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Modulation of IKKβ/NF-κB and TGF-β1/Smad via Fuzheng Huayu recipe involves in prevention of nutritional steatohepatitis and fibrosis in mice.

Objective(s): Fuzheng Huayu recipe (FZHY) exerts significant protective effects against liver fibrosis by strengthening the body’s resistance and removing blood stasis. However, the molecular mechanisms through which FZHY affects liver fibrosis are still unclear. In this study, we examined the expression levels of factors involved in the inhibitor κB kinase-β (IKK-β)/nuclear factor-κB (NF-κB) and transforming growth factor beta 1 (TGF-β1)/Smad signaling pathways to elucidate whether FZHY could attenuate nutritional steatohepatitis and fibrosis in mice. Materials and Methods: C57BL/6J mice were fed with methionine-choline deficient (MCD) diet for 8 weeks to induce fibrotic steatohepatitis. FZHY and/or heme oxygenase-1 (HO-1) chemical inducer (hemin) were administered to mice. The effects of FZHY alone and in combination with hemin were assessed by comparing the severity of hepatic injury, activation of hepatic stellate cells (HSCs), and the expression of oxidative stress, inflammation and fibrogenesis related genes. Results: Administration of FZHY, hemin and FZHY plus hemin significantly ameliorated liver injury. Additionally, our analysis indicated that administration of these agents significantly attenuated oxidative stress, downregulated the expression of pro-inflammatory and pro-fibrotic genes, including IKK-β, NF-κB, monocyte chemoattractant protein-1 (MCP-1), α-smooth muscle actin (α-SMA), TGF-β1, Smad3 and Smad4, and upregulated the expression of the antifibrogenic gene Smad7 (P< 0.001). Conclusion: FZHY-containing therapies prevented nutritional steatohepatitis and fibrosis through modulating the expression of factors associated with the IKKβ/NF-κB and TGF-β1/Smad signaling pathways and oxidative stress related genes.


Introduction
Non-alcoholic steatohepatitis (NASH) is now recognized as the most common cause of liver dysfunction worldwide. Active hepatic fibrogenesis is a crucial pathological process involved in the development of NASH due to its high prevalence and potential for severe hepatic outcomes, such as liver cirrhosis, liver failure, and hepatocellular carcinoma (1). From 2000 to 2010, the percentage of orthotopic liver transplants performed for NASH in the United States increased by 6.2% (2). Future projections estimate that in the next decade, NASH and related fibrosis will surpass viral hepatitis as the major cause of end-stage liver disease, becoming the primary indication for liver transplantation (3). According to the "two hit" hypothesis proposed by Day and James (4), the appearances of oxidative stress, overexpression of key pathogenic factors involved in the development of necro-inflammation and fibrosis, such as nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNF α), interleukin 6 (IL-6), α-smooth muscle actin (α-SMA) and transforming growth factor beta 1 (TGF-β1) are potentially major therapeutic targets in steatohepatitis and advanced fibrosis. However, the precise cellular and biochemical mechanisms of NASH are not fully understood, and specific antifibrotic drug targets have not yet been identified. Thus, it is very important to explore the mechanisms of NASH pathogenesis, especially in order to identify novel effective and curative therapies for NASH and related fibrosis.
Fuzheng Huayu recipe (FZHY) is comprised of 6 traditional Chinese medicines: Fructus Schisandrae chinensis (Wuweizi), Semen persicae (Taoren), Radix Salvia miltiorrhizae (Danshen), Fermentation Mycelium Powder (Dongchongxiacao), Pollen Pini (Songhuafen), and Gynostemma pentaphyllammak (Jiaogulan) (5). This concoction functions to treat imbalanced bodily functions and is used to nourish the liver, dissolve blood stasis, and tonify the spirit, etc. (6). Previous studies have shown that FZHY exerts protective effects against liver fibrosis in animal models through inhibiting collagen synthesis and restoring mitochondrial dysfunction (7). However, the molecular mechanisms mediating the therapeutic effects of FZHY are still unclear. Oxidative stress is an essential "second hit" in the pathogenesis of NASH, because increased free radicals can cause lipid peroxidation, which is a significant aspect of oxidative damage and oxidative modification of phospholipid polyunsaturated fatty acid residues in membrane structure and then induces the inflammatory response and activation of stellate cells further promoting collagen synthesis and fibrogenesis (8,9). Heme oxygenase-1 (HO-1) is a stress-responsive protein induced by various oxidative agents, and its induction is thought to be an adaptive cellular response to survive from exposure to environmental stresses. Our previous study demonstrated that upregulation of HO-1 provided a beneficial role in modulating oxidative stress, inflammatory insults and hepatic stellate cells (HSC) activation in livers with fibrosing steatohepatitis (10).
In the present study, we used the HO-1 chemical inducer hemin as a control drug to explore (1) whether FZHY plays a role in attenuating liver fibrosis in vivo, (2) the effects of FZHY on the expression of genes involved in the inhibitor-κB kinase-β (IKK-β)/NF-κB and TGF-β1/Smad signaling pathways, which also play critical roles in oxidative stress-induced steatohepatitis and fibrosis.

Animals and treatments
Eight-week-old male C57BL/6J mice (body weights, 20 to 25 g) were obtained from the Experimental Animal Center of the Chinese Academy of Medical Sciences and were bred in a temperaturecontrolled animal facility with a 12 hr light-dark cycle. They had free access to water and were allowed to adapt to their food and environment for 1 week before the start of the experiment. The mice were randomly divided into 5 groups (n = 6 mice per group): 1) MCD group, mice fed methionine-choline deficient diet (ICN, Aurora, Ohio); 2) control group, mice fed MCD diet supplemented with choline bitartate (2 g/kg) and DL-methionine (3 g/kg; ICN); 3) MCD + hemin group, mice fed MCD diet administered with hemin (30 μmol/kg) by intraperitoneal (IP) injection 3 times per week; 4) MCD + FZHY group, mice fed MCD diet supplemented with FZHY (15 g/kg/d, Huanghai Pharmaceutical Company Limited, Shanghai, China); 5) MCD + FZHY + hemin group, mice fed MCD diet combined with FZHY and hemin treatments, as described above. The experiment lasted up to 8 weeks (10,11). During the experiment, body weights and rate of food intake were recorded. All animals were killed after overnight fasting at the end of the experiment. Livers were weighed and fixed in 10% formalin for histological analysis or snap-frozen in lipid nitrogen followed by storage at -80 °C until use. All protocols and procedures were performed following the guidelines of the Hebei Committee for the Care and Use of Laboratory Animals and were approved by the Animal Experimentation Ethics Committee of Hebei Medical University.

Histological analysis
Formalin-fixed livers were embedded in paraffin, and 4-μm sections were stained with hematoxylin and eosin and Masson-trichrome stains. Two experienced hepatopathologists independently evaluated the slides and assigned scores for hepatic steatosis, inflammation, and fibrosis. The stage of fibrosis was assessed using a 4-point scale (1, mild/moderate zone 3 perisinusoidal fibrosis or portal fibrosis only; 2, zone 3 and portal/periportal fibrosis; 3, bridging fibrosis; and 4, cirrhosis).

Immunohistochemical evaluation of α-SMA and TGF-β1 expression
Immunostaining for α-SMA and TGF-β1 was performed in paraffin-embedded liver sections using specific antibodies recognizing α-SMA and TGF-β1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and an avidin-biotin complex (ABC) immunoperoxidase. Briefly, endogenous peroxidase activity was blocked by treating sections with 3% hydrogen peroxide. After blocking with 10% non-immunized rabbit serum, primary antibodies targeting α-SMA (dilution 1:100) and TGF-β1 (dilution 1:200) were applied. Primary antibodies were omitted, and nonimmunized goat serum was used as a negative control. After extensive rinsing, the biotinylated secondary antibody and ABC complex/horseradish peroxidase (HRP) were applied. Peroxidase activity was visualized with diaminobenzidine. The sections were then counterstained with hematoxylin. Quantitative analysis of α-SMA-and TGF-β1-stained liver sections was performed by morphometric analysis.

Western blotting analysis of hepatic protein expression
Total protein was extracted from liver tissues, and the protein concentrations were measured by the Bradford method (DC Protein Assay; Bio-Rad, Hercules, CA, USA) as previously described (12). Equal amounts of protein from each sample (100 μg/well) were loaded onto 10% sodium dodecyl sulfate polyacrylamide gels, and proteins were then transferred onto equilibrated polyvinylidene difluoride membranes (Amersham Biosciences, Buckinghamshire, UK) by electroblotting. The membranes were incubated with primary antibodies targeting Smad3, Smad4 or Smad7 (Santa Cruz Biotechnology) overnight at 4°C. Membranes were further incubated with secondary antibodies for 1 hr at room temperature. Proteins were detected by enhanced chemiluminescence (ECL; Amersham Corporation, Arlington Heights, CA, USA), and bands were quantified by scanning densitometry using a digital Kodak Gel Logic 200 system (Carestream Molecular Imaging, USA). β-actin was used as a loading control for protein normalization.

Statistical analysis
All data are presented as means±standard deviations (SDs). Statistical analysis was performed by one-way analysis of variance (ANOVA) and Student-Newman-Keuls test for evaluating differences between groups using SPSS 13.0 (v. 13.0; SPSS Inc., Chicago, IL, USA). A P-value of less than 0.05 was considered statistically significant.

Effect of FZHY on hepatic inflammation and fibrosis in mice fed with MCD diet
The liver sections from mice fed an MCD diet for 8 weeks exhibited severe macrosteatosis, spot or focal hepatocyte necrosis, inflammatory infiltration, portal fibrosis and fibrous septum. FZHY with or without hemin administration could significantly ameliorate the severity of liver injury, which have been demonstrated in our previous study (7).

FZHY attenuated hepatic oxidative stress and inhibited the expression of IKK-β/NF-κB signaling pathway genes in mice fed with MCD diet
To determine the mechanisms mediating the effects of FZHY on the liver, we investigated the mRNA expression levels of NF-κB-dependent inflammatory genes in the liver tissues, including IKK-β, NF-κB p65, MCP-1 and HO-1 (an anti-oxidative stress factor). Relative to control mice, mice fed MCD diet exhibited upregulation of hepatic IKK-β, NF-κB p65, MCP-1 and HO-1 genes (P< 0.001, Figure 1). Additionally, administration of FZHY decreased the expression of IKK-β, NF-κB p65 and MCP-1 mRNAs. Concomitant with the downregulation of inflammatory genes and improvement of liver histology, the expression of HO-1 was also reduced, indicating a reduction in oxidative stress-mediated liver injury.

FZHY suppressed the activation of hepatic stellate cells (HSCs) in MCD diet-induced fibrotic steatohepatitis
Next, we evaluated the role of FZHY in the development of liver fibrosis by assessing the hepatic expression of α-SMA, a well-known marker of activated HSCs. As shown in Figure 2A, increased α-SMA expression in activated HSCs, fibrotic areas, and vessel walls was observed in liver sections of MCDtreated mice, while little α-SMA was deposited in liver sections from control mice. FZHY and/or hemin treatment significantly suppressed α-SMA

Effects of FZHY on the activation of TGF-β1/Smad pathway genes in non-alcoholic fibrosing steatohepatitis induced by MCD diet
Immunohistochemistry analysis revealed increased expression of TGF-β1 in activated HSCs, inflammatory regions, and fibrotic areas in liver sections of MCDtreated mice compared with that of control mice. FZHY or hemin treatment significantly inhibited the upregulation of TGF-β1, and there was a further decrease in the expression of TGF-β1 in liver sections of mice treated with FZHY plus hemin ( Figure 2B).
To evaluate the detailed mechanisms through which FZHY alleviates liver fibrosis, we investigated the hepatic expression levels of fibrosis-related genes. As shown in Figure 3, the hepatic mRNA and protein expression levels of Smad3, Smad4, and Smad7 were markedly elevated in mice fed an MCD diet (P<0.001). The expression levels of Smad3 and Smad4 were significantly downregulated, while the expression of Smad7 was further upregulated in the livers of mice treated with FZHY or hemin compared with mice consuming an MCD diet alone. These effects were exacerbated in mice treated with FZHY plus hemin. Thus, FZHY may alleviate liver fibrosis through modulating the expression of genes in the TGF-β1/Smad pathway.

Discussion
The pathogenesis of NASH is thought to involve a multi-step process, for which oxidative stress is the most popular proposed mechanism of hepatocellular injury (13). More advanced liver damage correlates with greater degrees of oxidative stress. Excessive production of reactive oxygen species (ROS) is a characteristic of oxidative stress and can activate inflammatory pathways, enhance the production of both type I collagen and TGF-β, and promote hepatocyte injury, necro-inflammation, and hepatic fibrogenesis (15)(16)(17). Additionally, oxidative stress has been shown to stimulate the activation of Kupffer cells and HSCs; thus, reducing oxidative stress may also provide anti-inflammatory and antifibrotic effects. Our previous studies revealed increased tissue levels of oxidative stress markers and lipid peroxidation products such as hepatic microsomal fatty acid oxidizing enzyme cytochrome P450 2E1 (CYP2E1) and malondialdehyde (MDA), and these effects were significantly reduced by FZHY pretreatment (7). Furthermore, the expression of HO-1, an inducible microsomal enzyme, is upregulated in response to cellular stress and by pro-oxidative stimuli, and HO-1 has been shown to exert protective effects against ROS-mediated liver fibrosis (17). In the current study, hepatic HO-1 mRNA expression was increased with the progression of liver fibrosis, suggesting that there was enhanced oxidative stress in the livers of mice fed an MCD diet. FZHY administration for 8 weeks resulted in improved liver pathology, concomitant with a dramatic reduction in HO-1 expression. This effect may be due to the decreased expression of hepatic MDA and CYP2E1 following FZHY treatment.
Damaged hepatocytes and kupffer cells can also release inflammatory and fibrogenic mediators that recruit inflammatory cells and provoke HSC activation (15). Thus, inflammation is not merely associated with chronic liver disease, but also promotes disease progression (18). IKK-β is a proinflammatory kinase that phosphorylates the inhibitor of NF-κB, leading to NF-κB activation via its nuclear translocation (19). NF-κB is an inducible transcription factor and has critical roles in regulating the expression of multiple inflammatory genes, including TNF-α, IL-6 and MCP-1 (20,21). Growing evidence indicates that NASH patients exhibit high levels of NF-κB induction (22). Additionally, NF-κB has been shown to be activated by TNF-α and ROS. Conversely, suppression of NF-κB activation prevents the induction of proinflammatory cytokines (23,24). MCP-1 is a potent chemoattractant facilitating the recruitment of monocytes, lymphocytes, and activated HSC. Increased levels of MCP-1 can amplify inflammation in liver injury and are associated with the progression of fibrosis in chronic liver disease (25). Previous studies have shown that MCP-1 regulation is closely associated with the oxidative stresssensitive expression of NF-κB (26). Indeed, in this study, we demonstrated that the mRNA levels of IKKβ, NF-κB p65 and MCP-1 were significantly elevated in MCD-treated mice when compared with control mice. Moreover, pre-administration of FZHY or hemin inhibited the expression of IKK-β, NF-κB p65 and MCP-1, and reduced inflammation and necrosis in liver sections. These data indicated that consumption of MCD diet induced liver injury that could be prevented by FZHY through modulating the NF-κB signaling pathway related genes. Additionally, the observed suppression of NF-κB expression was likely due to the restoration of antioxidant defenses and inhibition of IKK-β expression.
Growing evidence has shown that NF-κB activation can regulate not only inflammatory signals elicited in macrophages and inflammatory cells in the liver, but also fibrogenic responses in HSCs (27,28). HSCs are the predominant cells in the liver and play a central role in the process of hepatic fibrosis. The persistence of oxidative stress and inflammatory stimuli accompanying sustained injury can lead to a perpetuation phase regulated by autocrine and paracrine stimulation (29) in which HSCs transform from a quiescent state to a myofibroblastlike phenotype characterized by increases in proliferation, extracellular matrix (ECM) accumu-lation, α-SMA expression, and retinoid loss (30)(31)(32). As shown in our study, the expression of α-SMA, a marker of activated HSCs, was very weak in liver sections of normal mice, but was considerably elevated upon MCD induction. Moreover, administration of FZHY or hemin resulted in a significant decrease in α-SMA expression in the livers of MCD-treated mice.
NF-κB activation can also enhance TGF-β signaling, which plays a major role in the process of HSC activation (33). Once activated, TGF-β1 binds to its cognate receptors on the cell surface, leading to phosphorylation of the intracellular mediator Smad3. TGF-β then recruits the common partner Smad4 to form a hetero-oligomer. The resulting complex finally translocates from the cytoplasm to the nucleus, further regulating specific TGF-β1 target genes and leading to collagen formation, HSC activation, and ECM synthesis (34)(35)(36). In this pathway, antagonistic Smad7 could combine with the Smad complex in the cytoplasm and prevent stimulating signals from being transmitted into the nucleus. Therefore, Smad7 functions in a negative feedback loop to terminate or reduce the strength of the signal (37). Disrupting TGF-β signaling pathways will reduce collagen synthesis, prevent scar formation, and accelerate matrix degradation, thereby inhibiting the progression of liver fibrosis (38). In this study, we observed a marked increase in the protein expression of TGF-β1 in mice fed MCD diet. Moreover, the mRNA and protein expression levels of Smad3, Smad4 and Smad7 were also dramatically increased in the livers of mice fed MCD diet compared with those in control mice. Treatment with FZHY or hemin significantly downregulated the expression of TGF-β1, Smad3 and Smad4, and further upregulated Smad7 expression. Therefore, FZHY might play a role in inhibiting the expression of factors involved in the IKK-β/NF-κB and TGF-β1/Smad pathways, hence interrupting the positive feedback loop in hepatic fibrogenesis. This effect was supported by observations that FZHY could significantly reduce collagen deposition, downregulate the protein expression of α-SMA and TGFβ1, and inhibit the nuclear translocation of Smad3 in fibrotic kidney tissue (39).

Conclusion
The present study provided evidence of the protective role of FZHY in ameliorating hepatic oxidative stress, inflammation and fibrosis in experimental nutritional steatohepatitis. Changes in the expression levels of pro-inflammatory and pro-fibrotic genes involved in the IKK-β/NF-κB and TGF-β1/Smad pathways may be responsible for the observed effects of FZHY on alleviating steatohepatitis and fibrosis.

Acknowledgment
Financial support of the Pearl Ocean Liver Fibrosis Grant (No. 001) and Project of Hebei Provincial Administration of Traditional Chinese Medicine (No. 2013015) are appreciatively acknowledged. "The results described in this paper were part of student thesis", Financial disclosures: none declared.