Role Of L-type Ca²⁺channels In Fe²⁺-induced Cytotoxicity In MES23.5 Cells

Parkinson’s disease（PD）,also known as tremor paralysis,is a central neurodegenerative disease characterized by the selective loss of dopaminergic（DA）neurons in the substantia nigra（SN）and the appearance of amyloid inclusion bodies containing a-synuclein（a-SYN）as the main component-Lewy bodies.However,the exact cause of PD pathological changes is unclear.Genetic and non-genetic factors including oxidative stress,protein aggregation,apoptosis,and abnormal accumulation of iron may be involved in the pathogenesis of PD.Studies in PD animal model and PD patients autopsy showed that iron selectively aggregates in the SN,therefore,excessive deposition of trace metal element iron in the brain and oxidative stress induced by iron accumulation are considered to be one of the main causes involved in neurodegeneration disease.Previous studies have shown that L-type Ca²⁺channels mediate iron entering into not only PC 12,N-2a and other cell lines,but also primary hippocampal neurons and other neurons.Iron may also competitively enter into these cells with calicum in the same rount.This suggests that in the case of iron overload,L-type Ca²⁺channels may be closely related to the pathogenesis and development of neurodegenerative diseases such as PD.Growing evidence indicates that L-type Ca²⁺channels in DA neurons may aggravate oxidative stress by affecting the entry of iron.Although iron overload is known to be associated with neurodegenerative diseases,the underlying mechanisms of iron-induced neurodegeneration in the pathogenesis of PD have not been fully understood.There is evidence that isradipine,a calcium channel blocker for dihydropyridines（DHPs）,can penetrate the blood-brain barrier,promote the maturation of DA neurons,repair damaged neurons and protect neurons against the toxic effects of neuromelanin.There is also evidence that isradipine plays a neuroprotective role in reducing neuronal damage by alleviating intracellular calcium load and mitochondrial metabolic stress in DA neurons in SN.So,we speculate that in the case of iron overload,L-type Ca²⁺channel blockers may serve as a new therapeutic strategy for relieving iron overload mediated neurotoxicity in PD.In order to investigate the role of L-type Ca²⁺channel in Fe²⁺-induced cytotoxicity,in the present study,we observed the effects of isradipine on Fe²⁺-induced cytotoxicity in MES23.5 cells.The 4-methylazozolium blue（MTT）colorimetry assay was used to measure cell viability,flow cytometry（FCM）was used to detect changes in mitochondrial membrane potential（△Ψm）and reactive oxygen species（ROS）,and Western blot was used to detect apoptosis-related Cleaved-caspase-3,Bcl-2 and Bax protein expression to preliminary assess protective effect of isradipine on Fe²⁺-induced cytotoxicity.We also detected the effect of Fe²⁺on the mRNA expressions of Cavl.2 and Cav1.3 calcium channel al subunit by Real-time PCR,and selectively interfered with the expression of Cav1.2 Ca²⁺channel al mRNA by miRNA interference technology and established stable infected cell lines.Then,the CCK8 assay was used to detect the survival rate of lentiviral infection cells,flow cytometry was used to detect ROS production,laser scanning confocal microscopy imaging technology was used to detect the changes of intracellular iron content in the lentivirus infected cells.The results are as follows:1.After treatment of MES23.5 cells with different concentrations of Fe²⁺（0,20,40,60,80,100 μmol/L）for 4 h,the △Ψm changes were detected by flow cytometry.The results showed that Fe²⁺dose-dependently decreased △Ψm.After treated with 40 μmol/L Fe²⁺,the △Ψm decreased by 13.18%compared with the control group,and the difference was statistically significant（P<0.001）.40 μmol/L Fe²⁺was used for subsequent experiments.2.MTT assay was used to detect the cell viability.The results showed that the cell viability in Fe²⁺treatment group was decreased to 73.90%of control,and the difference was statistically significant（P<0.001）.Pretreatment with isradipine significantly improves cell viability compared with Fe²⁺treatment（P<0.001）.3.By Hoechst staining,we observed the changes of nuclear morphology in different treatment groups.The nucleus in control group showed a round,large and uniform low-density fluorescence dispersion state.Compared with the control,the nucleus of Fe²⁺group showed obvious morphology changes such as nuclear pyknosis,fragmentation,and bright fluorescence（P<0.001）.Pretreatment with isradipine inhibit the nuclear morphology changes caused by Fe²⁺（P<0.01）.4.By flow cytometry,we detected △Ψm changes in the different treated MES23.5 cells.Compared with the control,the △Ψm in the Fe²⁺group significantly decreased by 11.28%,and the difference was statistically significant（P<0.001）.Compared with the Fe²⁺alone treatment group,the △Ψm in the isradipine pretreatment group significantly increased（P<0.001）5.By flow cytometry,we detected ROS changes in different treatment groups.The results showed that compared with the control,ROS production in the Fe²⁺group significantly increased,and the difference was statistically significant（P<0.001）.Pretreatment with isradipine significantly reduces the level of ROS compared with Fe²⁺alone group,and the difference is statistically significant（P<0.01）.6.The expressions of Bcl-2 and Bax protein in different treatment groups were measured by Western blot.Compared with the control,the ratio of Bcl-2/Bax was significantly decreased in the Fe²⁺group（p<0.01）.Pretreatment with isradipine partially improved the decrease of Bcl-2/B ax protein ratio（P<0.05）.7.The expressions of Cleaved caspase-3 protein in MES23.5 cells in different treatment groups were measured by Western blot.Compared with the control,the expression of Cleaved caspase-3 protein was markedly increased in the Fe²⁺group（p<0.01）.Pretreatment with isradipine partially blocked the increase in the expression of Cleaved caspase-3 protein caused by Fe²⁺（P<0.05）.8.The mRNA expression of Cav1.2 and Cavl.3 Ca²⁺channel al subunit were detected by Real-time PCR.The results showed that the mRNA expression of Cav1.2 Ca²⁺channel al subunit were up-regulated by 1.40 and 1.24 times at 2 h and 4 h,respectively,in the Fe²⁺treatment group compared with the control.The mRNA expression of Cavl.3 Ca²⁺channel al subunit was up-regulated by 1.31 and 1.34 times at 2 h and 4 h in the Fe²⁺treatment group compared with the control,and the difference was statistically significant（P<0.001,P<0.01）.9.After successful interference Cavl.2 channel al subunit by shRNA lentiviral vector,the CCK8 was used to detect cell viability in the different treated MES23.5 cells.The results showed that there was no statistical difference in cell viability between Cav1.2 shRNA lentivirus group and control group,which indicated that lentivirus had no effect on cell growth.10.Compared with the empty vector transfected cells,the cell viability decreased after treatment with 40 μmol/L Fe²⁺for 4 h（P<0.001）.Cav1.2 shRNA lentivirus transfection attenuates Fe²⁺-induced decreace in the cell viability（P<0.05）.11.Flow cytometry was used to detect the levels of reactive oxygen species ROS in different treatment groups.Compared with the empty vector transfected cells,the ROS content was markedly increased after treatment with 40 μmol/L Fe²⁺for 4 h（P<0.01）.Cav1.2 shRNA lentivirus transfection attenuates Fe²⁺-induced increase in the ROS production（P<0.05）.12.The cells in different treatment groups were perfused with 100 μmol/L Fe²⁺for 30 min.The fluorescence intensity of calcein in the cells reflected the intracellular iron content.The results showed that Cavl.2 shRNA lentivirus transfection may delay the fluorescence quenching induced by Fe²⁺perfusion.These data indicate that isradipine protects cells against Fe²⁺-induced cytotoxicity under iron overload conditions.L-type Cav1.2 Ca²⁺channels may induce cell damage by participating in abnormal iron accumulation.