MOBT Alleviates Pulmonary Fibrosis through an lncITPF-hnRNP-l-Complex-Mediated Signaling Pathway

Abstract

Pulmonary fibrosis is characterized by the destruction of alveolar architecture and the irreversible scarring of lung parenchyma, with few therapeutic options and effective therapeutic drugs. Here, we demonstrate the anti-pulmonary fibrosis of 3-(4-methoxyphenyl)-4-oxo-4H-1-benzopyran-7-yl(αS)-α,3,4-trihydroxybenzenepropanoate (MOBT) in mice and a cell model induced by bleomycin and transforming growth factor-β1. The anti-pulmonary fibrosis of MOBT was evaluated using a MicroCT imaging system for small animals, lung function analysis and H&E and Masson staining. The results of RNA fluorescence in situ hybridization, chromatin immunoprecipitation (ChIP)-PCR, RNA immunoprecipitation, ChIP-seq, RNA-seq, and half-life experiments demonstrated the anti-pulmonary fibrotic mechanism. Mechanistic dissection showed that MOBT inhibited lncITPF transcription by preventing p-Smad2/3 translocation from the cytoplasm to the nucleus, resulting in a reduction in the amount of the lncITPF-hnRNP L complex. The decreased lncITPF-hnRNP L complex reduced MEF2c expression by blocking its alternative splicing, which in turn inhibited the expression of MEF2c target genes, such as TAGLN2 and FMN1. Briefly, MOBT alleviated pulmonary fibrosis through the lncITPF-hnRNP-l-complex-targeted MEF2c signaling pathway. We hope that this study will provide not only a new drug candidate but also a novel therapeutic drug target, which will bring new treatment strategies for pulmonary fibrosis.

Keywords: MEF2c; hnRNP L; lncITPF; pulmonary fibrosis.

Figure 1

MOBT alleviated pulmonary fibrosis in vivo. (A) Molecular formula of MOBT and schematic illustration of MOBT administrated into mice. (B) The MicroCT imaging system for small animals displayed that the plain scan of the lung window in the sham group showed the texture of both lungs was clear, and no exudation or space occupying lesions were found in the lung parenchyma. The MOBT alone had no obvious changes compared with the sham group. However, both lower lungs of the mice in the BLM group showed diffused reticular blurring and interstitial changes. Meanwhile, the BLM + MOBT treatment group significantly reduced the lesion range of reticular changes. (C) The body weight monitoring revealed that mouse weight in the BLM group was significantly reduced compared with the sham group, whereas in the MOBT group it was increased compared with the BLM group. (D) The result of mice lung function showed that MOBT treatment improved the FVC. (E) H&E and Masson staining demonstrated that a portion of the alveolar structure was damaged, alveolar fusion, and collagen fibers were gathered in the lung of the BLM mice compared with the sham group, whereas MOBT alone group was similar to the sham. The BLM + MOBT treatment improved the alveolar structure and attenuated collagen deposition. (F) The Western blot demonstrated that MOBT inhibited the fibrotic protein expression, including collagen I, vimentin, and α-SMA compared with those in the BLM-treated group. (G) Western blot demonstrated that MOBT inhibited the expression of TGF-β1, CTGF, and VEGF, which are cytokines related to fibrosis, compared with those in the BLM group. Each bar represents mean ± SD, n = 6, * p < 0.05.

Figure 2

MOBT treatment reduced α-SMA expression. (A) The immunofluorescence result demonstrated that the BLM group expressed abundant α-SMA compared with the sham group, whereas the MOBT treatment significantly decreased the α-SMA expression. The red color indicates α-SMA. The blue color indicates nucleus. (B) The immunohistochemical result indicated that the sham group had distinct alveolar structures and thin alveolar walls. The alveolar structure in the BLM group was disordered and had thick alveolar walls. The MOBT treatment significantly improved the lung alveolar structure and decreased α-SMA deposition. The brown color indicates α-SMA.

Figure 3

MOBT inhibited lncITPF transcription via reducing Smad2/3 phosphorylation. (A) The Western blot analysis displayed that TGF-β1 increased the expression of collagen I, vimentin, and α-SMA, whereas MOBT decreased their expression. (B) qRT-PCR was used to discover the lncITPF expression level in the normal, TGF-β1, and MOBT + TGF-β1 groups. The results demonstrated that TGF-β1 increased the lncITPF expression, whereas MOBT caused a reduction. (C) The xCELLigence real-time cell analysis (RTCA) system was used to analyze the proliferation of MRC-5 cells. Overexpression of lncITPF increased the proliferation of activated fibroblasts, similar to the TGF-β1 effect. MOBT reduced the proliferation of activated fibroblasts compared with the TGF-β1-treated group. (D) The RTCA system was used to assay the migration of MRC-5 cells. Overexpression of lncITPF increased the migration of activated fibroblasts, similar to the TGF-β1 effect. MOBT reduced the migration of activated fibroblasts compared with the TGF-β1-treated group. (E) Single-molecule RNA-FISH revealed that the lncITPF (red) was mostly located in the nucleus. MOBT treatment reduced the lncITPF expression and did not lead to its translocation from the nucleus to the cytoplasm. 18S RNAs were used as cytoplasmic localization markers. U6s were used as nuclear localization markers. DAPI was used for the staining of fixed cell DNA (blue). (F) The Western blot analysis demonstrated that MOBT inhibited Smad2/3 and p-Smad2/3 expression. (G) ChIP-PCR was used to evaluate the binding of Smad2/3 in the lncITPF promoter region under MOBT treatment. The results reflected that MOBT decreased Smad2/3 protein enrichment in the lncITPF promoter. (H) Actinomycin D rapidly decreased the expression of lncITPF, and the half-life of lncITPF was approximately 50 min. The combination of actinomycin D and TGF-β1 increased the half-life of lncITPF (120 min). Meanwhile, the combination of actinomycin D, TGF-β1, and MOBT decreased the half-life of lncITPF (105 min). Each bar represents mean ± SD, * p < 0.05.

Figure 4

MOBT blocked lung fibrogenesis via inhibiting MEF2c stability. (A) The RIP assay showed that MOBT reduced the combination of pre-mMEF2c with hnRNP L. IgG was used as a control. (B) The Western blot analysis revealed that MEF2c expression increased in the TGF-β1-treated cells for different time points. (C) The Western blot analysis showed that 10 µg/mL MOBT decreased the MEF2c expression. (D) The immunofluorescence result demonstrated that the TGF-β1 group expressed abundant MEF2c compared with the normal group, whereas the TGF-β1 + MOBT group decreased the MEF2c expression. The green color indicates MEF2c. The blue color indicates the nucleus. (E) The immunofluorescence images showed that the MEF2c expression increased in IPF patient’s lung tissues compared with the normal tissue adjacent to lung cancer. The green color indicates MEF2c. The blue color indicates the nucleus. (F) Stability testing showed that MEF2c stability was promoted by TGF-β1 and decreased by MOBT treatment. The half-life in each group was 4.36, 11.65, and 5.96 h in the normal, TGF-β1, and TGF-β1 + MOBT groups, respectively. Each bar represents mean ± SD, * p < 0.05.

Figure 5

MOBT downregulated MEF2c expression through the lncITPF–hnRNP L complex. (A) The Western blot analysis showed that lncITPF overexpression promoted the expression level of MEF2c. (B) The Western blot analysis showed that si-lncITPF reduced the expression level of MEF2c. (C) The rescue experiments discovered that overexpression of lncITPF reversed the MOBT function, resulting in the blockage of MEF2c expression. (D) The rescue experiments demonstrated that si-hnRNP L reversed the joint action of MOBT and overexpression of lncITPF, resulting in a repressed MEF2c level. NC meant the negative control, BP meant the blank plasmid, and RP meant the overexpressed lncITPF recombinant plasmid. Each bar represents mean ± SD, * p < 0.05.

Figure 6

MOBT regulated the downstream target genes of MEF2c. (A) A ChIP-seq experiment was performed to identify genes binding with MEF2c. (B) RNA-seq was performed to analyze the differentially expressed genes. (C) Combined analysis revealed a total of 76 target genes in the ChIP-seq and RNA-seq data, with 11 upregulated and 65 downregulated genes. (D) The KEGG analysis revealed that the 76 target genes regulated by MEF2c were significantly enriched in the Hippo, Wnt, and MAPK signaling pathways. (E) The 44 significant differentially expressed genes that were selected from 76 target genes and listed in the hierarchical clustering of RNA-seq. (F) The binding sites for MEF2c in the TAGLN2 and FMN1 genes were analyzed.

Figure 7

MOBT regulated TAGLN2 and FMN1 via MEF2c. (A) The qRT-PCR and RNA-seq data demonstrated that TAGLN2 and FMN1 decreased under MOBT treatment compared with those in the TGF-β1 group. (B) The RIP experiment demonstrated that MEF2c bound to TAGLN2 and FMN1. (C) The Western blot result verified that MOBT inhibited TAGLN2 and FMN1. Each bar represents mean ± SD, * p < 0.05.

Figure 8

Anti-pulmonary fibrotic mechanism of MOBT. MOBT inhibited lncITPF transcription by preventing p-Smad2/3 translocation from the cytoplasm to the nucleus, resulting in the reduction of the lncITPF–hnRNP L complex. The decreased lncITPF–hnRNP L complex reduced MEF2c expression by blocking its alternative splicing, which in turn inhibited the expression of MEF2c target genes, such as TAGLN2 and FMN1.