ABSTRACT
Objective To investigate the effects of the rhein on the mitochondria fission and epithelial-mesenchymal transition(EMT)of breast cancer cells MDA-MB-231.Methods Human breast cancer cells were intervened with rhein,and the cells were divided into control group(0 μmol·L-1),low dose rhein group(100 μmol·L-1),and high dose rhein group(200 μmol·L-1).The proliferation activity of the cells was detected by CCK-8,and migrations was detected by Scratch-healing migration assay.The morphology and distribution of mitochondria were detected by transmission electron microscope,and the expression levels of Dynamin-related protein 1(Drp1),mitofusin2(Mfn2),E-cadherin,Vimentin proteins were detected by Western blot.Results Compared with control group,Rhein significantly reduced the protein expression of Drp1、Vimentin(P<0.05),and increased E-cadherin and Mfn2,thus down-regulating mitochondria fission,inhibiting cell proliferation and migration.High dose Rhein was better than low dose.Conclusion Rhein can inhibit the proliferation and migration of breast cancer cells by reducing the expression of Drp1,Vimentin and up-regulating Mfn2,E-cadherin proteins.
ABSTRACT
To explore whether the downregulation of protein phosphatase 2A catalytic subunit(PP2Ac)involved in the pathogenesis of mitochondria fission/fusion dynamics and functional imbalance induced by human tau accumulation. After cotransfection with mito-dsRed plasmids and pIRES-eGFP-tau40 plasmids 48 hours,the rat primary hippocampal neurons were observed with a laser scanning confocal microscope for their changes in shape and distribution of mitochondria.The expressions of mitochondria fission/fusion protein and PP2Ac and PP2Ab were detected by Western blotting.Furthermore,the shape and distribution of mitochondria of rat primary hippocampal neuron and wild type 293wt cells were assayed 48 hours after co-transfection with siPP2Ac-EGFP plasmids and mito-DsRed plasmids,and the fission/fusion dynamics of 293wt cells was captured with live cell time-lapse imaging after co-transfection with siPP2Ac plasmids and mito-Dendra2 plasmids.After transfection with siPP2Ac plasmids,the relative level of mitochondria fission/fusion protein of 293wt cells was assayed by Western blotting,and mitochondria membrane potential was detected by JC-1 staining,and the cellular viability was measured by CCK8 assay.Finally,the shape and distribution and membrane potential of mitochondria of HEK293 cells with stable transfection of htau40(293htau)were detected after co-transfection with PP2Ac and mito-dsRed plasmids. Human tau40 expression decreased distribution of mitochondria and significantly lowered PP2Ac level in primary hippocampal neuron(=4.814, =0.0086).Down-regulation of PP2Ac caused mitochondria elongation and perinuclear accumulation in primary hippocampal neuron and 293wt cells;in addition,down-regulation of PP2Ac in 293wt cells significantly increased mitochondria fusion rate(=2.857, =0.0074)and the levels of mitochondria fusion protein mitofusin(MFN)1(=6.768, =0.0025),MFN2(=3.121, =0.0035),and optic atrophy 1(=3.775, =0.0199);however,the levels of dynamin-like protein-1 and Fis1 remained unchanged.The down-regulation of PP2Ac in 293wt cells led to the significant decrease in mitochondria membrane potential(=2.300, =0.0270)and cell viability(=6.249, <0.0001).Finally,up-regulation of PP2Ac attenuated the abnormalities in the shape,distribution and function of mitochondria in the 293htau cells. Down-regulation of PP2Ac is involved in the abnormal shape and distribution of mitochondria and its dysfunction induced by human tau40 in rat primary hippocampal neurons and HEK293 cells.
Subject(s)
Animals , Humans , Rats , Catalytic Domain , Down-Regulation , HEK293 Cells , Mitochondria , Protein Phosphatase 2 , tau ProteinsABSTRACT
This study aimed to investigate the effect and mechanism of ligustilide, the main active ingredient in Ligusticum wallichii, on mitochondria fission after PC12 cell injury induced by oxygen and glucose deprivation/reperfusion(OGD/R). In the experiment, an OGD/R model was established in vitro, and PC12 cells were pre-treated with ligustilide for 3 h, and then the cell viability was detected by CCK-8 method. The effect of different concentrations of ligustilide on the morphology of PC12 cells after OGD/R injury was observed under an inverted microscope. Transmission electron microscopy was used to observe the mitochondrial fission of PC12 cells after OGD/R injury. DCFH-DA immunofluorescence staining method was used to detect intracellular reactive oxygen species(ROS) changes. Changes in mitochondria membrane potential(MMP) were detected by flow cytometry. Hochest 33258 was used to observe the apoptosis of PC12 cells. Western blot was used to detect changes in cytochrome C(Cyt C) content in mitochondria and cytoplasm, and mitochondrial fission-related proteins Drp 1 and Fis 1. All results showed that compared with the model group, ligustilide significantly increased the survival rate of PC12 cells and the number of cells. Further experiments showed that ligustilide inhibited the release of ROS and decline of mitochondrial membrane potential in PC12 cells after OGD/R injury. Moreover, ligustilide reduced the release of Cyt C and promoted the expressions of Drp1 and Fis1 in mitochondrial fission proteins. Verification experiments showed that mitochondrial fission inhibitor mdivi-1 decreased cell survival rate and inhibited fission. The results indicated that ligustilide exerted neuro-protective effects by promoting mitochondrial fission and reducing cell damage. It preliminary proves that the mechanism of ligustilide on ischemic brain injury may be related to the promotion of mitochondrial fission and the maintenance of cell homeostasis.
Subject(s)
Animals , Rats , 4-Butyrolactone , Apoptosis , Cell Survival , Glucose , Mitochondria , Oxygen , PC12 Cells , Reactive Oxygen Species , Reperfusion InjuryABSTRACT
Aim To investigate the protection mecha-nism of the extraction of the saffron crocus in ischemia/reperfusion rats. Methods Hematoxylin-eosin stai-ning, electron microscopy, and neurological assess-ments were performed in a transient middle cerebral ar-tery occlusion ( tMCAO ) rat model. The role of dy-namin-related protein 1 ( Drp1 ) and optic atrophy 1 ( Opa1 ) , the two key regulators of mitochondrial fis-sion and fusion in ischemic brain damage in vivo were observed. Results In ischemia/reperfusion rats, the extraction of the saffron crocus increased the level of protein Opa1 and decreased the level of protein Drp1 . Conclusions Inhibition of Drp1 and promotion of Opa1 , which means to maintain balancing mitochondri-al dynamics, is proposed as an efficient strategy for neuroprotection against ischemic brain damage.