Andrographolide Modulates Fibrogenic and Oxidative Stress Responses in Human Lung Fibroblasts

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  FIGURE 1. Effects of ANDRO on the expression of TGF‐β1‐induced gene and protein associated with fibrogenesis. MRC5 cells were pretreated with or without ANDRO (10 μM) for 4 h. Subsequently, exogenous TGF‐β1 (4 ng/mL) was added to the medium for 48 h. (A) The chemical structure of ANDRO was generated based on data from PubChem (CID: 5318517). (B) Gene expression of ACTA2, FN1, and CDH2 was detected through RT‐qPCR, and GAPDH was used as a loading control. (C) Protein expression of α‐SMA (encoded by ACTA2), fibronectin (encoded by FN1), and N‐cadherin (encoded by CDH2) was measured by Western blotting. One‐way ANOVA was used to analyze differences among groups, and the Tukey's multiple comparison test was performed for post hoc analysis. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05 versus control. #p < 0.05 versus TGF‐β1 treatment.

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  FIGURE 2. Schematic diagram of study design and analysis strategy. MRC5 cells were treated with or without 10 μM ANDRO for 4 h. Subsequently, 4 ng/mL of TGF‐β1 was added to the culture medium for 48 h. Total RNA was extracted and sequenced using RNA‐Seq to obtain gene expression profiles. We screened 3811 DEGs between the control and TGF‐β1 groups. Of these 3811 DEGs, 596 were significantly reversed by ANDRO treatment and were selected for further functional enrichment analysis using the ShinyGO 0.76. Significantly altered genes were analyzed, and the corresponding protein–protein interaction networks were generated using STRING 11.5. Validation of gene expression was performed by RT‐qPCR. Protein expression was detected by Western blotting. GSH level and ROS production were detected using the GSH assay and DCFH‐DA staining, respectively.

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  FIGURE 3. Clustering analysis of candidate DEGs associated with GSH metabolism in MRC5 cells. (A) Heatmap showing the hierarchical clustering of genes (GCLM, GSTA4, MGST1, GSR, MGST2, GSTM3, and G6PD) associated with GSH metabolism. (B) Network cluster analysis of protein–protein interactions for genes involved in GSH metabolism was performed using STRING software.

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  FIGURE 4. Validation of gene expression. Cells were pretreated with or without ANDRO (10 μM) for 4 h. Subsequently, exogenous TGF‐β1 (4 ng/mL) was added to the medium for 48 h. Gene expression of GCLM, GSR, and G6PD was measured in (A) MRC5 cells and (B) WI‐38 cells through RT‐qPCR. One‐way ANOVA was used to analyze differences among groups, and the Tukey's multiple comparison test was performed for post hoc analysis. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05 versus control. #p < 0.05 versus TGF‐β1 treatment.

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  FIGURE 5. Effects of ANDRO on expression of proteins related to GSH metabolism. MRC5 and WI‐38 cells were pretreated with or without ANDRO (10 μM) for 4 h, followed by the addition of exogenous TGF‐β1 (4 ng/mL) to the medium for 48 h. Expressions of GSH metabolism‐related proteins, including GCLM, GSR, and G6PD, were examined in (A) MRC5 cells and (B) WI‐38 cells by Western blotting. One‐way ANOVA was used to analyze differences among groups, and the Tukey's multiple comparison test was performed for post hoc analysis. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05 versus control. #p < 0.05 versus TGF‐β1 treatment.

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  FIGURE 6. Effects of ANDRO on TGF‐β1‐inhibited GSH level. MRC5 and WI‐38 cells were pretreated with or without ANDRO (10 μM) for 4 h. Subsequently, exogenous TGF‐β1 (4 ng/mL) was added to the medium for 48 h. Total GSH level was measured in MRC5 and WI‐38 cells by GSH assay. One‐way ANOVA was used to analyze differences among groups, and the Tukey's multiple comparison test was performed for post hoc analysis. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05 versus control. #p < 0.05 versus TGF‐β1 treatment.

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  FIGURE 7. Effects of ANDRO on TGF‐β1‐induced ROS production. MRC5 and WI‐38 Cells were pretreated with or without ANDRO (10 μM) for 4 h. Subsequently, exogenous TGF‐β1 (4 ng/mL) was added to the medium for 48 h. ROS levels were detected in MRC5 and WI‐38 cells by DCFH‐DA staining, and fluorescence intensity was measured using a microplate reader. One‐way ANOVA was used to analyze differences among groups, and the Tukey's multiple comparison test was performed for post hoc analysis. Data are presented as the mean ± SEM of three independent experiments. *p < 0.05 versus control. #p < 0.05 versus TGF‐β1 treatment.

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  FIGURE 8. Schematic diagram of ANDRO attenuating TGF‐β1‐induced fibrogenesis by modulating GSH metabolism in human lung fibroblasts. TGF‐β1 disrupts GSH homeostasis by downregulating key regulators of GSH metabolism, including GCLM, GSR, and G6PD, leading to excessive ROS accumulation and fibrotic responses. ANDRO reversed TGF‐β1‐modulated expression of genes and proteins associated with GSH metabolism. In addition, ANDRO restored TGF‐β1‐inhibited total GSH level and inhibited TGF‐β1‐induced ROS production. The involvement of ROS in TGF‐β1‐induced fibrogenic responses is supported by previous studies [28].