Consider The Effect Of Aconitine On The Phosphorylation State Of Phosphoprotein And Its Mechanism

【Objectives】Aconotine is the main component of alkaloids in Aconitum,which has a wide range of medicinal value.However,the therapeutic dose of aconitine is close to the lethal dose,poisoning can lead to serious arrhythmia and death.The toxicology and pharmacological mechanism of aconitine is complicated,and its cardiotoxicity mechanism is still unclear.The clinical manifestations of arrhythmia are varied and the etiology is complex.Ca2+plays an important role in the regulation of various functions of the body,especially at the cellular level.Calmodulin in cell membrane and sarcoplasmic reticulum have different functions and are regulated by various signal pathways.Phospholamban(PLN)is an important protein regulating calcium balance in cells.It changes its binding to sarcoplasmic reticulum calcium-ATPases(SERCAs)through phosphorylation modification,thus regulating SERCA2 a function.Since SERCA2 a is the only protein in the sarcoplasmic reticulum that can reabsorb Ca2+ in the cytoplasm,phosphorylation of PLN plays an important role in intracellular calcium balance.At present,studies on calcium balance disorders of cardiomyocytes are mainly focused on clinical medicine,and PLN protein is mainly explored from the perspective of cardiogenic diseases,such as cardiac hypertrophy,myocardial infarction,myocardial ischemia reperfusion,etc.However,in forensic medicine and toxicology,especially the study on Ca2+ regulation disorders of cells by aconitine,there is still a lack of corresponding in-depth exploration.Previous studies have confirmed that aconitine poisoning can cause intracellular calcium disturbances,leading to calcium overload.In the early stage,our research group used primary cultured rat ventricular myocytes to carry out experiments.In the process of experiments,the protein abundance of PLN was low,and the expression abundance of its phosphorylated protein was lower.The Western blot detection showed that the band was light,the background interference was strong,and the detection of phosphorylated protein could not be completed satisfactorily.As a result,the exploration of phosphorylation status and its mechanism encountered a bottleneck: it was difficult to detect by Western blot,and the follow-up analysis was difficult.Therefore,our group used the Flp-In system to construct a TRex293 cell line that can stably express human PLN protein,named hPLN-293 cell(related papers are being published and edited),The cell line can overexpress PLN protein under the induction of Doxycyline(Dox),and the cell line can proliferate,which is convenient for observing the influence of drug intervention on the phosphorylation state of PLN and conducting research on its cytotoxicity mechanism.The following experiments are accomplished:(1)Induce the expression of PLN on the first,fifth,and tenth generation hPLN-293 cells,and test the stability of the cells expressing PLN.(2)The morphological changes of cells induced by aconitine were observed by using various concentrations and varying times of exposure CCK8 method was used to determine the impression of aconitine on cell activity,to determine the appropriate conditions for subsequent experiments.(3)To detect the effect of aconitine exposure on the concentration of calcium ions in cells,and explore the effect of aconitine on the phosphorylation state of PLN protein and its Thr17 sites,Ser16 sites,as well as the phosphorylation status of its upstream phosphorylases Ca MKⅡand PKA and to further explore the cytotoxicity mechanism of aconitine.【Methods】(1)Construct hPLN-293 cell line and test the stability of its overexpression of PLN:Using the 1st,5th and 10 th generation hPLN-293 cells,and Dox was added to induce them to overexpress the transferred human PLN protein and no Dox induction group as control.Detect the expression of PLN by Western-blot protein printing method,and the differences between the two groups were compared.(2)Using hPLN-293 cells to observe the cytotoxic effect of aconitine intervention:The cells were cultured to six-well plates and treated with different concentrations of aconitine(0,0.5,1,10,100μmol/L)for different time(0,1,2,4 hours).The activity of cells after intervention with aconitine at different concentrations and time was detected by CCK8 method.Compare the differences of cell morphology in each group.(3)Observe the changes of intracellular calcium ion content before and after poisoning:Using Fluo-3 to label calcium ions,the calcium ions can be excited to emit green fluorescence under a laser confocal scanning microscope.The fluorescence intensity before and after cell infection can reflect the change of calcium concentration before and after aconitine infection.(4)Use Western-blot method to detect:(1)the effect of aconitine on the expression of total PLN protein and SERCA2 a protein;(2)changes in the phosphorylation status of Ser16-PLN of PLN and its upstream phosphorylase protein kinase A(PKA);(3)Changes in the phosphorylation status of threonine at position 17 of PLN(Thr17-PLN)and its upstream Ca MKⅡ;(4)the effect of PKA and Ca MKⅡ inhibitor intervention on the phosphorylation of PLN protein caused by aconitine poisoning.【Results】(1)After adding the inducer Dox,the expression of PLN protein in the cell line was significantly increased,which facilitated the detection and analysis of its expression.The cells can stably express PLN protein and are not affected by the number of passages,and have good stability.The cells can be used subsequent experiments.(2)The cell morphology did not change significantly after the intervention of aconitine(0.5,1μmol/L).The cell viability decreased after the intervention of aconitine(10μmol/L and 100 μ mol/L),and the morphology changed with the time prolonged after aconitine acted on the cell.Aconitine(1μmol/L)was used for 2 hours to carry out follow-up experiments.Aconitine(1μmol/L)interferes with cells leading to calcium overload.(3)After aconitine poisoning,the total protein expression of PLN and SERCA2 a increased in the cells,and the phosphorylation of Ser16-p-PLN and its upstream p-PKA decreased;Thr17-p-PLN and its upstream p-Ca MK Ⅱ phosphorylation also decreased;aconitine caused the phosphorylation level of Ser16 to decrease more significantly than that of Thr17;PKA and Ca MK Ⅱ inhibitors were treated with aconitine after intervention,resulting in a more significant decrease in phosphorylation of PLN protein.【Conclusions】The constructed hPLN-293 cells have good PLN protein expression abundance and stability,and can be used in cytological experiments for PLN phosphorylation research.Aconitine(1μmol/L)exposure causes intracellular calcium overload,and at the same time caused a feedback increase in PLN and SERCA2 a protein content.Aconitine may inhibit the phosphorylation pathway of PKA-Ser16-PLN and Ca MKⅡ-Thr17-PLN,reduce the phosphorylation status of PLN(more obvious effect on Ser16-PLN phosphorylation),thereby increasing the inhibitory effect of PLN on SERCA2 a,leading to a decrease in the ability of SERCA2 a to reabsorb calcium ions,causing calcium overload in the cytoplasm.