OBJECTIVE To investigate the mechanism of Xiaoai Jiedu Recipe in promoting ferroptosis of tumor cells and inhibiting the growth of transplanted tumors in hepatocellular carcinoma mice by proteomics method.
METHODS C57BL/6J mice were randomly divided into, model group, low- and high-dose groups of Xiaoai Jiedu Recipe, Xiaoai Jiedu Recipe combined with ferroptosis inhibitor group, ferroptosis inhibitor group and cisplatin group. The H22 mouse transplantation tumor model was constructed and the drug administration interventions were as follows: the low- and high-dose groups of Xiaoai Jiedu Recipe were given Xiaoai Jiedu Recipe by gavage at the doses of 10 and 20 g·kg-1·d-1; the ferroptosis inhibitor group was given Liproxstatin-1 by intraperitoneal injection at the dose of 10 mg·kg-1·d-1; the cisplatin group was given cisplatin by intraperitoneal injection at the dose of 10 mg·kg-1·d-1; the Xiaoai Jiedu Recipe combined with ferroptosis inhibitor group was given a gavage dose of 20 g·kg-1·d-1of Xiaoai Jiedu Recipe and an intraperitoneal injection of 10 mg·kg-1·d-1 of the ferroptosis inhibitor Liproxstatin-1; the model group was given an equal amount of saline by gavage. The drugs were administered for 11 d continuously. The tumors were stripped to calculate tumor inhibition rate. Pathological changes were observed by hematoxylin-eosin (HE). Mitochondrial structural changes were observed by transmission electron microscopy. Reactive oxygen species (ROS) levels were detected by flow cytometry. Serum was prepared and analysed by TMT peptide labelling combined with LC-MS/MS to find differential protein expression profiles, applying IPA software. Serum iron ions, glutathione (GSH) and malondialdehyde (MDA) levels were measured biochemically. Protein expression levels of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HMOX1), cystine/glutamate reverse transporter protein (SLC7A11) and glutathione peroxidase 4 (GPX4) were measured by protein Western blot.
RESULTS The tumor growth was inhibited in the low- and high-dose groups and the cisplatin group, with inhibition rates of 36.12%, 51.63% and 57.43%, respectively, while the tumor growth was promoted in the ferroptosis inhibitor group, with an inhibition rate of -45.56%, and the tumor size was reduced in the Xiaoai Jiedu Recipe combined with ferroptosis inhibitor group compared with the ferroptosis inhibitor group, with an inhibition rate of 18.11%. HE staining showed that apoptotic cells and a large number of vacuoles accumulated in the tumor tissues of the high-dose group and the cisplatin group. Transmission electron microscopy showed that the mitochondrial atrophy and membrane density increased to a certain extent in the low and high-dose groups of Xiaoai Jiedu Recipe. Flow cytometry results showed that ROS levels were significantly increased after the intervention of Xiaoai Jiedu Recipe (P < 0.01). Proteomic tests showed that there were 129 differential proteins, including 62 down-regulated proteins and 67 up-regulated proteins, in the high-dose group of Xiaoai Jiedu Recipe compared with the model group, and these differential expressions were involved in lipid metabolism, et al. The results of biochemical tests showed that the intervention of Xiaoai Jiedu Recipe increased the serum iron and MDA levels, and decreased the GSH level in mice. Western blot results showed that the protein expression levels of Nrf2 and HMOX1 increased, and those of SLC7A11 and GPX4 decreased after the intervention of the high-dose group of Xiaoai Jiedu Recipe (P < 0.01).
CONCLUSION Xiaoai Jiedu Recipe promotes ferroptosis in hepatocellular carcinoma cells by inducing the Nrf2/HMOX1 signaling pathway, regulating oxidative stress and elevating the level of lipid peroxidation, which may be one of its mechanisms of action in inhibiting the growth of transplanted tumors.
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