Leflunomide-induced liver injury in mice: Involvement of TLR4 mediated activation of PI3K/mTOR/NFκB pathway

Leflunomide is a disease modifying anti-rheumatic drug (DMARD) beneficial in refractory cases of rheumatoid arthritis. Since leflunomide approval, hepatotoxicity and instructions of liver function monitoring have been recommended. The current work aimed to explore the possible role of inflammation in leflunomide-induced hepatotoxicity with a focus on the TLR4-mediated stimulation of PI3K/mTOR/NFκB pathway.

Forty-eight male albino mice were allocated into four different groups (n; 12 mice/group). Group (i): normal mice, Group (ii-iv) mice received escalating dosed/s of leflunomide (2.5, 5 or 10 mg/kg, p.o.) every 48 h for eight weeks. At the end of the study, mice were sacrificed, and blood samples were collected for determination of liver enzymes. Liver samples were collected; (1) formalin-fixed for histologic examination, (2) frozen for PI3K and mTOR genes PCR assays.

Results indicated a significant elevation of liver enzymes in leflunomide-treated mice (10 mg/kg); AST and ALT activities were 218.17 ± 6.83 U/L and 99.83 ± 9.82 U/L versus 130.5 ± 12.79 U/L and 44.72 ± 3.58 U/L in the vehicle group. Additionally, histopathological examination revealed higher necro-inflammatory scores in leflunomide-treated mice. Immunohistochemistry indicated dose-dependent increased staining of TLR4 and caspase 3. Furthermore, leflunomide-treated mice (5 or 10 mg/kg) showed greater staining for NFκB compared to vehicle control. RT-PCR results revealed upregulations in genes expressing PI3K and mTOR by leflunomide.

The current study highlights the possible role of TLR4-PI3K/mTOR/NFκB in the pathogenesis of leflunomide-induced hepatic injury. A better understanding of mechanisms of leflunomide-induced hepatotoxicity may be of translational implication for the predictive, preventive and therapeutic purposes.