Combinational Therapy of Mesenchymal Stem Cells and Metformin in Bleomycin-Induced Idiopathic Pulmonary Fibrosis in Rat Model

Abstract

Idiopathic pulmonary fibrosis (IPF) is a progressive and severe lung disease characterized by the buildup of interstitial fibrosis, where excessive collagen accumulates, leading to airway obstruction. This condition is initiated by the abnormal proliferation of alveolar type II (AT2) cells. Metformin, an established antidiabetic drug, has gained attention for its repurposed use as an anti-fibrotic agent. Meanwhile, adipose-derived mesenchymal stem cells (ADMSCs) exhibit potent anti-inflammatory and regenerative properties, and they have been shown to reduce collagen deposition. In this study, we hypothesize that the combination of metformin and ADMSCs can synergistically alleviate IPF and promote healthy lung tissue regeneration in a rat model. The goal is to evaluate the safety and efficacy of this approach at multiple levels; biochemical, molecular, histopathological, and histochemical. To induce IPF, Wistar albino rats received a single intratracheal dose of bleomycin (5 mg/kg body weight). The therapeutic phase involved treatment with either metformin or ADMSCs or a combination of both. Metformin was administered intraperitoneally (65 mg/kg body weight) every other day, while ADMSCs were delivered intravenously (1 × 10⁶ cells/0.5 ml DMEM/rat) through the tail vein. Our results demonstrated the effectiveness of combinational therapy, especially in mitigating oxidative stress. This was evidenced by the restoration of oxidative stress biomarkers, malondialdehyde (MDA), and catalase (CAT), as well as the regulation of collagenase type IV (MMP9), bovine serum albumin (BSA), and total protein levels in lung tissues. Moreover, the therapy modulated the expression of key inflammatory and fibrotic genes, including the pro-fibrotic marker TGF-β1, proinflammatory cytokine IL-6, and anti-inflammatory cytokine IL-10. Histopathological and histochemical analyses further supported the therapeutic benefits, showing significant recovery from bleomycin-induced fibrosis in rats treated with either the single or combined therapy. The findings suggest that this combinational approach could be a promising strategy for IPF treatment by simultaneously reducing inflammation, oxidative stress, and fibrosis while promoting tissue regeneration.

Keywords: Bleomycin; Collagen deposition; Idiopathic pulmonary fibrosis; Mesenchymal stem cells; Metformin.

Fig. 1

Experimental design for induction and therapeutic phases. In the induction (I) phase, the negative control group is CI, and the bleomycin-induced group is BI. In the therapeutic (T) phase, the negative control group: CT, the bleomycin-induced untreated group: BT, the BT group treated with MSCs: BTS, the BT group treated with metformin: BTM, and the BT group treated with a combination of metformin and MSCs: BTMS. Intratracheally (I.T.)., intraperitoneally (I.P.), and intravenously (I.V.). Created in BioRender. Hassan, N. (2024) https://BioRender.com/l86f060

Fig. 2

Effect of metformin and/or MSCs on histopathological changes in the lungs of bleomycin-induced rats compared to the control group during the induction and therapeutic phases. In the induction (I) phase, the negative control group: CI, and the bleomycin-induced group: BI. In the therapeutic (T), the negative control group: CT, the bleomycin-induced group: BT, the BT group treated with metformin: BTM, the BT group treated with MSCs: BTS, and the BT group treated with a combination of metformin and MSCs: BTMS. A CI group showing normal alveolar structure. B BI group exhibiting severe peribronchial and perivascular inflammatory cell infiltration. C Lung tissue from the CT group showing normal alveolar structure. D BT group displaying severe interstitial pneumonia with fibroplasia. E BTM group demonstrating moderate interstitial pneumonia. F BTS group showing mild perivascular inflammatory cell infiltration. G BTMS group displaying normal lung parenchyma

Fig. 3

The effect of metformin and/or MSCs on collagen deposition in bleomycin-induced pulmonary fibrosis compared to control groups during the induction and therapeutic phases. In the induction (I) phase, the negative control group: CI, and the bleomycin-induced group: BI. In the therapeutic (T), the negative control group: CT, the bleomycin-induced group: BT, the BT group treated with metformin: BTM, the BT group treated with MSCs: BTS, and the BT group treated with a combination of metformin and MSCs: BTMS. A CI group showing normal lung tissue. B BI group showing extensive perivascular and peribronchial fibrosis. C CT group showing normal lung tissue. D BT group showing interstitial fibrosis. E BTM group showing reduced interstitial fibrosis. F BTS group showing reduced peribronchial fibrosis. G BTMS group showing nearly normal lung tissue

Fig. 4

Single and combined effects of metformin and MSCs on collagen levels in lung tissue after bleomycin-induced IPF. In the induction (I) phase, the negative control group: CI, and the bleomycin-induced group: BI. In the therapeutic (T), the negative control group: CT, the bleomycin-induced group: BT, the BT group treated with metformin: BTM, the BT group treated with MSCs: BTS, and the BT group treated with a combination of metformin and MSCs: BTMS. Statistical significance is indicated by different letters where groups with different letters are significantly different at P ≤ 0.05, while groups sharing the same letters are not significantly different at P > 0.05

Fig. 5

Detection of MSCs labeled with red PKH26 fluorescent dye in the fibrotic lung tissue, with phase contrast demonstrating their homing ability to the site of injury

Fig. 6

Single/combined effect of metformin and MSCs on A MDA, B SOD, C CAT, D BSA, E MMP9, and F total protein concentration (BCA) levels in lung homogenates after induction of IPF by bleomycin. In the induction (I) phase, the negative control group: CI, and the bleomycin-induced group: BI. In the therapeutic (T), the negative control group: CT, the bleomycin-induced group: BT, the BT group treated with metformin: BTM, the BT group treated with MSCs: BTS, and the BT group treated with a combination of metformin and MSCs: BTMS. Groups with different letters are significantly different (P ≤ 0.05), while groups with the same letters are non-significantly different (P > 0.05)

Fig. 7

Single/combined effect of metformin and MSCs on the expression of A TGF-β1, B IL-6, and C IL-8 genes from lung homogenate after induction of IPF by bleomycin. In the induction (I) phase, the negative control group: CI, and the bleomycin-induced group: BI. In the therapeutic (T), the negative control group: CT, the bleomycin-induced group: BT, the BT group treated with metformin: BTM, the BT group treated with MSCs: BTS, and the BT group treated with a combination of metformin and MSCs: BTMS. Groups with different letters are significantly different (P ≤ 0.05), while groups with the same letters are non-significantly different (P > 0.05)