Study On The Mechanism Of Aconitine-induced Mitochondrial Energy Metabolism Dysfunction In SH-SY5Y Cells

ObjectiveAconitine is the main toxic substance of Chinese materia medica,such as Radix Aconiti,Radix Aconiti Kusnezoffii,and Radix Aconiti Lateralis Preparata,affects the central nervous system and peripheral nervous system.However,the underlying mechanism of aconitine-induced neurotoxicity remains unclear.This study focuses on investigating the influences of aconitine on mitochondrial energy metabolism and the specific molecular mechanisms involved in neuronal cells.MethodsHuman neuroblastoma cells(SH-SY5Y)were used as the study model and aconitine was used as the test substance.DMSO solvent control,50,100,200 and 400μmol/L aconitine stained groups were set up.Cell viability was determined by CCK-8assay kit after 24 h of aconitine treatment,LDH release was assessed by Lactate Dehydrogenase(LDH)activity assay kit and SDH activity was measured by Succinate Dehydrogenase(SDH)activity assay kit.The fluorescent probe DCFH-DA was used to localise and intracellular ROS level was quantified by flow cytometry.Determination of mt DNA content by PCR.Seahorse XF mitochondrial pressure kit was used to detect mitochondrial respiratory function,including mitochondrial basal respiration,ATP production,maximum respiration and proton leakage.Observation of cellular mitochondrial morphology and number with laser confocal microscopy.Western Blot method was used to detect TFAM,p-AMPK/AMPK,OPA1,Mfn1/2,p-Drp1/Drp1,p-Mff/Mff protein expression.The AMPK activator AICAR(0.5mmol/L)pretreated the cells for 1 hour,and then 400 μmol/L aconitine was exposed for 24 hours,and then the mitochondrial respiratory function and p-AMPK/AMPK protein expression were measured again.Results1.Aconitine(400 μmol/L)exposure suppressed cell proliferation and led to increase in reactive oxygen species(ROS)and lactate dehydrogenase(LDH)release(P<0.01).2.Mitochondrial biosynthesis was inhibited after treatment with 400 μmol/L aconitine,which was characterized by a decrease in TFAM expression and a significant reduction in mitochondrial number and mt DNA copy number(P<0.001).3.Aconitine(400 μmol/L)induced abnormal mitochondrial energy metabolism was observed based on significant decrease in ATP production,basal respiration,proton leak,maximal respiration,and succinate dehydrogenase(SDH)activity(P<0.05).4.Aconitine(400 μmol/L)down-regulated mitochondrial fusion protein Mfn2,promoted significant down-regulation of mitochondrial fusion proteins OPA1 and Mfn1(P<0.01),and promoted significant up-regulation of mitochondrial fission proteins p-Drp1 and p-Mff(P<0.01).5.Phosphorylation of AMPK was significantly reduced in 200～400 μmol/L aconitine-treated SH-SY5 Y cells(P<0.05).AMPK activator AIACR pretreatment effectively promoted ATP production to ameliorate mitochondrial energy metabolism disorder caused by aconitine.Conclusion1.Aconitine affected mitochondrial energy metabolism through ATP production inhibition and aerobic respiratory disorder.2.Aconitine weakened mitochondrial respiration via the inhibition of AMPK signaling pathway.3.Reduction of mitochondrial fusion and enhancement of mitochondrial fission may also be the important mechanism of mitochondrial toxicity caused by aconitine.