The Role And Mechanism Of TRPM7 Channel In Cadmium-induced PC12 Cell Injury

Cadmium(Cd)is a toxic heavy metal,in the form of divalent ions.It is one of the most toxic environmental and industrial pollutants.The central nervous system disease caused by cadmium has become a medical health issue that has attracted much attention.Cd has strong biological toxicity,long-term exposure to Cd environment can cause a large amount of Cd to accumulate in kidneys,livers,lungs,pancreas,testes,bones and other tissues or organs,which cause serious organ dysfunction.In addition,Cd can also penetrate the blood-brain barrier to accumulate in brain neurons,causing neurons to produce excessive reactive oxygen species,triggering oxidative stress and even causing neuronal apoptosis.Cd is currently found to be an important cause of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease.Because Cd is not an essential element of the body,it is generally believed that Cd in the body may enter the through a transport mechanism specifically for essential metals.According to research,there are many types of vector channels that can transport Cd into cells in vivo,mainly including iron ion transport vectors,calcium ion channels,transient receptor potentials,and zinc regulatory proteins.Studies on human osteoblasts have found that TRPM7 is an important channel for Cd to enter the cell.Transient receptor potential melastatin 7(TRPM7)belongs to the melastatin-related subfamily of TRP channels.It is a non-selective cation channel with high permeability to Ca2+and Mg2+.TRPM7 is expressed in almost all tissues including brain tissues and participates in a variety of physiological processes.Therefore,we think that TRPM7 may also be an important way for Cd to enter central nervous cells.However,it has not been reported whether TRPM7 channels are involved in neuronal damage induced by cadmium.So we explored the role of TRPM7 in cadmium-induced neuronal apoptosis and its molecular mechanism.PC 12 cells derived from rat adrenal pheochromocytoma have the characteristics of sympathetic neurons and can synthesize and secrete catecholamine neurotransmitters.A large number of experimental studies confirm,PC 12 cells and neurons have a lot of physiology and pharmacology in common.Therefore,PC 12 cells are a widely used cell line for neuronal function research.In this study,a cadmium damage model of neurons in vitro was first constructed from PC 12 cells,on this basis a TRPM7 channel blocker NS8593 was administered.MTT and lactate dehydrogenase(LDH)cytotoxicity detection were used to observe the blocking of TRPM7 channels.Effects on cadmium-induced cell damage;intracellular reactive oxygen species(ROS),malonic dialdehyde(MDA),superoxide dismutase(SOD),and catalase(CAT)detection Cell oxidative stress levels;mitochondrial membrane potential(MMP),TUNEL and DAPI staining,and Western Blot to observe the effect of blocking TRPM7 channel on cadmium-induced PC 12 cell damage and its molecular mechanism.Research results:1.Construction of cadmium damage model in PC 12 cells.PC 12 cells were cultured with cell culture solutions containing different concentrations of CdCl2(0.5,1,5,10,and 20 pM)for 24 h,and the appropriate concentration of cadmium damage was selected.MTT results showed that when the cadmium concentration was 5 ?M and 10?M,the cell viability of PC 12 cells was(56.87 ± 0.02)%and(42.95 ± 0.01)%.Therefore,we chose Cd concentration of 5 ?M and 10 ?M for subsequent experiments.2.TRPM7 inhibitor NS8593 significantly inhibited PC 12 cell damage caused by cadmium.MTT results showed that compared with 24h of cadmium treatment,cadmium and 10,20 and 40 ?M NS8593 could significantly improve the decline of PC 12 cell viability caused by cadmium.We have selected 20 ?M NS8593 in subsequent experiments.LDH release test results also show that NS8593 significantly reduces cell damage caused by cadmium.3.TRPM7 inhibitor NS8593 significantly inhibited oxidative stress in PC12 cells induced by cadmium.ROS results showed that cadmium(5 ?M,10 ?M)treatment for 4 h,ROS levels in PC 12 cells increased significantly,while 20 ?iM NS8593 intervention can significantly reduce the ROS generation induced by cadmium.The results of cell MDA production,SOD and CAT activity showed that after 24 h of cadmium(5 ?M,10?M)treatment,MDA levels in PC 12 cells increased significantly,SOD and CAT activities were significantly reduced,and the intervention with 20 ?M NS8593 significantly reduced the levels of MDA Increased production,increased SOD,CAT enzyme activity.4.TRPM7 inhibitor NS8593 significantly inhibited PC 12 cell apoptosis induced by cadmium.MMP results showed that the mitochondrial membrane potential of PC 12 cells decreased significantly after 4 h of cadmium(5 ?M,10 ?M)treatment,and 20 ?M NS8593 significantly increased the mitochondrial membrane potential decrease caused by cadmium.Apoptosis was observed by TUNEL and DAPI staining.The results showed that 20 ?M NS8593 could significantly inhibit cadmium-induced apoptosis.To further explore the mechanism of NS8593 on cadmium-induced apoptosis,Western blot was used to observe the expression of Bcl-2 and Bax proteins,and to observe the changes of Akt/Erk signaling pathway.Western blot analysis showed that NS8593 significantly reduced the increase in Bax/Bcl-2 ratio induced by cadmium,and found that NS8593 significantly inhibited the increase in Akt/Erk phosphorylation induced by cadmium.Conclusion:1.TRPM7 channels are involved in cadmium uptake by PC 12 cells,and blocking TRPM7 channels can reduce PC 12 cell damage caused by cadmium.2.TRPM7 channel inhibits cadmium-induced oxidative stress and improves cadmium-induced neuronal apoptosis by activating the Akt/Erk signaling pathway,which may provide new ideas for the treatment of cadmium-induced neurodegenerative diseases.