Qufeng Jiejing formula (祛风解痉方) ameliorated the injury of airway smooth muscle cells induced by platelet-derived growth factor-BB through the transforming growth factor-β1/Smads signaling pathway

doi:10.19852/j.cnki.jtcm.2025.04.003

Abstract

Abstract:

OBJECTIVE: To explore the role and mechanism of Qufeng Jiejing (祛风解痉，QFJJ) formula in the asthma progression.

METHODS: The Bagg Albino/c mice treated with Ovalbumin and AL(OH)3, and airway smooth muscle cells (ASMCs) treated with platelet-derived growth factor (PDGF)-BB to establish a asthma model in vivo and in vitro. The cell morphology was observed with microscope and immunofluorescence staining. The cell viability was assessed with methyl thiazolyl tetrazolium assay. The tumor necrosis factor-αlpha (TNF-α), interleukin-1beta (IL-1β), laminin, fibronectin and collagen IV levels in the ASMCs were detected with corresponding enzyme linked immunosorbent assay kits. Transwell and wound healing assays were conducted to test the cell migration. The TGF-β1, Smad2 and Smad3 levels were measured with Western blot.

RESULTS: We found that QFJJ formula treatment dramatically decreased the cell viability, TNF-α, IL-1β, laminin, fibronectin and collagen IV levels in the PDGF-BB stimulated ASMCs. Additionally, the protein levels of TGF-β1, Smad2 and Smad3 in the PDGF-BB stimulated ASMCs were prominently depleted after QFJJ formula treatment. Besides, SRI treatment neutralized the role of QFJJ formula in the PDGF-BB stimulated ASMCs.

CONCLUSION: QFJJ formula effectively relieved the asthma progression through ameliorate the ASMCs function, which was achieved through suppressing the TGF-β1/Smads signaling pathway.

Key words: asthma, myocytes, smooth muscle, transforming growth factor beta1, Smad proteins, signal transduction, Qufeng Jiejing formula

FAN Changzheng, ZHANG Qiong, FAN Maorong, MENG Hongxu, CONG Xiaodong, FAN Yiling, YUAN Shasha, MIAO Qing. Qufeng Jiejing formula (祛风解痉方) ameliorated the injury of airway smooth muscle cells induced by platelet-derived growth factor-BB through the transforming growth factor-β1/Smads signaling pathway[J]. Journal of Traditional Chinese Medicine, 2025, 45(4): 730-738.

Figures/Tables 7

Figure 1 QFJJ alleviate injured ASMCs induced by PDGF-BB A: cell viability detected by MTT assay; B: cell migration detected by transwell assay. B1: 0 μM PDGF-BB; B2: 12 μM PDGF-BB; B3: 25 μM PDGF-BB; B4: 50 μM PDGF-BB; C: statistical analysis of transwell result; D: cell migration detected by wound healing assay. D1: 0 μM PDGF-BB for 0 h; D2: 12 μM PDGF-BB for 0 h; D3: 25 μM PDGF-BB for 0 h; D4: 50 μM PDGF-BB for 0 h; D5: 0 μM PDGF-BB for 24 h; D6: 12 μM PDGF-BB for 24 h; D7: 25 μM PDGF-BB for 24 h; D8: 50 μM PDGF-BB for 24 h; E: statistical analysis of wound healing result. PDGF-BB: platelet-derived growth factor-BB. QFJJ: Qufeng Jiejing; ASMCs: airway smooth muscle cells; MTT: methylthiazolyldiphenyl-tetrazolium bromide; ANOVA: analysis of variance. Each treatment was performed in triplicate, and values are represented as the mean ± standard deviation (n = 6). One-way ANOVA was used for comparison among multiple groups, and Duncan test was used for further pairwise comparisons. Compared with PDGF-BB (0 ng/mL) group, aP < 0.05.

Table 1 Effects of different concentrations of PDGF-BB on inflammatory cytokines and extracellular matrix components (pg/mL, $\bar{x} \pm s$)

Item	PDGF-BB (ng/mL)
Item	0 (n = 3)	12 (n = 3)	25 (n = 3)	50 (n = 3)
TNF-α	13.20±0.75	20.76±1.37^a	48.02±3.77^b	52.37±4.48^b
IL-1β	2.94±0.27	3.71±0.44	6.82±0.50^b	7.65±0.54^b
Laminin	18.34±1.70	23.74±2.25	45.00±5.64^b	50.23±5.26^b
Fibronectin	26.60±3.27	31.44±4.91	45.18±3.35^b	52.42±5.08^b
Collagen IV	20.90±3.43	27.67±1.86^a	37.45±3.63^b	42.85±1.49^b

Figure 2 QFJJ formula relieved the injury in ASMCs induced by PDGF-BB A: ASMCs were cultured (1 × 107/well) in 96-well plates, and pre-cultured with 50 ng/mL PDGF-BB, then treated with PAT or different concentrations of QFJJ (0.59, 1.18, and 2.36 g/mL), cell viability detected by MTT assay for 3.5 h; B: ASMCs were cultured (1 × 105/well) in transwell, and pre-cultured with 50 ng/mL PDGF-BB, then treated with PAT or different concentrations of QFJJ, the cell migration detected by transwell assays for 6 h; B1: Control; B2: 50 μM PDGF-BB; B3: 50 μM PDGF-BB and PAT; B4: 50 μM PDGF-BB and 0.59 g/mL QFJJ; B5: 50 μM PDGF-BB and 1.18 g/mL QFJJ; B6: 50 μM PDGF-BB and 2.36 g/mL QFJJ. C: statistical analysis of transwell result; D: ASMCs were cultured (2000 /well) in 6-well plates, and pre-cultured with 50 ng/mL PDGF-BB, then treated with PAT or different concentrations of QFJJ, the cell migration detected by wound healing assays. D1: Control for 0 h; D2: 50 μM PDGF-BB for 0 h; D3: 50 μM PDGF-BB and PAT for 0 h; D4: 50 μM PDGF-BB and 0.59 g/mL QFJJ for 0 h; D5: 50 μM PDGF-BB and 1.18 g/mL QFJJ for 0 h; D6: 50 μM PDGF-BB and 2.36 g/mL QFJJ for 0 h; D7: Control for 24 h; D8: 50 μM PDGF-BB for 24 h; D9: 50 μM PDGF-BB and PAT for 24 h; D10: 50 μM PDGF-BB and 0.59 g/mL QFJJ for 24 h; D11: 50 μM PDGF-BB and 1.18 g/mL QFJJ for 24 h; D12: 50 μM PDGF-BB and 2.36 g/mL QFJJ for 24 h; E: statistical analysis of wound healing result. QFJJ: Qufeng Jiejing; ASMCs: airway smooth muscle cells; MTT: methylthiazolyldiphenyl-tetrazolium bromide; PDGF-BB: platelet-derived growth factor-BB; Con: control; PAT: prednisone acetate tablets; L: low dose; M: medium dose; H: high dose. Each treatment was performed in triplicate, and values are represented as the mean ± standard deviation (n = 6). Compared with control group, aP < 0.05; compared with PDGF-BB (50 ng/mL) group, bP < 0.05.

Table 2 Effects of different concentrations of QFJJ formulation and PAT positive control on PDGF-BB-stimulated ASMCs inflammatory cytokines and extracellular matrix components (pg/mL, $\bar{x} \pm s$)

Item	Con (n = 3)	PDGF-BB
Item	Con (n = 3)	- (n = 3)	PAT (n = 3)	L (n = 3)	M (n = 3)	H (n = 3)
TNF-α	12.60±0.80	51.08±4.61^a	23.52±3.73^b	46.89±4.18	26.20±0.95^b	18.66±4.27^b
IL-1β	3.11±0.10	7.40±0.50^a	4.18±0.47^b	6.08±0.25^b	3.88±0.71^b	3.38±0.16^b
Laminin	20.40±3.45	42.28±3.48^a	28.10±4.92^b	34.67±1.64^b	27.21±4.73^b	23.28±2.55^b
Fibronectin	24.65±1.67	47.43±0.67^a	29.91±2.03^b	38.45±3.68^b	29.33±1.96^b	25.98±1.95^b
Collagen IV	22.22±2.52	40.81±4.18^a	25.05±2.07^b	34.54±1.07	23.93±1.63^b	20.73±2.46^b

Figure 3 Activation of TGF-β1/Smads signaling pathway reversed the QFJJ formula roles in the PDGF-BB induced ASMCs A1: Western blot analysis was performed to detect protein expression; A2: gray analysis of the protein levels of TGF-β1; A3: gray analysis of the protein ratio of p-smad2/smad2; A4: gray analysis of the protein ratio of p-smad3/smad3; B: cell viability detected by MTT assay for 3.5 h; C: cell migration detected by transwell assays for 6 h. C1: Control; C2: 50 ng/mL PDGF-BB; C3: 50 ng/mL PDGF-BB and 1.18 g/mL QFJJ; C4: 50 ng/mL PDGF-BB, and 1.18 g/mL QFJJ and SRI. D: statistical analysis of transwell result; E: cell migration detected by wound healing assays. E1: Conrol for 0 h; E2: 50 ng/mL PDGF-BB for 0 h; E3: 50 ng/mL PDGF-BB and 1.18 g/mL QFJJ for 0 h; E4: 50 ng/mL PDGF-BB, 1.18 g/mL QFJJ and SRI for 0 h; E5: Control for 24 h; E6: 50 ng/mL PDGF-BB for 24 h; E7: 50 ng/mL PDGF-BB and 1.18 g/mL QFJJ for 24 h; E4: 50 ng/mL PDGF-BB, 1.18 g/mL QFJJ and SRI for 24 h; F: statistical analysis of wound healing result. TGF-β1: transforming growth factor-β 1; QFJJ: qufengjiejing; ASMCs: airway smooth muscle cells; MTT: methylthiazolyldiphenyl-tetrazolium bromide; PDGF-BB: platelet-derived growth factor-BB; Con: Control; M: medium dose of QFJJ; SRI: SRI-011381 hydrochloride; ANOVA: analysis of variance. Each treatment was performed in triplicate, and values are represented as the mean ± standard deviation (n = 6). One-way ANOVA was used for comparison among multiple groups, and Duncan test was used for further pairwise comparisons. Compared with control group, aP < 0.05; compared with PDGF-BB (50 ng/mL) group, bP < 0.05; compared with PDGF-BB (50 ng/mL) and QFJJ (1.18 g/mL) group, cP < 0.05.

Table 3 Effects of TGF-β1/Smads signaling pathway on QFJJ-treated PDGF-BB stimulation of ASMCs inflammatory cytokines and extracellular matrix components (pg/mL, $\bar{x} \pm s$)

Item	Con (n = 3)	PDGF-BB (n = 3)	PDGF-BB+M (n = 3)	PDGF-BB+M+SRI (n = 3)
TNF-α	13.00±1.79	52.96±2.94^a	25.29±5.98^b	41.34±3.73^c
IL-1β	3.32±0.47	6.91±0.45^a	4.18±0.14^b	5.76±0.45^c
Laminin	23.17±3.48	39.74±1.16^a	25.13±0.93^b	33.51±2.99^c
Fibronectin	24.35±4.83	43.94±3.74^a	28.55±3.07^b	38.17±3.97^c
Collagen IV	15.88±5.19	40.15±2.59^a	23.49±4.03^b	36.66±2.58^c

Figure 4 QFJJ formula inhibited the progression of asthma in vivo A: sections of left lung tissue from mice were used for HE staining; A1: Control; A2: OVA mice; A3: OVA mice treated with PAT; A4: OVA mice treated with 0.59 g QFJJ; A5: OVA mice treated with 1.18 g QFJJ; A6: OVA mice treated with 2.36 g QFJJ. B: statistical analysis of the inflammation score for each mice; C: statistical analysis of the WAi/Pbm for each mice; D: statistical analysis of the WAm/Pbm for each mice; E: the mRNA levels of TGF-β1, Smad2 and Smad3 were detected with PCR assay; E1: TGF-β1; E2: smad2; E3: smad3; F: the protein levels of TGF-β1, p-Smad2 and p-Smad3 were analyzed with western blot assay; F1: electrophoretic bands; F2: gray analysis of TGF-β1 protein levels; F3: gray analysis of the protein ratio of p-smad2/smad2; F4: gray analysis of the protein ratio of p-smad3/smad3. QFJJ: Qufeng Jiejing; HE: hematoxylin-eosin staining; PDGF-BB: platelet-derived growth factor-BB; OVA: ovalbumin; WAm/Pbm: Wall area of smooth muscle / perimeter of bronchial membrane；WAi/Pbm: wall area index / perimeter of bronchial membrane; PAT: prednisone acetate tablets; Con: control; L: low dose; M: medium dose; H: high dose; PCR: Polymerase chain reaction; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; TGF-β1: transforming growth factor-beta 1; ANOVA: analysis of variance. Each treatment was performed in triplicate, and values are represented as the mean ± standard deviation (n = 6). One-way ANOVA was used for comparison among multiple groups, and Duncan test was used for further pairwise comparisons. Compared with control group, aP < 0.05; compared with OVA group, bP < 0.05.

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