RESUMO
Aim To explore the internal mechanism of NK2 activation of NK cells from the perspective of "mitochondrial dysfunction-abnormal cell activation".Methods NK-92MI cells were divided into blank group, TSLP group, 1, 5, and 10 μmol·L-1 Mdivi-1 dose groups.The levels of IL-4, IL-5, and IFN-γ in the supernatant of each group were determined by ELISA; The expression of p-Drp1 and MnSOD protein in each group was determined by Western blotting; the ROS level of each group was detected by DHE staining and flow cytometry; mitochondrial morphology was observed by confocal laser in each group of cells.Results ELISA showed that compared with control group, the levels of IL-4 and IL-5 in cell supernatant of TSLP group significantly increased, and the level of IFN-γ was down-regulated(P<0.05); Compared with TSLP group, the levels of IL-4 and IL-5 in cell supernatant of 5 and 10 μmol·L-1 Mdivi-1 group decreased, and the IFN-γ concentration of the 10 μmol·L-1 Mdivi-1 group rose(P<0.05).DHE staining and flow cytometry showed that ROS level of cells in TSLP group was significantly higher than control group.Compared with TSLP group, ROS level of the 5 and 10 μmol·L-1 Mdivi-1 groups decreased(P<0.05).The laser confocal results showed that after TSLP stimulation, a large number of spherical mitochondria were formed in cells.This phenomenon was improved to a certain extent after the intervention of 5, 10 μmol·L-1 Mdivi-1.Western blot analysis showed that the p-Drp1 level of NK-92MI cells in TSLP group was significantly up-regulated, and the expression of MnSOD decreased, while the intervention of Mdivi-1 effectively reversed the changes in the expression of the above-mentioned molecules.Conclusions Mitochondrial dynamic imbalance may be one of the internal mechanisms of abnormal activation of NK cells, and it may be an important target for regulating NK2 activation of NK cells and improving the allergic inflammatory response mediated by it.
RESUMO
Objective: To observe the apoptosis and mitochondrial fission of human ovarian cancer SKOV3 cells after treated by myricetin and dynamin related protein 1 (DRP1) inhibitor mdivi-1 alone or combined, and to explore the mechanism of myricetin in inducing the apoptosis of SKOV3 cells. Methods: The SKOV3 cells were cultured in vitro and randomly divided into control group, mdivi-1 group, myricetin group and combined group. The cells in mdivi-1 group were treated with 50 μmol · L-1 madivi-1 for 1 h followed by common culture medium for 23 h; the cells in myricetin group were treated with 50 g · L-1 myricetin for 24 h; the cells combined group were treated with 50 jumol · L-1 midiv-1 for 1 h followed by 50 g · L-1 myricetin for 23 h. The survival rates of cells in various groups were detected by MTT assay. The apoptoic rates of cells in various groups were detected by Muse14 apoptosis detection kit. The expression levels of Cyt C, caspase3, DRP1 and FIS1 were observed by Western blotting method. The mitochondrial fission of cells in various groups was observed with MitoTracker® Red. Results; Compared with control group, the survival rate of cells in myricetin group was decreased significantly (P<0. 05); compared with myricetin group, the survival rate of cells in combined group was increased significantly (P<0. 05). Compared with control group, the apoptotic rate of cells in myricetin group was increased (P<0. 05); compared with myricetin group, the apoptotic rate of cells in combined group was decreased (P<0. 05). Compared with control group, the expression levels of Cyt C and caspase3 proteins in the cells in myricetin group were increased (P<0. 05); compared with myricetin group, the expression levels of Cyto C and caspase3 proteins in the cells in combined group were decreased (P<0. 05). Compared with control group, the degree of mitochondrial fission of the cells in myricetin group was increased; compared myricetin group, the degree of mitochondrial fission of the cells in combined group was decreased. Compared with control group, the expression levels of DRP1 and FIS1 proteins in the cells in myricetin group were increased (P<0. 05); compared with myricetin group, the expression levels of DRP1 and FIS1 proteins in the cells in combined group were decreased (P<0. 05). Conclusion: Myricetin can induce the apoptosis of human ovarian cancer SKOV3 cells by promoting the DRPl-dependent mitochondrial fission.
RESUMO
Objective To detect the expression of mitochondrial dynamics proteins (Mfn2 and Drp1) in thyroid squa?mous carcinoma cell line SW579 and the effects of Mitochondrial division inhibitor, Mdivi-1, on proliferation, apoptosis and invasion of SW579. Methods In SW579 and Nthy-ori 3-1 cell lines, the expression levels of Mfn2 and Drp1 were deter?mined by western blot while the transcription level of Mfn2 and Drp1 mRNA were measured by RT-PCR. Then, SW579 cells were divided into control group (DMSO, 0.1%) and Mdivi-1 low, medium and high dose groups (Mdivi-1 of 15,30 and 45μmol/L were incubated with cells for 16 hours respectively). Then the ability of cell proliferation was detected using MTT assay, the mitochondrial membrane potential was determined by fluorescence spectrophotometer, the expression levels of cy?tochrome C and Caspase-3 were quantified by Western blot and the transcription level of the Cyt C and Caspase-3 mRNA were determined by RT-PCR. The ability of invasion in each group was measured with Transwell assays. Results Com?pared with Nthy-ori 3-1, the mRNA transcription and protein expression levels of the Mfn2 was remarkably decreased, while the mRNA transcription and protein expression of the Drp1 was significantly increased in SW579 cells (P<0.01). Compared with control group, the cell survival rates and mitochondrial membrane potential of SW579 were decreased dramat?ically (P<0.01). The mRNA transcription and protein expression of the cytochrome C and Caspase-3 were increased dra?matically (P<0.01) and the capability of invasion was markedly decreased in all the Mdivi-1 groups in a dosage dependent manner compared with those in control groups (P<0.01). Conclusion Abnormal mitochondrial dynamics may be involved in thyroid squamous cell carcinoma SW579 cells;Mdivi-1 can inhibit the cell proliferation and invasion as well as induce apoptosis.
RESUMO
Objective To detect the effects of the selective mitochondrial fission inhibitor-Mdivi-1 on the malondi-alolehyde (MDA), glutathione (GSH) as well as cytochrome C (Cyt-C) in neuronal mitochondria and neuronal apoptosis. Methods Thirty-six adult female SD rats (250-300 g) were randomly divided into 3 groups (n=12):sham operation (Sham) group, single spinal cord injury (SCI) group and Mdivi-1 pretreatment (1.20 mg/kg, Mdivi-1) group. In sham group, the rats’ spinal cord was exposed, but no hit. The rat model of spinal cord injury was established by Allen’s method in SCI group and Mdivi-1 group. In Mdivi-1 group, rats were given Mdivi-1 through the tail vein 15 min before spinal cord injury, and SCI group received the same amount of dimethyl sulfoxide (DMSO). Rats in Sham group were sacrificed 8 h after exposing spinal cord. Rats in SCI group and Mdivi-1 group were sacrificed at 8 h after the spinal cord injury, then were removed the spinal cord T9-11. The contents of MDA and GSH in mitochondria of spinal cord tissues were detected with spectrophotometer. The expressions of Cyt-C protein in the mitochondria and cytoplasm were detected by Western blot assay. The neuronal apoptosis was assessed by TUNEL staining. Results Compared with Sham group, levels of Cyt-C and GSH in mitochondria were decreased significantly (P<0.01), while levels of MDA in mitochondria, Cyt-C in cytoplasm and the neuronal apopto-sis were increased significantly in SCI group (P<0.01). Compared with SCI group, Cyt-C and GSH levels in mitochondria were increased significantly in Mdivi-1 group (P<0.01), however, MDA in mitochondria,Cyt-C in cytoplasm and the neuro-nal apoptosis were significantly reduced (P<0.01). Conclusion Mdivi-1 can relieve neurons from mitochondrial oxidative damage, inhibit the release of cytochrome C and neuronal apoptosis after acute spinal cord injury, which plays a role in pro-moting the recovery of spinal cord function.