Naloxone is a non-selective opiate receptors antagonist (Van Dorp et al. 2007), that is mainly used in treatment of opiate addiction and alcoholism (Brewer and Wong 2004). Recently,opiate antagonist has been shown to provide protection against a variety of inflammation related diseases such as Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis when used off label but within a specific dosage window (Medina-Rodriguez et al. 2020). Here, in this study, we attempted to look into possible LD NX hepatoprotective properties against Con A-induced AIH.
In the current investigation, liver function tests showed that rats given Con A alone had significantly higher blood levels of ALT, AST, and bilirubin. In addition, Con A group showed a considerable fall in blood levels of albumin and evident histological abnormalities, demonstrating a deterioration in liver function caused by Con A's hepatotoxic effects. These data are in agreement with previous studies (El- Kashef and Abdelrahman 2020; Mounieb et al.2017). Interestingly, these findings were significantly improved in animals pre-treated with LD NX, indicating that LD NX has the ability to attenuate liver damage caused by Con A.
Reactive oxygen species (ROS) have been linked in several studies (Shirin et al. 2010; Zhuang et al. 2016) to acute liver injury caused by Con A. The current investigation shows that administration of Con A induces hepatic oxidative stress as indicated by a significant increase in hepatic MDA and significant depletion in liver GSH content as well as a clear reduction in SOD and CAT activities in rats treated with a single dose injection of Con A. These findings are consistent with earlier research (Zhuang et al. 2016). Our results demonstrate that pre-treatment with LD NX before Con A injection reveal a high protection against Con A-induced oxidative stress as shown by a definite decline in hepatic MDA and restoration of the hepatic antioxidants GSH, SOD and CAT. These findings corroborated prior study that opioids antagonist are powerful antioxidants (Ebrahimkhani et al. 2006). The Nrf2/HO-1 pathway has been shown to play a significant role in the regulation of several cytoprotective genes including antioxidant ones that protects against oxidative damage (Khodir et al. 2017). In this study, pre-treatment with LD NX boosted the cellular antioxidant defense mechanism as shown by an increase in Nrf2 and HO-1 expression.
TLR4 is known to be involved in drug-induced liver damage, which can be resolved by blocking it (Ishida et al. 2021). Several investigators found that TLR4 was key in the pathophysiology of Con A-induced liver injury (Sahin et al. 2013). It is essential for the inflammatory signaling responses to diverse stimuli, which results in the transcription of a variety of inflammatory genes in NF-κB-dependent pathway (Gargiulo et al. 2015). A recent demonstration has shown that Chinese propolis protects vein endothelial cells from inflammation by interfering with MAPK/NF-κB signaling pathway (Xuan et al. 2019). Moreover, it has been reported that TLR4/NF-κB signaling pathway's activation is crucial for the start of the innate immune response and the subsequent release of inflammatory molecules (Gargiulo et al. 2015). Con A has been shown to have the capacity to activate T cells to release a variety of of hepatotoxic cytokines, including TNF-a, IFN-g, IL-1b, and IL-6 (Mounieb et al. 2017; Tiers et al.1998). Previously, it has been reported that TNF-α and ROS may both function as positive feedback signals to activate NF-κB (Shirin et al. 2010). Also, it has been demonstrated that Nrf2 regulates NF-κB expression. Notably, NF-κB can also regulate Nrf2 expression, implying a complex interdependence or bidirectional interaction between these pathways (Que et al. 2023). In our study, the expression of TLR4 was elevated in the Con A group, however, LD NX pre-treatment before Con A injection dramatically reversed this overexpression. Furthermore, pre-treatment with LD NX significantly attenuated the release of the inflammatory molecules, TNF-α, IFN-γ, IL-1β and IL-6. Moreover, NF-κB expression was highly attenuated in animals pre-treated with LD NX before Con A injection. Recently, it has been shown that curcumin has anti-inflammatory properties via blocking NF-κB and JNK signaling pathways (Ruan et al. 2022). Furthermore, it has been demonstrated that JNK plays a significant role in the development of hepatitis (Das et al. 2009). In our study, JNK expression induced by Con A was significantly reduced in rats pre-treated with LD NX before administration of Con A.
Considering that TLR4 plays a key role in controlling inflammation, we hypothesized that LD NX exerts its hepatoprotective impact in this model, at least in part, via inhibition of TLR4 expression. Supporting our data, Hutchinson and his colleagues stated that naloxone has the ability to block TLR4 signaling (Hutchinson et al. 2008). Moreover, TLR4 antagonism has contributed to various effects of naloxone that include inflammation that were noted before the drug was discovered to be a TLR4 antagonist. In animal models, naloxone, for example, demonstrated an anti-inflammatory impact against sepsis (Medina-Rodriguez et al. 2020). LD NX is thought to exert its anti-inflammatory effects through non-opioid antagonist pathways (Li et al. 2018).
According to our findings, LD NX significantly stimulated the Nrf2/HO-1 pathway which may then suppress the activation of the TLR4/NF-κB pathways and inhibit the production of inflammatory cytokines. So, its hepatoprotective effect may account for its anti-inflammatory impact and anti-oxidant effect.
Finally, our results show for the first time that LD NX pre-treatment before Con A administration significantly reduced all hepatic damage markers and improved liver function, indicating that LD NX has the capacity to protect rats from Con A-induced hepatitis via modulation of TLR4/NF-κB and Nrf2/HO-1 pathways.