Protective effect of Bufei Yishen Formula against cigarette smoke extract-induced human bronchial epithelial cell damage and its mechanism

doi:10.12122/j.issn.1673-4254.2025.07.03

Abstract

Abstract:

Objective To evaluate the protective effect of Bufei Yishen Formula (BYF) against cigarette smoke extract (CSE)-induced injuries in human bronchial epithelial BEAS-2B cells and explore the underlying mechanism. Methods BEAS-2B cells exposed to CSE were treated with normal rat serum, BYF-medicated rat serum at low or high doses, pyrrolidine dithiocarbamate (PDTC, a NF-κB inhibitor), PDTC combined with high-dose BYF-medicated serum, or S-carbomethyloysteine (S-CMC, as the positive control). CCK-8 assay was used to determine the optimal concentration and treatment time of CSE, BYF-medicated serum and S-CMC. The treated cells were examined for inflammatory factor levels in the supernatant and cellular expressions of MUC5AC and MUC5B using ELISA, cell ultrastructural changes with transmission electron microscopy, and cell apoptosis rate using flow cytometry. The expression levels of TLR4/NF‑κB pathway-associated mRNAs and proteins were determined by qRT-PCR and Western blotting. Results CSE exposure significantly increased secretions of IL-1β, IL-6 and TNF-α, mRNA and protein expressions of MUC5AC and MUC5B, and early and total apoptosis rates in BEAS-2B cells, where the presence of apoptotic bodies was detected. CSE also significantly enhanced the mRNA and protein expressions of TLR4, I-κB, and NF-κB and reduced mRNA and protein expressions of AQP5. Treatments of the CSE-exposed cells with BYF-medicated serum, PDTC and S-CMC all significantly lowered inflammatory factor levels, MUC5AC and MUC5B expressions, and early and total cell apoptosis rates, and partly reversed the changes in cellular ultrastructure and mRNA and protein expressions of the TLR4/NF-κB pathway, and the effects were the most conspicuous following the combined treatment with high-dose BYF-medicated serum and PDTC. Conclusion BYF can inhibit cell apoptosis, inflammation and mucus hypersecretion in CSE-induced BEAS-2B cells by inhibiting the TLR4/NF-κB signaling pathway.

Key words: inflammation response, cigarette smoke extract, Bufei Yishen Formula, mucin hypersecretion, TLR4/NF?κB signaling pathway

Zhengyuan FAN, Zihan SHEN, Ya LI, Tingting SHEN, Gaofeng LI, Suyun LI. Protective effect of Bufei Yishen Formula against cigarette smoke extract-induced human bronchial epithelial cell damage and its mechanism[J]. Journal of Southern Medical University, 2025, 45(7): 1372-1379.

Figures/Tables 10

Tab.1 Treatment protocols in each group

Group	Intervention methods
Control	DMEM+10% blank serum
CSE	DMEM+10%CSE+10% blank serum
BYL	DMEM+10%CSE+5% medicated serum+5%blank serum
BYH	DMEM+10%CSE+10% medicated serum
PDTC	DMEM+10%CSE+10% blank serum+10 μmol/L PDTC
BYH+PDTC	DMEM+10%CSE+10% medicated serum+10 μmol/L PDTC
S-CMC	DMEM+10%CSE+10% blank serum+10 μmol/L S-CMC

Tab.2 Primer sequence for RT-qPCR

Gene	Primer sequence (5'-3')	Length(bp)
TLR4	Forward: CTCCCTGGTGTTGGATTTTACG Reverse: CTCGTTTCTCACCCAGTCCTCA	226
I-κB	Forward: CCCAAGTACCCGGATACAGCA Reverse: GTCATCGTAGGGCAACTCATCTT	133
NF-κB	Forward: GTATTGCTGTGCCTTCCCGA Reverse: CAAGGCCTGGTTTGAGATCTG	146
AQP5	Forward: AGCCCCTCTCACTGGGTCTT Reverse: CTCTCGATGATCTTCCCAGTCC	165
MUC5AC	Forward: GGTCTTCTACTTCCCTGGTCTGTG Reverse: GTCAGCTCAACAACTAGGCCATC	159
MUC5B	Forward: CAGGCTAGTCCTCAACTTCCTGT Reverse: GTGTTGTGGGCGTAGAACTCATT	141

Fig.1 Survival rate of BEAS-2B cells treated with different concentrations of cigarette smoke extract (CSE). *P<0.05 vs Control; #P<0.05 vs 10% CSE for 6 h.

Fig.2 Survival rate of BEAS-2B cells treated with BYF-medicated serum and S-CMC. *P<0.05, **P<0.01 vs Control.

Fig.3 Expressions of IL-6, IL-1β and TNF-α in BEAS-2B cells with different treatments. A: Control group. B: CSE treatment group. C: Low-dose BYF-medicated serum group. D: High-dose BYF-medicated serum group. E: PDTC treatment group. F: High-dose BYF-medicated serum and PDTC treatment group. G: S-CMC treatment group. **P<0.01 vs A; ##P<0.01 vs B; ▲▲P<0.01 vs C; △P<0.05, △△P<0.01 vs E; ●●P<0.01 vs G.

Fig.4 Relative mRNA and protein expressions of MUC5AC and MUC5B in BEAS-2B cells with different treatments. A: Control group. B: CSE treatment group. C: Low-dose BYF-medicated serum group. D: High-dose BYF-medicated serum group. E: PDTC treatment group. F: High-dose BYF-medicated serum and PDTC treatment group. G: S-CMC treatment group. **P<0.01 vs A; ##P<0.01 vs B; ●P<0.05 vs G.

Fig.5 Transmission electron microscopy for examining ultrastructural changes in BEAS-2B cells following different treatments (Scale bar=2 μm). A: Control group. B: CSE treatment group. C: Low-dose BYF-medicated serum group. D: High-dose BYF-medicated serum group. E: PDTC treatment group. F: High-dose BYF-medicated serum and PDTC treatment group. G: S-CMC treatment group.

Fig. 6 Flow cytometry of apoptosis rates of BEAS-2B cells in different groups. A: Control group. B: CSE treatment group. C: Low-dose BYF-medicated serum group. D: High-dose BYF-medicated serum group. E: PDTC group. F: High-dose BYF-medicated serum+PDTC group. G: S-CMC group. **P<0.01 vs A; #P<0.05, ##P<0.01 vs B; ▲▲P<0.01 vs C.

Fig.7 Relative mRNA expressions of TLR4, I-κB, NF-κB and AQP5 in BEAS-2B cells in different groups. A: Control group. B: CSE treatment group. C: Low-dose BYF-medicated serum group. D: High-dose BYF-medicated serum group. E: PDTC group. F: High-dose BYF-medicated serum+PDTC group. G: S-CMC group. **P<0.01 vs A; ##P<0.01 vs B; ▲P<0.05, ▲▲P<0.01 vs C; △△P<0.01 vs E; ●P<0.05, ●●P<0.01 vs G.

Fig.8 Relative protein expressions of TLR4, I-κB, NF-κB and AQP5 of BEAS-2B cells in different groups. A: Control group. B: CSE treatment group. C: Low-dose BYF-medicated serum group. D: High-dose BYF-medicated serum group. E: PDTC group. F: High-dose BYF-medicated serum+PDTC group. G: S-CMC group. **P<0.01 vs A; #P<0.05, ##P<0.01 vs B; ▲P<0.05, ▲▲P<0.01 vs C; △P<0.05, △△P<0.01 vs E; ●P<0.05, ●●P<0.01 vs G.

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