Current research defines the mechanism of hepatocyte apoptosis as being controlled by the redox modulation system, which accounts for ethanol-induced liver injury. We demonstrated in this study that initial depletion of Grx1 brings about Fas s-glutathionylation during ethanol exposure, which promotes the aggregation and binding of Fas with FasL, and further activates caspases, resulting in apoptosis; these effects all occur via inactivation of the NF-κB/Akt pathway. To our knowledge, we observed the involvement of Grx1 in Fas-induced hepatocyte apoptosis for the first time. Moreover, our results showed that depletion of Grx1 in mice plays a critical role in ethanol-induced liver injury.
Oxidative stress is the main pathogenesis of ethanol-induced liver injury and exhibits a significant effect on the regulation of signaling proteins . Oxidative stress modulates glutathione to the disulfide form[22, 23], and some reports have documented that ethanol exposure decreases GSH concentration  and restores GSH to normalize ALT in vivo [25, 26], suggesting that the imbalance in GSH due to oxidative stress contributes to hepatocyte injury. In the present study, we found a decrease in the GSH level and an increase in the GSSG-to-GSH ratio during ethanol-induced liver injury in conjunction with oxidative stress. Furthermore, the increase in GSSG favored s-glutathionylation through catalysis by the protein thiol oxidoreductase Grx1, which reduces mixed disulfide bonds, especially protein s-glutathionylation (PSSG) [27, 28]. Here, we reported that ethanol exposure induced Grx1 activity in liver tissue that specifically reduced GSH mixed disulfides, whereas Grx1 depletion promoted PSSGs, which might be associated with increases in apoptosis.
Grx1 has been implicated in various diseases, including chronic pulmonary disease, lung inflammation, Pseudomonas aeruginosa pneumonia, and hepatic warm I/R injury[29, 30, 31, 32]. The reducing power of Grx1 reverses protein oxidation, thereby potentially inhibiting the subsequent deleterious influence of the protein. We previously demonstrated enhanced Grx1 activity in intestinal tissue after the induction of NEC [22, 23] and that ablation of Grx1 resulted in enhanced intestinal injury . Collectively, the current evidence [11, 33] demonstrated that Grx1 and s-glutathionylation function in diverse settings and play complex roles in disease pathogenesis. Because Grx1 has antioxidant and anti-inflammatory functions, it would be expected that Grx1 depletion would promote the susceptibility of the liver to injury. Herein, we demonstrated that Grx1 deletion promoted ethanol-induced liver injury in mice that manifested as increased plasma ASL and ALT levels and increased caspase activities.
Fas-induced apoptosis has been proposed to function in various liver diseases, such as ischemia/reperfusion injury, viral hepatitis, fulminant hepatic liver failure, and nonalcoholic and alcoholic steatohepatitis[34, 35, 36, 37]. Previous research suggested that s-glutathionylation of Fas results in robust Fas activation, which activates FasL-induced signaling and apoptosis [16, 38]. To date, the role of Grx1 in modulating Fas protein s-glutathionylation during ethanol stress has not been studied. In the present study, we demonstrated that after Grx1 ablation, increased s-glutathionylation of Fas overlapped with accelerated apoptosis that was consistent with ethanol-induced liver injury. We also demonstrated that these increases were normalized by FasL antibody, indicating an association with Fas s-glutathionylation and suggesting the involvement of the Grx1/Fas s-glutathionylation signaling axis in ethanol–induced liver injury.
In addition to the antioxidant effect, the Grx1/Fas axis might regulate the inflammatory signaling cascades that facilitate the function of Grx1 in ethanol-induced liver injury. Previous work demonstrated that nuclear factor-κB (NF-κB) was also dampened by s-glutathionylation, and Grx1 ablation further inhibited NF-κB activation [39, 40, 41]. In the current setting, NF-κB activation was reduced in ethanol-treated Grx1-ablated mice compared with that of WT mice, supporting the effect of Grx1 on NF-κB activity in the pathogenesis of ethanol-induced liver injury. NF-κB is well known as a proinflammatory transcription factor[42, 43, 44], and it could be inferred that NF-κB s-glutathionylation is responsible for the phenotype and is involved in the molecular modulation observed in the present study. We observed that NF-κB blockade promoted Fas-induced hepatocyte apoptosis in WT mice but was unable to further enhance the effect of Grx1 ablation, which supported the role of the NF-κB redox state in mediating the antiapoptotic effects of Grx1. In addition, Akt has also been linked to the regulation of Grx1[45, 46], which was also observed in the present study. TNF-α is a downstream proinflammatory factor of NF-κB, which was confirmed to be necessary for liver injury induced by ethanol . In the current study, Grx1 ablation further consolidated ethanol-induced increases in TNF-α mRNA and protein levels, which was normalized by blocking NF-κB, confirming the current hypothesis. Given the observations that the various inflammatory mediators mentioned above were regulated by Grx1 under ethanol exposure, we hypothesized that Grx1 normalizes rather than eliminates inflammatory cytokine activation, which may be a useful therapeutic target for ethanol exposure.
Under ethanol exposure, injured apoptotic cells are mostly hepatocytes, and there is the possibility that other immune cells within the liver may also undergo activation, including macrophages, infiltrating immune cells, and activated stellate cells. Here, we evaluated this hypothesis and demonstrated the hepatic macrophages accumulation and activation in ethanol-treated Grx1-/- mice. A previous report suggested that macrophage alternation in Fas-induced apoptosis accounted for increased inflammatory cytokines . A low level of early proinflammatory cytokines (i.e., TNF-α) exhibited here was in accordance with the above scenario. Furthermore, inhibition of inflammatory cell apoptosis would result in an exacerbated inflammatory or fibrotic response[49, 50]. To our knowledge, this is the first report to explore Grx1 ablation-modulation of macrophage enrichment in the liver. However, our current study was limited in that we did not address the cellular specificity of immune cell apoptosis. Additional studies are required to assess these effects, which might be beyond the scope of the current research.
In conclusion, our study illuminated a new dimension of knowledge regarding Grx1, which plays an essential role in modulating ethanol-induced liver injury. The underlying mechanisms are related to redox-based regulation of Fas/FasL-induced apoptosis by multiple roles of Grx1 in the pathogenesis of ethanol exposure. Correspondingly, Grx1 might be a critical therapeutic target for treatment of ethanol-induced liver injury.