Stevioside attenuates bleomycin-induced pulmonary fibrosis by activating the Nrf2 pathway and inhibiting the NF-κB and TGF-β1/Smad2/3 pathways

Abstract

Objective: This study aimed to investigate the effects of stevioside (STE) on pulmonary fibrosis (PF) and the potential mechanisms. Methods: In this study, a mouse model of PF was established by a single intratracheal injection of bleomycin (BLM, 3 mg/kg). The experiment consisted of four groups: control group, BLM group, and STE treatment groups (STE 50 and 100 mg/kg). ELISA and biochemical tests were conducted to determine the levels of TNF-α, IL-1β, IL-6, NO, hydroxyproline (HYP), SOD, GSH, and MDA. Histopathological changes and collagen deposition in lung tissues were observed by HE and Masson staining. Immunohistochemistry was performed to determine the levels of collagen I-, collagen III-, TGF-β1- and p-Smad2/3-positive cells. Western blot analysis was used to measure the expression of epithelial-mesenchymal transition (EMT) markers, including α-SMA, vimentin, E-cadherin, and ZO-1, as well as proteins related to the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, nuclear transcription factor-κB (NF-κB) pathway, and TGF-β1/Smad2/3 pathway in lung tissues. Results: STE significantly alleviated BLM-induced body weight loss and lung injury in mice, decreased HYP levels, and reduced the levels of collagen I- and collagen III-positive cells, thereby decreasing extracellular matrix (ECM) deposition. Moreover, STE markedly improved oxidative stress (MDA levels were decreased, while SOD and GSH activity were enhanced), the inflammatory response (the levels of TNF-α, IL-1β, IL-6, and NO were reduced), and EMT (the expression of α-SMA and vimentin was downregulated, and the expression of E-cadherin and ZO-1 was upregulated). Further mechanistic analysis revealed that STE could activate the Nrf2 pathway and inhibit the NF-κB and TGF-β1/Smad2/3 pathways. Conclusion: STE may alleviate oxidative stress by activating the Nrf2 pathway, suppress the inflammatory response by downregulating the NF-κB pathway, and inhibit EMT progression by blocking the TGF-β1/Smad2/3 pathway, thereby improving BLM-induced PF.

Keywords:

• Stevioside
• pulmonary fibrosis
• oxidative stress
• inflammatory response
• epithelial-mesenchymal transition

Declaration of interest

No potential conflict of interest was reported by the authors.

Ethics approval and consent to participate

The authors declare that the data supporting the findings of this study are available within the article. All procedures were performed in accordance with the protocol outlined in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and were approved by the Committee on the Ethics of Animal Experiments of Wannan Medical College.

Data availability statement

The data set used and analyzed in this study can be obtained from the corresponding author upon reasonable request.

Additional information

Funding

This work was supported by the Innovation Training Program for College Students in Anhui Province in 2021 (No. S202110368079).