# Combined Therapy with Pirfenidone, Metformin, and Mesenchymal Stem Cells Attenuates Bleomycin-Induced Pulmonary Fibrosis in Rats

## TL;DR

A combination of pirfenidone, metformin, and stem cells reduced lung scarring in rats with pulmonary fibrosis, showing better results than individual treatments.

## Contribution

The novel contribution is demonstrating the synergistic therapeutic effect of combining pirfenidone, metformin, and mesenchymal stem cells in treating pulmonary fibrosis.

## Key findings

- Combination therapy significantly reduced oxidative stress and inflammatory markers in lung tissues.
- The treatment downregulated fibrogenic genes like MMP-9 and Col1α1 while improving antioxidant capacity.
- Histopathological analysis showed minimal fibrosis and inflammation in the combination treatment group.

## Abstract

Background/Objectives: Pulmonary fibrosis is a chronic, progressive lung disease marked by scarring and inflammation, leading to impaired respiratory function. This study aimed to investigate the combined therapeutic effects of pirfenidone (PFD), metformin (MET), and bone marrow-derived mesenchymal stem cells (BM-MSCs) on bleomycin (BLM)-induced pulmonary fibrosis in rats. Methods: Forty-eight Western Albino rats were divided into six groups: normal control, BLM-positive control, and four treatment groups receiving PFD, MET, BM-MSCs, and their combination. Treatments were administered for four weeks starting on day 21 post-BLM instillation. Lung tissues were analyzed for oxidative stress markers, inflammatory cytokines, apoptotic markers, and fibrogenic gene expression. Histopathological changes were assessed using hematoxylin and eosin (H&E) and Masson’s trichrome staining. Results: The combination therapy significantly reduced oxidative stress and inflammatory markers while enhancing antioxidant capacity. It decreased pro-apoptotic Bcl-2-associated X protein (BAX) and increased anti-apoptotic B-cell lymphoma 2 (Bcl-2) levels. Additionally, anti-inflammatory interleukin-10 (IL-10) was elevated, while tumor necrosis factor-alpha (TNF-α) and transforming growth factor-beta 1 (TGF-β1) levels were markedly lowered. Gene expression analysis showed a significant downregulation of matrix metalloproteinase-9 (MMP-9) and collagen type 1 alpha 1 (Col1α1). Histologically, the combination treatment group exhibited minimal fibrosis and inflammation, closely resembling normal lung tissue. Conclusions: The combination of PFD, MET, and BM-MSCs offered superior therapeutic efficacy in treating BLM-induced pulmonary fibrosis compared to individual treatments. This multimodal approach effectively targets oxidative stress, inflammation, apoptosis, and fibrosis, suggesting strong potential for future clinical application.