坏死性凋亡对肾小管上皮细胞晶体黏附的影响及聚乙二醇的干预作用

目的 考察坏死性凋亡抑制剂GSK-872以及聚合物PEG-4000对小鼠肾小管上皮细胞(TCMK-1)表面一水草酸钙晶体黏附沉积的影响。方法 实验一用200、400、600、800ug/ml一水草酸钙粉末状晶体作用于TCMK-1细胞,另一组使用RIPK3抑制剂GSK-872作用于TCMK-1细胞,再加入相同质量浓度一水草酸钙晶体处理细胞,空白对照按照相同容积加入等量PBS,孵育12h后倒置显微镜观察表面晶体黏附情况。在400、800ug/ml一水草酸钙晶体浓度下,用CCK-8法检测细胞增殖活性、DCFH探针法检测细胞氧化应激水平,蛋白质免疫印迹法检测坏死性凋亡相关蛋白RIPK1,RIPK3,p-MLKL的表达,电感耦合等离子体发射光谱法(ICP)检测细胞表面晶体黏附量。实验二a组用800ug/ml一水草酸钙粉末状晶体作用于TCMK-1细胞。b组用10%容积浓度PEG-4000作用于TCMK-1细胞并混匀,再加入800ug/ml一水草酸钙粉末状晶体作用于细胞。c组用800ug/ml一水草酸钙粉末状晶体作用于TCMK-1细胞,再加入10%容积浓度PEG-4000并混匀。各组孵育12h后倒置显微镜观察表面晶体黏附情况,CCK-8法检测细胞增殖活性、DCFH探针法检测细胞氧化应激水平,ICP法检测细胞表面晶体黏附量。结果 实验一采用400ug/ml 一水草酸钙晶体浓度时,与一水草酸钙晶体处理组相比, GSK-872预处理组中TCMK-1细胞晶体黏附量减少,细胞增殖活性增强,氧化应激水平降低(P均＜0.05)。使用800ug/ml 一水草酸钙晶体浓度时,与一水草酸钙晶体处理组相比, GSK-872预处理组中TCMK-1细胞晶体黏附量无明显变化(P>0.05),细胞增殖活性增强,氧化应激水平降低,p-MLKL表达减少(P＜0.05)。实验二与一水草酸钙晶体处理组相比,TCMK-1细胞在PEG-4000预处理组晶体黏附量明显减少,细胞增殖活性增强,氧化应激水平降低(P均＜0.05)。而PEG-4000后加入组与一水草酸钙晶体处理组相比,晶体黏附量、细胞增殖活性、氧化应激水平均无明显变化(P均>0.05)。结论 坏死性凋亡抑制剂GSK-872可以一定程度减少一水草酸钙晶体在细胞表明黏附沉积,但在较高的晶体负荷下晶体可在细胞表面聚集形成不定型沉淀,预使用聚合物PEG-4000能够保持一水草酸钙微晶粒在悬液中保持悬浮稳定,减少晶体聚集沉积及细胞黏附,减轻细胞氧化应激损伤。而充分接触一水草酸钙晶体后的TCMK-1细胞加入PEG-4000不能逆转晶体细胞黏附聚集沉淀。

Effect of necrotizing apoptosis on crystal adhesion of renal tubular epithelial cells and the intervention of polyethylene glycol

Objective To explore the effects of necrotizing apoptosis inhibitor GSK-872 and polymer PEG-4000 on the adhesion and deposition of calcium oxalate monohydrate on the surface of mouse renal tubular epithelial cells (TCMK-1).Methods In experiment 1, TCMK-1 cells were treated with 200,400,600,800ug/ml calcium oxalate monohydrate(COM) powder crystal. In the other group, RIPK3 inhibitor GSK-872 was pre-treated with the same concentration of COM. In the blank control group, the same amount of PBS was added according to the same volume.After incubation for 12h, the surface crystal adhesion was observed under the inverted microscope. In 400, 800 ug/ml concentration of COM crystal, with CCK-8 method to detect cell proliferation activity, DCFH probe method to detect cell oxidative stress levels, protein immunoblot method to detect the expression of necrotic apoptosis related proteins RIPK1, RIPK3, p-MLKL.Inductively coupled plasma emission spectroscopy (ICP) was used to detect the crystal adhesion on cell surface. In experiment 2, TCMK-1 cells were treated with 800ug/ml calcium oxalate monohydrate powder crystal in group a. In group b, TCMK-1 cells were treated with 10% volumetric concentration PEG-4000 and mixed, and 800ug/ml calcium oxalate monohydrate powder crystal was added to the cells.In group c, TCMK-1 cells were treated with 800ug/ml calcium oxalate monohydrate powder crystal, followed by 10% volumetric concentration of PEG-4000.After incubation for 12h, the surface crystal adhesion was observed under an inverted microscope. The cell proliferation activity was detected by CCK-8 method, the oxidative stress level was detected by DCFH probe method, and the surface crystal adhesion was detected by ICP method.Results In experiment 1 of 400ug/ml concentration of COM crystal, compared with calcium oxalate monohydrate crystal treatment group, TCMK-1 cells in GSK-872 pretreatment group decreased crystal adhesion, increased cell proliferation activity and decreased oxidative stress level (all p < 0.05).When COM concentration was used at 800ug/ml, the crystal adhesion of TCMK-1 cells in GSK-872 pretreated group had no significant change compared with COM crystal treatment group (P>0.05), while cell proliferation activity increased, oxidative stress level decreased, and P-MLKL expression decreased (p < 0.05).In experiment 2, compared with COM crystal treatment group, the crystal adhesion amount of TCMK-1 cells in PEG-4000 pre-treatment group was significantly decreased,with the cell proliferation activity enhanced, and the oxidative stress level decreased (all P < 0.05).Compared with COM crystal treatment group, PEG-4000 added group had no significant changes in crystal adhesion, cell proliferation activity and oxidative stress level (all P >0.05).Conclusion The necrotic apoptosis inhibitor GSK-872 can reduce the adhesion deposition of COM crystals in cells to a certain extent, but the crystals can aggregate on the cell surface to form amorphous precipitates under high crystal load. The pre-application of polymer PEG-4000 can maintain the suspension stability of COM micrograins in suspension.It can reduce the accumulation and deposition of crystals and cell adhesion, and reduce the oxidative stress damage of cells. However, the addition of PEG-4000 to TCMK-1 cells after full exposure to COM crystals could not reverse the adhesion, aggregation and precipitation of crystal cells.