| Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome that has the similar pathological changes with alcoholic liver disease, but the patients suffer from it without excessive alcohol intake. Non-alcoholic steatohepatitis/fibrosing steatohepatitis is the key stage in the spectrum of NAFLD which ranges from simple fatty liver disease to liver cirrhosis. Up to now, the pathogenesis of NAFLD leading to disease progression remains poorly understood and definitive therapy for these patients are lacking.Heme oxygenase-1 (HO-1), an antioxidant defense enzyme, can cleave heme into equimolar amounts of carbon monoxide, biliverdin/bilirubin, and free iron. Recent reports indicated that HO-1 plays a vital role in many aspects, such as suppression of oxidative stress, inflammation, cell proliferation and microcirculation improvement in various pathological conditions. However, there are few investigations regarding the protective effect of HO-1 on NAFLD. Importantly, no evidence is available for whether the expression of HO-1 contributes to the pathophysiological changes in the development of nutritional steatohepatitis/fibrosing steatohepatitis.In this study, we aimed to establish a non-alcoholic steatohepatitis/ fibrosing steatohepatitis model caused by high fat, methionine and choline deficient (MCD) diet, to elucidate the effect of HO-1 induction on the pathogenesis of non-alcoholic steatohepatitis/fibrosing steatohepatitis by targeting genetic modulation, which will provide scientific evidence for HO-1 as a new therapeutic target of NAFLD.Part 1 Establishment of animal non-alcoholic steatohepatitis/fibrosing steatohepatitis model and the expression of HO-1 in NAFLD Objective: To establish the experimental models of non-alcoholic steatohepatitis/fibrosing steatohepatitis and explore the role and potential molecular mechanism of HO-1 in progressive NAFLD in mice.Methods: Experimental non-alcoholic steatohepatitis/fibrosing steatohepatitis models were established by feeding mice with MCD diet (MCD group). Control animals were fed with choline-methionine supplemented diet (control group). Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined by enzymic method with automatic biochemistry analyzer. Hepatic steatosis was observed by SudanⅣstaining; Hepatic inflammation and fibrosis were graded under HE and Masson staining. The stage of steatosis and fibrosis was assessed according to the guidelines for diagnosis and treatment of non-alcoholic fatty liver diseases. The mRNA and protein expressions of HO-1 in hepatic tissue were measured by real-time quantitative PCR, Western blot and immunohistochemical staining, respectively.Results:1 Serum ALT and AST levels of experimental mice Animal models of non-alcoholic steatohepatitis and fibrotic steatohepatitis were established by feeding mice MCD diet for 4 and 8 weeks, serum ALT (267.03±31.50U/L, 695.87±73.96U/L) and AST (517.43±20.84U/L, 986.93±23.69U/L) levels of the model mice were higher than that of the control mice (31.43±4.76U/L, 32.43±13.54U/L and 34.73±6.16U/L, 33.20±14.00U/L) respectively, the differences of them are significant, P<0.001.2 The liver anatomy and histopathology2.1 Observation with the naked eyes, livers of the control mice looks mahogany, bright and shiny. In MCD fed mice, the liver volume decreased slightly and presented yellowish and greasy at week 4, and then the liver volume decreased obviously, the liver edge became blunt and cohesive to adjacent tissue, and the sections of liver were more greasy and dim at week 8.2.2 Observations under optical microscopy, normal liver histology were presented in mice fed with the control diet for 4 and 8 weeks. However, mice fed with the MCD diet developed steatohepatitis with hepatocyte ballooning changes, scattered lobular inflammatory cells infiltration, and inflammatory foci at week 4 and severe steatohepatitis, portal and perisinusoidal fibrosis at week 8, as detected by HE and Masson trichrome staining. The frozen sections were stained by SudanⅣwhich showed occasional lipid droplet in mice fed with control diet, and a great quantity of red lipid droplet distributed in hepatic cells in MCD feeding mice at week 4. The quantity of lipid droplet increased slightly in MCD fed mice at week 8 and had no significant difference with mice at week 4.3 Hepatic HO-1 expression3.1 Expression level of HO-1 gene in mice hepatic tissues was determined by real-time quantitative PCR. The mRNA expression level of HO-1 were 5.49±1.26, 4.66±0.67 (at week 4 and 8, respectively), all were significantly higher than that in the control group (0.86±0.12, 0.79±0.20), (P<0.01).3.2 Expression level of HO-1 protein in mice hepatic tissues was also measured with Western blot. Compared with mice fed methionine-choline supplemented diet (0.22±0.03, 0.26±0.094), the HO-1 protein expression in MCD fed mice (0.66±0.05, 0.53±0.08) enhanced significantly as aggravation of hepatic injury (P<0.01).3.3 Immunohistochemical staining for HO-1 expression showed that HO-1 expression was significantly increased (1.13±0.18, 0.91±0.15) in the liver sections of the model mice at week 4 and 8 by compared with that of the control mice (0.09±0.01, 0.04±0.00), which expressed mainly both in the nuclei and cytoplasm in the hepatocytes and kupffer cells (P<0.01).Conclusion:1 The non-alcoholic steatohepatitis/fibrosing steatohepatitis models could be established successfully by feeding mice with MCD for 4 and 8 weeks, respectively. And the pathological characteristic of the models were very similar to that of human, are suitable for investigation of pathogenesis and novel therapies of non-alcoholic steatohepatitis/fibrosing steatohepatitis. 2 The expression of hepatic HO-1 mRNA and protein increased significantly in non-alcoholic steatohepatitis/fibrosing steatohepatitis models, which are considered as a compensatory mechanism of the organism. HO-1 may be involved in the pathological progress of steatohepatitis and fibrosis, and plays a potential protective efficacy on the livers.Part 2 Heme oxygenase-1 prevents non-alcoholic steatohepatitis through suppressing hepatocyte apoptosis in miceObjective: To elucidate the effect of HO-1 on hepatocellular apoptosis in nutritional steatohepatitis in mice.Methods: C57BL/6J mice were fed with MCD diet for 4 weeks to induce steatohepatitis. HO-1 inducer (hemin), HO-1 inhibitor zinc protoporphyrin IX (ZnPP-IX) and/or adenovirus carrying HO-1 gene (Ad-HO-1) were administered to mice, respectively. HE stain was used for the observation of steatosis, inflammatory response in liver sections; Thiobarbituric acid reactive substances analyzed malondialdehyde (MDA) content; The distribution and expression of HO-1 in hepatic tissue were measured by immunohistochemical staining, real-time quantitative PCR and Western blot, respectively; Hepatocyte apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay;The mRNA and protein expression of apoptosis related genes were assayed by real-time quantitative PCR and Western blot.Results:1 Serum ALT and AST levels of experimental miceSerum ALT (267.03±31.50U/L, 268.27±30.44U/L) and AST (517.43±20.84U/L, 509.73±16.57U/L) of mice adminstrated with MCD diet with or without Ad-GFP significantly increased by compared with that of mice fed the control diet (31.43±4.76U/L, 34.73±6.16U/L). A significant reduction of ALT (109.16±16.49U/L) and AST (235.38±14.37U/L) levels was noted after hemin treatment for 4 weeks compared to MCD diet fed mice. A similar effect was observed by Ad-HO-1 gene transfer (110.12±9.12U/L, 211.34±19.86U/L) compared to mice fed the MCD diet administered Ad-GFP. The combination of hemin and Ad-HO-1 failed to show an additive effect on suppressing ALT (103.24±6.07U/L) and AST (205.86±13.37U/L) levels. However, the levels of ALT (499.74±14.09U/L) and AST (775.12±63.89U/L) were markedly increased in mice treated with ZnPP-IX than those of the mice fed MCD diet only.2 Routine pathologic examinationsMice fed the control diet showed normal liver histology at week 4. However, mice fed with the MCD diet and mice fed the MCD diet administered Ad-GFP exhibited moderate or severe hepatic macrovesicular steatosis and inflammatory infiltratory, which were in line with part 1 (NAS 6~7S1). Treatment with HO-1 inducer hemin or Ad-HO-1 markedly reduced the severity of hepatic steatosis and inflammatory infiltration (NAS2~3S0). Treatment with hemin and Ad-HO-1 gave similar effect without further improvement. In contrast, hepatic steatosis and inflammatory infiltration were aggravated with treatment of HO-1 inhibitor ZnPP-IX (NAS7~8S1~2).3 Effect of HO-1 induction on hepatocyte apoptosisTUNEL-positive cells appeared occasionally in the liver sections of control mice (0.43±0.25%), but were frequently observed in mice fed with the MCD diet alone or with Ad-GFP (3.59±1.02%, 4.17±1.04%). TUNEL- positive cells were decreased by hemin (1.63±0.78%) or Ad-HO-1 (1.53±0.45%) administration. Treatment with hemin plus Ad-HO-1 did not further reduce the number of apoptotic cell (1.33±0.53%) in the liver sections of the mice. In contrast, TUNEL-positive cells were markedly increased in the licer section of the mice received ZnPP-IX (6.38±0.80%) compared with that of mice with the MCD diet alone.4 Effect of HO-1 induction on hepatic oxidative stressMice fed with MCD diet with or without Ad-GFP resulted in a prominent increase in MDA level (5.09±1.01nmol/mg, 4.95±0.75nmol/mg) compared with that of the control mice (1.68±0.32nmol/mg). A significant reduction of MDA content was noted after hemin treatment for 4 weeks (3.50±0.67 nmol/mg) compared to MCD-treated mice. A similar effect was observed by Ad-HO-1 gene transfer (3.69±0.78nmol/mg) compared to mice administered Ad-GFP. The combination of hemin and Ad-HO-1 failed to show an additive effect on suppressing MDA levels (3.13±0.66nmol/mg). However, the level of MDA was markedly increased in mice treated with ZnPP-IX than those fed MCD diet only.5 Effect of hemin and/or Ad-HO-1 on hepatic HO-1 expressionIn MCD feeding mice, mRNA and protein expression of HO-1 had a elevation. The mRNA and protein expression of HO-1 were further up-regulated by hemin or Ad-HO-1 administration. Co-administration of hemin and Ad-HO-1 had no better effect on up-regulation of HO-1 expression. In contrast, the expression of HO-1 was down-regulated by ZnPP-IX compared with mice fed a MCD diet. Immunohistochemical staining for HO-1 followed the same trend and appeared to be in hepatocytes and kupffer cells both in the nuclei and cytoplasm.6 Effect of HO-1 gene modulation on the expression of genes related to apoptosisIn MCD feeding mice, mRNA and protein expression of CYP2E1, Cyt-c, Fas, FasL, caspase-3, caspase-9 and Bax had a marked elevation and the anti-apoptosis gene Bcl-2 was dramatically decreased. The expression of CYP2E1, Cyt-c, Fas, FasL, caspase-3, caspase-9 and Bax were down- regulated and Bcl-2 was up-regulated by hemin or Ad-HO-1 administration. No further effect on regulating apoptosis genes expression was observed in administration of hemin and Ad-HO-1. In contrast, the expression of CYP2E1, Cyt-c, Fas, FasL, caspase-3, caspase-9 and Bax were further up-regulated and Bcl-2 was further down-regulated by ZnPP-IX compared with mice fed a MCD diet.Conclusions:1 Oxidative stress existed in nutritional steatohepatitis in mice, which might trigger cell apoptosis and play an important role in the development of apoptotic injury.2 The expression of HO-1 mRNA and protein increased significantly by hemin or Ad-HO-1 administration, which was associated with attenuatation of liver injury.3 Increase the expression of HO-1 can attenuate hepatic statosis and necroinflammation through suppressing oxidative stress and hepatocyte apoptosis in mice.Part 3 Effect of HO-1 induction on the fibrogenic related genes in nutritional fibrosing steatohepatitis in miceObjective: To elucidate the effect and the mechanism of HO-1 in nutritional fibrosing steatohepatitis in mice.Methods:Male C57BL/6J mice were fed with MCD diet for 8 weeks to induce fibrosing steatohepatitis. Mice were grouped and the administrative interventions were given in accordance with part 2. Liver injury was assessed by serum ALT, AST levels and histological examination; hepatic hydroxyproline was detected to quantify collagen content; hepatic lipid peroxides levels were determined; the expression of inflammatory factors tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and suppressor of cytokine signaling-1 (SOCS-1) were assayed by RT-PCR and real-time quantitative PCR; the expression levels of pro-fibrotic genes alpha-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGF-β1), matrix metallopeptidase-2 (MMP-2) and matrix metallopeptidase-9 (MMP-9) were assayed by real-time quantitative PCR and Western blot.Results:1 Measurement of serum ALT and ASTMice fed an MCD diet alone or with Ad-GFP administration showed significantly higher serum ALT levels (695.87±73.96U/L, 643.93±45.01U/L) and AST (986.93±23.70U/L, 918.90±127.74U/L), which indicated hepatic inflammation. A significant reduction of serum ALT (234.28±35.36U/L, 229.96±38.86U/L) and AST (308.80±29.20U/L, 291.00±20.53U/L) was noticed after hemin or Ad-HO-1 treatment. Co-administration of hemin and Ad-HO-1 gave similar effect without further improvement. Conversely, mice treated with ZnPP-IX showed the highest ALT and AST levels. 2 Routine pathologic observationThe liver sections from mice fed with MCD diet for 8 weeks exhibited disordered lobule structure, macrosteatosis in Zone 3, spot or focal hepatocyte necrosis, inflammatory infiltration and portal and perisinusoidal fibrosis (NAS 7~8S1~2). More pronounced liver injury was noted in mice treated with ZnPP-IX (NAS7~8S2~3). However, hemin and/or Ad-HO-1 administration could notably ameliorate hepatic steatosis, necrotic inflammation and progressive fibrosis (NAS3~4S0~1).3 Effect of HO-1 induction on hepatic hydroxyproline content Mice in MCD group and Ad-GFP group showed significantly higher hepatic hydroxyproline level (196.67±21.93μg/mg, 316.67±44.01μg/mg) than the control mice, and a significant reduction was noticed after hemin and/or Ad-HO-1 treatment (263.00±14.73μg/mg, 271.67±13.32μg/mg, 258.67±20.21μg/mg). Conversely, the ZnPP-IX group showed the highest hepatic hydroxyproline content (446.00±24.42μg/mg). Quantitation of collagen by measuring the amount of hepatic hydroxyproline content supported the observation that treatment with hemin and/or Ad-HO-1 reversed liver fibrosis induced by MCD feeding.4 Effect of HO-1 induction on hepatic MDAMDA was analyzed as an indicator of lipid peroxidation. Mice intake of the MCD diet resulted in a significant increase in hepatic MDA content (4.81±0.98nmol/mg) compared with that of the control mice (1.49±0.40nmol/mg), indicating hepatic oxidative stress. An impressive reduction of hepatic MDA (3.57±0.63nmol/mg) was noted after hemin treatment for 8 weeks compared to MCD-treated mice. A similar effect was observed by Ad-HO-1 gene transfer (3.43±0.49nmol/mg) compared to mice administered Ad-GFP (4.63±0.92nmol/mg). The combination of hemin and Ad-HO-1 failed to show an additive effect on suppressing MDA concentrations. Reduction of hepatic MDA effected by hemin and/or Ad-HO-1 was completely blocked by treatment of ZnPP-IX.5 Up-regulation of HO-1 by hemin and/or Ad-HO-1 The mRNA and protein expressions of HO-1 assayed by quantitative real-time PCR and Western blot are following the same trend as part 2.6 Effect of HO-1 on the expression of pro-inflammatory genesCompared to control mice, hepatic TNF-α, IL-6 and SOCS-1 genes were up-regulated in MCD diet-fed mice, which were significantly blunted by the treatment with hemin or Ad-HO-1. The combination of hemin and Ad-HO-1 caused similar effects on preventing the TNF-α, IL-6 and SOCS-1 mRNA expression. Whereas, supplement with ZnPP-IX led to a further increase of TNF-α, IL-6 and SOCS-1 expression compared with mice fed MCD diet only.7 Effect of HO-1 on the expression of fibrosis related genesMice fed a MCD diet showed enhanced expression of hepatic mRNA and protein ofα-SMA, TGF-β1, MMP-2 and MMP-9, which was significantly blunted by the treatment with hemin or Ad-HO-1. A similar effect was observed in hemin plus Ad-HO-1 group. Conversely, hepatic mRNA and protein expressions ofα-SMA, TGF-β1, MMP-2 and MMP-9 were further up-regulated by ZnPP-IX treatment as compared to MCD group.Conclusions:1 Up-regulation of HO-1 provided a beneficial role in inhibiting oxidative stress, inflammatory, HSCs activation and fibrogenic cytokines expression in the livers with fibrosing steatohepatitis.2 Targeted genetic modulation of HO-1 might serve as a therapeutic approach for fibrotic steatohepatitis.
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