Ginkgolic Acids Impair Mitochondrial Function By Decreasing Mitochondrial Biogenesis And Promoting Mitophagy

Objective: Ginkgolic acid(GA),a natural component extracted from the nuts,leaves,and testa of Ginkgo biloba,has been shown to be SUMOylation inhibitor and histone acetyltransferase inhibitor.GA also shows a wide range of biological activities,including anti-tumor,anti-bacterial,pro-apoptosis effects.Mitochondria harbor the processes of biological oxidative and energy transduction in eukaryotic cells.Mitochondrial dysfunction is associated with a broad spectrum of pathologies,including cancer,aging,neurodegenerative disease,obesity and diabetes.Previous study showed that genes related to the mitochondria present significantly changes in GA treated mouse bone marrow stromal cells,suggesting that GA may play an important role in mitochondria.However,the exact role and mechanism of GA regulation of mitochondrial function are still unclear.The aim of this study was to identify the effects of GA on mitochondrial function and its regulatory mechanism.Methods:(1)The copy number changes of mitochondrial DNA(mt DNA)and the transcription level changes of some related genes was determined by q PCR experiments.(2)The expression changes of mitochondrial related proteins and other related proteins was determined by western blot experiments.(3)Immunofluorescence was used to detect the morphology of mitochondria and the co-localization of mitochondria with autophagosome or lysosome.(4)The mitochondrial membrane potential was detected by JC-1.(5)The oxygen consumption rate was measured by Seahorse XF experiment.(6)The ATP level was determined by luciferase.(7)The differences on gene expression profiling was determined by RNA-sequencing(RNA-seq)analysis and gene sets enrich analysis.(8)CUCKOO Workgroup was used to predict the sites of SUMOylation and acetylation.Result:(1)After the treatment with GA in He La cells,RNA-seq analysis showed that plenty of mitochondrial-related genes were significantly changed,and,at the same time,mitochondria was fragment,mt DNA copy numbers and mitochondrial-related proteins mass were decreased,mitochondrial oxygen consumption rate and the ATP levels were declined.Therefore,the mass and functions of mitochondria were impaired upon GA treatment.(2)The q PCR experiments showed the decrease of mitochondrial related genes and PGC1?(is known as the major regulator of mitochondrial biogenesis),the protein level of PGC1? was also decreased after GA treatment.The inhibitor SR18292 and the activator ZLN005 of PGC1? separately treatment with GA,western blot to examine the mitochondrial proteins demonstrated GA regulates mitochondrial biogenesis through inhibiting PGC1?,further resulting in mitochondrial mass loss.(3)The colocalizations of mitochondria and autophagosomes/lysosomes were increased in GA-treated He La cells compared with the DMSO treatment.Autophagy inhibition by ATG7-knocked out or autophagy inhibitor CQ can restore GA-induced mitochondrial mass loss.These results demonstrated GA decreases mitochondrial mass by inducing mitophagy.(4)The BNIP3 and FUNDC1 knockdown cells were constructed.The knockdown of BNIP3 and FUNDC1 cells and Parkin-overexpressed cells were treated by DMSO or GA,and then examined mitochondrial proteins,mt DNA and mitochondrial membrane potential.The data showed GA induces mitophagy through FUNDC1 pathway.Src kinase and CK2,the phosphorylation-regulated proteins of FUNDC1,were decreased after treatment by GA,which were examined by western blot and q PCR,SUMOylation or acetylation sites prediction were performed by using the GPS website of the CUCKOO Workgroup.The result could hypothesized that GA may regulate the SUMOylation or acetylation of FUNDC1.Conclusions: This study confirmed GA impaired mitochondrial function by inhibiting PGC1?-dependent mitochondrial biogenesis and promoting FUNDC1-dependent mitophagy.This study may provide new mechanistic insights of GA in its future clinical applications for cancers or other diseases and increased the understanding of GA.