In order to closely mimic human liver fibrosis and cirrhosis, the organosulfur chemical Thioacetamide (C2H5NS) is widely used to induce efficient animal models for acute and long-term liver damage [23]. Via the CYP450, TAA is changed into the extremely hazardous reactive metabolite TAA-sulfur dioxide (TAA-S-dioxide), which attaches to liver parenchyma and macromolecules, triggering necrosis and activating HSCs [24]. TAA S-dioxide compromises the integrity and stability of cellular membranes, triggering them to become more permeable and allowing the escape of liver enzymes, AST and ALT [25]. Our study results demonstrated that TAA significantly damaged the liver, showed by a significant rise in serum ALT and AST activities and a marked drop in albumin level. These results agreed with those of earlier investigations [26–28]. The lowered enzyme activity and restored serum albumin level show that treatment with LC significantly preserved hepatocyte integrity in a dose-dependent manner. Our data proved that LC offers protection against liver damage caused by TAA. Our findings are consistent with earlier research [15, 29].
Under normal physiological conditions, endogenous antioxidant defense mechanisms, which include antioxidant enzymes (SOD, CAT, etc.) and non-enzymatic (GSH, etc.), kept oxidative stress formation and clearance in a cellular homeostasis [30]. The development of numerous hepatic disorders is ultimately caused by a pro-oxidant/antioxidant imbalance that results in the depletion or inactivation of antioxidants in hepatocytes [31]. In our investigation, TAA triggered a significant oxidative stress, which was demonstrated by a marked elevation in liver MDA level and a noticeably lower levels of SOD and GSH. These results are consistent with prior investigations [32, 33]. Inhibiting lipid peroxidation and increasing SOD activity and GSH levels in LC treated groups allowed us to assess the antioxidant ability of LC against oxidative stress. Previous studies are consistent with our findings [34, 35].
An essential mechanism for controlling TAA-induced oxidative damage and inflammation is the Nrf2/HO-1 pathway. Inhibiting oxidative stress and the inflammatory response is a defense mechanism that is mediated by Nfr2 and its downstream antioxidant genes [36]. Specific genes for antioxidant defense, including SOD, CAT, HO-1, and GSH, are regulated by Nrf2-ARE pathway activation [37]. When Nrf2 is stimulated and translocates to the nucleus, it activates the transcription of anti-oxidant genes through phosphorylation of AKT by activated phosphatidylinositol kinase (PI3K) [38]. According to this study, the hepatic Nrf2/HO-1, and PI3K expressions were downregulated in the TAA group but were upregulated in LC treated groups. These findings are consistent with earlier ones [21]. In HL7702 hepatocytes treated with hydrogen peroxide (H2O2), LC pretreatment was found to improve Nrf2 nuclear translocation, DNA binding activity, and HO-1 expression [39], which agrees with our findings. Therefore, an increase in nuclear Nrf2 expression that coincides with an increase in endogenous antioxidant defense may constitute LC's protective mechanism in TAA-induced liver fibrosis. These results demonstrated the critical contribution of the Nrf2/HO-1 pathway to the antifibrotic activity of LC.
The major pathway involved in the production and release of inflammatory mediators during inflammation, is the TLR4/NF-κB signaling pathway [40, 41]. TLR4 is crucial for controlling inflammation, HSC activation, and liver fibrosis [42]. TNF-α and IL-1β play a role in the pathogenesis of inflammatory liver diseases [43]. In the TAA group, TLR4 and its downstream target cytokines, IL-1β and TNF-α, were increased. The findings of this study are consistent with earlier researches [44, 45]. LC treated groups showed a marked reduction in TLR4 level and expression, which was in parallel with the decrease in the levels of its downstream inflammatory cytokines, IL-1β and TNF-α. Previous studies are consistent with our findings [46–48]. It's possible to explain LC's anti-inflammatory effects by the fact that it suppressed TLR4 pathway.
In the fibrogenic liver, HSCs proliferate, migrate, and transformed into myofibroblasts like cells [49]. Smooth muscle actin (α-SMA) is the most common myofibroblasts marker [50]. Immunohistochemical evaluations of liver sections in our investigation revealed increased α-SMA positive cells in the TAA group. Due to liver injury, HSCs become more active, which causes an increase in α-SMA release [51, 52]. As a result, monoclonal α-SMA antibody immunohistochemical staining indicates the presence of activated HSCs. Expression of α-SMA was significantly downregulated by LC treatment. This outcome is consistent with prior research [53, 54].
Additionally, TAA caused an increase in caspase-3, a sign of apoptosis [55]. Caspase-3 is associated with liver fibrosis and is a sensitive indication of liver damage [56]. The increased caspase-3 expression in the TAA group is thought to indicate the death of hepatocytes that resulted from the inflammation. Both dosages of LC considerably reduced the increase of caspase-3, indicating a protective and anti-apoptotic effect of LC. This outcome is consistent with other investigations [57, 58].
Histological analysis in the current investigation verified that LC had a protective effect against TAA's harmful effects. When combined, the findings of this research indicate that LC may activate the hepatic Nrf2/HO-1 signaling pathway, which may help to lessen the negative consequences of oxidative stress induced via TAA. Additionally, The TLR4 signaling pathway may be an important target for LC in the prevention of liver fibrosis.