The effects of alpha pinene on methotrexate induced intestinal damage and permeability increase in rats

Objective: Studies to reduce the gastrointestinal side effects of methotrexate (MTX), a chemotherapeutic and immunosuppressive agent, are still ongoing. This study investigated the potential protective effect of α-pinene, a natural compound with antioxidant and anti-inflammatory properties, against MTX-induced intestinal damage and increased permeability. Materials and Methods: In the study, 35 young adult male Spraque Dawley rats were divided into 5 groups as Control, DMSO, Mtx, αP and αP+Mtx (n=7). Single dose MTX (20 mg/kg, subcutaneous) injection was performed on day 12 of the two-week experimental protocol. Injection of α-pinene (50 mg/kg) and DMSO, the solvent of α-pinene, was performed subcutaneously for 14 days and animals were sacrificed on the 15th day. LPS and IFABP levels in serum samples were measured using ELISA method. In jejunum and colon tissue homogenates, ZO-1 and occludin levels were measured by ELISA and MDA level was measured spectrophotometrically. After haematoxylin-eosin staining, histological evaluation and damage scoring were performed. Results: A single dose of MTX caused a statistically significant increase in serum LPS and IFABP levels (p<0.001 compared to the Control and DMSO groups). There was no significant change in ZO-1 or occludin levels in jejunum or colon tissue in the MTX group. However, MDA levels in the same tissues were significantly higher than in the Control and DMSO groups. Histological examination revealed MTX-induced tissue damage in both the jejunum and colon. Two weeks of α-pinene pretreatment significantly reduced the increases in serum LPS and IFABP induced by MTX (p < 0.01 and p < 0.05, respectively). However, parameters indicating intestinal permeability did not completely return to control levels. In the αP+Mtx group, no significant change was detected in tissue ZO-1 and occludin levels. However, both jejunum (p < 0.05) and colon (p < 0.05) MDA levels were significantly lower than in the Mtx group. In this group, MDA levels were similar to those in the Control and DMSO groups. α-Pinene pretreatment prevented MTX-induced tissue damage in the jejunum and colon to a great extent (p < 0.001). Yet, the histological damage score did not return to control levels in either tissue. Conclusions: This study revealed that α-pinene exhibits significant protective effects against MTX-induced intestinal toxicity. Histopathological and biochemical findings revealed that α-pinene provided partial protection to the intestinal mucosa and suppressed oxidative stress. However, the protective effect was limited, and an increase in damage and permeability could not be completely prevented. These results suggest that α-pinene has potential as a supportive treatment agent, but further research is needed. Keywords: methotrexate, alpha pinene, jejunum, colon, intestinal permeability, LPS, IFABP, ZO-1, occludin