OBJECTIVE To explore the mechanism of Qingyi Hufei Formula (QHF) in treating radiation-induced pneumonia (RP) based on transcriptomics.

METHODS SPF mice were randomly divided into blank control group, model group, low-dose QHF group, high-dose QHF group, and glycyrrhizic acid (GA) group. A mouse model of RP was established by whole-chest irradiation with a dose of 15 Gy, and the drug intervention was given continuously for 2 weeks from the first day of modeling. The lung coefficient of mice was evaluated, and the pathological damage of lung tissue was observed by HE staining and quantified by Szapiel score. The levels of HMGB1, TNF-α, IL-1β and IL-6 in bronchoalveolar lavage fluid (BALF) were detected by ELISA. Differentially expressed genes in lung tissues were screened by transcriptomics, and GO and KEGG enrichment analysis were performed. The protein expression of HMGB1, TLR4, NF-κB and p-NF-κB in lung tissue was detected by Western blot and immunohistochemistry. Human bronchial epithelial cells (BEAS-2B) were irradiated with 15Gy X-ray to construct RP cell model, and the expression of HMGB1 was regulated by transfection technology. Cell viability was detected by CCK-8 assay. The release of lactate dehydrogenase (LDH) was measured. The levels of TNF-α, IL-1β, and IL-6 in the supernatant were detected by ELISA. The expression of HMGB1/TLR4/NF-κB pathway related proteins was detected by Western blot and immunofluorescence.

RESULTS Compared with the control group, the model group had thickening of alveolar wall, obvious infiltration of inflammatory cells around the bronchus, and significant increases in lung coefficient, Szapiel score, and inflammatory factors such as HMGB1 in BALF. The lung coefficient, Szapiel score and inflammatory indexes in the low and high dose Qingyihufei Formula groups and GA group were significantly decreased and the destruction of alveolar structure and inflammatory infiltration were alleviated. Transcriptome gene enrichment analysis showed that the differentially expressed genes in the prognosis of Qingyihufei Formula were significantly enriched in Toll-like receptor and NF-κB signaling pathways. Western blot and immunohistochemistry results demonstrated that QHF could significantly reduce the protein expression of HMGB1, p-Nf-κB p65/NF-κB p65 and TLR4 in lung tissue. In the in vitro experiments, QHF significantly increased the cell viability, decreased the release of LDH, decreased the secretion of inflammatory factors, and inhibited the protein expression of HMGB1/TLR4/NF-κB pathway. However, overexpression of HMGB1 reversed the above protective effects of Qingyihufei Formula.

CONCLUSION Qingyihufei Formula can effectively treat RP possibly by inhibiting the activation of HMGB1/TLR4/NF-κB signaling pathway and reducing inflammatory response.