| Objective:The following experiments were performed to understand the cytotoxic effects of ellagic acid,an active ingredient of Chinese herbal medicine,on normal renal tubular epithelial cells and mitochondrial function,and to provide a basis for investigating the safety of ellagic acid in the treatment of diabetic nephropathy.Methods:(1)In vitro cultured renal tubule epithelial cells(HK-2 cells)were treated with different concentrations of 0,50,100,150 and 200 μM ellagic acid for 24 and 48 hours to establish a model of renal tubule epithelial cell damage.(2)Cells from each group were harvested under sterile conditions,stained with Annexin V-FITC,and apoptosis was counted by flow cytometry to build an apoptosis model.Expression of apoptosis-related proteins bcl-2 and bax was detected in each group by Western blot.(3)To understand the effects of ellagic acid excess on mitochondrial functional status,cells from each group were harvested under sterile conditions and fluorescent probe DCFH-DA was used to detect ROS by flow cytometry and mitochondrial membrane potential changes by jc-1 staining,ROS,mitochondrial membrane potential changes by confocal microscopy,q RT-.PCR was performed to detect mRNA expression of PINK-1,Parkin,BNIP3,FUNDC1,LCB3 and P62,which are related to mitochondrial function.(4)To better understand the potential mechanisms of apoptosis induced by excess ellagic acid in HK-2 cells,an apoptotic group(100μM ellagic acid,48h)and a normal group were selected or examined for transcriptome sequencing analysis,the differences in gene expression by KEGG and GO functional enrichment analysis,and the differences in apoptosis-related and mitochondria-related genes for correlation analysis.The differences between apoptosis-related and mitochondria-related genes were respectively selected for correlation analysis to investigate the potential mechanisms and pathways involved in renal tubule epithelial cell apoptosis due to excess ellagic acid and the effects of excess ellagic acid on mitochondrial function.Results:Effect of excess ellagic acid on cell viability and apoptosis of HK-2 cells(1)After treatment of HK-2 cells with 50,100,150,200μM ellagic acid for 24 hours,48 hours,CCK-8results showed that compared with the normal group:cell viability of the group treated with 50μM,100μM,150μM,200μM ellagic acid in the intervention time of 24 hours,48 hours.The decrease in cell viability was dose-dependent and all were significantly different(p<0.05),in addition,the decrease in cell viability also changed with time.(2)After treatment of HK-2 cells with 50,100,150,200 μ M ellagic acid for 48 hours,Annexin V-FITC staining showed that apoptosis occurred in HK-2 cells in the groups treated with 100μM,150μM and 200μM ellagic acid in a dose-dependent manner compared with the normal group at the intervention time of 48 hours,and all were significantly different(p<0.0001).Western blot results showed that the expression of anti-apoptotic protein bcl-2 was reduced in the 100μM,150μM and200 μ M ellagic acid-treated groups compared with the normal group,with significant differences(p<0.05).The expression of apoptotic protein bax increased in the 150μM and 200μM ellagic acid-treated groups,with significant differences(p<0.05).Effect of excess ellagic acid on mitochondrial function of HK-2 cells.(1)After treatment of HK-2 cells with50,100,150,200 μ M ellagic acid for 24 hours,jc-1 staining showed that the mitochondrial membrane potential of cells in the 50,100,150,200μM ellagic acid-treated group decreased compared with the normal group,with significant differences(p<0.0001).Confocal microscopy showed that the cell morphology in the ellagic acid-treated group changed compared to the normal group,with elongated tails and increased nuclear fragmentation.The decreased expression of red fluorescence and increased green fluorescence predicted a decrease in mitochondrial membrane potential.(2)After treatment of HK-2 cells with 50,100,150 and 200μM ellagic acid for 6 h,the results of reactive oxygen species(ROS)efflux pattern showed increased ROS production in the 50,100,150 and 200 μ M ellagic acid-treated groups compared with the normal group,with significant differences(p<0.05).Confocal microscopy showed that the green fluorescence of HK-2 cells increased and the size and morphology of cells changed in the ellagic acid-treated group compared with the normal group.(3)After treating HK-2 cells with 50,100,150,200 μ M ellagic acid for 24 hours,q RT-PCR results showed that compared with the normal group:PINK1 mRNA expression increased in the ellagic acid-treated group,but there was no significant difference(p>0.05);Parkin mRNA expression decreased,with significant difference in the 150 μ M ellagic acid-treated group(p<0.05);BNIP3 mRNA expression increased and all were significantly different(p<0.05);FUNDC1 mRNA expression decreased,with significant differences in the150,200 μ M ellagic acid-treated groups(p<0.05);LC3B mRNA expression increased,with significant differences in the 100,200 μ M ellagic acid-treated groups(p<0.05).P62 mRNA expression increased,with significant differences between the 100,150 and 200 μ M ellagic acid-treated groups(p<0.05).Transcriptomic sequencing correlation analysis of the potential mechanism of apoptosis induced by excess ellagic acid(1)There were 5079 differentially expressed genes in the apoptosis group compared with the normal group,of which 2277 genes were upregulated and 2802 genes were downregulated,all of which were significant(p<0.05).(2)The molecular functions of the differentially expressed genes in GO annotation analysis focus on extracellular regions,plasma membrane structures,organelles,and intracellular protein complexes,as well as cellular immune response processes,cellular responses to external stimuli,cellular developmental processes,and biosynthesis of cellular components.The differentially expressed genes analyzed with KEGG annotation are mainly related to hμMan disease development,biological systems,cellular processes,intracellular processing of environmental information,intracellular processing of genetic information,and intracellular metabolism.The functional enrichment analyses of KEGG mainly involve the pathways associated with immunity,the pathways associated with cell proliferation and metabolism,the pathways induced by hypoxia,nucleotide metabolism,the diseases associated with disruption of cell proliferation,infectious diseases,autotoxemia and autotoxemia.KEGG is enriched for pathways related to immunity and pathways related to cell proliferation and metabolism,hypoxia-induced pathways,nucleotide metabolism,cell proliferation disorders,infectious diseases,autoimmune diseases,apoptosis,and autophagy.(3)The results of GO functional enrichment analysis of apoptosis-related genes show that apoptosis-related genes are mainly involved in the regulation of apoptosis signaling pathways,regulation of mitochondrial organization,regulation of outer mitochondrial membrane reeabilization,the upregulation of protein localization activation of cysteine endopeptidase activity and upregulation of peptidase activity,regulation of protein localization on the membrane,exogenous apoptosis signaling pathways,apoptosis The analysis of the enrichment of KEGG signaling pathways showed that the differential genes related to apoptosis were mainly enriched in the TNF,NF-KB,MAPK,and FOXO signaling pathways.(4)GO function enrichment analysis of mitochondria-related genes revealed that the differential expression of mitochondria-related genes is mainly enriched in mitochondrial metabolic homeostasis,mitochondrial autophagy,apoptosis,etc.expressed genes are mainly enriched in reactive oxygen species formation,oxidative phosphorylation processes,fatty acid synthesis and metabolism,neurodegenerative diseases related to mitochondrial dysfunction,diseases related to glucose and lipid metabolism,and nucleic acid metabolism.Diseases related to glucose and lipid metabolism and nucleotide metabolism Cluster analysis of mitochondria-related genes with different expression levels based on certain criteria showed that upregulated genes with different expression levels were mainly responsible for the induction of DNA damage,mitochondrial autophagy,transcriptional expression under hypoxia,and positive regulation of mitochondrial apoptosis pathway under hypoxic conditions,cell growth arrest,and cellular immune activation.Down-regulated genes were found to be mainly involved in mitochondrial respiratory chain organization.Down-regulated genes with different expression levels are mainly involved in the organization of the mitochondrial respiratory chain complex,respiratory chain electron transport,fatty acid metabolism,nucleotide metabolism,glycolysis,ATP synthesis,DNA damage repair and protection,mitochondrial binding proteins,mitochondrial translation,and protein metabolism.Conclusion:Excess ellagic acid is cytotoxic to HK-2 cells,resulting in decreased cell viability,increased cellular mitochondrial reactive oxygen species,decreased membrane potential,impaired mitochondrial autophagy,and the onset of apoptosis,with the onset of apoptosis possibly related to mitochondrial dysfunction. |
PDF Full Text Request