TLRs-JNK/ NF-κB Pathway Underlies the Protective Effect of the Sulfide Salt Against Liver Toxicity

Abstract

Hydrogen sulfide (H₂S) is an endogenously gas transmitter signaling molecule with known antioxidant, anti-inflammatory, and cytoprotective properties. Although accumulating evidence shows the therapeutic potential of H₂S in various hepatic diseases, its role in cyclophosphamide (CP)-induced hepatotoxicity remains elusive. The present study was undertaken to investigate the impact of endogenous and exogenous H₂S on toll-like receptors (TLRs)-mediated inflammatory response and apoptosis in CP-induced hepatotoxicity. Either an H₂S donor (NaHS (100 μM/kg) or an H2S blocker [dl-propargylglycine (PAG) (30 mg/kg, i. p.)], was administered for 10 days before a single ip injection of CP (200 mg/kg). NaHS attenuated conferred hepatoprotection against CP-induced toxicity, significantly decreasing serum hepatic function tests and improving hepatic histopathology. Additionally, NaHS-treated rats exhibited antioxidant activity in liver tissues compared with the CP group. The upregulated hepatic levels of TLR2/4 and their downstream signaling molecules including c-Jun N-terminal kinase (JNK) and nuclear factor-kappa B (NF-κB) were also suppressed by NaHS protective treatment. NaHS showed anti-inflammatory and antiapoptotic effects; reducing hepatic level tumor necrosis factor-alpha (TNF-α) and caspase-3 expression. Interestingly, the cytotoxic events induced in CP-treated rats were not significantly altered upon the blocking of endogenous H₂S. Taken together, the present study suggested that exogenously applied H₂S rather than the endogenously generated H₂S, displayed a hepatoprotective effect against CP-induced hepatotoxicity that might be mediated by TLRs-JNK/NF-κB pathways.

Keywords: hepatotoxicity; hydrogen sulfide; inflammatory response; oxidative stress; toll-like receptors.

FIGURE 1

Effect of NaHS and PAG on the serum levels of ALT (A) and AST (B) in CP-induced induced hepatotoxicity in rats. Data are represented as mean ± SEM. *,+,° are significantly different from control, CP and NaHS groups, respectively, where n = 6 and p < 0.05. CP; cyclophosphamide,PAG; DL-propargylglycine, ALT; alanine aminotransferase, AST; aspartate aminotransferase.

FIGURE 2

Effect of NaHS and PAG on rats’ liver tissues stained with hematoxylin and eosin (H&E) in CP- induced hepatotoxicity (×200 and ×400). Liver tissue of control group (A) showed normal morphological features of hepatic parenchyma with many apparent intact radiating hepatocytes. Liver tissues of rats treated with CP (B) showed sever diffuse hepatocellular vacuolar degeneration with karyopyknosis (arrow) accompanied with moderate dilatation of hepatic blood vessels (star) and mild records of periportal inflammatory cells infiltrates (red arrow). Liver tissues of NaHS rat group) showing intact histological structure of hepatic lobule with few degenerative changes (black arrow) mild hepatic blood vessel dilatation (star) or inflammatory cells infiltrates (C). microscopic examination of liver tissues of PAG rat group (D) showed wide diffuse areas of vacuolar degenerative changes of most of hepatocytes (arrow) with many dilated, congested hepatic blood vessels (star) and mild records of focal perivascular inflammatory cells infiltrates (red arrow).

FIGURE 3

Effect of NaHS and PAG on the severity of histopathological lesions in CP-induced hepatotoxicity in rats. All parameters were represented as mean score. Kruskal–Wallis and then Dunn’s test was applied for comparison. * is significantly different from control where p < 0.05. CP; cyclophosphamide, PAG; DL-propargylglycine.

FIGURE 4

Representative Western blot analysis of the effect of NaHS and PAG on hepatic TLR4 (A) and p-JNK (B) protein expressions in CP-induced hepatotoxicity in rats, showing protein bands of each group (upper panel) and graphs present their densitometric analysis (lower panel). Data are represented as mean ± SEM. *,+,° are significantly different from control, CP and NaHS groups, respectively, where n = 6 and p<0.05. CP; cyclophosphamide, PAG; DL-propargylglycine, TLR4; Toll-like receptor4, p-JNK; phosphorylated Jun N-terminal kinase.

FIGURE 5

The effect of NaHS and PAG on hepatic level of TLR-2 (A) and TNF-α (B) in CP-induced hepatotoxicity in rats. Data are represented as mean ± SEM. *,+,° are significantly different from control, CP and NaHS groups, respectively, where n = 6 and p<0.05. CP; cyclophosphamide, PAG; DL-propargylglycine, TLR2; Toll-like receptor 2, TNF-α; tumor necrosis factor-alpha.

FIGURE 6

Representative photomicrographs of immunohistochemical analysis of hepatic NF-κB protein expression (A) Control group, (B) CP group, (C) NaHS group, (D) and PAG group. All reactive hepatocytes are labeled with red arrows, while black arrows indicate negative reactive hepatocytes. (E) A semi-quantitative analysis of NF-κB in rat’s liver tissue. Data are represented as mean ± SEM. *, +, ° are significantly different from control, CP and NaHS groups, respectively, where n = 6 and p < 0.05. CP; cyclophosphamide, PAG; DL-propargylglycine, NF-κB; nuclear factor kappa B.

FIGURE 7

Representative photomicrographs of immunohistochemical analysis of hepatic caspase-3 protein expression (A) Control group, (B) CP group, (C) NaHS group, and (D) PAG group. All reactive hepatocytes are labeled with red arrows, while black arrows indicate negative reactive hepatocytes. (E) A semi-quantitative analysis of caspase-3 in rat’s liver tissue. Data are represented as mean ± SEM. *, +, ° are significantly different from control, CP and NaHS groups, respectively, where n = 6 and p < 0.05. CP; Cyclophosphamide, PAG; DL-propargylglycine.