RESUMO
Background: Fuzheng Nizeng Decoction (FZNZ) has a history of decades in gastric precancerous lesions (GPL) treatment, which has shown clear clinical efficacy. Blocking GPL is a key measure to reduce the incidence of gastric cancer (GC). Therefore, we aim to investigate the mechanism of FZNZ-induced ferroptosis and endoplasmic reticulum (ER) in MNNG-induced gastric precancerous lesion (MC) cells, which has been rarely studied in Traditional Chinese Medicine (TCM). Methods: First, CCK8 and lactate dehydrogenase assays were conducted to study the potential effect of FZNZ on MC cells. Second, combined transcriptomic and metabolomic analysis were used to explore the effect and mechanism of FZNZ. Functionally, the occurrence of ferroptosis was assessed by transmission electron microscopy morphological observation and measurement of ferrous iron levels, lipid peroxidation, and glutathione levels. Finally, the expression levels of mRNAs or proteins related to ferroptosis and ER stress were determined by qPCR or western blot assays, respectively. Results: FZNZ inhibited MC cells viability and induced cell death. By metabolomics coupled with transcriptomics analysis, we found that the mechanism of FZNZ treatment induced ferroptosis and was related to glutathione metabolism and ER stress. We then, for the first time, found that FZNZ induced ferroptosis, which contributed to an increase in intracellular ferrous iron, reactive oxygen species, and malondialdehyde and a decrease in glutathione. Meanwhile, the protein level of glutathione peroxidase 4 (GPX4) was decreased. The mRNA levels of ATF3/CHOP/CHAC1, which are related to ferroptosis and ER stress, were also upregulated. Conclusion: Our results elaborate that FZNZ could induce ferroptosis and ER stress in MC cells, and reduce GPX4/GSH. ATF3/CHOP/CHAC1 may play a crosstalk role, which provides a new molecular mechanism for the treatment of GPL.
RESUMO
Attempts to develop gossypol and steroidal hormones alone as a male contraceptive have been tested for many years; however, both caused undesirable side effects that have prevented their acceptance. In this study, we formulated a regimen of combined gossypol at a low dose of 12 mg/kg or a high dose of 50 mg/kg plus methyltestosterone 20 mg/kg and ethinylestradiol 100 g/kg daily (12 mg G+H and 50 mg G+H) administered for 6 weeks in adult rats. The possible roles of germ cell apoptosis and related genes expression were studied by techniques of TdT-mediated dUTP nick end-labeling (TUNEL), agarose gel electrophoresis of low-molecular-weight DNA, in situ hybridization and reverse transcription-polymerase chain reaction detection. Results showed that germ cell apoptosis and related genes expression were significantly induced after combined drug administration. The apoptosis index increased 3.86- and 9.65-fold in the 12-mg and 50-mg G+H-treated groups, respectively, as compared to the control group. DNA ladder formation on the agarose gel further validated the findings of TUNEL-stained apoptotic cells. The apoptosis-related genes fas mRNA expression levels increased 0.44- and 1.39-fold, bax mRNA 0.74- and 2.56-fold, caspase-3 mRNA 0.60- and 1.29-fold, and caspase-9 mRNA 2.50- and 4.08-fold, respectively, in the 12-mg and 50-mg G+H-treated groups vs. the control group. These results indicated that our drug regimen applied as a contraceptive could induce rat germ cell apoptosis. The apoptotic process involved fas system, bax and caspase family genes and the apoptotic extent and cell types were gossypol dose-dependent.