| Maduramicin frequently causes severe cardiotoxicity in target animals and non-target animals in veterinary clinic.Apoptotic and non-apoptotic cell death mediate the cardiotoxicity induced by maduramicin.However,the mechanism of non-apoptotic cell death induced by maduramicin is unclear.Recent study found that a novel non-apoptotic cell death ‘methuosis’ may mediate maduramicin-caused cardiotoxicity,but its morphological characteristics and molecular mechanism need to be elucidated.In current study,rat cardiomyocyte(H9c2)was used as in vitro model to explore the morphogenesis and mechanism of methuosis in H9c2 cells caused by maduramicin.This study will provide new ideas for understanding the cardiotoxicity of maduramicin and provide a theoretical foundation for guaranteeing human and animal health by detoxication of maduramicin.This study was divided into the following four sections:1 Morphological study of methuosis induced by maduramicin in H9c2 cellsTo investigate the effect of maduramicin on the morphological changes of H9c2 and dissect the relationship between morphology and methuosis,rat cardiomyocyte line H9c2 was used as an in vitro model in this study.H9c2 cells were treated with different concentrations of maduramicin(0,0.15625,0.0625,0.25,1,and 5 μg/m L)for 24,48,and72 h,and the cell morphology and cytoplasmic vacuoles were observed.The changes in cell viability were determined by performing cell counting kit-8(CCK-8)method.After H9c2 cells were treated with maduramicin(1 μg/m L)for 12-36 h,maduramicin was removed to determine whether the severe cytoplasmic vacuolation caused by maduramicin was reversible.In addition,after H9c2 cells were exposed to maduramicin(1 μg/m L)for 24 h,Golgi-Tracker Red,ER-Tracker Red,Lyso-Tracker Red,Mito-Tracker Green and other probe markers were used to clarify the characteristics of vacuoles in cardiomyocytes and its colocalization relationship with organelles.H9c2 cells were treated with maduramicin(1μg/m L)for 48 h,and attached and detached cells were collected to observe the ultrastructure of H9c2 cells by performing transmission electron microscopy,which to investigate the morphological changes of nuclei,mitochondria,endoplasmic reticulum and the characteristics of intracytoplasmic vacuoles,and to determine the similarities and differences of maduramicin-induced methuosis with other cell death types.The results showed that maduramicin triggered severely cytoplasmic vacuolization of H9c2 cells,along with cell death in a time and concentration-dependent manner.Intrerestingly,maduramicin-induced cytoplasmic vacuolization of H9c2 cells was reversible when the remove of maduramicin.The organelle staining results indicated that the cytoplasmic vacuoles induced by maduramicin did not originate from the swelling of organelles such as mitochondria,endoplasmic reticulum,and Golgi apparatus,however,maduramicin-caused cytoplasmic vacuoles were partially co-localized with lysosomes.Transmission electron microscopy observation revealed that the cytoplasmic vacuoles were phase-lucent and surrounded by a single-layer membrane.The obove findings demonstrated that cytoplasmic vacuolization induced by maduramicin is a typical morphological characteristic of methuosis.2 Study on the effects of inhibitors of cellular vacuolization on madramicin-induced cytoplasmic vacuolization in H9c2 cellsIn order to explore the origin of maduramicin-induced cytoplasmic vacuoles in H9c2 cells,after H9c2 cells culturing for a certain period of time,EHT1864,Cytochalasin-D,Bafilomycin A1 and other inhibitors were used to co-incubate with H9c2 cells for a certain period of time,followed by maduramicin exposure(0,0.25,1 μg/m L)for 48 h.Then,the CCK-8 method was performed to determine cell viability.In addition,to analyze the origin of maduramicin-induced cytoplasmic vacuoles in H9c2 cells,an inverted phase-contrast microscope was used to observe the formation of vacuoles in H9c2 cells,investigating whether the number of cytoplasmic vacuoles was reduced,and whether the diameter became smaller,and whether the intracytoplasmic vacuoles disappeared.The results showed that EHT1864,a specific Rac1 inhibitor,significantly inhibited mduramicin-caused cell death rather than the production of cytoplasmic vacuoles;Cytochalasin-D and bafilomycin A1 can significantly inhibited both cell death and cytoplasmic vacuoles induced by maduramicin.These above results suggest that cytoplasmic vacuoles in H9c2 cells induced by maduramicin may originate from micropinocytosis,Rac1 mediates maduramicin-induced cardiotoxicity.3 Mechanism of methuosis mediates maduramicin-induced cytotoxicity of H9c2 cellsTo understand the toxic mechanism of methuosis-mediated H9c2 cell death caused by Maduramicin,H9c2 cells were treated with maduramicin for 24 h,48 h and 72 h.At the same time,H9c2 cells were pre-incubated with EHT1864 and then treated with maduramicin for 48 h.After that,RNA was extracted by Trizol method,and after quality evaluation,RNA was converted into c DNA by reverse transcription,followed by RT-q PCR to analyze the gene expression changes of ras and rac1.At the same time,total cell protein was extracted and determined by BCA method for protein concentration detection,followed by western blot to understand the expression pattern of H-Ras and Rac1.To further clarify the location relationship between cytoplasmic vacuoles and organelles,H9c2 cells were treated with maduramicin for 24 h,and cardiomyocytes were pretreated with cytoplasmic vacuole tracer dextran-alexa flour 488 for 1 h.At the same time,immunofluorescence technology and laser scanning confocal microscopy were carried out to label LAMP1,Rab7,LC3 B,EEA1 and other organelle markers.Moreover,to determine the expression of activated Rac1 protein,the Rac1 activation assay biochem kit was used to detect the Rac1-GTPase protein.The results indicated that the gene and protein expressions of H-Ras and Rac1 in H9c2 cells were increased significantly after maduramicin treatment for 24 h to72 h,and the expression of activated Rac1 was also increased significantly.After pretreatment of H9c2 cells with EHT1864,the expression of Rac1 was decreased significantly.In addition,fluorescence analysis showed that dextran-alexa flour 488 was incorporated into cytoplasmic vacuoles in H9c2 cells.At the same time,maduramicin-induced cytoplasmic vacuoles showed no obvious colocalization with early endosomal marker protein EEA1,late endosomal marker protein Rab7,lysosomal marker protein LAMP1 and autophagosome marker protein LC3 B.In conclusion,Rac1 is a key protein in maduramicin-induced cytotoxicity of H9c2 cells,and the cytoplasmic vacuoles in H9c2 cells caused by maduramicin are derived from macropinocytosis.4 Effect of si RNA silencing Rac1 on methuosis of H9c2 cells induced by maduramicinTo clarify whether Rac1 protein mediates the methuosis of H9c2 cells induced by madomicin,si RNA interference technology was used to knock down the expression of Rac1,investigating the morphological changes and cytotoxicity of H9c2 cells after maduramicin exposure.si RNA design software was used to design multiple si RNA sequences of the target gene rac1,and fluorescent labeled si RNAs were used to optimize the transfection conditions of rac1 in H9c2 cells.After transfection of rac1 si RNA into H9c2 cells,RT-q PCR was carried out to detect the m RNA expression of rac1.When the rac1 si RNA was successfully transfected into cells,H9c2 cells were exposed to maduramicin for 48 h,followed by cell viability assay using CCK-8 method and observation of the changes of intracellular vacuoles by performing inverted phase-contrast microscope.The results showed that after successfully silencing the rac1 gene,the cell death rate induced by maduramicin was significantly reduced.At the same time,cell density after knocking down Rac1 was significantly higher than that of the control group,while the cytoplasmic vacuoles were not decreased.In summary,regulating the expression of Rac1 protein can partially alleviate the cytotoxicity rather than methuosis of H9c2 cells induced by maduramicin. |
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