[Mechanism of Sangpi Zhike Formula to regulate balance of "oxidation-reduction" axis and alleviate ferroptosis and inflammatory injury for improving post-infection cough based on UPLC-QE-MS and experimental verification].

This study explored the mechanism of Sangpi Zhike Formula in regulating the "oxidation-reduction" balance, reducing the accumulation of reactive oxygen species(ROS), thereby alleviating inflammatory injury and interfering with the process of ferroptosis, and relieving post-infection cough(PIC). The potential pharmacological mechanism was investigated using ultrahigh performance liquid chromatography-Q Exactive-high resolution mass spectrometry(UPLC-QE-MS) and network pharmacology. A total of 70 SPF-grade SD rats(half male and half female) were randomly divided into a normal control group, a model group, a montelukast sodium(western medicine) group, a Ferrostatin-1(ferroptosis inhibitor) group, and low-, medium-, and high-dose Sangpi Zhike Formula(TCM) groups, with 10 rats in each group. The dosages of Sangpi Zhike Formula for the low-, medium-, and high-dose TCM groups were 2.43, 4.85, and 9.70 g·kg~(-1)·d~(-1), respectively. Montelukast sodium was administered at 0.5 mg·kg~(-1)·d~(-1), while Ferrostatin-1 was injected intraperitoneally at 5 mg·kg~(-1). The treatment period lasted 14 d. Pathological damages in lung lobes was assessed by HE staining, ROS accumulation was observed by immunofluorescence, target protein expression was examined by immunohistochemistry, and lung index was calculated to evaluate pulmonary infiltration and congestion. Biochemical assays were performed to measure the levels of total superoxide dismutase(T-SOD), catalase(CAT), malondialdehyde(MDA), total antioxidant capacity(T-AOC), reduced glutathione(GSH), glutathione peroxidase(GSH-PX), oxidized glutathione(GSSG), and total iron in lung homogenates. ELISA was used to quantify tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), and interleukin-6(IL-6). Western blot and qRT-PCR were employed to determine protein and mRNA levels of tumor protein p53(p53), cystine/glutamate transporter(SLC7A11), glutathione peroxidase 4(GPX4), long-chain acyl-CoA synthetase 4(ACSL4), lysophosphatidylcholine acyltransferase 3(LPCAT3), heme oxygenase-1(HO-1), NFE2 related factor 2(Nrf2), NLR family pyrin domain containing 3(NLRP3), Toll-like receptor 4(TLR4), and nuclear factor-kappa light chain enhancer of activated B cells p65(NF-κB p65) in lung tissue. Network pharmacology analysis indicated that the Sangpi Zhike Formula might exert therapeutic effects by targeting TNF, AKT1, IL-6, and TP53, thereby modulating ferroptosis-and pyroptosis-related signaling pathways. Experimental verification showed that compared with the normal group, the model group had obvious inflammatory cell infiltration, structural disorders, significant ROS accumulation in lung tissue, with a significantly increased fluorescence intensity(P<0.01), significantly increased levels of MDA, CAT, GSSG, T-AOC, and total iron, decreased GSH level, and significantly upregulated expressions of p53, ACSL4, LPCAT3, HO-1, NLRP3, TLR4, NF-κB proteins and mRNAs(P<0.01), and significantly downregulated expressions of SLC7A11, GPX4, Nrf2 proteins and mRNAs(P<0.01). Compared with the model group, the medium-dose TCM group and the ferroptosis inhibitor group showed significant differences in the above indicators(P<0.01); other drug intervention groups also showed significant differences in the above indicators compared with the model group(P<0.05). These results suggest that the Sangpi Zhike Formula may alleviate PIC symptoms by adjusting the "oxidation-reduction" balance in lung tissue, reducing ROS accumulation, and thereby alleviating inflammatory injury and interfering with the ferroptosis process.