Effects of salviainolic acid A (SA‐A) on liver injury: SA‐A action on hepatic peroxidation

Abstract: Background/Aims: This study aimed to investigate the actions of salviainolic acid A (SA‐A), an antiperoxidative component of Salvia miltiorrhiza (Sm), on rat liver injury and fibrosis. Methods: Acute and chronic rat liver injury models were established using carbon tetrachloride (CCl₄). After 48 h (acute) or during 6 weeks of CCl₄ injection, rats were further divided and treated with biphenyl dimethyl‐dicarboxylate (BDD) or colchicine, as a control antifibrotic treatment, with Sm, a herbal compound, or SA‐A, a water‐soluble extract of Sm. Liver function was investigated by assessing alanine transaminase (ALT) and aspartate transaminase (AST) activities, histological analysis, hydroxyproline (Hyp) and malondiadehyde (MDA) content. In vitro, isolated cultured hepatocytes were injured with CCl₄ gas for 24 h, followed by treatment with either vitamin E or various concentrations of SA‐A. The extent of hepatocyte injury was monitored by analyzing various lipid peroxidative parameters such as superoxide dismutase (SOD), glutathione (GSH), catalase (CAT), lactase dehydrogenase (LDH), and glutathione peroxidase (GSH‐PX) levels in hepatocyte supernatants. Results: SA‐A significantly decreased abnormal serum ALT activity both in acutely and chronically injured rat livers, decreased abnormal serum AST activity, Hyp and MDA content and attenuated hepatic collagen deposition. After CCl₄ incubation and injury, the activities of AST, ALT CAT, GSH‐PX and LDH and MDA content in hepatocyte supernatants increased significantly, but GSH levels decreased significantly. SA‐A markedly improved these pathological changes in a dose‐dependent manner. 10^?4 mol/l SA‐A had stronger inhibitory action than vitamin E. Conclusions: Our studies suggest that SA‐A has antiperoxidative effects on injured hepatocytes in liver injury and fibrosis induced by CCl₄.     

Melatonin promotes hepatic differentiation of human dental pulp stem cells: clinical implications for the prevention of liver fibrosis

Melatonin's effect on hepatic differentiation of stem cells remains unclear. The aim of this study was to investigate the action of melatonin on hepatic differentiation as well as its related signaling pathways of human dental pulp stem cells (hDPSCs) and to examine the therapeutic effects of a combination of melatonin and hDPSC transplantation on carbon tetrachloride (CCl₄)‐induced liver fibrosis in mice. In vitro hepatic differentiation was assessed by periodic acid‐Schiff (PAS) staining and mRNA expression for hepatocyte markers. Liver fibrosis model was established by injecting 0.5 mL/kg CCl₄ followed by treatment with melatonin (5 mg/kg, twice a week) and hDPSCs. In vivo therapeutic effects were evaluated by histopathology and by means of liver function tests including measurement of alanine transaminase (ALT), aspartate transaminase (AST), and ammonia levels. Melatonin promoted hepatic differentiation based on mRNA expression of differentiation markers and PAS‐stained glycogen‐laden cells. In addition, melatonin increased bone morphogenic protein (BMP)‐2 expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. Furthermore, melatonin activated p38, extracellular signal‐regulated kinase (ERK), and nuclear factor‐κB (NF‐κB) in hDPSCs. Melatonin‐induced hepatic differentiation was attenuated by inhibitors of BMP, p38, ERK, and NF‐κB. Compared to treatment of CCl₄‐injured mice with either melatonin or hDPSC transplantation alone, the combination of melatonin and hDPSC significantly suppressed liver fibrosis and restored ALT, AST, and ammonia levels. For the first time, this study demonstrates that melatonin promotes hepatic differentiation of hDPSCs by modulating the BMP, p38, ERK, and NF‐κB pathway. Combined treatment of grafted hDPSCs and melatonin could be a viable approach for the treatment of liver cirrhosis.     

N-methyl-4-isoleucine cyclosporine attenuates CCl -induced liver fibrosis in rats by interacting with cyclophilin B and D

Background and Aim: N‐methyl‐4‐isoleucine cyclosporine (NIM811), a new analogue of cyclosporine A, can inhibit collagen deposition in vitro and reduce liver necrosis in a bile‐duct‐ligation animal model. However, whether NIM811 effects on CCl₄‐induced rat liver fibrosis, and the related mechanism has not been determined. Methods: A liver fibrosis model was induced in Wistar rats using CCl₄ for 6 weeks. Meanwhile, two different doses of NIM811 (low‐dose 10 mg/kg and high‐dose 20 mg/kg) were given to the CCl₄‐treated rats. Liver fibrosis was then evaluated according to histopathological scoring and liver hydroxyproline content. Serum alanine aminotransferase, aspartate aminotransferase and albumin levels, expression of matrix metalloproteinase‐13, tissue inhibitor of metalloproteinase‐1, α‐smooth muscle actin and cyclophilin B and D in liver tissue were determined. Cyclophilin B and D were also studied in an hepatic stellate cell line. Results: Hydroxyproline content was decreased in both NIM811 groups compared with the model (P < 0.05). Liver necrosis and fibrosis were also attenuated in the NIM811 groups. NIM811 suppressed the expression of tissue inhibitor of metalloproteinase‐1, transforming growth factor beta mRNA and α‐smooth muscle actin protein in liver tissue. Expression of cyclophilin B in the fibrosis model was increased compared with the normal group (P < 0.05), and was decreased significantly in the low‐dose NIM811 treatment group (P < 0.05), which indicated that cyclophilin B might have a profibrotic effect. In vitro studies revealed that cyclophilin B and/or D knockout were associated with collagen inhibition. Conclusions: NIM811 attenuates liver fibrosis in a CCl₄‐induced rat liver fibrosis model, which may be related to binding with cyclophilin B and D.     

Steatosis correlates with hepatic expression of death receptors and activation of nuclear factor‐κB in chronic hepatitis C

Background: Steatosis is recognized as a predictor of the severity as well as the progression of fibrosis in chronic hepatitis C. The mechanisms that cause increased hepatocellular injury associated with steatosis remain largely unknown. Methods: We studied the correlation of hepatic expression of death receptors: Fas and tumour necrosis factor‐α receptor 1 (TNF‐R1), and downstream caspase (caspase‐3) with hepatic steatosis by immunohistochemical study in chronic hepatitis C and determined the role of nuclear factor‐κB (NF‐κB). Results: Ninety patients (49 males and 41 females, mean age of 50.5 ± 10.4 years, genotype 1 or 2) with chronic hepatitis C virus infection were recruited. The factors associated with steatosis grade were body mass index (P=0.004) and fibrosis stage (P=0.034). Moderate/severe steatosis was an independent variable associated with advanced fibrosis stage by stepwise logistic regression analysis. The expression of immunoreactivity for Fas, TNF‐R1 and active caspases‐3 in liver tissues was significantly correlated with the steatosis grade (P<0.001, P<0.001 and P<0.001 respectively). The extent of active caspases‐3 correlated significantly with the expression of Fas (r=0.659, P<0.001) and TNF‐R1 (r=0.617, P<0.001). NF‐κB p65 expression correlated significantly with the extent of Fas (r=0.405, P<0.001), TNF‐R1 (r=0.448, P=0.002) and active caspase‐3 (r=0.313, P=0.003), and correlated with steatosis grade (P<0.001) but not with inflammatory and fibrosis scores. Conclusion: Our observations suggest a mechanism whereby steatosis contributes to the progression of liver injury in chronic hepatitis C through upregulation of death receptors and activation of NF‐κB.     

Overexpression of NK2 promotes liver fibrosis in carbon tetrachloride‐induced chronic liver injury

Background/Aims: Hepatocyte growth factor (HGF) inhibits liver fibrosis induced by carbon tetrachloride (CCl₄) in animal models. NK2 is a natural splice variant of HGF, but its in vivo function remains to be elucidated. We investigated the in vivo effects of NK2 on CCl₄‐induced liver fibrosis. Methods: NK2 transgenic mice and wild‐type (WT) mice were injected intraperitoneally with CCl₄ twice a week. The extent of hepatic fibrosis was evaluated by Azan–Mallory staining. Expression levels of mRNAs of transforming growth factor‐β1 (TGF‐β1) and matrix metalloproteinase‐13 (MMP‐13) were examined by real‐time polymerase chain reaction. The protein levels of α‐smooth muscle actin (α‐SMA), c‐Met and its phosphorylation were determined by Western blot analysis. Results: Liver fibrosis was significantly more severe in NK2 transgenic mice than in WT mice. CCl₄ administration increased the expression levels of TGF‐β1 mRNA and α‐SMA protein, and decreased the expression of MMP‐13 mRNA in livers of NK2 transgenic mice compared with those of WT mice. c‐Met protein expression in the liver was compatible with the degree of fibrosis. As for c‐Met activation, no difference was found between NK2 and WT livers. Conclusion: Overexpression of NK2 acts as an antagonist of HGF and promotes liver fibrosis in CCl₄‐induced chronic liver injury.     

Pharmacological actions of curcumin in liver diseases or damage

Since 1900 bc , several therapeutic activities have been attributed to the rhizomes of the plant Curcuma longa for a variety of diseases, including liver disorders. Curcumin, the main active compound obtained from this plant, was first isolated two centuries ago and its structure as diferuloylmethane was determined in 1910. Curcumin has shown anti‐inflammatory, anti‐oxidant, antifungal, antibacterial and anticancer activities. The pharmacological properties of curcumin were reviewed recently and focused mainly on its anticancer properties. However, its beneficial activity on liver diseases (known centuries ago, and demonstrated recently utilizing animal models) has not being reviewed in depth until now. The curcumin ability to inhibit several factors like nuclear factor‐κB, which modulates several pro‐inflammatory and profibrotic cytokines as well as its anti‐oxidant properties, provide a rational molecular basis to use it in hepatic disorders. Curcumin attenuates liver injury induced by ethanol, thioacetamide, iron overdose, cholestasis and acute, subchronic and chronic carbon tetrachloride (CCl₄) intoxication; moreover, it reverses CCl₄ cirrhosis to some extent. Unfortunately, the number of studies of curcumin on liver diseases is still very low and investigations in this area must be encouraged because hepatic disorders constitute one of the main causes of worldwide mortality.     

Hepatoprotective effect of the selective mineralocorticoid receptor antagonist, eplerenone against carbon tetrachloride-induced liver injury in rats

Background. Eplerenone is a selective mineralocorticoid receptor (MR) antagonist, and its potential protective role in cardiovascular injury has been reported by several studies. However, whether and how this drug can ameliorate hepatic injury in rats is unknown.Material and methods. The present study was conducted to investigate effect of eplerenone against liver injury induced by carbon tetrachloride (CCl₄) in rats. The biochemical liver function tests and oxidative stress parameters including malondialdehyde (MDA), reactive oxygen species (ROS), in addition to the reduced glutathione (GSH) levels were evaluated. Moreover, serum tumor necrotic factors (TNF-α) level and histopathological changes were examined.Results. Our results show that pre-treatment with eplerenone (4 mg/kg per day for 4 weeks) revealed attenuation in serum activities of alanine aminotransferase (ALT), aspartate aminotransferase, (AST), alkaline phosphatase (ALP) and bilirubin levels that were enhanced by CCl₄. Further, pre-treatment with eplerenone inhibited the elevated hepatic MDA content and restored hepatic GSH to its normal level. The enhanced hepatic ROS production in CCl₄-treated group was markedly decreased by eplerenone administration. Eple-renone pre-treatment significantly attenuated the inflammatory responses caused by CCl₄ as evident by the decreased serum TNF-α level. Histopathological studies showed that eplerenone alleviated the liver damage and reduced the lesions caused by CCl₄.Conclusion. Collectively, the present study provides a proof to hepatoprotective actions of eplerenone via reducing oxidative stress and inflammatory responses in CCl₄-induced liver damage in rat model.     

All‐trans‐retinoic acid ameliorates carbon tetrachloride‐induced liver fibrosis in mice through modulating cytokine production

Background/Aims: Liver fibrosis with any aetiology, induced by the transdifferentiation and proliferation of hepatic stellate cells (HSCs) to produce collagen, is characterized by progressive worsening in liver function, leading to a high incidence of death. We have recently reported that all‐trans‐retinoic acid (ATRA) suppresses the transdifferentiation and proliferation of lung fibroblasts and prevents radiation‐ or bleomycin‐induced lung fibrosis. Methods: We examined the impact of ATRA on carbon tetrachloride (CCl₄)‐induced liver fibrosis. We performed histological examinations and quantitative measurements of transforming growth factor (TGF)‐β1 and interleukin (IL)‐6 in CCl₄‐treated mouse liver tissues with or without the administration of ATRA, and investigated the effect of ATRA on the production of the cytokines in quiescent and activated HSCs. Results: CCl₄‐induced liver fibrosis was attenuated in histology by intraperitoneal administration of ATRA, and the overall survival rate at 12 weeks was 26.5% without ATRA (n=25), whereas it was 75.0% (n=24) in the treatment group (P=0.0187). In vitro studies disclosed that the administration of ATRA reduced (i) the production of TGF‐β1, IL‐6 and collagen from HSCs, (ii) TGF‐β‐dependent transdifferentiation of the cells and IL‐6‐dependent cell proliferation and (iii) the activities of nuclear factor‐κB p65 and p38mitogen‐activated protein kinase, which stimulate the production of TGF‐β1 and IL‐6, which could be the mechanism underlying the preventive effect of ATRA on liver fibrosis. Conclusions: Our findings indicate that ATRA ameliorates liver fibrosis. As the oral administration of the drug results in good compliance, ATRA could be a novel approach in the treatment of liver fibrosis.     

Beta-carotene (provitamin A) decreases the severity of CCl4-induced hepatic inflammation and fibrosis in rats

Earlier data from experiments in rats have shown that administration of retinyl esters (vitamin A) strongly influences the effects of CCl₄ on the liver. The accumulation of collagen was inhibited, but an increase in CCl₄-toxicity with high mortality was observed. The present study was conducted to examine the effects of β-carotene (provitamin A) on CCl₄-related general and hepatic toxicity in rats. Oral administration of β-carotene during CCl₄-treatment resulted, biochemically, in a significantly lower increase in the hydroxyproline liver content and, histopathologically, in less severe liver fibrosis as compared with the liver of rats not treated with β-carotene. The study also showed that β-carotene administration could prevent the long-term loss of retinoids from the CCl₄-injured liver. No significant toxic effects of β-carotene, as previously found with retinyl esters (vitamin A), were observed. This experimental study suggests that β-carotene has the therapeutic potential to decrease the severity of liver fibrosis without marked toxicity.     

Transplantation of bone marrow cells reduces CCl4‐induced liver fibrosis in mice

Background: We investigated the reversibility of liver fibrosis induced with a CCl₄ injection and the role of stem cells in reversing the hepatic injury. Furthermore, the most effective cell fraction among bone marrow cells (BMCs) in the repair process was analysed. Methods: C57BL/6 mice were divided into four groups after 5 weeks of injection of CCl₄: control, sacrificed after 5 weeks, sacrificed at 10 weeks and sacrificed 5 weeks later after GFP‐donor BM transplantation. Liver function tests and real‐time polymerase chain reaction (PCR) of markers indicating liver fibrosis were compared between the groups. To identify the most effective BMC fraction that repairs liver injury, the mice were divided into three groups after the injection of CCl₄ for 2 days: granulocyte colony stimulating factor (G‐CSF) only, mononuclear cell (MNC) transplantation and Lin‐Sca‐1+c‐kit+haematopoietic stem cell (HSC) transplantation. Eight days after transplantation, the mice were harvested and morphometric, immunohistochemical analyses were performed to compare the expression of extracellular matrix and liver fibrosis‐related factors. Results: The liver fibrosis induced by CCl₄ was not spontaneously recovered but was persistent until 10 weeks, but the group injected with BMCs had less fibrosis and better liver function. Mobilization with G‐CSF increased the recovery of the injured liver and the best results were seen in those mice administered the MNC fraction and Lin‐Sca‐1+c‐kit+HSC fraction, with no difference between the two groups. Conclusion: BMC transplantation and stem cell mobilization with G‐CSF effectively treats liver injury in mice. These are promising techniques for autologous transplantation in humans with liver fibrosis.     

Calcium staining by the glyoxal-bis-(2-hydroxyanil)-method in the livers of rats treated with CCl4, diltiazem,and with both agents together

ABSTRACT— We studied the histochemistry of Ca in livers treated with CCl₄, diltiazem (one of the Ca antagonists), and both agents together to determine whether hepatocytes or other parts of the liver in liver lesions show Ca staining and whether the grade or location of Ca in these injuries varies. For Ca staining, cryostat sections were treated by the glyoxal-bis-(2-hydroxyanil) (GBHA)-method using O.C.T. imbedding compound instead of paraffin. Diltiazem-treated rats showed Ca granules in the bile canaliculi around the terminal hepatic veins and Kupffer cells 6 h after intragastric injection. Rats treated with CCl₄ showed fine red granules in the cytoplasm of the hepatocytes around the terminal hepatic veins as soon as 5 min mildly and 2 h moderately after intraperitoneal injection. Hepatocytes around the terminal hepatic veins showed positive Ca granules 6 to 30 h after intragastric injection of CCl₄. Hepatocytes stained by Ca showed acidophilic degeneration and coagulative necrosis. The hepatocytes of rats treated with both diltiazem and CCl₄ revealed fewer Ca granules than those treated with CCl₄ alone. In summary, Ca was stained by the GBHA method from the early stage of liver injury by CCl₄ and was closely involved in acidophilic degeneration and coagulative necrosis of hepatocytes. The Ca staining in liver cells in CCl₄-treated rats was decreased by diltiazem.     

Antifibrotic effect of heparin on liver fibrosis model in rats

AIM: To evaluate the effect of chronic thrombin inhibition by heparin on experimentally induced chronic liver injury (liver fibrosis) in rats.METHODS: Chronic liver injury (liver fibrosis) was induced in Wistar rats by oral administration of carbon tetrachloride (CCl₄) for 7 wk, an animal model with persistent severe hepatic fibrosis. Intravenous administration of the thrombin antagonist (heparin) started 1 wk after the start of CCl₄ intoxication for 6 wk. After completion of treatment (7 wk), markers of hepatic dysfunction were measured and changes evaluated histopathologically.RESULTS: Higher serum glutamate oxaloacetate transaminase (SGOT), serum glutamate pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total, direct and indirect bilirubin levels, as well as lower fibrinogen levels, were found in CCl₄ intoxicated rats. Heparin, silymarin and combination of drug (heparin and silymarin) treatment for 6 wk prevented a rise in SGOT, SGPT, ALP, total, direct and indirect bilirubin levels and improved fibrinogen levels. Deterioration in hepatic function determined by the fibrosis area was retarded, as evident from hepatic histopathology. Total protein levels were not changed in all groups.CONCLUSION: Heparin, a thrombin antagonist, preserved hepatic function and reduced severity of hepatic dysfunction/fibrogenesis. Combination of heparin and silymarin produced additional benefits on liver fibrosis.     

A voluntary oral ethanol-feeding rat model associated with necroinflammatory liver injury

Background: The intragastric (IG) ethanol infusion model results in fatty liver, necrosis, inflammation and fibrosis. This model was utilized to study the pathogenesis of alcoholic liver disease (ALD). Disadvantages of the IG model include maintenance of the animals and equipment expense. To develop a voluntary feeding model for ALD, we took advantage of two important observations in the IG model: (i) female rats demonstrate greater severity of alcohol‐induced liver injury than males and (ii) rats fed fish oil as a source of fatty acids develop more severe alcoholic liver injury than rats fed other fatty acids with ethanol. Methods: Female Wistar rats (205 to 220 g) were fed for 8 weeks a diet containing 8% ethanol, fish oil (30% of calories), protein, and dextrose. Pair‐fed controls (FD) received dextrose in amounts isocaloric to ethanol. The following measurements were made: liver pathology [fatty liver (0 to 4), necrosis, inflammation and fibrosis by Sirius Red], endotoxin and alanine aminotransferase (ALT) in plasma, urine ethanol, lipid peroxidation, nuclear factor kappa‐B (NF‐κB) and mRNA levels for tumor necrosis factor‐alpha (TNF‐α), cyclooxygenase‐2 (COX‐2), and inducible nitric oxide synthase (iNOS). Protein levels for iNOS and nitrotyrosine were evaluated by immunohistochemistry and Western Blot analysis. Liver proteasome and cytochrome P450 2E1 activity and protein levels of asialoglycoprotein receptor (ASGPR) were also evaluated. In addition, mRNA levels of fibrogenic markers were assessed. Results: All animals lost weight for the initial 2 to 3 weeks but then gained weight until killing at 8 weeks. There was, however, a significant difference (p < 0.05) in weight between the ethanol‐fed (Etoh) and (FD) groups at the end of the experiment. The mean urine ethanol levels ranged between 190 and 240 mg/dl. The severity of pathological changes was greater (p < 0.01) in Etoh vs. FD: fatty liver, 3.0 ± 1.2 vs. 1.2 ± 0.4; necrosis (foci/mm²), 3.9 ± 2.3 vs. 0.4 ± 0.3; inflammation (cells/mm²), 19.0 ± 6.3 vs. 1.8 ± 0.6. Centrilobular collagen deposition (% area), assessed by Sirius Red staining, was greater in Etoh vs. FD. Levels of endotoxin, ALT, CYP2E1 and lipid peroxidation markers were also higher (p < 0.01) in Etoh vs. FD. Levels of NF‐κB and mRNA of pro‐inflammatory mediators (TNF‐α, COX‐2, iNOS) and procollagen‐I were increased (p < 0.05) in ethanol‐fed rats. Immunohistochemical analysis showed more intense staining for both iNOS and nitrotyrosine in the centrilobular areas in the Etoh vs. FD groups. The greater area of positive staining for iNOS and nitrotyrosine in Etoh vs. FD was confirmed by Western Blot analysis. An increase in the expression of mRNA for profibrogenic genes (p < 0.05) was seen in ethanol‐fed rats. Conclusions: A voluntary feeding regimen consisting of fish oil and ethanol in female rats is technically less demanding yet produces pathological and biochemical changes similar to those observed with the IG model. Pathological changes include fatty liver, necrosis and inflammation. Increased NF‐κB and mRNA and protein levels of the pro‐inflammatory mediators TNF‐α, COX‐2 and iNOS, coincided with the presence of necroinflammatory changes. The voluntary feeding regimen is proposed as an alternative to the IG model in the study of alcoholic liver injury.