The Effect Of Nickel (?) On The Apoptosis Of Hacat Cells And The Preliminary Study On Its Mechanism

Background and purpose:Nickel is a natural and abundant element in the earth and widely used in construction,food,and medical equipments.Nickel-containing casting alloys are often used to be temporary and permanent dental restorations and(?thodontic devices due to their superior mechanical properties.In general,medical nickel-containing materials are safe to be used in the human body.However,domestic and foreign reports recently revealed that nickel-containing alloys endanger the health of periodontal and mucous tissues in the oral cavity.After using nickel-containing alloys,nickel ions in the urine of patients will increase significantly.Significantly higher nickel concentrations can be found in oral mucosal cells in patients with fixed orthodontic appliances.This shows that nickel-containing alloys continuously release nickel ions to the oral environment.It is closely related to the complex micro-environment of the oral cavity,temperature changes,saliva and food related electrochemical corrosion.The use of nickel-conta:ining alloys for long-time restorations in dentistry has long been controversial.Metallic nickel is toxic and it is origeninally identified that nickel may result in allergenic and carcinogenic response.The lesions of gums and oral mucosal caused by nickel-containing alloy materials has been controversial.Some scholars believe that oral nickel-containing alloy materials can reduce cell activity through oxidative stress reaction and inflammation.Recent studies have shown that nickel ions induce apoptosis in multiple cell types and inhibit cell proliferation.Although the pathogenic mechanism is not yet clear,nickel-induced oxidative stress in cells has become a consensus.The purpose of this study was to identify the effect of nickel on the growth of human epithelial tissue cells.Hacat was used as a subject to study the effect of divalent nickel ions(Ni²⁺)on the apoptosis of Hacat cells,and the mechanism of action was preliminarily studied.Methods:1.The Hacat cells were treated with different concentrations of Ni²⁺?0,100 ?M,200?M,400?M 600 ?M,800 ?M?and time gradients?0,24 h,48 h,72 h?to observe changes in cell morphology.The cell apoptosis was detected by flow cytometry using Annexin V and PI double staining.2.The Hacat cells were treated with different concentrations of Ni²⁺?0,100 ?M,200?M,400 ?M,600 ?M,800 ?M?and time gradients?0,24 h,48 h,72 h,and 96 h?,and cell proliferation inhibition was detected by MTT assay.3.The Hacat cells were treated with different concentration gradients and time gradients of Ni²⁺.The ROS content of O2·-,H2O2,and OH in the cells was detected by flow cytometiy using ROS detection reagent?DCFHDA dye?.4.The Hacat cells were treated with different concentrations of Ni²⁺?0.100 ?M,200?M,400 ?M,600 ?M,800 ??for 24 h and 48 h.Western blot was used to detect the expression of Akt,caspase 3,Bax,Bcl-xL,P38 and other molecules in Hacat cells.Results:1.Ni²⁺ induce apoptosis in Hacat cells,and the apoptosis is enhanced with increasing concentration and time.2.Ni²⁺ inhibit the proliferation of Hacat cells,and the inhibitory effect increases with increasing concentration and time.3.The level of reactive oxygen species in Hacat cells was increased after the induction of Ni²⁺,and this effect is coGcentration-dependent.4.The expression of p-Akt,cleaved caspase3 and other molecules in Hacat cells increased after Ni²⁺ treatment,and the expression levels of Bcl-xL,Bax and phosphorylated P38?p-P38?were not significantly different.Conclusions:1.Ni²⁺ induce apoptosis of Hacat cells and inhibits proliferation of Hacat cells in a dose-and time-dependent manner.2.The mechanism of apoptosis induced by Ni²⁺ in Hacat cells may be related to the increase of intracellular reactive oxygen species and the activation of Akt,caspase3 and other molecules,while the effect of Bcl-xL,Bax and P38 proteins on the apoptosis of Hacat cells induced by Ni²⁺ is not obvious.