[Regulatory effects of serum containing Hedysari Radix Praeparata Cum Melle-vinegar processed Curcumae Rhizoma on mitochondrial function and mitophagy in HT-29 cells].

This study aimed to investigate the effects of the serum containing Hedysari Radix Praeparata Cum Melle-vinegar processed Curcumae Rhizoma(HRCR) on mitochondrial damage and mitophagy in human colon cancer HT-29 cells. The serum containing HRCR was prepared, and a blank group, a 5-fluorouracil(5-FU) group, and serum containing HRCR groups(low, medium, and high doses) were established. The optimal intervention concentration and time of HRCR and 5-FU on HT-29 cells were screened by the cell counting kit-8(CCK-8) method. Changes in the mitochondrial morphology and ultrastructure were observed by transmission electron microscopy and laser confocal microscopy. Mitochondrial membrane potential was detected by the JC-10 fluorescent probe. Intracellular reactive oxygen species(ROS) level was determined by the 2',7'-dichlorodihydrofluorescein diacetate(DCFH-DA) method. Adenosine triphosphate(ATP) content was detected by the ATP kit. Apoptosis rate was detected by Annexin V-FITC/PI double staining flow cytometry. Mitophagy was observed by laser confocal microscopy, and the expression levels of mitochondrial autophagy-related proteins were detected by Western blot. The results showed that after 24, 48, and 72 h of intervention with serum containing HRCR, HT-29 cell proliferation was significantly inhibited in groups at all concentrations. The cell proliferation inhibition of the 5-FU group showed concentration and time dependence. After intervention by 10% HRCR-containing serum for 72 h, the mitochondrial morphology and structure of HT-29 cells were significantly damaged and deformed, with decreased mitochondrial membrane potential, increased intracellular ROS level, decreased ATP content, and increased total apoptosis rate. Under laser confocal microscopy, it was observed that there was almost no co-localization of mitochondria and lysosomes after HRCR intervention. Additionally, HRCR could inhibit the expression of Parkin protein(Parkin), PTEN-induced putative kinase 1(PINK1), and microtubule-associated protein 1A/1B-light chain 3(LC3) in cells, thus inhibiting mitophagy. Through combined treatment of the mitophagy inhibitor Mdivi-1 and HRCR, the inhibitory effect of HRCR on cell proliferation could be further enhanced to promote HRCR-induced apoptosis. In conclusion, the serum containing HRCR can induce apoptosis through the inhibition of HT-29 cell proliferation and mitophagy, which are achieved by damaging mitochondrial morphology, reducing mitochondrial membrane potential, increasing ROS level, decreasing ATP content, and inhibiting the Parkin, PINK1, and LC3 signaling pathways, thereby exerting anti-colon cancer effects.