both P<0.01) and attenuated by valsartan treatment (both P<0.01). CONCLUSION: Pretreatment with AT1 receptor blocker valsartan attenuates left ventricular remodeling and failure in a rat model of adriamycin-induced dilated cardiomyopathy.     

Ganoderma lucidum extract protects rat cerebral cortical neurons from hypoxia/reoxygenation injury in vitro

AIM: To investigate the neuroprotective effect of Ganoderma lucidum extract (GLE) in an in vitro model of primary cultured neurons with oxygen and glucose deprivation (OGD). METHODS: Neuronal injury was induced by oxygen and glucose deprivation/reoxygenation (OGD/R). The neuronal injury and viability were determined by LDH leakage and XTT assay at 0 h,3 h,6 h,12 h,24 h,48 h and 72 h after OGD/R. Neuronal apoptosis was detected by flow cytometry (FCM). The expression of apoptosis-related proteins was analyzed by Western blotting.RESULTS: The viability of the neurons increased with exposure to GLE (0.1 mg/L,1 mg/L and 10 mg/L)after OGD/R. The LDH releases were also significantly reduced. GLE significantly inhibited OGD/R-induced apoptosis of cultured rat cortical neurons in a concentration-dependent and time-dependent manner(P<0.05). GLE at concentrations of 0.1 mg/L,1 mg/L and 10 mg/L inhibited the expression of caspase-3 and caspase-8 proenzyme. Additionally,GLE at concentration of 10 mg/L suppressed the expression of caspase-9 proenzyme.CONCLUSION: Our findings provide the evidence that the GLE has neuroprotective effect on cerebral ischemia. The mechanisms are related to the inhibition of caspase-3,-8 and-9 activations. GLE may be a novel and effective reagent for treating ischemic stroke.     

Insulin-like growth factor I protects neonatal rat cardiomyocytes from apoptosis through improvement of mitochondrial function

AIM：To investigate whether mitochondrial mechanism is involved in the anti-apoptotic effect of insulin-like growth factor I (IGF-I) on cardiomyocytes. METHODS：Primary neonatal rat cardiomyocytes (NRCMs) were cultured and treated with 200 μmol/L hydrogen peroxide (H2O2) to induce apoptosis. Kruppel-like factor 9 (KLF9)-specific siRNA was transfected into the cells by Lipofectamine 2000. The mitochondrial function was measured by JC-1 mitochondrial membrane potential (MMP) assay. The mitochondrial morphology was observed by transmission electron microscopy. Myocardial cell apoptosis was detected by Annexin V-FITC/PI dual staining, caspase-3 activity assay, DNA-ladder analysis and Hoechst 33258 staining. RESULTS：The apoptosis of NRCMs was induced by H2O2, with MMP decreased by (24.0±1.6)% compared with control group. The fall rates of MMP in IGF-I group and KLF9 siRNA group were (18.3±1.2)% and (15.2±1.2)%, respectively (both P<0.01 vs H2O2 group), and improved mitochondrial morphology, decreased caspase-3 activity, attenuated DNA fragmentation and reduced apoptotic bodies were also observed in these two groups. The apoptotic rates of NRCMs in IGF-I group and KLF9 siRNA group were (22.4±4.2)% and (32.5±3.5)%, respectively, both lower than that in H2O2 group ［(42.5±1.8)%, P<0.01］. The anti-apoptotic effect of KLF9 silencing on NRCMs was consistent with that of IGF-I treatment. CONCLUSION：IGF-I protects NRCMs from apoptosis through down-regulating KLF9 expression and improving mitochondrial function.