Trigonelline hydrochloride attenuates silica-induced pulmonary fibrosis by orchestrating fibroblast to myofibroblast differentiation

Abstract

Background: Silicosis represents a paramount occupational health hazard globally, with its incidence, morbidity, and mortality on an upward trajectory, posing substantial clinical dilemmas due to limited effective treatment options available. Trigonelline (Trig), a plant alkaloid extracted mainly from coffee and fenugreek, have diverse biological properties such as protecting dermal fibroblasts against ultraviolet radiation and has the potential to inhibit collagen synthesis. However, it's unclear whether Trig inhibits fibroblast activation to attenuate silicosis-induced pulmonary fibrosis is unclear.

Methods: To evaluate the therapeutic efficacy of Trig in the context of silicosis-related pulmonary fibrosis, a mouse model of silicosis was utilized. The investigation seeks to elucidated Trig's impact on the progression of silica-induced pulmonary fibrosis by evaluating protein expression, mRNA levels and employing Hematoxylin and Eosin (H&E), Masson's trichrome, and Sirius Red staining. Subsequently, we explored the mechanism underlying of its functions.

Results: In vivo experiment, Trig has been demonstrated the significant efficacy in mitigating SiO₂-induced silicosis and BLM-induced pulmonary fibrosis, as evidenced by improved histochemical staining and reduced fibrotic marker expressions. Additionally, we showed that the differentiation of fibroblast to myofibroblast was imped in Trig + SiO₂ group. In terms of mechanism, we obtained in vitro evidence that Trig inhibited fibroblast-to-myofibroblast differentiation by repressing TGF-β/Smad signaling according to the in vitro evidence. Notably, our finding indicated that Trig seemed to be safe in mice and fibroblasts.

Conclusion: In summary, Trig attenuated the severity of silicosis-related pulmonary fibrosis by alleviating the differentiation of myofibroblasts, indicating the development of novel therapeutic approaches for silicosis fibrosis.

Keywords: Fibroblast; Pulmonary fibrosis; Silicosis; Trigonelline.

Fig. 1

Silicon dioxide and BLM-induced mouse model administration times. A Chemical structure of trigonelline hydrochloride; (B) silica-induced pulmonary fibrosis model and administration time; (C) BLM-induced mouse pulmonary fibrosis model and administration time

Fig. 2

Trig attenuates silica-induced pulmonary fibrosis in mice. A Western blot analysis of fibronectin, collagen 1 after Trig intervention. Left panel: representative western blot results. Right panel: bar graph of western blot results; (B, C) RT-PCR analysis of Fn1 (B) and Col1a1 (C) expression. D Representative images for H&E (top), Masson's stain (middle) and Sirius red (bottom) in mice after BLM induction. E A bar graph showing the fibrosis foci; (F-I) representative immunofluorescence images and analysis of fibronectin (F), collagen I (G) and α-SMA (H) in lung tissue sections, magnification × 400. 5–6 mice per group. **p < 0.01, *** p < 0.001, **** p < 0.0001

Fig. 3

Trig ameliorated BLM-induced pulmonary fibrosis in mice. A Representative images for H&E (top), Masson's stain (middle) and Sirius red (bottom) in mice after BLM induction. Magnification × 200; (B) Ashcroft scores for the severity of fibrosis; (C) Western blot of fibronectin, collagen 1 expression after Trig infection; Left panel: representative western blot results. Right panel: bar graph of western blot results; (D, E) RT-PCR analysis of Fn1 (D) and Col1a1 (E) expression. expression expansion; F-I Representative immunofluorescence images and analysis of fibronectin (F), collagen I (G) and α-SMA (H) in lung tissue sections. Magnification × 400. 5–6 mice per group. *p < 0.05, **p < 0.01, **** p < 0.0001

Fig. 4

Effect of Trig on fibroblast activation, proliferation and migration. A WB analysis of fibronectin, collagen 1 and α-SMA expression in fibroblasts. Left panel: representative western blot results. Right panel: bar graph of the western blot results; (B) Immunofluorescence of fibronectin, collagen 1 and α-SMA in fibroblasts; (C) Representative data in fibroblasts with EdU staining; (D) Histogram data of EdU staining; (E) Representative data of fibroblast scratch assay. L-Trig: 40 µM /mL of Trig, H-Trig: 80 µM /mL of Trig. * p < 0.05, **p < 0.01, **** p < 0.0001

Fig. 5

Impacts of Trig on TGF-β-stimulated Smad signaling. A Trig reduced TGF-β-induced Smad signaling. Left panel: representative western blot result for p-Smad2, p-Smad3, Smad2/3 at 1 h TGF-β stimulation. Right panel: figures showing the data with three replicates B RT-PCR analysis of SMAD 7expression. C Trig had no effect on PI3K/AKT signaling pathway. * p < 0.05

Fig. 6

Toxicity and safety analysis of Trig as a therapeutic drug. A cytotoxicity analysis; (B, C) AST, ALT representing liver function; (D, E) CR, UREA representing renal function; (F) H&E staining of various organ tissues: The sequence begins with heart tissue (far left), progresses to liver tissue (second from the left), centers on kidney tissue, and concludes with sections of intestine (first from the right) and spleen tissue (far right)

Fig. 7

Schematic diagram of the mechanism by which Trig affects pulmonary fibrosis. Trig attenuates pulmonary fibrosis by inhibiting the TGF-β/Smad signaling pathway in vitro, reducing the differentiation of fibroblasts to myofibroblasts and decreasing ECM deposition