Early morphological changes of porcine serum-induced hepatic fibrosis

When porcine serum is used to induce hepatic fibrosis in rats, linear septal fibrosis develops and outlines the periphery of Rappaport's functional liver acinus. A sequential morphological study was done to clarify the histogenesis of this hepatic fibrosis, especially in the early stages. Under light microscopic examination, the fibrotic septa developed as a thin fibrotic strand connecting terminal hepatic veins without any massive necrosis. Electron microscopic examination, however, revealed a thickening of the walls of the portal veins with narrowing lumen and damage to hepatocytes prior to the development of fibrosis. There was only mild inflammation and examination showed three types of mesenchymal cells; Ito cells, myofibroblasts, and fibroblasts. These changes suggested that this fibrosis is a result of hepatic damage, which in turn was possibly caused by disturbance of the portal vein.     

Remote delivery and expression of soluble type II TGF-beta receptor in muscle prevents hepatic fibrosis in rats

Transforming growth factor-beta (TGF-beta) has been implicated in the process of hepatic fibrosis, and stimulates production of extracellular matrix in hepatic stellate cells, which play a major role in the process. It has been recently reported that blockage of TGF-beta signaling prevents hepatic fibrosis. We evaluated a strategy for anti-TGF-beta gene therapy for hepatic fibrosis by transfecting plasmids expressing an entire extracellular domain of human TGF-beta type II [soluble type II TGF-beta receptor (sTGF-betaIIR)] into skeletal muscle in a rat experimental model of dimethylnitrosamine- (DMN-) induced fibrosis. sTGF-betaIIR treatment decreased significantly the occurrence of DMN-induced hepatic fibrosis, evaluated by computed image analysis and by measurement of hydroxyproline content of the liver, and reduced the expression of collagen and alpha-smooth muscle actin. The treatment also caused a significant decrease in hepatic levels of interleukin- (IL-) 12 (Th1 cytokine) and an increase in those of IL-10 (Th2 cytokine), indicating a change in the Th1/Th2 cytokine balance in the liver. In conclusion, blockade of TGF-beta after intramuscular transfer of the soluble type II TGF-beta receptor gene suppressed hepatic fibrosis, suggesting that this strategy may be useful for gene therapy of hepatic fibrosis.     

Anti-monocyte chemoattractant protein-1 gene therapy prevents dimethylnitrosamine-induced hepatic fibrosis in rats

Monocyte chemoattractant protein-1 (MCP-1) has been implicated in the process of hepatic inflammation, recruiting monocytes and lymphocytes during liver injury. MCP-1 also activates directly hepatic stellate cells, which play a major role in hepatic fibrosis. However, it remains unclear whether blockage of MCP-1 signaling could prevent hepatic fibrosis in vivo. We evaluated a strategy for anti-MCP-1 gene therapy against hepatic fibrosis by transfecting an amino-terminal deletion mutant, lacking the amino-terminal codons 2 to 8 of the human MCP-1 gene and designated 7ND, into skeletal muscle in a rat experimental model of dimethylnitrosamine (DMN)-induced fibrosis. Anti-MCP-1 gene therapy decreased significantly the occurrence of DMN-induced hepatic fibrosis, evaluated by computed image analysis and by measurement of hydroxyproline contents of the liver, accompanied by a reduction in the expressions of alpha-smooth muscle actin. This treatment also caused a significant decrease in hepatic tissue levels of interleukin (IL)-12 (Th1 cytokine) and an increase in those of IL-10 (Th2 cytokine), indicating a change in the Th1/Th2 cytokine balance in the liver. In conclusion, blockade of MCP-1 after intramuscular transfer of the 7ND gene suppressed hepatic fibrosis, and this strategy may be a useful and feasible gene therapy against hepatic fibrosis.     

Captopril reduces collagen and mast cell and eosinophil accumulation in pig serum-induced rat liver fibrosis

The effect of captopril on the development of hepatic septal fibrosis in a specific experimental model produced by repeated injections of whole pig serum into the peritoneal cavity of rats was studied. The results afforded four basic conclusions. First, the experimental model used seems to be a pure form of septal fibrosis, which depends on active tissue fibroplasia, without hepatocyte necrosis. The fibrotic septa, located between limiting plates of adjacent classic hepatic lobules, and delimiting the classic liver lobule, consisted of collagen fibers infiltrated by eosinophils, mast cells, fat-storing cells (Ito cells), transitional cells and interstitial fibro-blasts. Second, the angiotensin-converting enzyme inhibitor captopril attenuated the hepatic fibrosis induced by pig serum administration, as proven by a decrease In hepatic hydroxyproline concentration and histological examination of the liver. Third, this attenuation of hepatic fibrosis might be related, at least in part, to diminished mast cell and eosinophil accumulation in the hepatic tissue. Finally, these data may indicate a novel action of angiotensin-converting enzyme inhibitor in general, and for captopril in particular, as drugs potentially capable of reducing eosinophils in fibrotic processes.     

柴芪益肝颗粒防治四氯化碳诱导大鼠肝纤维化的作用

目的观察柴芪益肝颗粒治疗四氯化碳诱导的大鼠肝纤维化的疗效及其对Leptin、TGF—β1和IL-13因子的影响。方法将50只SD大鼠随机分为模型组、柴芪益肝颗粒组、复方鳖甲软肝片组、秋水仙碱组和空白对照组,每组10只。大鼠腹腔注射四氯化碳诱导肝纤维化,光镜观察肝组织病理学改变,放射免疫分析法检测肝纤四项,液相芯片检测血清细胞因子。结果与模型组比较,柴芪益肝颗粒组肝脏病理变化显著减轻,肝小叶结构基本清晰,细胞索、肝窦无明显异常;与模型组比较,透明质酸（P=0．001）、层粘连蛋白（P=0．005）和Ⅳ型胶原（P=0．000）水平显著降低,瘦素（P=0．012）、转化生长因子131（TGF-β1）（P=0．000）和白介素13（IL-13）（P=0．016）水平亦显著降低,差异均有统计学意义。结论柴芪益肝颗粒显著改善肝纤维化,可能与其通过降低瘦素、TGF—β1和IL-13水平,抑制细胞外基质沉积有关。     

Dimethyl sulfoxide inhibits dimethylnitrosamine-induced hepatic fibrosis in rats

We studied the preventive effects of dimethyl sulfoxide (DMSO) on experimental hepatic fibrosis induced by dimethylnitrosamine (DMN) in rats. Treatment with DMN caused a significant decrease in body and liver weight. Oral DMSO (2 ml/kg daily for 4 weeks) essentially prevented this DMN-induced body and liver weight loss with no major side effects. DMSO suppressed the induction of hepatic fibrosis, as determined by histological evaluation, and reduced hepatic hydroxyproline. It also suppressed the expression of mRNA for type I collagen in the liver. Because hepatic stellate cells (HSC) are the major cellular source of the collagen in hepatic fibrosis, we examined the effects of DMSO on collagen production in vitro using rat primary HSC culture. However, it was found that DMSO did not inhibit the collagen production in vitro. We next evaluated the effects of DMSO on tumor necrosis factor alpha (TNFalpha) and nitric oxide (NO) production by Kupffer cells, because these factors represent major activator of HSC, and because monocyte-macrophage infiltration has been implicated as being pathogenetically important for hepatic fibrosis induced by DMN. DMSO inhibited lipopolysaccharide (LPS)-induced TNFalpha and NO production, and reduced TNFalpha mRNA levels. DMSO also suppressed the LPS-induced nuclear factor kappa B activation in a murine macrophage-like cell line. These results suggest that the inhibitory effects of DMSO on hepatic fibrosis may be primarily exerted via blocking of DMN-induced inflammation. These results also implied that DMSO may be potentially useful for preventing the development of hepatic fibrosis.     

Rosuvastatin treatment prevents progressive kidney inflammation and fibrosis in stroke-prone rats

Salt-loaded, spontaneously hypertensive stroke-prone rats show progressive increases in blood pressure and proteinuria and accumulate acute-phase proteins in body fluids, modeling events during renal damage. The aim of this study was to assess the pathological events occurring in the kidney of spontaneously hypertensive stroke-prone rats over time and evaluate the effects of statin treatment, which is known to improve renal and cardiovascular outcomes. Kidneys of male spontaneously hypertensive stroke-prone rats euthanized at different stages of proteinuria showed progressive inflammatory cell infiltration, the accumulation of alpha-smooth muscle actin-positive cells, degenerative changes in podocytes, and severe fibrosis. These were accompanied by an imbalance in the plasminogen/plasmin and metalloprotease systems characterized by the increased renal expression of plasminogen activator inhibitor-1, tissue plasminogen activator, and urokinase plasminogen activator; the net result was an increase in plasmin and matrix metalloproteinase (MMP)-2 and a reduction in MMP-9 activity. Chronic treatment with the hydrophilic rosuvastatin had renoprotective effects in terms of morphology and inflammation and prevented the changes in plasmin, MMP-2, and MMP-9 activity. These effects were independent of the changes in blood pressure and plasma lipid levels. Treatment with the lipophilic simvastatin was not renoprotective. These data suggest that rosuvastatin may have potential utility as a therapeutic option in renal diseases that are characterized by inflammation and fibrosis.     

Effects of Hochu-ekki-to and Ninjin-youei-to, traditional Japanese medicines, on porcine serum-induced liver fibrosis in rats

In this study, we estimated the effects of traditional Japanese medicines on liver fibrosis in Wistar rats injected with porcine serum twice a week for 8 weeks. The rats were orally administered Hochu-ekki-to, Ninjin-youei-to (100 and 300 mg/kg/day) or Sho-saiko-to (300 mg/kg/day) 5 days per week. Serum and liver samples were obtained 2 days after the last porcine serum injection. Hochu-ekki-to and Ninjin-youei-to showed significant suppressive effects on the increase in hepatic hydroxyproline, namely total collagen. Further, Ninjin-youei-to significantly suppressed the increases of type IV collagen localized in the basement membrane and prolyl 4-hydroxylase, a collagen synthesis enzyme, in serum or liver. Hochu-ekki-to showed a similar trend. Although Sho-saiko-to did not significantly suppress the increase in hepatic hydroxyproline, it intensely suppressed serum type IV collagen. Further, Hochu-ekki-to, Ninjin-youei-to, and Sho-saiko-to inhibited the production of fibrogenic cytokines, namely TGF-β1 and IL-13, in the serum and liver. Additionally, we showed that IL-13 levels were positively correlated with hydroxyproline contents in the liver. These results suggest that Ninjin-youei-to as well as Hochu-ekki-to suppress porcine serum-induced liver fibrosis more effectively than Sho-saiko-to. The effects of these three medicines probably depend on the inhibition of fibrogenic cytokine production, resulting in the suppression of collagen synthesis and deposition in the liver, though different mechanisms underlie their anti-fibrogenic effects.     

Changes in TIMP-1 and -2 expression in the early stage of porcine serum-induced liver fibrosis in rats

It is widely recognized that tissue inhibitors of metalloproteinases (TIMPs), especially TIMP-1 and -2, play a key role in the progression of hepatic fibrosis. In the present study, we examined the changes in TIMP-1 and -2 expressions in the early stage of porcine serum (PS)-induced liver fibrosis in Brown Norway (BN) and Wistar rats. The rats were injected intraperitoneally with 0.5 ml/head of PS twice a week for up to 8 weeks and examined at 2, 4 and 8 weeks. Hepatic fibrosis and inflammatory cell infiltration developed at 4 and 8 weeks in BN and Wistar rats, respectively, and formation of pseudolobules was detected at 8 weeks in rats of both strains. The expression of liver TIMP-1 and -2 mRNAs significantly increased at 8 weeks in rats of both strains. At the same time, TIMP-1 and -2 activities were also detected in the liver of both strains. On the other hand, the expression of serum TIMP-1 and -2 proteins increased earlier (at 4 weeks for TIMP-1 and at 2 or 4 weeks for TIMP-2) than that of liver TIMP-1 and -2 mRNAs did. Although there are some reports suggestive of why the elevation of serum TIMP-1 and -2 proteins preceded that of liver TIMP-1 and -2 mRNAs, the exact reason is still obscure. In conclusion, the present study showed for the first time the mode of TIMP-1 and -2 expression and activity in the early stage of PS-induced rat liver fibrosis model.     

Serological and immunohistochemical studies on porcine-serum-induced hepatic fibrosis in rats

We previously reported that the strain difference in the development of porcine-serum (PS)-induced rat hepatic fibrosis was closely related to the difference in the mode of MHC class-II-related genes expression. This study was carried out to clarify the serological and immunohistochemical changes in this hepatic fibrosis model. Six-week-old male Brown Norway (BN) and Wistar rats were injected with 0.5 ml of sterile PS twice a week for up to 8 weeks. The serum levels of PS-specific IgG1, IgG2a, and IgM were elevated more prominently in BN rats than Wistar rats. In the liver, significant increases in the numbers of PS-, OX-6 (RT1.B)-, CD4-, CD8, ED1-, and ED2-positive cells occurred earlier in BN rats than Wistar rats. At 8 weeks, deposition of PS and immunoglobulins was observed in hepatic fibrous septa and renal glomerular mesangium, and IgG1- and IgG2a-positive cells were found in the white pulp of the spleen. The present results suggest that humoral immunity probably regulated by MHC class II molecules and inflammatory cells may be involved in PS-induced hepatic fibrosis in rats.     

P物质受体拮抗剂对缺氧大鼠模型肝功能及肝细胞凋亡的影响

将30只雄性SD大鼠(体重240~300 g)随机分为3组,分别为对照组(不进行缺氧处理)、低氧组及NK-1R拮抗剂组,建立大鼠缺氧模型,NK-1R拮抗剂组腹腔注射P物质受体(NK-1R)拮抗剂(S3144)进行干预。观察各组大鼠肝组织病理学变化,检测肝功能指标及Tunnel法测肝凋亡细胞百分比,探讨P物质受体(NK-1R)拮抗剂对缺氧大鼠模型肝损伤的影响及其机制。结果显示:(1)缺氧模型的大鼠肝细胞肿胀,排列紊乱,气球样变性较多,并有点状坏死灶,损伤较NK-1R拮抗剂组严重。(2)NK-1R拮抗剂组GGT[(6.750 0±1.669 1)U/]明显低于低氧组[(9.000 0±1.414 2)U/L)](P<0.05);ALT、AST、ALP各组之间比较无明显差异(P>0.05)。(3)NK-1R拮抗剂组肝细胞凋亡百分比[(26.626 3±10.934 8)%]明显低于低氧组[(44.581 4±17.324 5)%](P<0.05)。研究表明:P物质可能与缺氧性肝损伤有关,通过P物质受体(NK-1R)拮抗剂可能减轻缺氧性肝损伤。     

Double intratracheal instillation of keratinocyte growth factor prevents bleomycin-induced lung fibrosis in rats

Alveolar re-epithelialization is necessary in the repair of damaged alveolar epithelium after lung injury. Keratinocyte growth factor (KGF) has been shown to be a potent proliferation and differentiation factor for rat alveolar type II cells. The present study examined whether KGF would prevent bleomycin-induced lung fibrosis. Adult rats were anaesthetized and recombinant human KGF (rhKGF) (150 μg/kg) or saline was injected intratracheally at 48 h before and 24 h after bleomycin (Bleo, 5 mg/kg) instillation. Seven and 14 days after the last administration, rat lungs were processed for lung physiology, immunohistochemistry, and in situhybridization. Double instillation of KGF prevented the loss of body weight and reduction in total lung capacity (TLC) due to Bleo, and markedly attenuated the protein accumulation and mRNA expression of collagen types I and III and the decreased expression of surfactant protein mRNAs in the fibrotic lesions of Bleo-treated rats. KGF may play an important role in maintaining alveolar epithelium and repairing the damaged epithelium after lung injury. © 1998 John Wiley & Sons, Ltd.     

An interleukin-1 receptor antagonist decreases fibrosis induced by dimethylnitrosamine in rat liver

The main pathological feature of liver fibrosis is the accumulation of extracellular matrix associated with hyperplasia and activation of perisinusoidal (Ito) cells (PSC) to myofibroblast-like cells. Interleukin-1 enhances collagen synthesis by increasing the proliferative activity of cultured PSC and has been implicated in the pathogenesis of hepatic fibrosis.Interleukin-1 receptor antagonist (IL-1ra) can block the binding of IL-1 to its receptors and act as a natural inhibitor of IL-1. We have examined whether the administration of IL-1ra can interfere with the development of experimental cirrhosis induced by dimethylnitrosamine (DMN). Rats were divided in three groups and received respectively DMN, DMN+IL-1ra and IL-1ra. For each group the collagen content of the hepatic tissue and the volume density of the inflammatory infiltrate were measured. Immunostaining for laminin and alpha-smooth muscle actin were also performed.In animals given DMN+IL-1ra we observed a decreased deposition of laminin and collagen, and a decreased number of laminin-positive PSC and of alpha-smooth muscle actin reactive cells, compared with animals receiving DMN alone. The present findings suggest that the early activation of PSC in vivo is at least in part mediated by IL-1 and confirm that the administration of IL-1ra may be of interest in modifying the biological effects of IL-1.     

Treatment of fulminant hepatic failure in rats using a bioartificial liver device containing porcine hepatocytes producing interleukin-1 receptor antagonist

Fulminant hepatic failure (FHF) is a serious clinical condition that is associated with high mortality. There is evidence that FHF is an inflammatory disease, which is supported clinically by elevated serum levels of cytokines. In an effort to develop hepatocytes with additional functions for use in our bioartificial liver (BAL) device, we focused on interleukin-1 (IL-1) blockade as a therapeutic modality. Primary porcine hepatocytes were isolated from the livers of miniature swine and then transfected with an adenoviral vector encoding human interleukin-1 receptor antagonist (AdIL-1Ra). The transfected hepatocytes secreted human IL-1Ra. These transfected hepatocytes were incorporated into a flat-plate BAL device to evaluate their efficacy in treating D-galactosamine (GalN)- induced FHF in a rat model. After extracorporeal perfusion with the BAL device containing the transfected hepatocytes, there were significant reductions in the plasma levels of hepatic enzymes (aspartate aminotransferase and alanine aminotransferase) and cytokines (IL-1 and IL-6), indicating a beneficial effect. Animal survival was significantly improved in the treated group compared to the control group. These experiments demonstrate that combining inflammatory cytokine blockade with a functional BAL device may be an effective therapeutic option in the treatment of FHF.     

干扰素α-2a与肝纤维化大鼠肝星状细胞的凋亡

背景：干扰素α-2a改善肝纤维化的机制直到目前仍尚未阐明。 目的：进一步验证干扰素α-2a对 CCl₄诱导大鼠肝纤维化模型中肝星状细胞凋亡的影响。 方法：建立CCl₄诱导肝纤维化模型,健康SD雌性大鼠50只,采用随机对照原则将SD大鼠分成5组,即生理盐水对照组、纤维化模型组、6×10⁴ U/kg 干扰素α-2a干预组、12×10⁴ U/kg干扰素α-2a干预组及6×10⁴ U/kg干扰素α-2a对照组。造模8周时取肝组织标本,分别进行肝纤维化指标检测;RT-PCR分析肝组织bcl-2、bax的表达;免疫组织化学染色用a平滑肌肌动蛋白对活化的肝星状细胞进行标记。 结果与结论：肝组织病理形态显示CCl₄诱导肝纤维化成功建立,表现为纤维化模型组汇管区周围纤维化明显,有芒状纤维和纤维间隔形成,各干扰素α-2a干预组肝纤维化有不同程度缓解。纤维化模型组有大量a平滑肌肌动蛋白阳性表达,6×10⁴ U/kg干扰素α-2a干预组a平滑肌肌动蛋白阳性表达较纤维化模型组减少,12×10⁴ U/kg干扰素α-2a干预组更少,6×10⁴ U/kg干扰素α-2a对照组未见a平滑肌肌动蛋白阳性表达。结果提示干扰素α-2a能下调CCl₄诱导肝纤维化bcl-2的表达,及上调bax的表达。提示干扰素α-2a阻断CCl₄诱导肝纤维化机制存在通过调节bcl-2、bax的表达,诱导肝星状细胞凋亡途径,该调节作用可能与干扰素α-2a剂量相关。     

ROCK inhibition prevents fetal serum-induced alteration in structure and function of organ-cultured mesenteric artery

Chronic treatment with fetal bovine serum (FBS) causes contractility reduction, morphological alteration and DNA synthesis in organ-cultured vascular tissues. Here, we tested the hypothesis that chronic inhibition of ROCK has a protective effect on FBS-induced alterations in small arteries. Rabbit mesenteric arterial rings were cultured in FBS-supplemented culture medium with or without Y-27632, a reversible ROCK inhibitor. Chronic Y-27632 treatment prevented FBS-induced gradual arterial constriction, wall thickening, reduced contractility, and increased ROCK-specific MYPT1 Thr853 phosphorylation. Treatment with Y-27632 also prevented decreased eNOS mRNA expression, and reduced acetylcholine-induced relaxation. Sudden application of Y-27632 to pre-cultured rings reduced MYPT1 phosphorylation and re-widened the constricted rings. Chronic treatment with Y-27632, however, rather augmented than reduced the FBS-induced RhoA over-expression, also increased ROCK1 and MYPT1 expression and averted the FBS-induced reduction of MLC expression, suggesting a compensation of inhibited RhoA/ROCK activity. Sudden removal of Y-27632 caused a rebound in MYPT1 phosphorylation and vasoconstriction in rabbit mesenteric artery. To test which ROCK isoform has greater involvement in FBS-induced contraction, haploinsufficient Rock1 ^+/− and Rock2 ^+/− mouse mesenteric arterial rings were subjected to organ-culture. FBS-induced contraction and RhoA over-expression in either heterozygous animal was not different from wild-type animals. These results suggest that FBS-induced contraction is mediated by up-regulation of RhoA and subsequent activation of ROCK. In conclusion, chronic ROCK inhibition produces some effects that protect against FBS-stimulated vasoconstriction and remodeling. There are also negative effects that a sudden withdrawal of ROCK inhibitor might cause a stronger vasoconstriction than before it was used.     

The N-methyl-D-aspartate (NMDA) receptor antagonist, dextrorphan, prevents the neurotoxic effects of 3,4-methylenedioxymethamphetamine (MDMA) in rats

Using the systemically active, non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dextrorphan, we explored the role of the NMDA receptor-calcium channel complex in the toxic mechanism of action of 3,4-methylenedioxymethamphetamine (MDMA). Rats were treated with MDMA, dextrorphan, or the combination of MDMA and increasing doses of dextrorphan, and then killed 10 days later for the assay of serotonin and dopamine in the striatum, hippocampus, and cortex. Dextrorphan totally prevented the serotonin-depleting effects of MDMA in the straitum, with a lessened but still significant blockade noted in the hippocampus and cortex. These findings may provide a clue to the molecular events underlying MDMA-induced neurotoxicity.     

Interferon-alpha gene therapy prevents aflatoxin and carbon tetrachloride promoted hepatic carcinogenesis in rats

Retrovirus-mediated interferon alpha (IFN-alpha) gene transfer was evaluated with regard to its possible protective effects against aflatoxin B1 (AFB1)-initiated and carbon tetrachloride (CCl4)-promoted hepatic carcinogenesis in rats. To our knowledge, this is the first time an experimental in vivo gene therapy trial was conducted in Egypt. Two genes were examined in liver tissue by RT-PCR: the first was glutathione-S-transferase placental (GST-P) isoenzyme, as an early marker to detect hepatic malignancy; the second was IFN-alpha gene expression to detect the efficiency of gene uptake and its persistence after transduction. Forty male rats, divided equally into 4 groups, were included in the study: the first group was the control; the second group received CCl4 0.2 ml subcutaneously twice weekly for 12 weeks and AFB1 0.25 mg/kg body wt intraperitoneally twice weekly for 6 weeks; the third group received IFN-alpha (10(8) pfu) intravenously in the tail vein prior to the start of CCl4 and AFB1 injections; and the fourth group received IFN-alpha (10(8) pfu) by intrahepatic injection under ultrasonography guide after termination of the CCl4 and AFB1 injection schedule. The results showed that IFN-alpha has a marked and significant protective effect against hepatic fibrogenesis as well as hepatic carcinogenesis. Pathological examination of liver tissue proved that IFN-alpha minimized both fibrotic and cirrhotic processes. The amount of fibrosis was less in both groups receiving IFN-alpha, with more protection in the group that received IFN-alpha intravenously prior to CCl4 and AFB1. The results of RT-PCR showed that the IFN-alpha gene was significantly expressed in both groups receiving IFN-alpha, with a more intense expression in the group that received IFN-alpha by intrahepatic injection after termination of CCl4 and AFB1 injections. The IFN-alpha gene was detected after three months of gene transduction in rats receiving IFN-alpha intravenously prior to CCl4 and AFB1 and after one month of gene transduction in the post CCl4 and AFB1 rats. IFN-alpha gene was not expressed in the two groups that did not undergo gene transfer. Histopathological signs of premalignant macronodules were evident in the group receiving CCl4 and AFB1, but not IFN-alpha as well as in the group that received IFN-alpha at the end of the experiment. GST-P gene expression was also detected in these two groups, confirming early malignant transformation. In conclusion, IFN-alpha exerts significant protective effects, but more so when the gene is administered before fibrogenic and carcinogenic induction in hepatic tissues. IFN-alpha gene therapy may be justified in clinical trials for high-risk candidates with hepatic carcinogenesis.     

Acetaldehyde prevents nuclear factor-kappa B activation and hepatic inflammation in ethanol-fed rats.

Acetaldehyde has been proposed as one of the mediators of liver injury in alcoholic liver disease. We investigated whether increased acetaldehyde levels affected the development of alcoholic liver injury. Male Wistar rats were fed a liquid diet containing fish oil and ethanol by intragastric infusion. Sustained elevations of acetaldehyde were achieved by daily treatment with two inhibitors of aldehyde dehydrogenase (ALDH): disulfiram and benzcoprine. Pathologic changes, plasma and liver acetaldehyde, nuclear factor-kappa B (NF-kappaB) and I kappa B alpha (I kappaB alpha) protein, tumor necrosis factor-alpha (TNF-alpha) and cyclooxygenase 2 (COX-2) mRNA were evaluated. Treatment with the ALDH inhibitors led to increased acetaldehyde in liver and plasma but prevented necrosis and inflammation. Steatosis was not affected. Both inhibitors decreased activation of NF-kappaB and down-regulated TNF-alpha and COX-2 expression. Decreased activation of NF-kappaB was accompanied by I kappaB alpha preservation. Acetaldehyde probably inhibits NF-kappaB activation through I kappaB alpha preservation. Down-regulation of TNF-alpha and COX-2 occur secondary to inhibition of NF-kappaB and account for the absence of necrosis and inflammation in the ALDH inhibitor-treated groups.     

A new Agkistrodon halys venom-purified protein C activator prevents myocardial fibrosis in diabetic rats

Aim

To assess the effects of protein C activator (PCA) from Agkistrodon halys snake venom on cardiac fibrosis in streptozotocin (STZ) induced diabetic rat model, and investigate the mechanisms of its action.

Methods

PCA was identified by one-dimensional reversed phase liquid chromatography – mass spectrometry/mass spectrometry. Male Sprague-Dawley rats (120-140 g) were randomly assigned to negative control (NC) and diabetic group. Diabetes was induced by STZ in high-fat diet fed rats. Diabetic group was subdivided into three groups: diabetic group (DM), diabetic group treated with PCA (0.5, 2, and 8 mg/kg), and diabetic group treated with metformin (5 mg/kg, positive control). NC and DM groups received the same volume of distilled water. Left ventricular mass index (LVWI) and collagen volume fraction were measured by hematoxylin and eosin and Masson staining. Transforming growth factor beta-1 (TGF-β1) and interleukin 1 beta (IL-1β) levels were determined by enzyme-linked immunosorbent assay.

Results

The diabetic rat model was successfully established by STZ induction and high-fat diet. Glucose level, LVWI, TGF-β1 and IL-1β level, and collagen volume fraction were significantly reduced in diabetic rats treated by PCA in a dose-dependent manner (P < 0.050), especially in the high dose (8 mg/kg) group (P < 0.010), compared to diabetes group. The high dose PCA had the same effect as metformin positive control in reducing the level of fasting blood glucose. PCA decreased the expression of MMP-2 and reduced that of TIMP-2.

Conclusion

Our results indicate that PCA has anti-fibrotic effects and that it may be used to treat myocardial fibrosis.The main complications and leading causes of death among patients with diabetes mellitus are coronary artery disease (CAD), hypertension, and diabetic cardiomyopathy (DCM), a direct adverse effect of diabetes on the heart (1). Epidemiological data showed that the risk for heart failure is respectively 2.4-fold and 5-fold higher in diabetic men and women than in non-diabetic individuals (2). Indeed, diabetes is a significant risk factor for heart failure and an independent risk factor for increased mortality among individuals with heart failure (3). Myocardial fibrosis (MF) plays a major role in the pathophysiology of DCM (4). It is also strongly associated with some angiocardiopathy diseases such as hypertension, DCM, rheumatic heart disease, and myocardial infarction (5,6).Fibrosis is a complex process characterized by cardiac fibroblast accumulation and excess extracellular matrix deposition of collagen I and III (7), which is a leading cause of diabetes and heart failure. Therefore, in diabetes and many cardiovascular diseases, it is vital to develop appropriate drugs to prevent MF. Angiotensin-converting enzyme inhibitors (ACEI) have been shown in animal models to prevent and treat MF in early stage; clinically, ACEI improve ventricular remodeling and the prognosis of heart failure (8). However, they cause adverse reactions such as cough, hypotension, and nausea, and increase the treatment costs, which all indicates the need for novel candidates in MF treatment.Snake venom contains multiple proteins and peptides with different structure and function (9), and is an extremely rich source of pharmacologically active molecules with a considerable clinical and medical potential (10-14). Many toxins are being explored and developed for treatment of hypertension, thrombosis, and cancer. In recent years, special attention has been paid to proteins affecting hemostasis, in order to design new therapeutic agents for blood coagulation and other hematological disorders (15). Protein C activators (PCA) are proteases that activate protein C in the mammalian coagulation system in vitro. PCA activity was first reported in venom extracts of the snake Agkistrodon contortrix contortrix in 1985 (16). The reptilian PCA has attracted increasing attention in both clinical and basic research (17). Other functions of snake venom PCA have to be further studied. Therefore, we hypothesized that snake venom PCA could protect against cardiac fibrosis in DCM. We established a type 2 diabetes model to investigate the cardio-protective properties of PCA in DCM and determine the underlying mechanisms of its action.