Hydrogen sulfide (H₂S) is a well-known gas transmitter that mediates various physiology and signaling in various human tissues (Li et al., 2011). The liver is an important site for endogenous H₂S production that is mediated by cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE) which is the primary enzyme for H₂S generation in the liver tissues (Fiorucci et al., 2005; Mustafa et al., 2009). Multiple studies have shown that H₂S displays a significant role in regulating hepatic physiology and pathology (Li X. et al., 2019; Wu et al., 2019). Accumulated data pointed that the protective effect mediated by H₂S in the hepatic pathology and toxicity is mainly orchestrated by it is anti-oxidative, anti-inflammatory effects (Fouad et al., 2020; Liu et al., 2020).

Hepatotoxicity is considered one of the major side effects that limit the clinical use of cyclophosphamide (CP) as a potent alkylating agent against various human malignancies and immunological disorders (Taslimi et al., 2019). The hepatotoxicity associated with CP therapy is mainly attributed to the major toxic acrolein produced as a result of CP hepatic bioactivation (King and Perry, 2001; Mahmoud et al., 2017). Acrolein depletes cellular antioxidant defenses and increases the generation of reactive oxygen species (ROS) and oxidative stress in hepatocytes (Mohammad et al., 2012). Consequently, it is currently believed that oxidative stress might represent the main driver of hepatotoxicity associated with CP therapy (Mahmoud et al., 2017; ALHaithloul et al., 2019).

ROS triggers the activation of critical signaling molecules including toll-like receptors (TLRs) which have a critical role in regulating innate immunity and inflammatory responses (Li et al., 2014; Li Y. et al., 2019). TLRs are expressed on different hepatocytes specially Kupffer cells and their activity is strongly correlated to the hepatic stress reaction (Gustot et al., 2006; Ojaniemi et al., 2006). Moreover, TLRs ligation is associated with initiating proinflammatory pathways resulting in activation of both c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB) which have a prominent role in cellular apoptosis (Li et al., 2020). Although TLRs-mediated signals have been implicated in various liver diseases, the significance of TLRs activation and their two different downstream pathways in CP-induced hepatotoxicity are yet to be evaluated.

Previous reports illustrated the therapeutic potential of H₂S in several hepatic diseases, including hepatic ischemia/reperfusion (I/R) injury (Kang et al., 2009), nonalcoholic steatohepatitis (Li et al., 2018), liver fibrosis (Song et al., 2015), and liver cancer (Yin et al., 2012), But in the term of CP-induced hepatotoxicity, the role of H₂S is still unclear and needs more investigation. Our recent study showed that H₂S can protect renal cells against CP-induced oxidative damage (Waz et al., 2021). Additionally, earlier reports showed that H₂S and CSE biosynthesis during inflammation have displayed TLR/NF-κB and TLR/JNK-dependent manner (Kandil et al., 2010; Zheng et al., 2013; Huang et al., 2016). In this regard, the current study aims to investigate the impact of endogenous and exogenous H₂S on TLRs pathways in CP-induced hepatotoxicity. A pre-treatment with either NaHS, an exogenous H₂S donor, or DL-propargylglycine (PAG), an irreversible CSE inhibitor, was investigated in CP-treated rats with hepatotoxicity in terms of modulation of TLRs-mediated inflammatory response, oxidative stress, and apoptosis.

Hepatotoxicity is one of the major side effects that limit the therapeutic use of CP in the clinical setting. Hepatotoxicity associated with CP antineoplastic action is mainly attributed to acroline, the toxic CP hepatic metabolite, which evolves ROS and highly interferes with the antioxidant defense system. Indeed, oxidative stress is the main detrimental factor that has been formerly incriminated in the pathogenesis of liver injury and dysfunction induced by CP. Findings from the present study showed that CP-treated rats showed a liver injury evident by altered hepatic histological structure and increased serum liver markers. The observed liver injury in CP-treated rats is associated with increased oxidative stress in terms of increased MDA and NO_x ⁻ levels along with decreased GSH levels in the liver.

Accumulating evidence supported the beneficial effect of H₂S-based therapy in different models of liver injury (Kang et al., 2009; Zhang et al., 2013; Wu et al., 2015; Wu et al., 2019; Fouad et al., 2020). Current data showed that pretreatment with H₂S donor (NaHS) suppressed lipid peroxidation and elevated levels of GSH in liver tissues of CP-induced rats. The potent anti-oxidant effect that is recorded in the NaHS group went along the marked improvement in liver function and pathological changes. The current results are consistent with previous reports showed H2S regulation of redox reaction and exert ROS scavenger effect (Mani et al., 2011; Huang et al., 2016). Lee et al., 2014) showed that activation of cysteine/cystine transporters mediated by H2S leads to elevating GSH production which protects against ROS-mediated damage.

Interestingly, pretreatment with PAG, an irreversible inhibitor of CSE, in CP-injured rats did not show any alterations in liver function, structure, or oxidative status compared to CP-treated animals. Although hepatic expression of CSE exceeds that of CBS by nearly 60 folds (Singh and Banerjee, 2011), a study by Mani et al. (2011) showed that CSE knockouts were not associated with any indices of hepatic disorders including ALT, AST, and albumin levels. The neutral effect of CSE inhibition in CP-treated animals on hepatic function could be justified by the insignificant role of CSE on oxidative status. Previous results showed that increased plasma homocysteine levels are associated with CBS deletion in the liver but not CSE deletion (Robert et al., 2005; Yang et al., 2008). As elevated homocysteine plasma triggers ROS production and impairs GSH-related anti-oxidant defense (Liu et al., 2013), reduced hepatic oxidative stress is suggested to be linked to CBS, not CSE.

In the liver tissues, TLRs are extensively expressed in hepatocytes and other cells, and they showed a critical role in liver physiological function (Schwabe et al., 2006; Chen and Sun, 2011). The most prominent hepatic TLRs are TLR2 and TLR4 have been shown to be important for the production of the inflammatory response observed in different models of experimental hepatic injury such as hepatic ischemia, inflammation, and acute hepatic failure (Tu et al., 2012; Mahmoud et al., 2014; Qiu et al., 2018). Importantly, activation of TLRs signaling pathways is reported to be regulated during oxidative stress (Gustot et al., 2006). Additionally, it was reported that intracellular ROS production is a potential activator for TLR2/4 expressions (Huang et al., 2011). In our data, the hepatic level of TLR2/4 showed a significant increase upon CP-treatment. These elevated levels were detected along with oxidative stress exhibited in this group, which conceivably showed that ROS may be the trigger for TLR4/2 expression. Equally important, previous studies showed that TLR2/4, mediated inflammatory and oxidative stress activities, are initiated in response to damaged cells in the liver tissue (Chang and Toledo-Pereyra, 2012; Mahmoud et al., 2014). Hence, it is suggested that liver injury induced by CP-toxicity may be a direct stimulator for TLR2/4 expression which, in turn, can induce ROS production and causes oxidative stress. However, this hypothesis requires further investigation.

In conjunction with oxidative stress, previous studies reported significant increments of inflammatory cytokines including NF-κB and TNF-α in the serum of CP-treated rats (Mahmoud et al., 2017). It is well known that TLR2/4 induced MyD88-dependent and independent signaling cascade to initiate translocation of NF-κB and subsequent activation of TNF-α (Matsumura et al., 2000; Chang and Toledo-Pereyra, 2012). These findings went along with our results which showed elevated levels of NF-κB and TNF-α in the liver tissues of CP-treated animals along with increased hepatic TLR2/4 detected in the same group.

Data of the present study showed substantial decrease of TLR2/4 and NF-κB expressions in the liver tissue of the NaHS group that could be explained by the ROS quenching effect of exogenous H₂S. The current data is supported by previous studies showed that H2S exert an anti-inflammatory effect via multiple mechanisms including upregulation of antioxidant defense and regulating inflammatory signal transduction (Pan et al., 2011; Li et al., 2016). Hence, it is also plausible to attribute the insignificant change in hepatic TLR2/4 levels observed along with PAG pretreatment to it is neutral effect on the oxidative status of CP-treated rats. Furthermore, the results of (Huang et al., 2016) consolidate the data of the present study as it concluded that exogenous H₂S can mediate its anti-inflammatory effect via a direct suppression of activated TLR4/NF-κB pathway in hyperglycemic-injured cardiomyocytes (Huang et al., 2016).

A recent study by Mohammed et al. (2020) showed the involvement of basic inflammatory pathways such as JNK in an animal model of CP-induced hepatotoxicity (Mohammed et al., 2020). The aforementioned study showed that oxidative and nitrosative stress induced by CP contributes to JNK activation in the liver tissues, which results in piling up the ROS in the cell. Moreover, other previous results showed that JNK is activated by ROS generated as a result of GSH-depleted mitochondria and peroxynitrite formation in liver-injured animal models (Hanawa et al., 2008; Kandil et al., 2010). On the other hand, other studies suggested that TLR2/4-induced JNK stimulates the production of pro-inflammatory cytokines mainly TNF-α, besides ROS which is believed to be a major contributor to liver injury (Chang and Toledo-Pereyra, 2012; Li W. et al., 2019; Gong et al., 2019). Here, we demonstrated an upregulation of JNK expression in CP-injured liver tissues along with increased hepatic TLR2/4 protein levels and oxidative stress. More importantly, the results of the present study showed a significant decrease in hepatic JNK expression along with NaHS pretreated CP-induced injured rats. The accumulated data point to the role of exogenous H₂S in modulating mitogen-activated protein kinases (MAPKs) signaling and JNK as a dominant effector of MAPKs in liver tissues. In fact, the reported data showed an ameliorating effect on oxidative stress and cell injury along with H₂S supplement that may be secondary to alleviating MAPK/JNK signaling pathway (Xu et al., 2011; Yuan et al., 2017; Li X. et al., 2019). In harmony, NaHS pretreatment showed protection against oxidative stress in CP-treated animals along with decreased hepatic protein expression of p-JNK. Although CSE is responsible for more than 90% of hepatic biosynthesis capacity (Singh and Banerjee, 2011), it is interesting to find out that hepatic p-JNK expression in the PAG group did not change in comparison to CP-treated rats. Such findings reinforced the probability that endogenous H₂S may not be adequately abated due to the existence of alternative routes of H₂S production including the action of CBS and 3-mercaptopyruvate sulfurtransferase (3-MST).

ROS and pro-inflammatory cytokines could potentially trigger apoptosis and cell death in liver tissue (Heeba and Mahmoud, 2014; Hamza et al., 2020). Here, CP treatment induced apoptotic cell death as shown by increased hepatic caspase-3 expression along with enhanced oxidative stress which coincided with previous studies (Caglayan et al., 2018; Aladaileh et al., 2019). The present results showed that CP-mediated excessive ROS levels are associated with activated TLR2/4 signaling pathways which are suggested to have a central role in initiating an apoptotic response. Indeed, it is believed that TLR2/4 can activate the initiator of the apoptotic cascade via interaction with the Fas-associated death domain protein (FADD) through MyD-88 which leads to ultimately activated caspase-3 (Li et al., 2014; Liu et al., 2015). The anti-apoptotic effect of H₂S donner was emphasized in our work via detecting a significant reduction of hepatic caspase-3 expression in the NaHS-treated group. In harmony, Tan et al. (2015) reported the anti-apoptotic and anti-inflammatory actions of H₂S in renal I/R injury via ameliorating the activated TLR2/4 (Tan et al., 2015). Conspicuously, inhibiting TLR2/4 signaling averts translocation of NF-κB and activation of JNK which would eventually lead to preventing cell apoptosis (Li et al., 2014). Additionally, another study showed that the anti-apoptotic and anti-inflammatory effects of exogenous H₂S may be mediated by inhibiting JNK phosphorylation (Li X. et al., 2019), downregulation of NF-κB (Li et al., 2016), or by direct inhibition of ROS production (Spassov et al., 2017). Consequently, it is possible that the neutral effect of PAG pretreatment on hepatic caspase-3 expression might be linked to its non-significant effect on the oxidative status of CP-treated rats.

Taken together, these findings suggested that exogenous H₂S plays an important role in the protection against CP-induced hepatotoxicity via alleviating oxidative stress, inflammatory and apoptotic responses in liver tissues of CP-treated animals. The present study also indicated that ameliorating ROS generation and suppression of TLRs-JNK/NF-κB signaling pathways are the proposed molecular mechanisms underlying the hepatoprotective effect of NaHS in this model. Further investigations are warranted to explore the clinical application of H2S donors against CP-induced hepatotoxicity.