These authors contributed equally to this work.
Steatohepatitis/Metabolic Liver Disease
Article first published online: 25 AUG 2011
Copyright © 2011 American Association for the Study of Liver Diseases
Volume 54, Issue 3, pages 846–856, 2 September 2011
How to Cite
Miller, A. M., Wang, H., Bertola, A., Park, O., Horiguchi, N., Hwan Ki, S., Yin, S., Lafdil, F. and Gao, B. (2011), Inflammation-associated interleukin-6/signal transducer and activator of transcription 3 activation ameliorates alcoholic and nonalcoholic fatty liver diseases in interleukin-10–deficient mice. Hepatology, 54: 846–856. doi: 10.1002/hep.24517
Potential conflict of interest: Nothing to report.
Supported by the intramural program of the National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health.
- Issue published online: 25 AUG 2011
- Article first published online: 25 AUG 2011
- Accepted manuscript online: 2 JUL 2011 03:32PM EST
- Manuscript Accepted: 13 JUN 2011
- Manuscript Received: 23 MAR 2011
- intramural program of the National Institute on Alcohol Abuse and Alcoholism
- National Institutes of Health
Alcoholic and nonalcoholic steatohepatitis are characterized by fatty liver plus inflammation. It is generally believed that steatosis promotes inflammation, whereas inflammation in turn aggregates steatosis. Thus, we hypothesized the deletion of interleukin (IL)-10, a key anti-inflammatory cytokine, exacerbates liver inflammation, steatosis, and hepatocellular damage in alcoholic and nonalcoholic fatty liver disease models that were achieved via feeding mice with a liquid diet containing 5% ethanol for 4 weeks or a high-fat diet (HFD) for 12 weeks, respectively. IL-10 knockout (IL-10−/−) mice and several other strains of genetically modified mice were generated and used. Compared with wild-type mice, IL-10−/− mice had greater liver inflammatory response with higher levels of IL-6 and hepatic signal transducer and activator of transcription 3 (STAT3) activation, but less steatosis and hepatocellular damage after alcohol or HFD feeding. An additional deletion of IL-6 or hepatic STAT3 restored steatosis and hepatocellular damage but further enhanced liver inflammatory response in IL-10−/− mice. In addition, the hepatic expression of sterol regulatory element-binding protein 1 and key downstream lipogenic proteins and enzymes in fatty acid synthesis were down-regulated in IL-10−/− mice. Conversely, IL-10−/− mice displayed enhanced levels of phosphorylated adenosine monophosphate-activated protein kinase and its downstream targets including phosphorylated acetyl-coenzyme A carboxylase and carnitine palmitoyltransferase 1 in the liver. Such dysregulations were corrected in IL-10−/−IL-6−/− or IL-10−/−STAT3Hep−/− double knockout mice. Conclusion: IL-10−/− mice are prone to liver inflammatory response but are resistant to steatosis and hepatocellular damage induced by ethanol or HFD feeding. Resistance to steatosis in these mice is attributable to elevation of inflammation-associated hepatic IL-6/STAT3 activation that subsequently down-regulates lipogenic genes but up-regulates fatty acid oxidation-associated genes in the liver. (HEPATOLOGY 2011; 54:846–856)
Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are the two most prominent causes of chronic liver diseases worldwide, leading to liver fibrosis, cirrhosis, and hepatocellular carcinoma. Both diseases are histologically similar and are characterized microscopically by steatosis, hepatocellular damage, pericellular fibrosis, and inflammation with predominantly polymorphonuclear granulocytes.1-3 At present, it is not clear why only a small percentage of patients with alcoholic and nonalcoholic fatty liver develop inflammation in the liver.4, 5 Gut-derived LPS-TLR4-Kupffer cells-tumor necrosis factor α (TNF-α) axis is generally believed to play a key role in inducing inflammation in both ASH and NASH.5-10 Both ASH and NASH patients have elevated levels of several proinflammatory cytokines in the liver and serum, including interleukin (IL)-8 and IL-17, which function as critical chemoattractants and activators for neutrophils and contribute to liver inflammation and injury in these diseases.11-13 Furthermore, lipid accumulation in hepatocytes induces the production of proinflammatory cytokines14-16 and hepatic lipotoxicity that promote hepatocellular damage, Kupffer cell activation, and inflammation,6, 17, 18 suggesting that steatosis promotes liver inflammation. However, the effects of inflammation on steatosis and hepatocellular damage still remain obscure. Inflammation has been implicated in promoting steatosis and liver injury through production of proinflammatory cytokines such as TNF-α and IL-1β in mice.19, 20 The lipogenic effects of TNF-α and IL-1β are mediated through up-regulation of the master lipid synthesis transcription factor sterol regulatory element-binding protein 1c (SREBP1c)21 and the key triglyceride synthesis enzyme diacylglycerol acyltransferase,20 respectively.
In addition to producing TNF-α and IL-1β, inflammatory cells also produce hepatoprotective cytokines (such as IL-6 and IL-22) and anti-inflammatory cytokines (such as IL-10 and adiponectin) that ameliorate hepatocellular damage.22, 23 Among them, IL-10 has been shown to play the most significant role in ameliorating liver inflammation in many models.24, 25 The roles of IL-10 in ASH and NASH have been investigated, but with controversial results. Hill et al.26 reported that feeding IL-10−/− mice with alcohol in drinking water for 7 weeks enhanced LPS-induced liver inflammation and injury. Although the steatosis was not thoroughly examined in this study, the authors stated alcohol feeding induced fat accumulation in 50%-75% of both wild-type (WT) and IL-10−/− mice and that LPS treatment attenuated steatosis in both groups.26 Collective results on the role of IL-10 in high-fat diet (HFD)-induced steatosis and insulin resistance have been controversial. It was reported that disruption of the IL-10 gene has no effect on insulin resistance, yet another study has shown that blockage of IL-10 aggregates insulin resistance in HFD-fed mice.27, 28 Furthermore, den Boer et al.27 reported that IL-10−/− mice were more susceptible to steatosis induced by feeding with a HFD diet (40% calories from fat) compared with WT mice. The results from clinical studies on the association of IL-10 polymorphisms and ASH are also inconsistent. For example, Grove et al.29 reported that heavy drinkers with the −627*A allele in the IL-10 promoter were associated with an increased risk of development of advanced ASH, whereas others did not find such an association.30 Therefore, to further clarify the role of IL-10 in ASH and NASH, IL-10−/− and WT mice were fed a liquid diet containing 5% ethanol for 4 weeks or a HFD diet (60% calories from fat) for 12 weeks. As expected, our results show that IL-10−/− mice had greater liver inflammation, but surprisingly had less steatosis and liver injury compared with WT mice. We also found that in response to alcohol or HFD feeding, IL-10−/− mice produce markedly greater levels of IL-6/signal transducer and activator of transcription 3 (STAT3) activation in hepatocytes that subsequently attenuate steatosis and liver injury.
Materials and Methods
Mice, Ethanol, and HFD Feeding Models.
Eight- to 10-week-old male mice were used in this study. IL-10−/−, IL-6−/−, and WT control C57BL/6J mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Generation of hepatocyte-specific STAT3 knockout (STAT3Hep−/−) mice, IL-10−/−IL-6−/−, and IL-10−/−STAT3Hep−/− double knockout (dKO) mice is described in the Supporting Information.
For the chronic alcohol feeding model, mice were fed a Lieber-DeCarli diet containing 5% ethanol (ETOH) or pair-fed control diet as described in the Supporting Information. For the HFD feeding model, mice were fed a HFD (60 kcal % saturated lard) obtained from Research Diets, Inc. (New Brunswick, NJ) for 12 weeks or a standard diet (STD) (10 kcal % fat) as a control.
Data are expressed as means ± SEM. Eight to 10 mice/per group were used. To compare values obtained from two groups, a Student t test was performed. To compare values obtained from three or more groups, one-way analysis of variance was performed followed by Tukey's post hoc test. P < 0.05 was considered statistically significant. Statistical analyses between STD and HFD groups in Figs. 2-6 were not performed and labeled due to too many parameters.
Additional methods are described in the Supporting Information.
IL-10−/− Mice Are Prone to ETOH and HFD-Induced Hepatic Inflammatory Response and IL-6/STAT3 Activation but Are Resistant to Steatosis Development.
To investigate the role of IL-10 in ASH and NASH, WT and IL-10−/− mice were fed an ETOH diet and a HFD and their corresponding control diets. A small percentage (<10%) of IL-10−/− mice developed colitis with rectal prolapse during feeding and were removed from the experiments. The remaining IL-10−/− mice gained similar body weight during feeding (Supporting Figs. 1 and 2). In addition, IL-10−/− and WT mice had similar levels of serum ETOH concentrations after gavage (Supporting Fig. 3).
As shown in Fig. 1A-C, in WT mice, ETOH feeding induced significantly greater steatosis, liver injury (serum alanine aminotransferase [ALT] levels), and elevation of hepatic IL-6 levels compared with pair-fed groups. Hepatic phosphorylated STAT3 (pSTAT3) levels also tended to be higher in ETOH-fed mice compared with pair-fed groups, but did not reach a statistically significant difference. Surprisingly, despite increased liver inflammatory responses (Supporting Figs. 4 and 5), IL-10−/− mice were resistant to ETOH-induced steatosis and elevation of serum ALT (Fig. 1A,B) compared with WT mice. In addition, hepatic IL-6 messenger RNA (mRNA) and pSTAT3 protein levels were markedly higher in IL-10−/− mice versus WT mice (Fig. 1B,C).
Results from HFD feeding are shown in Fig. 1D-F. In WT mice, HFD feeding induced markedly greater fatty liver, and elevation of ALT, hepatic IL-6, and pSTAT3 activation compared with STD feeding. IL-10−/− mice were prone to HFD-induced liver inflammatory response (Supporting Figs. 2 and 3 and Supporting Table 1) and elevation of hepatic IL-6/pSTAT3 but were resistant to HFD-induced steatosis and elevation of serum ALT compared with WT mice.
Elevation of IL-6 Contributes to the Resistance of IL-10−/− Mice to HFD- and ETOH-Induced Steatosis and Hepatic Injury.
The hepatoprotection of IL-6/STAT3 in ameliorating fatty liver diseases has been well-documented in many rodent models.22 Therefore, we hypothesized that the elevated IL-6/STAT3 activation is responsible for the resistance of IL-10−/− mice to ETOH- or HFD-induced steatosis. To test this hypothesis, we made an additional deletion of IL-6 in IL-10−/− mice to generate IL-10−/−IL-6−/− dKO mice.
Four lines of mice were fed a STD or HFD for 12 weeks. As shown in Fig. 2, compared with WT mice, HFD-induced steatosis and serum ALT elevation were exacerbated in IL-6−/− mice but diminished in IL-10−/− mice, and the additional deletion of IL-6 restored the HFD-induced steatosis and serum ALT elevation in IL-10−/− mice to levels comparable to WT mice as verified by way of histological analysis (Fig. 2A) and by measuring hepatic triglyceride and serum ALT (Fig. 2B) levels.
Hepatic expression of several inflammatory markers and cytokines is shown in Fig. 2C,D. In general, compared with WT mice, IL-6−/− mice had comparable expression levels whereas IL-10−/− mice had higher expression levels of these genes in both the STD and HFD groups. Expression of most of these genes was exacerbated in IL-10−/−IL-6−/− dKO mice compared with IL-10−/− mice.
Fig. 2E shows the serum cytokine levels. Compared with WT mice, IL-6−/− mice had similar levels of serum TNF-α and interferon-γ (IFN-γ), whereas IL-10−/− mice had higher levels of these cytokines in both the STD and HFD groups. Serum levels of IFN-γ were further elevated in IL-10−/−IL-6−/− dKO mice versus IL-10−/− mice after HFD feeding. Finally, serum levels of IL-6 were higher in IL-10−/− mice than those in WT mice. As expected, IL-6 levels were not detected in IL-6−/− and IL-10−/−IL-6−/− dKO mice.
Four lines of mice were also fed an ETOH diet and pair-fed for 4 weeks, and analyzed similarly to the studies shown in Fig. 2. In general, findings similar to the HFD model were seen in the ETOH feeding model and are described in Supporting Fig. 4.
Activation of Hepatocyte STAT3 Contributes to the Resistance of IL-10−/− Mice to HFD- and ETOH-Induced Steatosis and Hepatic Injury.
As shown in Fig. 3A,B, IL-10−/− mice were resistant to HFD-induced steatosis and serum ALT elevation compared with WT mice, which was partially restored in IL-10−/−STAT3Hep−/− dKO mice. This suggests that enhanced hepatic STAT3 activation is responsible for the reduced steatosis and liver injury in IL-10−/− mice after HFD feeding.
Furthermore, Fig. 3C,D shows that hepatic mRNA levels of several inflammatory markers and cytokines were highest in IL-10−/−STAT3Hep−/− mice, followed by IL-10−/− mice and WT mice in both the STD and HFD-fed groups. Serum levels of TNF-α, IFN-γ, and IL-6 were also higher in IL-10−/−STAT3Hep−/− mice than those in IL-10−/− mice (Fig. 3E). Experiments similar to the HFD model described in Fig. 3 were also performed in the ETOH model. Similar changes, albeit to a lesser extent, were observed in the ETOH model (Supporting Fig. 5).
An Additional Deletion of IL-6 or Hepatic STAT3 Diminishes STAT3 Activation but Increases STAT1 Activation in HFD-Fed IL-10−/− Mice.
To further understand the mechanisms by which IL-10−/− mice are prone to inflammatory response but resistant to steatosis induced by HFD or ETOH diet, we examined the activation of STAT3 (pSTAT3) and pSTAT1, which play an important role in controlling steatosis and liver inflammation.31 Because the HFD model induces more dramatic phenotypes compared with the ETOH model, the studies on the underlying mechanisms were predominately focused in this HFD model.
As shown in Fig. 4A, in both the STD and HFD groups, hepatic levels of pSTAT3 were lower in IL-6−/− mice but higher in IL-10−/− versus WT mice. Compared with IL-10−/− mice, IL-10−/−IL-6−/− mice had significantly lower levels of hepatic activated pSTAT3 expression, while expression of STAT3 was comparable in these two groups. Additionally, expression of pSTAT1 and STAT1 protein was higher in the HFD-fed group versus the STD group, with the greatest expression in IL-10−/− IL-6−/− mice.
IL-6 and Its Downstream Signal STAT3 Are Responsible for the Down-regulation and Up-regulation of Hepatic Lipogenic and Fatty Acid Oxidation Genes, Respectively, in HFD-Fed IL-10−/− Mice.
In agreement with previous reports, HFD feeding up-regulated hepatic protein expression of nuclear mature SREBP-1c, a master transcription factor that controls lipogenic gene expression, and the expression of its target genes acetyl-coenzyme A carboxylase (ACC1) and fatty acid synthase (FAS) (WT in the HFD group versus the STD group [Fig. 5A]). Hepatic expression of SREBP-1c, ACC1, and FAS was higher in IL-6−/− mice but lower in IL-10−/− mice compared with those in WT mice (Fig. 5A,B). Reduced expression of these genes in IL-10−/− mice was partially reversed in IL-10−/−IL-6−/− dKO mice (Fig. 5A,B).
Activation of adenosine monophosphate-activated protein kinase (AMPK) plays a key role in controlling lipid metabolism by phosphorylating and subsequently inhibiting ACC and suppressing the expression of ACC and FAS through down-regulation of SREBP-1c.32 ACC is an important enzyme for fatty acid synthesis, which catalyzes the first step in de novo fatty acid biosynthesis by converting acetyl coenzyme A to malonyl coenzyme A. Malonyl coenzyme A acts as a potent inhibitor of fatty acid oxidation by inhibiting carnitine palmitoyltransferase 1 (CPT-1), which transports fatty acids into the mitochondria for oxidation.33, 34 As shown in Fig. 5, expression of activated (i.e., phosphorylated) AMPK (pAMPK) was significantly higher in IL-10−/− mice than that in WT mice in both the STD and HFD groups, whereas such up-regulation was diminished in IL-10−/−IL-6−/− mice. Expression of pAMPK was comparable between IL-6−/− mice and WT mice. Consistent with the elevated levels of pAMPK, IL-10−/− mice had higher levels of inhibited (i.e., phosphorylated) ACC1 (pACC1) compared with WT mice. Such elevated phosphorylated ACC1 was reduced in IL-10−/−IL-6−/− mice versus IL-10−/− mice. In addition, hepatic expression of CPT1 was higher in HFD-fed IL-10−/− mice compared with WT mice. An additional deletion of IL-6 reduced hepatic CPT1 expression in IL-10−/−IL-6−/− mice versus IL-10−/− mice.
Expression of these lipid metabolism-associated genes were also examined in WT, IL-10−/−, and IL-10−/− STAT3Hep−/− mice (Fig. 6). Compared with WT mice, IL-10−/− mice had reduced expression of SREBP-1c, ACC1, and FAS but enhanced expression of pAMPK, pACC1, and CPT-1 in the liver. These dysregulations were partially corrected in IL-10−/− STAT3Hep−/− mice.
In this article, we have demonstrated that IL-10−/− mice have greater liver inflammatory response but less steatosis and liver injury compared with WT mice after feeding with an ETOH or HFD diet. Our data suggest that in our models, inflammatory response reduces rather than promotes steatosis through activation of hepatic IL-6/STAT3, which subsequently inhibits the expression of lipogenic genes (SREBP-1c, ACC1, and FAS). In concert, IL-6 up-regulates the expression of CPT-1 and activates AMPK, which in turn further attenuates the expression of SREBP-1c and its target genes and inhibits ACC1. We have integrated our findings in a model depicting the effects of inflammation on steatosis in IL-10−/− mice (Fig. 7).
Several lines of evidence suggest that inflammation-associated elevation of IL-6/STAT3 contributes to reduced steatosis in ETOH- or HFD-fed IL-10−/− mice. First, serum and hepatic IL-6 levels and activation of hepatic STAT3 were higher in IL-10−/− mice versus WT mice (Figs. 1-4 and Supporting Figs. 4 and 5). Second, the hepatoprotection of IL-6/STAT3 in steatosis has been well-documented in both ETOH and HFD models.31, 35 Third, an additional deletion of IL-6 or hepatic STAT3 restores steatosis and liver injury in IL-10−/− mice, providing conclusive evidence that elevated IL-6/STAT3 activation contributes to the reduced steatosis and hepatocellular damage in IL-10−/− mice. Finally, it is well established that the antisteatotic effects of IL-6/STAT3 are mediated through the inhibition of lipogenic genes (SREBP-1c, ACC, and FAS) and stimulation of fatty acid oxidation genes (pAMPK and CPT-1) in the liver.35-37 Our results revealed that expression of these lipogenic genes and fatty acid oxidation genes were down-regulated and up-regulated, respectively, in IL-10−/− mice and that these dysregulations were corrected after an additional deletion of IL-6 or hepatic STAT3 in dKO mice, suggesting that IL-6/STAT3 activation is responsible for inhibition of lipogenic genes and up-regulation of fatty acid oxidation genes in IL-10−/− mice. The mechanism by which the IL-6/STAT3 activation mediates the decrease in lipogenic gene expression may involve the interaction of STAT3 and SREBP-1c promoter. Numerous studies have shown that activated STAT3 inhibits SREBP-1c promoter activity in hepatocytes38 and results in decreased SREBP-1c protein expression,35-37 suggesting that STAT3 activation can directly inhibit SREBP-1c promoter activity and subsequently attenuate SREBP-1c–controlled lipogenic genes. However, how STAT3 inhibits SREBP-1c promoter activity remains unknown.
Whereas IL-10 is a well-documented anti-inflammatory cytokine,39 IL-6 acts as a proinflammatory cytokine in various conditions.40 In the liver, IL-6 is implicated in promoting liver inflammation through activation of hepatic STAT3 and subsequent production of acute phase proteins in various liver injury models.41 Interestingly, an additional deletion of IL-6 or hepatic STAT3 exacerbated rather than reduced liver inflammatory response in IL-10−/− mice (Figs. 1-3), suggesting that IL-6 acts as an anti-inflammatory cytokine through activation of hepatocyte STAT3 in IL-10−/− mice in our models. By using hepatocyte-specific IL-6 receptor knockout mice, Wunderlich et al.42 recently also reported that IL-6 acts as an anti-inflammatory cytokine by targeting hepatocytes. One potential explanation for the anti-inflammatory effect of IL-6/STAT3 in our models is its hepatoprotection in reducing steatosis and liver injury, subsequently preventing steatosis/injury-associated inflammation. Another explanation for the anti-inflammatory effect of IL-6/STAT3 is mediated by the inhibition of hepatic STAT1, a key signaling pathway to promote liver inflammation.43 As shown in Fig. 4, hepatic expression of activated pSTAT1 was markedly higher in HFD-fed IL-10−/−IL-6−/− and IL-10−/−STAT3Hep−/− dKO mice compared with IL-10−/− mice, indicating that IL-6/STAT3 activation is responsible for inhibiting STAT1 activation.
In conclusion, IL-10−/− mice displayed greater liver inflammatory response but less steatosis after ETOH or HFD feeding compared with WT mice, and inflammation-associated IL-6/STAT3 activation contributes to the reduced steatosis in these mice. Interestingly, hepatic IL-6/STAT3 is also activated in WT mice after ETOH or HFD feeding, but to a lesser extent compared with IL-10−/− mice (Figs. 1-3). This finding suggests that endogenous IL-10 plays an important role in inhibiting hepatic IL-6/STAT3 activation, which may account for the weak activation of this signaling pathway in the liver in WT mice during ETOH or HFD feeding. It has been reported that hepatic levels of IL-10 were elevated in mice after an 8 weeks of HFD feeding;28 however, we did not observe hepatic IL-10 up-regulation in WT mice after 12 weeks of HFD or 4 weeks of ETOH feeding. In contrast, we observed marked up-regulation of hepatic IL-10 mRNA in mice fed with HFD for 1 year (unpublished data). Furthermore, it has been reported that hepatic expression of IL-10 mRNA is not up-regulated in obese individuals without fatty liver but markedly up-regulated in those with fatty liver, which is further increased in individuals with NASH.44 This suggests that hepatic IL-10 is elevated after long-term HFD consumption in patients and in mice, which may play a compensatory role in preventing inflammation in fatty liver disease.
The fact that IL-10−/− mice had greater liver inflammatory response but less steatosis suggests that inflammation, as reported previously, may not only promote the development of fatty liver by producing TNF-α and IL-1 but may also ameliorate the fatty liver by producing cytokines (such as IL-6) that activate STAT3. Therefore, the overall effect of inflammation on hepatic steatosis is determined by the balance between detrimental cytokines that promote steatosis and hepatoprotective cytokines that prevent steatosis. It is of keen interest to explore the effect of inflammation on steatosis in patients with ASH and NASH. Recently, Bertola et al.44 reported that the liver of obese patients without obvious steatosis (S0) was associated with elevation IL-6 but not TNF-α and IL-1β. It is plausible that such elevation of inflammation-associated IL-6 plays a compensatory role in preventing the development of steatosis in the early stage of nonalcoholic fatty liver in obese patients. The liver of obese patients with severe steatosis (S3) and NASH was associated with highest fold induction of IL-6, followed by TNF-α and IL-1β. It is probable that the steatosis in these patients is modulated negatively by IL-6 but positively by TNF-α and IL-1β. Modulation of the balance between these cytokines may be a therapeutic option to treat nonalcoholic fatty liver disease and NASH.
Additional Supporting Information may be found in the online version of this article.
|HEP_24517_sm_SuppInfoFig1.tif||649K||Fig. SI. Body weight. Various strains of mice were fed a HFD and a control diet for 12 weeks. Body weight measured and recorded weekly.|
|HEP_24517_sm_SuppInfoFig2.tif||645K||Fig. S2. Body weight. Various strains of mice were fed an ETCH diet and pair fed diet for 4 weeks . Body weight was measured before and after feeding.|
|HEP_24517_sm_SuppInfoFig3.tif||633K||Fig. S3. Blood ethanol concentrations after gavage of ethanol. Various strains of mice were gavaged with ethanol (59/kg body weight). Blood ethanol concentrations were measured 2 hours later.|
|HEP_24517_sm_SuppInfoFig4.tif||3599K||Supplemental Fig. S4. An additional deletion of IL-6 restores ETOH-induced steatosis and elevation of serum ALT in IL-104 mice. Male C57BL/6J (WT), IL-lO-′-, IL-6 ′-, IL-10′1L-6′ mice were fed ETCH diet or pair-fed for 4 weeks. (A) H&E staining of liver sections from ETCH- and pair-fed mice. (B) Liver triglyceride levels (upper panel) and serum ALT levels (lower panel). (C) Real-time PCR analyses of hepatic inflammatory markers CCR2 and F480. (D) Real-time PCR analyses of hepatic cytokines. (E) Serum levels of proinflammatory cytokines. *p<005 **D<QQ in comparison with corresponding WT group; # P<0.05, ## P<0.05 in comparison with corresponding IL-i 0′ group.|
|HEP_24517_sm_SuppInfoFig5.tif||3031K||Supplemental Fig. S5. An additional deletion of hepatocyte STAT3 restores ETOHinduced steatosis and elevation of serum ALT in IL-104 mice. WT, IL-10′, IL-10′ STAT3HeP-/- mice were fed ETCH diet or pair-fed for 4 weeks. (A) H&E staining of liver sections from ETCH and control fed mice. (B) Liver triglyceride levels (upper panel) and serum ALT levels (lower panel). (C) Real-time PCR analyses of hepatic inflammatory markers CCR2 and F4180. (D) Real-time PCR analyses of hepatic cytokines. (E) Serum levels of proinflammatory cytokines. # P<0.05, P<0.05. Statistical analyses were only performed between IL-i OSTAT3HeP′ dKC and IL-i 0′ mice. # P<0.05, ## P<0.01.|
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