Pingchuanning Formula suppresses airway inflammation in a rat model of asthmatic cold syndrome by regulating the HMGB1/Beclin-1 axis-mediated autophagy

doi:10.12122/j.issn.1673-4254.2025.06.05

Abstract

Abstract:

Objective To explore the mechanism of Pingchuanning Formula (PCN) for inhibiting airway inflammation in rats with asthmatic cold syndrome. Methods A total of 105 SD rats were randomized equally into 7 groups, including a control group, an asthmatic cold syndrome model group, 3 PCN treatment groups at high, medium and low doses, a Guilong Kechuanning (GLCKN) treatment group, and a dexamethasone (DEX) treatment group. In all but the control rats, asthma cold syndrome models were established and daily gavage of saline, PCN, GLCKN or DEX was administered 29 days after the start of modeling. The changes in general condition, lung function and lung histopathology of the rats were observed, and inflammatory factors in the alveolar lavage fluid (BALF), oxidative stress, lung tissue ultrastructure, cytokine levels, and expressions of the genes related to the HMGB1/Beclin-1 axis and autophagy were analyzed. Results The rat models had obvious manifestations of asthmatic cold syndrome with significantly decreased body mass, food intake, and water intake, reduced FEV_0.3, FVC, and FEV_0.3/FVC, obvious inflammatory cell infiltration in the lung tissue, and increased alveolar inflammation score and counts of neutrophils, eosinophils, lymphocytes, macrophages, and leukocytes in the BALF. The rat models also had significantly increased MDA level and decreased SOD level and exhibited obvious ultrastructural changes in the lung tissues, where the expressions of HMGB1, Beclin-1, ATG5, TNF-α, IL-6,IL-1β, and IL-13 and the LC3II/I ratio were increased, while the levels of Bcl-2 and IFN-γ were decreased. PCN treatment significantly improved these pathological changes in the rat models, and its therapeutic effect was better than that of GLKCN and similar to that of DEX. Conclusion PCN can effectively alleviate airway inflammation in rat models of asthmatic cold syndrome possibly by modulating the HMGB1/Beclin-1 signaling axis to suppress cell autophagy, thereby attenuating airway inflammatory damages.

Key words: bronchial asthma, Pingchuanning Formula, cell autophagy, lung function, airway inflammation, asthmatic cold syndrome

Xinheng WANG, Xiaohan SHAO, Tongtong LI, Lu ZHANG, Qinjun YANG, Weidong YE, Jiabing TONG, Zegeng LI, Xiangming FANG. Pingchuanning Formula suppresses airway inflammation in a rat model of asthmatic cold syndrome by regulating the HMGB1/Beclin-1 axis-mediated autophagy[J]. Journal of Southern Medical University, 2025, 45(6): 1153-1162.

Figures/Tables 11

Tab.1 Composition of Pingchuanning Formula (PCN)

Scientific name	Family	Drug name
Ephedra sinica stapf	Ephedraceae	Chinese ephedra
Prunus armeniaca L.	Rosaceae	Apricot
Citrus reticulata blanco	Rutaceae	Satsuma orenge
Bulbus fritillariae thunbergii	Liliaceae	Bulb of thunberg fritillary
Asarum heterotropoides Fr. Schmidt var.	Aristolochiaceae	Manchur wildginger
Perilla frutescens (L.) Britt.	Lamiaceae	Common perilla
Astragali radix	Fabaceae	Milkvetch root
Radix pseudoxtellariae	Caryophyllaceae	Heterophylly falsestarwort root
Pheretima	Megascolecidae	Earthworm
Pinelliae rhizoma	Araceae	Pinellia ternata
Saposhnikoviae radix	Umbelliferae	Divaricate saposhniovia root

Fig.1 Schematic diagram of the establishment of the rat model and drug administration.

Tab.2 Primers for amplification of the target mRNAs

Gene	Amplicon size (bp)	Forward primer (5'→3')	Reverse primer (5'→3')
GAPDH	127	CAAGGCTGAGAATGGGAAGC	GAAGACGCCAGTAGACTCCA
Beclin-1	134	GAATGAGGGCGACAGTGAAC	CCTGGACCTTCTCCAGGTTT
ATG5	71	TCCTGCCACAGTAAGGGAAG	TCACAGTCAATGGCAGGAGT
HMGB1	85	CCTAAGAAGCCGAGAGGCAA	CATCCGGGTGCTTCTTCTTG-3'
Bcl-2	84	GCCTTCTTTGAGTTCGGTG	GCCAGGAGAAATCAAACAGAG

Fig.2 Comparison of body mass (A), food intake (B), and water intake (C) of the rats among the groups. *P<0.05 vs control group, #P<0.05 vs model group.

Fig.3 Comparison of lung function of the rats among the groups. A: FEV0.3; B: FVC; C: FEV0.3/FVC; *P<0.05 vs control group, #P<0.05 vs model group.

Fig.4 Lung histopathology and inflammation scores of the rats in each group (HE staining, scale bar=40 μm). *P<0.05 vs control group, #P<0.05 vs model group (n=6).

Fig.5 Comparison of inflammatory cell counts in the BALF and oxidative stress factors among the groups. A: Total white cell counts. B: Neu cell counts. C: Eos cell counts. D: Lym cell counts. E: Mac cell counts. F: MDA level. G: SOD level. *P<0.05 vs control group, #P<0.05 vs model group

Fig.6 Ultrastructure of lung tissue of rats in each group observed by TEM (Original magnification:×10 000, ×25 000). AP: Autophagosome; AL: Autophagolysosome.

Fig.7 Expression of autophagy-related mRNAs in the lung tissue of the rats in each group. A: HMGB1 mRNA expression; B: Beclin-1 mRNA expression; C: Bcl-2 mRNA expression; D: ATG5 mRNA expression. *P<0.05 vs control group; #P<0.05 vs model group.

Fig.8 Expression levels of autophagy-associated proteins in the lung tissue of the rats in the groups. A: HMGB1 expression; B: Western blotting for analyzing HMGB1, Beclin-1, Bcl-2, LC3II/LC3I, and ATG5 expressions in the lung tissue; C: Beclin-1 expression; D: Bcl-2 expression; E: LC3II/LC3I ratio; F: ATG5 expression. *P<0.05 vs control group; #P<0.05 vs model group.

Fig.9 Comparison of lung tissue inflammatory factors among the group. A: IFN-γ expression; B: TNF-α expression; C: IL-1β expression; D: IL-6 expression; E: IL-13 expression. *P<0.05 vs control group; #P<0.05 vs model group (n=8)

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