Expression Of CaSR In Neurons And Cardiomyocytes Incubated With Magnesium Free Extracellular Fluid And Its Relationship With MAPK Pathway

Objective: In order to simulate clinical epilepsy cases,using a classical animal model of epilepsy,in which neurons were incubated in extracellular media without magnesium,and induce abnormal discharges.Then calcium-sensing receptor agonists and inhibitors were used to interfere with the expression of CaSR.To investigate the expression of calcium sensitive receptor(CaSR)and the mechanism of MAPK pathway regulating the proliferation and apoptosis of neurons and cardiomyocytes after co-incubation with magnesium free extracellular fluid.Methods: The epileptic model of neurons and cardiomyocytes co-culture were established by incubating neurons and cardiomyocytes with magnesium free extracellular medium for 3 hours,and then replacing normal DMEM culture medium for 6 hours.The survival rate and shape of neurons and cardiomyocytes were detected by MTT colorimetric,HE staining and transmission electron microscopy.The expression of CaSR protein was observed by Western blot.The MAPK pathway was detected by Western blot with the agonist of CaSR(spermidine)and the inhibitor of CaSR(calhex231).Exploring the expression of CaSR in neurons and cardiomyocytes and the role of MAPK pathway.Results: HE staining and transmission electron microscopy showed that the neurons and cardiomyocytes of the control group are complete,in the magnesium free group,the cell morphology was incomplete,the cells swelled and broken,and the outline was blurred.After adding agonist spermine,the cells became edematous and the degree of rupture became more serious.MTT results showed that the cell survival rate of the control group was higher than other groups,except magnesium free group(p<0.05),and magnesium free+spermine group's cell survival rate was lower than other treatment group,except magnesium free+spermine+ calhex231 group(p<0.05).Compared with the control group,the expression of Bcl-2 protein in magnesium free group was decreased(p<0.01),and spermine could decrease the expression of Bcl-2 and P-ERK in magnesium free group(p<0.05).Spermidine could promote the expression of CaSR induced by magnesium free extracellular fluid(p<0.01),and increase the expression of P-JNK and P-P38 MAPK protein in magnesium free group(p<0.01).Conclusions: The magnesium free extracellular fluid can increase the expression of CaSR in neurons and cardiomyocytes,and can induce abnormal discharge of neurons to damage neurons and cardiomyocytes.Increased expression of CaSR can activate MAPK signal transduction pathway.