Rosmarinic acid ameliorates radiation-induced intestinal injury through PI3K/AKT/NF-κB/MMP9 axis inhibition: From network pharmacology prediction to experimental validation

Background: Radiation-induced intestinal injury (RIII) is a common complication following radiotherapy for abdominal and pelvic tumors, and there is currently a lack of highly effective and specific preventive or therapeutic drugs. Rosmarinic acid (RA), as a natural polyphenolic compound, possesses anti-inflammatory and antioxidant activities, but its specific therapeutic effects and mechanisms on RIII remain poorly understood.

Methods: Target prediction was performed using network pharmacology combined with molecular docking and molecular dynamics simulation. Subsequently, in an in vivo experiment, C57BL/6 mice that subjected to 25 Gy of local abdominal irradiation were orally administered 200 mg/kg rosmarinic acid (RA). Intestinal injury was assessed by histological examination, serum biomarkers including intestinal fatty acid-binding protein (I-FABP), diamine oxidase (DAO), transforming growth factor-β1 (TGF-β1), leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5)-positive cells and Ki67 immunostaining, 16S rRNA Sequencing, and Western blotting. Activation of the phosphatidylinositol 3-kinase/protein kinase B/nuclear factor-κB (PI3K/Akt/NF-κB) signaling pathway was also detected. In vitro experiments, MODE-K cells were pre-treated with 25 μM RA before irradiation, and cell viability, malondialdehyde (MDA) levels, as well as the expression of target molecules at both mRNA and protein levels were analyzed.

Results: Network pharmacology identified MMP9, EGFR, and MMP2 as core targets of RA against RIII, which was further validated by the strong binding affinities observed in molecular docking and MD simulations. In vivo, RA ameliorated intestinal histopathological damage, maintained barrier integrity, reduced serum I‑FABP by 20% (P < 0.05) and DAO by 8.91% (P < 0.0001), increased TGF‑β1 level by 84.65% (P < 0.001), inhibited villus breakage and crypt destruction, protected LGR5 + stem cells (increased by 253.34% compared with the radiation group, P < 0.0001), and decreased Ki67 + proliferating cells by 13.29% (P < 0.05). Additionally, RA partially reversed the radiation-induced loss of gut microbial diversity. In vitro, RA pretreatment increased the viability of irradiated MODE-K cells by 44.21% (P < 0.0001) and suppressed MDA levels by 58.61% (P < 0.0001). Meanwhile, RA downregulated the mRNA expression of IL-18, TNF-α, CASP3, MMP2, and MMP9 by 50.88%, 75.52%, 82.89%, 50.25%, and 60.07%, respectively (all P < 0.0001). Western blot results showed that radiation upregulated the protein levels of p‑PI3K, p‑AKT, p‑P65, and MMP9 in cells, while RA treatment markedly attenuated this upregulation of these proteins (P < 0.01).

Conclusions: Collectively, these data demonstrate that RA inhibits radiation‑induced activation of the PI3K/Akt/NF‑κB/MMP9 axis, reduces oxidative stress, inflammation and Apoptosis, and preserves the intestinal stem cell niche. These data identify RA as a promising radioprotective candidate for the prevention and treatment of RIII.