蛋白磷酸酶2A在氯化镉致小鼠海马神经元细胞tau蛋白过度磷酸化及β淀粉样蛋白沉积中的作用

Effect of protein phosphatase 2A in tau protein hyperphosphorylation andβ-amyloid protein deposition of mouse hippocampal neuron cells induced by cadmium chloride

目的探讨蛋白磷酸酶2A(PP2A)在氯化镉致小鼠海马神经元HT-22细胞tau蛋白过度磷酸化及β淀粉样蛋白(Aβ)沉积中的作用。方法采用不同浓度(0μmol/L、1.25μmol/L、2.50μmol/L、5.00μmol/L、10.00μmol/L、20.00μmol/L)氯化镉对HT-22细胞进行染毒72 h,采用CCK-8法测定细胞活力,以筛选后续实验的染毒浓度。将HT-22细胞分为对照组、低剂量组、中剂量组、高剂量组,分别给予0μmol/L、1.25μmol/L、2.50μmol/L、5.00μmol/L氯化镉染毒,72 h后在倒置显微镜下观察细胞形态,并采用Western blot检测细胞中β淀粉样蛋白前体(APP)、tau蛋白、磷酸化tau蛋白181(Thr181)、PP2A-Aα蛋白、PP2A-Aβ蛋白、PP2A-C蛋白表达水平。结果(1)染毒72 h后,与经0μmol/L氯化镉染毒后的细胞相比,经不同浓度氯化镉染毒后的细胞活力均下降(均P<0.05)。选择细胞存活率为80%左右的氯化镉浓度作为后续实验最高染毒浓度。(2)染毒72 h后,镜下观察发现对照组细胞呈梭形,不同剂量组细胞呈梭形及多边形;与对照组相比,不同剂量组细胞间隙逐渐增宽,且在高剂量组中可见少量漂浮细胞。与对照组相比,低剂量组、中剂量组HT-22细胞的tau蛋白表达水平升高,中剂量组、高剂量组HT-22细胞的Thr181和APP蛋白表达水平升高,各剂量组PP2A-C蛋白表达水平均降低,高剂量组PP2A-Aα、PP2A-Aβ蛋白表达水平下降(均P<0.05)。结论镉可能通过下调PP2A亚基的表达,以诱导小鼠海马神经元细胞tau蛋白过度磷酸化和Aβ生成,进而产生神经毒性。

Objective To investigate the effect of protein phosphatase 2A(PP2A)in tau protein hyperphosphorylation and β-amyloid protein(Aβ)deposition of mouse hippocampal neuron HT-22 cells induced by cadmium chloride.Methods Cadmium chloride with different concentrations(0μmol/L,1.25μmol/L,2.50μmol/L,5.00μmol/L,10.00μmol/L,and 20.00μmol/L)was used to performed toxication on HT-22 cells for 72 hours.Cell activity was measured by the CCK-8 method for screening toxication concentration of subsequent experiments.HT-22 cells were assigned to control group,low-dose group,medium-dose group,or high-dose group,and toxicated by 0μmol/L,1.25μmol/L,2.50μmol/L,5.00μmol/L cadmium chloride,respectively,and then cellular morphology was observed under the inverted microscope after 72 hours.The protein expressions of β-amyloid protein precursor(APP),tau,phospho-tau protein 181(Thr181),PP2A-Aα,PP2A-Aβ,and PP2A-C in cells were detected by the Western blot.Results(1)After 72-hour toxication,compared with cells after toxication by 0μmol/L cadmium chloride,the activity of cells toxicated by different concentrations of cadmium chloride declined(all P<0.05).Cadmium chloride concentration with a cell survival rate of about 80% was selected as the highest toxication concentration in subsequent experiments.(2)After 72-hour toxication,cells in the control group observed via microscope presented as fusiformis,and cells in the different doses groups presented as fusiformis and polygon.Compared with the control group,cells in the different doses groups presented as space broadening,and small number of floating cells in the high-dose group.Compared with the control group,the protein expression of tau in HT-22 cells of the low-and medium-dose groups was elevated,the protein expressions of Thr181 and APP in HT-22 cells of the medium-and high-dose groups were elevated,the protein expression of PP2A-C declined in various doses groups,and the protein expressions of PP2A-Aαand PP2A-Aβ declined in the high-dose group(all P<0.05).Conclusion Cadmium may induce hyperphosphorylation of tau protein and Aβ generation in mouse hippocampal neuron cells by down-regulating the expression of PP2A subunit,resulting in neurotoxicity.