Basic ScienceActivation of the STING-IRF3 pathway promotes hepatocyte inflammation, apoptosis and induces metabolic disorders in nonalcoholic fatty liver disease
Introduction
In recent decades, the rapid increase in the prevalence of obesity has become a serious global health problem [1]. Nonalcoholic fatty liver disease (NAFLD) is common in obesity and metabolic syndrome [2], and has become a main cause of liver disease in industrialized countries, including China [3], [4], [5]. Hepatic insulin resistance and type 2 diabetes are considered sequelae of NAFLD [6]. In brief, NAFLD increases the risk of insulin resistance and leads to obesity-related glucose metabolic disorders [1], yet the pathogenesis of NAFLD is complex and not well understood. The two-hit hypothesis [7] has been proposed to explain NAFLD pathogenesis. In this hypothesis, steatosis represents the “first hit”, increasing the vulnerability of the liver to various “second hits” including immune-related reactions, oxidative stress, lipid peroxidation damage, etc. [8], [9], [10], finally leading to hepatic inflammation and cellular death. These mechanisms are not well understood, and effective measures for preventing and treating NAFLD are lacking.
Stimulator of interferon genes (STING) is an important protein in the innate immune signaling pathway, newly discovered in 2008 [11]. The majority of previous studies have focused on the effects of activated STING signaling upon viral infections in immune cells. After infection, the pathogenic DNA is always present in the cytoplasm, and is sensed by cytosolic DNA sensors to initial innate immune responses [12], [13], [14], [15], [16]. However, under certain pathological conditions, self DNA also can be recognized by innate immune sensors causing several autoimmune and auto-inflammatory diseases [17]. STING, as a mediator of the innate immune signaling pathway, plays a role in linking upstream DNA sensors to downstream factors [11]. A recent study suggests that STING and interferon (IFN) regulatory factor 3 (IRF3), a downstream factor of STING, play an important role in early alcoholic liver disease [18]. Furthermore, chronic activation of STING can lead to cell metabolic disorders in an autoimmune/auto-inflammatory disease mouse model [19]. However, the specific molecular mechanism of STING in NAFLD remains poorly understood.
Given the importance of STING in inflammatory response, we explored the role of STING in NAFLD and its possible regulatory mechanism in hepatic inflammation, apoptosis, and glucose and lipid metabolic disorders using an obese mouse model with a high-fat diet (HFD) in vivo and the L-O2 human liver cell line in vitro.
Section snippets
Materials
The primary antibodies were as follows: STING (Abcam, USA, Cat: ab131604), phospho-IRF3 (S386) (Abcam, USA, Cat: ab76493), IRF3 (Abcam, USA, Cat: ab76409), IFN-β (Abcam, USA, Cat: ab176343), phospho-NF-κB p65 (CST, USA, Cat: 3033), NF-κB p65 (CST, USA, Cat: 3034), TNF-alpha (Abcam, USA, Cat: ab183218), IL-1β (BIOSS, China, Cat: bs-0812R), IL-6 (BIOSS, China, Cat: bs-0782R). Bax (Abcam, USA, Cat: ab32503), Bcl-2 (Abcam, USA, Cat: ab182858), Cleaved/total caspase-3 (CST, USA, Cat: 9915),
Evaluation of the Obese Mouse Model
The BW of C57BL/6 mice with the HFD were markedly higher than those on the NCD from 4 to 12 weeks (Fig. 1A). The body fat content (%) in mice given HFD treatment was double that of mice fed the NCD (Fig. 1B). The IPGTT (2 g/kg) showed that NCD-fed mice could tolerate glucose, whereas HFD-fed mice seemed to have impaired tolerance. The levels of fasting blood glucose (FBG) of HFD-fed mice were higher than those of NCD-fed mice. The AUC also supported this conclusion (Fig. 1C). Whole-body insulin
Discussion
In the present study, we found augmented expression of STING and phospho-IRF3 in the livers of an HFD-induced obese mouse model, as well as in the FFA-induced L-O2 cell line. After inhibiting STING and IRF3 with corresponding siRNAs, the FFA-induced levels of p-p65/p65, inflammatory cytokines, and apoptosis signaling were significantly downregulated, and both glucose and lipid metabolism tended to be normal. Therefore, the STING-IRF3 pathway might be involved in NAFLD by regulating
Author Contributions
J.T. Qiao, X.G. Hou and L. Chen conceived the study and designed the experiments. J.T. Qiao, C. Cui, L. Qing and T.Y. He performed the experiments. J.T. Qiao collected the data, analyzed the results and wrote the manuscript. X.G. Hou and L. S. Wang analyzed the data, and drafted and revised the manuscript. All other authors revised the manuscript critically for important intellectual content and approved the final version to be published. L. Chen is the guarantor of this work.
Funding
This work was supported by the National Natural Science Foundation of China (grant numbers 81370943, 81400769, and 81670706); Science and Technology Department of Shandong Province-Independent Innovation and Achievement Transformation Program (grant number 2014ZZCX02201), Jinan Science and Technology Development Plan (grant number 201503008), State Science and Technology Commission (grant number 201502007), and Discipline Development Project of Qilu Hospital of Shandong University (grant number
Conflict of Interest
The authors have no conflicts of interest to disclose.
Acknowledgements
We thank LetPub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript.
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