Journal of Traditional Chinese Medicine ›› 2022, Vol. 42 ›› Issue (4): 520-529.DOI: 10.19852/j.cnki.jtcm.20220519.001
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Shenqihuatan formula (参七化痰方) reduces inflammation by inhibiting transforming growth factor-beta-stimulated signaling pathway in airway smooth muscle cells
CHEN Jingjing1, WANG Yuanyuan2, ZHANG Nianzhi3(
), XUE Xiaoming1
- 1 Shanxi Provincial Traditional Chinese Medicine Hospital, Taiyuan 030012, China
2 Anhui University of Traditional Chinese Medicine, Hefei 230038, China
3 Department of Respiratory, the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China
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Received:2021-04-29Accepted:2021-07-02Online:2022-08-15Published:2022-07-12 -
Contact:ZHANG Nianzhi -
About author:Prof. ZHANG Nianzhi, Department of Respiratory, the First Affiliated Hospital of Anhui University of Traditional Chinese Medicine, Hefei 230031, China. zhangnz@ahtcm.edu.cn, Telephone: +86-13505615645
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Supported by:Study on the Mechanism of the Method of Yiqi Huoxue Huata regulating autophagy in airway epithelial cells of COPD based on SIRT1/mTOR signaling pathway(82174312);Study on the mechanism of the method of YIQI HUOXUE HUATA intervention in COPD airway remodeling based on RhoA/ROCK signaling pathway(81473675)
Cite this article
CHEN Jingjing, WANG Yuanyuan, ZHANG Nianzhi, XUE Xiaoming. Shenqihuatan formula (参七化痰方) reduces inflammation by inhibiting transforming growth factor-beta-stimulated signaling pathway in airway smooth muscle cells[J]. Journal of Traditional Chinese Medicine, 2022, 42(4): 520-529.
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Figure 1 SQHT inhibits TGF-β-stimulated cell processes A: CCK-8 results of the viability of ASMCs; B1: representative micrographs show the migration of ASMCs via crystal violet staining (×100); B2: histogram of Transwell assay; C: representative micrographs of F-actin staining demonstrat the F-actin formation and cytoskeletal reorganization of ASMCs via immunofluorescence (×100). Scale bar, 20 μm. aP < 0.01, vs Control; bP < 0.05, cP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; TGF-β: transforming growth factor-beta; CCK-8: cell counting kit (CCK)-8; ASMCs: airway smooth muscle cells.
Figure 1 SQHT inhibits TGF-β-stimulated cell processes A: CCK-8 results of the viability of ASMCs; B1: representative micrographs show the migration of ASMCs via crystal violet staining (×100); B2: histogram of Transwell assay; C: representative micrographs of F-actin staining demonstrat the F-actin formation and cytoskeletal reorganization of ASMCs via immunofluorescence (×100). Scale bar, 20 μm. aP < 0.01, vs Control; bP < 0.05, cP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; TGF-β: transforming growth factor-beta; CCK-8: cell counting kit (CCK)-8; ASMCs: airway smooth muscle cells.
Figure 2 SQHT drives apoptosis in stimulated ASMCs A1: representative micrographs of flow cytometry show apoptosis; A2: histogram of flow cytometry shows apoptosis; B1: representative immunoblotting bands of Western blotting results show the relative protein expression levels of Bcl-2 and Bax; B2: histogram of Western blotting results; C1: ELISA results of the concentration of IL-1β; C2: ELISA results of the concentration of IL-6. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; ASMCs: airway smooth muscle cells; Bcl-2: B cell lymphoma 2; Bax: Bcl-2-Associated X; ELISA: enzyme-linked immuno sorbent assay; IL-1β: Interleukin-1β; TGF-β: transforming growth factor-beta.
Figure 2 SQHT drives apoptosis in stimulated ASMCs A1: representative micrographs of flow cytometry show apoptosis; A2: histogram of flow cytometry shows apoptosis; B1: representative immunoblotting bands of Western blotting results show the relative protein expression levels of Bcl-2 and Bax; B2: histogram of Western blotting results; C1: ELISA results of the concentration of IL-1β; C2: ELISA results of the concentration of IL-6. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; ASMCs: airway smooth muscle cells; Bcl-2: B cell lymphoma 2; Bax: Bcl-2-Associated X; ELISA: enzyme-linked immuno sorbent assay; IL-1β: Interleukin-1β; TGF-β: transforming growth factor-beta.
Figure 3 SQHT exhibits comparable inhibition to Fasudil on the cell processes A: CCK-8 results of the cell proliferation; B1: representative micrographs of Transwell results show the migration of ASMCs via crystal violet staining (×100); B2: histogram of Transwell results; C: representative micrographs of F-actin staining reveal the F-actin formation and cytoskeletal reorganization of ASMCs via immunofluorescence (×100). Scale bar, 20 μm; D1: representative images of flow cytometry show apoptosis in the groups; D2: histogram of flow cytometry shows apoptosis in the groups; E1: representative immunoblotting band of Western blotting results of the relative protein expression levels of Bcl-2 and Bax. E2: histogram of Western blotting results. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; CCK-8: Cell counting kit (CCK)-8; ASMCs: airway smooth muscle cells; Bcl-2: B cell lymphoma 2; Bax: Bcl-2-Associated X; TGF-β: transforming growth factor-beta.
Figure 3 SQHT exhibits comparable inhibition to Fasudil on the cell processes A: CCK-8 results of the cell proliferation; B1: representative micrographs of Transwell results show the migration of ASMCs via crystal violet staining (×100); B2: histogram of Transwell results; C: representative micrographs of F-actin staining reveal the F-actin formation and cytoskeletal reorganization of ASMCs via immunofluorescence (×100). Scale bar, 20 μm; D1: representative images of flow cytometry show apoptosis in the groups; D2: histogram of flow cytometry shows apoptosis in the groups; E1: representative immunoblotting band of Western blotting results of the relative protein expression levels of Bcl-2 and Bax. E2: histogram of Western blotting results. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; CCK-8: Cell counting kit (CCK)-8; ASMCs: airway smooth muscle cells; Bcl-2: B cell lymphoma 2; Bax: Bcl-2-Associated X; TGF-β: transforming growth factor-beta.
Figure 4 SQHT or Fasudil inhibit the RhoA and ERK1/2 pathway in TGF-β-stimulated ASMCs A1: representative immunoblotting band of western blotting results of the relative protein expression levels of RhoA, ROCK1 and 2; A2: histogram of western blotting results; B: qRT-PCR results of the relative mRNA expression levels of RhoA, ROCK1 and 2; C1: representative immunoblotting band of western blotting results of the relative protein expression levels of p-ERK1/2 and ERK1/2; C2: histogram of western blotting results of the relative protein expression levels of p-ERK1/2 and ERK1/2; C3: histogram of Western blotting results of the ratio of p-ERK/ERK. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; RhoA: Ras homolog gene family, member A; ERK1/2: extracellular regulated protein kinases 1/2; TGF-β: transforming growth factor-beta; ASMCs: airway smooth muscle cells; ROCK: recombinant rho associated coiled coil containing protein kinase; qRT-PCR: quantitative real-time polymerase chain reaction.
Figure 4 SQHT or Fasudil inhibit the RhoA and ERK1/2 pathway in TGF-β-stimulated ASMCs A1: representative immunoblotting band of western blotting results of the relative protein expression levels of RhoA, ROCK1 and 2; A2: histogram of western blotting results; B: qRT-PCR results of the relative mRNA expression levels of RhoA, ROCK1 and 2; C1: representative immunoblotting band of western blotting results of the relative protein expression levels of p-ERK1/2 and ERK1/2; C2: histogram of western blotting results of the relative protein expression levels of p-ERK1/2 and ERK1/2; C3: histogram of Western blotting results of the ratio of p-ERK/ERK. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; RhoA: Ras homolog gene family, member A; ERK1/2: extracellular regulated protein kinases 1/2; TGF-β: transforming growth factor-beta; ASMCs: airway smooth muscle cells; ROCK: recombinant rho associated coiled coil containing protein kinase; qRT-PCR: quantitative real-time polymerase chain reaction.
Figure 5 SQHT inhibits the Snail/Slug signaling pathway A: histogram of qRT-PCR results of the relative mRNA expression levels of Snail and Slug, respectively; B1: representative immunoblotting band of Western blotting results of the relative protein expression levels of Snail and Slug, respectively; B2: histogram of Western blotting results; C1: ELISA results of the concentration of MMP-9; C2: ELISA results of the concentration of TIMP-1. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; qRT-PCR: quantitative real-time polymerase chain reaction; ELISA: enzyme-linked immune sorbent assay; MMP-9: matrix metalloproteinase-9; TIMP-1: tissue inhibitor of metalloproteinase.
Figure 5 SQHT inhibits the Snail/Slug signaling pathway A: histogram of qRT-PCR results of the relative mRNA expression levels of Snail and Slug, respectively; B1: representative immunoblotting band of Western blotting results of the relative protein expression levels of Snail and Slug, respectively; B2: histogram of Western blotting results; C1: ELISA results of the concentration of MMP-9; C2: ELISA results of the concentration of TIMP-1. aP < 0.01, vs Control; bP < 0.01, vs TGF-β group. SQHT: Shenqihuatan formula; qRT-PCR: quantitative real-time polymerase chain reaction; ELISA: enzyme-linked immune sorbent assay; MMP-9: matrix metalloproteinase-9; TIMP-1: tissue inhibitor of metalloproteinase.
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