OBJECTIVE To elucidate the effective substances and mechanism of action of Xingbei Zhike Granules in treating acute bronchitis.

METHODS The potential effective substances and action mechanisms of Xingbei Zhike Granules were predicted through network pharmacology. Subsequently, the effective substances were enriched and purified by macroporous adsorption resin and separated. The anti-inflammatory effective components in the effective substances were screened through an in vitro inflammatory cell model, and a group of effective components (ECPs) was established. Finally, a mouse model of acute bronchitis was constructed by intranasal instillation of LPS to evaluate the pharmacological effects of Xingbei Zhike Granules, effective substances, and ECPs. RT-qPCR was used to detect the expression of main mRNAs and Western blot was used to detect the proteins of inflammatory mediators and the NLRP3 pathway, and the diversity of intestinal flora in mouse feces was analyzed through microbiota analysis.

RESULTS Network pharmacology predicted that the flavonoid components of Xingbei Zhike Granules had anti-inflammatory effects and might treat acute bronchitis through the NLRP3 signaling pathway. The 60% ethanol elution fraction obtained by macroporous adsorption resin was the total flavonoid fraction (XBZK-60). Five flavonoid compounds were isolated from the total flavonoid fraction, namely liquiritin, oroxin A, chrysin-7-O-β-D-glucuronide, chrysin-7-O-gentiobioside, and oroxin B. Oroxin A, chrysin-7-O-β-D-glucuronide and oroxin B were screened as ECPs through an in vitro inflammatory cell model. The in vivo pharmacodynamic results showed that compared with the model group, the Xingbei Zhike Granules group, XBZK-60 group, and ECPs group could significantly reduce the content of IL-6 and IL-1β in the bronchoalveolar lavage fluid of mice with acute bronchitis (P<0.05, P<0.01). RT-qPCR results indicated that each administration group could significantly reduce the mRNA expression of IL-6, IL-1α, IL-1β and TNF-α in the lung tissues of mice with acute bronchitis (P<0.01). Western blot results showed that each administration group could significantly down-regulate the protein expression of Cleaved Caspase-1, ASC, NLRP3, and TNF-α in the lung tissues of mice with acute bronchitis (P<0.05, P<0.01). The gut microbiota analysis showed that compared with the model group, the abundance and richness of gut microbiota in each administration group were significantly increased, and the numbers of beneficial bacteria Lachnospiraceae_NK4A136_group, Prevotellaceae_UCG-001, and Colidextribacter in the gut microbiota were significantly higher than those in the model group.

CONCLUSION Total flavonoid components are the effective substances of Xingbei Zhike Granules in the treatment of acute bronchitis, and they can achieve anti-inflammatory effects by regulating the NLRP3 signaling pathway. Among them, oroxin A, oroxin B and chrysin-7-O-β-D-glucuronide are the key components of the total flavonoids.