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.     

Splenocyte apoptosis in Plasmodium berghei ANKA infection: possible role of TNF‐α and TGF‐β

Cerebral malaria is associated with the circulating levels of tumour necrosis factor alpha (TNF‐α) and transforming growth factor β (TGF‐β), but association between these two cytokines and implications in splenocyte apoptosis remain largely obscured. We have evaluated the outcome of TGF‐β and TNF‐α production in the context of splenocyte apoptosis during Plasmodium berghei ANKA (PbA) infection. Blood‐stage PbA infection confirmed blood–brain barrier disruption, disarray of white pulp, increase in percentage of sub‐G0/G1 and splenocyte apoptosis. Flow cytometric analysis reveals up‐regulation of Fas‐L followed by caspase‐8 and caspase‐3 activation and signifies possible involvement of Fas‐L‐mediated splenocyte apoptosis. We have observed down‐regulation of TGF‐β and up‐regulation of TNF‐α in tissue and serum level, respectively, during PbA infection. Association between the production of TGF‐β and the severity of malaria infection in splenocytes was verified with TGF‐β inhibitor that exacerbated the apoptotic process. In contrary, TNF‐α inhibitor causes significant delay in apoptotic process, but could not alter the lethality of parasite. Thus, results from this study suggest that the critical balance between TGF‐β and TNF‐α might have a key role on Fas‐L‐mediated splenocyte apoptosis during experimental cerebral malaria.     

Inhibition of hepatic tumour necrosis factor‐α attenuates the anandamide‐induced vasoconstrictive response in cirrhotic rat livers

Background: Increased anandamide, an endocannabinoid that interacts with both cannabinoid CB₁ and CB₂ receptors, can induce hepatic vasoconstrictive responses that contribute to the increased intrahepatic resistance (IHR) in cirrhotic rats. Chronic endotoxaemia and the subsequent release of tumour necrosis factor‐α (TNF‐α) are suggested to result in increased anandamide in cirrhotic livers. Thalidomide, which inhibited TNF‐α effectively, has been used clinically in states of chronic TNF‐α elevation with encouraging results. Aims: This study explores the possible effects of thalidomide on hepatic endocannabinoids and microcirculation of cirrhotic rats. Methods: Portal venous pressure (PVP), superior mesenteric arterial blood flow (SMA BF), hepatic TNF‐α, interleukin (IL‐6), protein expression of CB₁ and CB₂ receptor and thromboxane synthase (TXS) were measured in bile duct‐ligated (BDL) rats receiving 1‐month of vehicle (BDL‐V) or thalidomide (BDL‐thalido). The degree of hepatic fibrosis was also assessed. In the liver perfusion system, IHR and concentration–response curves of the portal perfusion pressure to anandamide were evaluated. Results: In BDL‐thalido rats, PVP, IHR and hepatic levels of TNF‐α and IL‐6, protein expression of CB₁ receptors, TXS and hepatic fibrosis were lower than in BDL‐V rats. In BDL‐thalido rat livers, the attenuation of the vasoconstrictive response to anandamide was associated with an upregulation of the CB₂ receptor and a downregulation of the CB₁ receptor. Nevertheless, SMA BF was not different between BDL‐thalido and BDL‐V rats. Conclusions: Thalidomide decreased the PVP and IHR through the attenuation of anandamide‐induced constrictive response, decreasing the production of TNF‐α, IL‐6 and TXA₂ in the liver and the suppression of hepatic fibrogenesis of rats with biliary cirrhosis of this study.     

Insulin and IGF1 enhance IL‐17‐induced chemokine expression through a GSK3B‐dependent mechanism: a new target for melatonin's anti‐inflammatory action

Obesity is a chronic inflammation with increased serum levels of insulin, insulin‐like growth factor 1 (IGF1), and interleukin‐17 (IL‐17). The objective of this study was to test a hypothesis that insulin and IGF1 enhance IL‐17‐induced expression of inflammatory chemokines/cytokines through a glycogen synthase kinase 3β (GSK3B)‐dependent mechanism, which can be inhibited by melatonin. We found that insulin/IGF1 and lithium chloride enhanced IL‐17‐induced expression of C‐X‐C motif ligand 1 (Cxcl1) and C‐C motif ligand 20 (Ccl20) in the Gsk3b^+/+, but not in Gsk3b^?/? mouse embryonic fibroblast (MEF) cells. IL‐17 induced higher levels of Cxcl1 and Ccl20 in the Gsk3b^?/? MEF cells, compared with the Gsk3b^+/+ MEF cells. Insulin and IGF1 activated Akt to phosphorylate GSK3B at serine 9, thus inhibiting GSK3B activity. Melatonin inhibited Akt activation, thus decreasing P‐GSK3B at serine 9 (i.e., increasing GSK3B activity) and subsequently inhibiting expression of Cxcl1 and Ccl20 that was induced either by IL‐17 alone or by a combination of insulin and IL‐17. Melatonin's inhibitory effects were only observed in the Gsk3b^+/+, but in not Gsk3b^?/? MEF cells. Melatonin also inhibited expression of Cxcl1, Ccl20, and Il‐6 that was induced by a combination of insulin and IL‐17 in the mouse prostatic tissues. Further, nighttime human blood, which contained high physiologic levels of melatonin, decreased expression of Cxcl1, Ccl20, and Il‐6 in the PC3 human prostate cancer xenograft tumors. Our data support our hypothesis and suggest that melatonin may be used to dampen IL‐17‐mediated inflammation that is enhanced by the increased levels of insulin and IGF1 in obesity.     

Polymorphisms in the transforming growth factor‐β1 gene and the risk of asthma: A meta‐analysis

Background and objective: Polymorphisms in the transforming growth factor‐β1 (TGF‐β1) gene have been implicated in susceptibility to asthma, but a large number of studies have reported inconclusive results. A meta‐analysis was performed to investigate the association between polymorphisms in the TGF‐β1 gene and asthma susceptibility. Methods: Searches were performed of Medline (Ovid), PubMed, the Chinese Biological Medicine Database (CBM), the Chinese Journals Full‐text Database (CNKI), the Cochrane Library Database and the Web of Science, covering all papers published up to 30 April 2009. Statistical analysis was performed using Revman4.2.8 and STATA10.0 software. Results: Two polymorphisms (?509C/T and 915G/C(G25C)) were investigated in 14 studies, involving 2979 asthma patients and 4941 control subjects. The results showed that individuals carrying the ?509T allele (TT+TC) had a 36% increased risk of asthma, when compared with homozygotes (?509CC) (OR 1.36, 95% CI: 1.12–1.65). However, there was no significant association with risk of asthma in carriers of the 915C allele (GC+CC) compared with 915GG homozygotes (OR 1.05, 95% CI: 0.65–1.70). In a subgroup analysis by ethnicity, the risk of asthma associated with the ?509T allele was significantly elevated among Asians (OR 1.50, 95% CI: 1.04–2.17) but not Caucasians (OR 1.16, 95% CI: 1.00–1.36). In a subgroup analysis by age, the ?509T allele was associated with a significantly elevated risk of asthma among adults (OR 1.45, 95% CI: 1.09–1.92) but not children (OR 1.19, 95% CI: 0.96–1.46). Conclusions: This meta‐analysis suggested that the ?509C/T polymorphism in the TGF‐β1 gene may be a risk factor for asthma. To further evaluate gene–gene and gene–environment interactions between polymorphisms in the TGF‐β1 gene and asthma susceptibility, more studies involving thousands of patients are required.     

Effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuornide in rats

Aim: Genipin is reported to stimulate the insertion of multidrug resistance protein 2 (Mrp2) in the bile canalicular membrane, thereby causing choleresis by the increased the biliary excretion of glutathione, which has been considered to be a substrate of Mrp2. In the present study, we examined the effect of genipin on cholestasis induced by estradiol‐17β‐glucuronide and lithocholate‐3‐O‐glucuronide, Mrp2 substrates, in rats. Further, the effect of genipin on the biliary excretion of substrates of P‐glycoprotein (P‐gp), vinblastine and erythromycin, was also studied. Methods: The effect of genipin infusion at the rate of 0.5 µmol/min/100 g on cholestasis induced by estradiol‐17β‐glucuronide (0.075 µmol/min/100 g for 20 min) and lithocholate‐3‐O‐glucuronide (0.15 µmol/min/100 g for 40 min) was studied. The effect of genipin infusion on the biliary excretion of a tracer dose of vinblastine and erythromycin infused at the rate of 0.1 µmol/min/100 g was also studied. Results: Genipin relieved estradiol‐17β‐glucuronide‐induced cholestasis, and cumulative biliary estradiol‐17β‐glucuronide excretion for 120 min was increased from 50 ± 20%–81 ± 20% dose. In contrast, genipin had no effect on lithocholate‐3‐O‐glucuronide‐induced cholestasis. Biliary excretion of a tracer dose of vinblastine and the maximum biliary excretion of erythromycin were significantly decreased by genipin. Conclusions: Genipin protected estradiol‐17β‐glucuronide‐induced cholestasis. The mechanism of the protection of cholestasis by genipin is unknown, but it is speculated to be due to a conformational change of P‐gp by genipin, in addition to the stimulation of Mrp2 insertion into the bile canaliculi.     

β‐Glucuronidase inhibitor tectorigenin isolated from the flower of Pueraria thunbergiana protects carbon tetrachloride‐induced liver injury

Background/Aim: To understand the relationship between the fluctuation in serum β‐glucuronidase and hepatotoxicity, an inhibitor of β‐glucuronidase was isolated from the flowers of Pueraria thunbergiana and its hepatoprotective activity was measured. Method: Tectorigenin was isolated from the flowers of pueria thunbergiana as an inhibitor of β‐glucuronidase, and serum ALT, AST and biological parameters as markers for its hepatoprotective activity were measured on CCl₄‐induced liver injury in mice. The relationship between serum β‐glucuronidase and hepatoprotective activities in mice was measured. Results: When tectorigenin at a dose of 100 mg/kg was intraperitoneally administered on CCl₄‐induced liver injury in mice, it significantly inhibited the increase of plasma ALT, AST and LDH activities. The inhibitory effect of tectorigenin is much more potent than that of dimethyl diphenyl bicarboxylate (DDB), which has been used as a commercial hepatoprotective agent. When tectoridin transformed to tectorigenin by intestinal bacteria was orally administered to mice, it showed hepatoprotective activity. However, when tectoridin was intraperitoneally administrated to mice, it did not exhibit hepatoprotective activity. Moreover, orally administered tectoridin not only inhibited β‐glucuronidase but also increased GSH content and GST activity on CCl₄‐induced hepatotoxicity of mice. Conclusion: We insist that an inhibitor of β‐glucuronidase tectorigenin may be hepatoprotective and tectoridin should be a prodrug transformed to tectorigenin.