Polymethylated Phloroglucinol Meroterpenoids from Rhodomyrtus tomentosa and Their Antibacterial and Acetylcholinesterase Inhibitory Effects.

Rhotomentodiones C-E, three new polymethylated phloroglucinol meroterpenoids with diverse configurations, were isolated from the twigs and leaves of Rhodomyrtus tomentosa. Their structures and absolute configurations were established mainly by means of comprehensive spectroscopic data and electron circular dichroism (ECD) calculation. Among them, Rhotomentodione D (2) exhibited both antibacterial activity with an MIC value of 12.5 µg/mL against Propionibacterium acnes and AChE inhibitory activity with an IC50 value of 22.9 µm.

Fuzheng Huayu recipe prevents nutritional fibrosing steatohepatitis in mice.

BACKGROUND: Fuzheng Huayu recipe (FZHY), a compound of Chinese herbal medicine, was reported to improve liver function and fibrosis in patients with hepatitis B virus infection. However, its effect on nutritional fibrosing steatohepatitis is unclear. We aimed to elucidate the role and molecular mechanism of FZHY on this disorder in mice. METHODS: C57BL/6 J mice were fed with methionine-choline deficient (MCD) diet for 8 weeks to induce fibrosing steatohepatitis. FZHY and/or heme oxygenase-1 (HO-1) chemical inducer (hemin) were administered to mice, respectively. The effect of FZHY was assessed by comparing the severity of hepatic injury, levels of hepatic lipid peroxides, activation of hepatic stellate cells (HSCs) and the expression of oxidative stress, inflammatory and fibrogenic related genes. RESULTS: Mice fed with MCD diet for 8 weeks showed severe hepatic injury including hepatic steatosis, necro-inflammation and fibrosis. Administration of FZHY or hemin significantly lowered serum levels of alanine aminotransferase, aspartate aminotransferase, reduced hepatic oxidative stress and ameliorated hepatic inflammation and fibrosis. An additive effect was observed in mice fed MCD supplemented with FZHY or/and hemin. These effects were associated with down-regulation of pro-oxidative stress gene cytochrome P450 2E1, up-regulation of anti-oxidative gene HO-1; suppression of pro-inflammation genes tumor necrosis factor alpha and interleukin-6; and inhibition of pro-fibrotic genes including α-smooth muscle actin, transforming growth factor beta 1, collagen type I (Col-1) and Col-3. CONCLUSIONS: Our study demonstrated the protective role of FZHY in ameliorating nutritional fibrosing steatohepatitis. The effect was mediated through regulating key genes related to oxidative stress, inflammation and fibrogenesis.

Adenovirus-mediated peroxisome proliferator activated receptor gamma overexpression prevents nutritional fibrotic steatohepatitis in mice.

OBJECTIVE: The pathogenesis of non-alcoholic steatohepatitis is still unclear. We have demonstrated previously that peroxisome proliferator activated receptor gamma (PPARγ) ligand protects against inflammation and fibrogenesis in experimental non-alcoholic steatohepatitis. We aim to elucidate the effect and the mechanism of PPARγ itself on nutritional fibrotic steatohepatitis in mice. METHODS: C57BL/6J mice were fed with methionine-choline deficient (MCD) diet for 8 weeks to induce fibrotic steatohepatitis. Mice fed the MCD diet were treated with adenovirus carrying PPARγ (Ad-PPARγ), Ad-PPARγ plus PPARγ agonist rosiglitazone, or PPARγ antagonist 2-chloro-5-nitrobenzaniliden (GW9662), respectively. The effects of up-regulation of PPARγ in the presence or absence of its agonist/or antagonist were assessed by comparing the severity of hepatic injury, activation of hepatic stellate cells and the expression of adiponectin, heme oxygenase-1, and fibrogenic related genes. RESULTS: Mice fed with MCD diet for 8 weeks showed severe hepatic injury including hepatic steatosis, inflammatory infiltration, and fibrosis. Administration of Ad-PPARγ significantly lowered serum alanine aminotransferase level and ameliorated hepatic steatosis, necroinflammation, and fibrosis. These effects were associated with enhanced expression of PPARγ, up-regulated expression of adiponectin and heme oxygenase-1, and down-regulated expression of tumor necrosis factor alpha, interleukin-6, α-smooth muscle actin, transforming growth factor beta 1, matrix metallopeptidase-2, and -9. Administration of GW9662 promoted the severity of liver histology. CONCLUSIONS: The present study provided evidences for the protective role of overexpressing PPARγ in ameliorating hepatic fibrosing steatohepatitis in mice. Modulation of PPARγ expression might serve as a therapeutic approach for fibrotic steatohepatitis.

[The role of heme oxygenase-1 in non-alcoholic steatohepatitis].

OBJECTIVE: To investigate the potential role of heme oxygenase-1 on preventing non-alcoholic steatohepatitis (NASH) in mice. METHODS: Experimental models of NASH were established by feeding male C57BL/6J mice with choline-methionine deficient diet (MCD) for four weeks. Control animals were fed with choline-methionine supplemented diet. The treatment groups were fed with MCD diet combined with HO-1 inducer hemin or inhibitor zinc protoporphyrin IX (ZnPP-IX). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were tested by enzymic method with automatic biochemistry analyzer. The degree of hepatic steatosis, inflammation and fibrosis were examined under HE staining. The hepatic mRNA and protein expressions of HO-1, TNFalpha and IL-6 were analyzed by RT-PCR and Western blot respectively. MCD fed mice showed increased serum ALT and AST levels and moderate to severe hepatic steatosis with inflammatory infiltration, hepatic spot or focal necrosis, light portal and sinus hepaticus fibrosis in the liver sections, which associated with enhanced expression of HO-1, TNFalpha and IL-6 mRNA and protein (1.13+/-0.11, 1.74+/-0.05; 0.20+/-0.01, 1.92+/-0.10; 0.58+/-0.02, 2.06+/-0.05 vs 0.43+/-0.02, 0.75+/-0.05; 0.08+/-0.00, 0.59+/-0.02; 0.22+/-0.01, 0.91+/-0.02). Administration of hemin significantly decreased serum ALT and AST levels and attenuated hepatic steatosis and necroinflammation which associated with up-regulation of antioxidative gene HO-1 and down-regulation of pro-inflammatory cytokines TNFalpha and IL-6 (P < 0.01). A contrary effect on serum aminotransferase levels and liver histopathology was observed in mice injected with ZnPP-IX (P < 0.01). CONCLUSIONS: The effect was associated with suppressed HO-1 expression and increased TNFaLPHA and IL-6 expression. The data provided a biochemical, morphological and molecular biological evidence for the protective role of HO-1 in ameliorating hepatic steatosis, necroinflammation in experimental nutritional steatohepatitis.

Antioxidants vitamin E and 1-aminobenzotriazole prevent experimental non-alcoholic steatohepatitis in mice.

OBJECTIVE: Hepatic oxidative stress plays a key role in the development of non-alcoholic steatohepatitis (NASH). However, the protective effects of antioxidants on NASH are largely unknown. The aim of this study was to elucidate the effect and mechanism of antioxidants on NASH in mice. MATERIAL AND METHODS: C57BL6/J mice were fed a methionine-choline-deficient (MCD) diet for 10 days or 3 weeks to induce steatohepatitis. Antioxidants (vitamin E, ABT, or vitamin E plus ABT) were supplemented in mice fed a MCD diet, respectively. The effect of antioxidants on oxidative stress and apoptosis was assessed, and activation of adiponectin and expressions of inflammatory factors, apoptosis-related genes, and fibrosis-related genes were assayed. RESULTS: MCD feeding in mice showed increasing serum alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) levels, and progressive hepatic injury including hepatic steatosis and inflammatory infiltration. Administration of antioxidants vitamin E and/or ABT significantly lowered serum ALAT and ASAT levels (p<0.001) and ameliorated hepatic steatosis and necroinflammation. These effects were associated with repressed hepatic lipid peroxides through reducing hepatic MDA content and enhancing hepatic superoxide dismutase (SOD) activity; down-regulated inflammatory factor COX-2, lowered activity of NF-kappaB, up-regulated anti-apoptotic gene Bcl-2, and down-regulated pro-apoptotic gene Bax suppressed expression of the fibrotic genes TGF-beta1 and MMP2. Moreover, expression of the anti-inflammatory factor adiponectin was also induced by vitamin E or ABT. A combination of vitamin E and ABT showed an additive effect on preventing liver injury. CONCLUSIONS: The present study provides morphological and molecular biological evidence for the protective role of the antioxidant vitamins E and ABT in ameliorating oxidative stress, hepatic apoptosis, and necroinflammation in experimental nutritional steatohepatitis.

[The role of apoptosis and the related genes in non-alcoholic steatohepatitis].

OBJECTIVE: To study the role of apoptosis and the expression of apoptosis-related genes Fas ligand (FasL), Fas, caspase-3 and caspase-8 in an animal model of non-alcoholic steatohepatitis (NASH). METHODS: An experimental progressive NASH model was established by feeding male C57BL6/J mice with a high fat, methionine-choline deficient (MCD-) diet for two days, five days, ten days, three weeks and eight weeks. Control mice were fed methionine-choline supplemented (MCD+) diet. Hepatic steatosis, inflammation and fibrosis were graded by examining their H and E stained liver sections. Hepatocyte apoptosis was detected by TUNEL assay. Expressions of mRNA and protein of FasL, Fas and caspase-8 were performed by quantitative real time RT-PCR and Western blot. Caspase-3 activity assay was conducted using ApoAlert caspase-3 assay kit. RESULTS: In MCD- mice, minimal hepatic steatosis was observed at day 5, and by day 10, mild steatosis with inflammatory infiltration was found. Severe steatohepatitis was noted at week 3, and fibrosis at week 8. TUNEL assay showed that apoptotic index in MCD- group was higher than that in MCD+ group at week 3 (15.59%+/-4.87% vs 5.17%+/-3.19%, P less than 0.05) and at week 8 (11.29%+/-3.22% vs 5.41%+/-1.54%, P less than 0.05). Compared to MCD+ group, the expression of FasL was dramatically increased on day 10 and in week 3 in MCD- mice both at the mRNA and protein levels (P less than 0.05 and P less than 0.01). Expression of Fas mRNA was up-regulated in weeks 3 and 8 (P less than 0.01), and expression of Fas in protein level was higher at week 8 (P less than 0.01) in MCD- group. Expression of caspase-8 significantly increased at the mRNA level at week 3 and week 8 (P less than 0.01 and P less than 0.05 respectively) and at the protein level at week 8 (P less than 0.05) in MCD- group. In all of the time points except for day 5, caspase-3 activities were significantly more enhanced in MCD- group than that in MCD+ group (P less than 0.05). CONCLUSIONS: In our experimental NASH model, hepatic apoptosis was frequently detected. Increased apoptosis was probably attributable to up-regulation of apoptosis-related genes, such as FasL/Fas system, and activation of the caspase pathway. These changes may provoke hepatic apoptosis and the development of inflammation and fibrosis.