Dihydromyricetin Modulates Nrf2 and NF-κB Crosstalk to Alleviate Methotrexate-Induced Lung Toxicity

Abstract

Background: Methotrexate (MTX) is an effective anticancer, anti-inflammatory, and immunomodulatory agent. However, it induces a serious pneumonitis that leads to irreversible fibrotic lung damage. This study addresses the protective role of the natural flavonoid dihydromyricetin (DHM) against MTX-induced pneumonitis via modulation of Nrf2/NF-κB signaling crosstalk.

Methods: Male Wistar rats were divided into 4 groups: control, which received the vehicle; MTX, which received a single MTX (40 mg/kg, i.p) at day 9 of the experiment; (MTX + DHM), which received oral DHM (300 mg/kg) for 14 days and methotrexate (40 mg/kg, i.p) on the 9th day; and DHM, which received DHM (300 mg/kg, p.o) for 14 days.

Results: Lung histopathological examination and scoring showed a decline in MTX-induced alveolar epithelial damage and decreased inflammatory cell infiltration by DHM treatment. Further, DHM significantly alleviated the oxidative stress by decreasing MDA while increasing GSH and SOD antioxidant levels. Additionally, DHM suppressed the pulmonary inflammation and fibrosis through decreasing levels of NF-κB, IL-1β, and TGF-β1 while promoting the expression of Nrf2, a positive regulator of antioxidant genes, and its downstream modulator, HO-1.

Conclusion: This study identified DHM as a promising therapeutic target against MTX-induced pneumonitis via activation of Nrf2 antioxidant signaling while suppressing the NF-κB mediated inflammatory pathways.

Keywords: HO-1; IL-1β; NF-κB; Nrf2; TGF-β1; dihydromyricetin; methotrexate.

Figure 1

Effect of treatment with dihydromyricetin (DHM, 300 mg/kg, p.o) on methotrexate (MTX, 40 mg/kg, i.p)-induced histopathological changes in lung tissues. Photomicrographs showing lung sections of all groups (n = 6, each): control group (a,b) showed normal structure of the lung tissues; numerous alveoli (stars) are separated by delicate septa of connective tissue (arrows). Higher magnifications show several connected clear alveoli (stars) lined by pneumocyte type I (arrows) and type II (dashed arrows). MTX group (c,d) showed disturbed structures; decreased alveoli (stars) with areas of consolidation (*), coalescence of numerous alveoli (stars) with degeneration of their alveolar septa (arrowhead), marked increase in the thickness of inter-alveolar septa (arrows) with inflammatory cell infiltration (curved arrow and insets), and markedly congested blood vessels. Notice interstitial hemorrhage (IH) and inflammatory cells mainly lymphocytes (double head arrows) and eosinophils (circles). The DHM + MTX group (e,f) showed marked morphological improvement, with few areas showing thickening of the alveolar septa (curved arrow and inset). The DHM group (g,h) showed normal morphological changes, with numerous alveoli (stars) separated by delicate septa of connective tissue (arrows); H&E (a,c,e,g) ×10; scale bar: 100 µm, (b,d,f,h) ×40; scale bar: 50 µm; and insets ×100 scale bar: 20 µm.

Figure 2

Effects of 2 weeks of treatment with dihydromyricetin (DHM, 300 mg/kg, p.o) on oxidative stress parameters in methotrexate (MTX, 40 mg/kg, i.p)-induced lung toxicity. (A) MDA, (B) SOD, and (C) GSH. Data are presented as means ± SEM (n = 6). * and # indicate significant differences from the control and MTX groups, respectively, at (p < 0.05).

Figure 3

Effect of 2 weeks of treatment with dihydromyricetin (DHM, 300 mg/kg, p.o) on pulmonary levels of (A) Nrf2, (B) HO-1, and (C) NF-κB in methotrexate (MTX, 40 mg/kg, i.p)-induced lung toxicity. Data are presented as means ± SEM (n = 6). * and # indicate significant differences from the control and MTX groups, respectively, at (p < 0.05).

Figure 4

Effect of 2 weeks of treatment with dihydromyricetin (DHM, 300 mg/kg, p.o) on pulmonary levels of (A) IL-1β and (B) TGF-β1 in methotrexate (MTX, 40 mg/kg, i.p)-induced lung toxicity. Data are presented as means ± SEM (n = 6). * and # indicate significant differences from the control and MTX groups, respectively, at (p < 0.05).