横纹肌溶解致急性肾损伤肾小管细胞的凋亡机制
摘要
目的：探讨肌红蛋白是否能引起小管细胞凋亡及其凋亡途径。
方法：(1)以不同浓度的维生素C还原100umol/L肌红蛋白,Mb与VitC的比例分别为1：0.63;1：1.25；1：2.5;1：5;1：10；1：15,利用分光光度计测量VitC还原肌红蛋白的最佳浓度。(2)设定单纯Mb组、Mb+VitC组、VitC组(Mb 200umol/L,Vit C 2 mmol/L)、Con组,刺激HK2细胞时间为分别0h,24h,48h,96h,通过MTT法来检测增殖情况,使用自动生化分析仪测量LDH值。(3)按照不同浓度还原态肌红蛋白分组,分组如下：Mb 200uM+VitC2mM组、Mb 100uM+VitC1mM组、Mb 50uM+VitC500uM组、Mb 25uM+VitC 250uM组、Mb 12.5uM+VitC 125uM组、Con组,刺激HK2细胞时间分别为0h、24h、48h、96小时,通过MTT法来检测HK2细胞增殖情况,使用自动生化分析仪测量LDH值。(4)流式细胞仪检测正常培养基(Con)组及Mb 200uM+VitC 2mM组,刺激HK2细胞48h后细胞凋亡情况。(5)以Mb 200uM+VitC 2mM为刺激物,根据刺激时间分为7组,分别为0h(Con)组、3h组、6h组、12h组、24h组、36h组、48h组,检测蛋白质水平GRP78、CytC、Caspase 9、Caspase 8、Caspase4/5。
结果：(1)当Mb与VitC浓度比为1：10时,在分光光度计检测还原态继红蛋白的620nm处达高峰。(2)Mb+VitC组细胞增殖能力较Con组明显降低,Mb+VitC组LDH较Con组升高。(3)Mb 200uM+VitC 2mM组细胞增殖能力较Con组明显降低,Mb 200uM+VitC 2mM组LDH较Con组升高。(4)流式细胞仪检测Mb 200uM+VitC 2mmM组凋亡早期细胞百分数较正常培养基(Con)组显著增高。(5)GRP78蛋白水平表达在3h、6h、12h、24h组较Con组显著增加,CytC蛋白水平表达在12h、24h、36h、48h组较Con组显著增加,Caspase9蛋白水平表达在6h、12h、48h组较Con组显著增加,Caspase8、4/5蛋白水平表达无明显变化。
结论：(1)维生素C可以还原肌红蛋白且维生素C与肌红蛋白的浓度比例为10：1时,可将肌红蛋白充分还原。(2)在细胞内起损伤作用的肌红蛋白为还原态。(3)200umol/L还原态肌红蛋白刺激48h可以引起HK2细胞凋亡。(4)肌红蛋白刺激通过线粒体途经引起细胞凋亡。
Objective:Verify whether Mb can lead to apoptosis in renal tubular epithelial cells and to explore the underling singal transduction pathway.
Methods:(1)100umol/L Mb was deoxygenized by VitC with different concentrations 1:0.63; 1:1.25; 1:2.5; 1:5; 1:10; 1:15,spectrophotometer was used to measure the optimal ratio between Mb and VitC. (2) HK2 cells were cultured in the medium added Mb and/or Vit C、Vit C (200umol/L Mb, mmol/L Vit C and in normal culture media for 0h,24h,48h,96h, MTT was applied to measure the proliferations of intervened cells, LDH was analysized by automatic biochemistry analyzer. (3) HK2 cells were cultured in the medium added 200umol/L Mb+2mmol/L VitC、100umol/L Mb+ 1 mmol/L VitC、50umol/L Mb+500umol/L VitC、25umol/L Mb+250umol/L VitC、12.5umol/L Mb+125umol/L VitC and in normal culture media for 0h,24h,48h,96h, MTT was applied to measure the proliferations of intervened cells, LDH was analysized by automatic biochemistry analyzer. (4)HK2 cells were cultured in the medium added 200umol/L Mb+2mmol/L VitC and normal culture medium for 48h,annexinⅤ/PI double-staining and flow cytometry were employed to detect the apoptosis changes in HK2 cells. (5) HK2 cells were cultured in the medium added 200umol/L Mb+2mmol/L VitC for selected durations(0h、3h、6h、12h、24h、36h、48h),then extracted celluar proteins GRP78、Caspase9、CytC、Caspase8、4/5 were analysized by Western blot.
Results:(1) The optimal dosage ratio between Mb and VitC was determined at the point of 1:10. (2) The proliferations of HK2 cells was diminished and LDH was decreased by deoxygenized Mb. (3) The proliferations of HK2 cells was diminished and LDH was decreased by 200umol/L Mb+2mmol/L VitC. (4) Cultured by Mb (200umol/L Mb+2mmol/L VitC) for 48h the apoptotic cells were more than the cells cultured by normal culture medium. (5) GRP78 at the protein level was found to decrease with the procession of intervening duration(3h、6h、12h、24h), while CytC and Caspase9 were found to reach the peak at 48h.On the contrary, Caspase8 and Caspase 4/5 were detected to show no obvious changes.
Conclusions:(1) Vit C can deoxygenize Mb and 10 times Vit C can completely deoxygenize Mb.(2)The deoxygenized Mb can injure HK2 cells. (3) The apoptosis was induced by deoxygenized Mb (200umol/L Mb+2mmol/L VitC) when cultured 48h. (4) The apoptosis is transmitted by the mitochondria apoptosis signal pathway when HK2 cells cultured by Mb.
方法：(1)以不同浓度的维生素C还原100umol/L肌红蛋白,Mb与VitC的比例分别为1：0.63;1：1.25；1：2.5;1：5;1：10；1：15,利用分光光度计测量VitC还原肌红蛋白的最佳浓度。(2)设定单纯Mb组、Mb+VitC组、VitC组(Mb 200umol/L,Vit C 2 mmol/L)、Con组,刺激HK2细胞时间为分别0h,24h,48h,96h,通过MTT法来检测增殖情况,使用自动生化分析仪测量LDH值。(3)按照不同浓度还原态肌红蛋白分组,分组如下：Mb 200uM+VitC2mM组、Mb 100uM+VitC1mM组、Mb 50uM+VitC500uM组、Mb 25uM+VitC 250uM组、Mb 12.5uM+VitC 125uM组、Con组,刺激HK2细胞时间分别为0h、24h、48h、96小时,通过MTT法来检测HK2细胞增殖情况,使用自动生化分析仪测量LDH值。(4)流式细胞仪检测正常培养基(Con)组及Mb 200uM+VitC 2mM组,刺激HK2细胞48h后细胞凋亡情况。(5)以Mb 200uM+VitC 2mM为刺激物,根据刺激时间分为7组,分别为0h(Con)组、3h组、6h组、12h组、24h组、36h组、48h组,检测蛋白质水平GRP78、CytC、Caspase 9、Caspase 8、Caspase4/5。
结果：(1)当Mb与VitC浓度比为1：10时,在分光光度计检测还原态继红蛋白的620nm处达高峰。(2)Mb+VitC组细胞增殖能力较Con组明显降低,Mb+VitC组LDH较Con组升高。(3)Mb 200uM+VitC 2mM组细胞增殖能力较Con组明显降低,Mb 200uM+VitC 2mM组LDH较Con组升高。(4)流式细胞仪检测Mb 200uM+VitC 2mmM组凋亡早期细胞百分数较正常培养基(Con)组显著增高。(5)GRP78蛋白水平表达在3h、6h、12h、24h组较Con组显著增加,CytC蛋白水平表达在12h、24h、36h、48h组较Con组显著增加,Caspase9蛋白水平表达在6h、12h、48h组较Con组显著增加,Caspase8、4/5蛋白水平表达无明显变化。
结论：(1)维生素C可以还原肌红蛋白且维生素C与肌红蛋白的浓度比例为10：1时,可将肌红蛋白充分还原。(2)在细胞内起损伤作用的肌红蛋白为还原态。(3)200umol/L还原态肌红蛋白刺激48h可以引起HK2细胞凋亡。(4)肌红蛋白刺激通过线粒体途经引起细胞凋亡。
Objective:Verify whether Mb can lead to apoptosis in renal tubular epithelial cells and to explore the underling singal transduction pathway.
Methods:(1)100umol/L Mb was deoxygenized by VitC with different concentrations 1:0.63; 1:1.25; 1:2.5; 1:5; 1:10; 1:15,spectrophotometer was used to measure the optimal ratio between Mb and VitC. (2) HK2 cells were cultured in the medium added Mb and/or Vit C、Vit C (200umol/L Mb, mmol/L Vit C and in normal culture media for 0h,24h,48h,96h, MTT was applied to measure the proliferations of intervened cells, LDH was analysized by automatic biochemistry analyzer. (3) HK2 cells were cultured in the medium added 200umol/L Mb+2mmol/L VitC、100umol/L Mb+ 1 mmol/L VitC、50umol/L Mb+500umol/L VitC、25umol/L Mb+250umol/L VitC、12.5umol/L Mb+125umol/L VitC and in normal culture media for 0h,24h,48h,96h, MTT was applied to measure the proliferations of intervened cells, LDH was analysized by automatic biochemistry analyzer. (4)HK2 cells were cultured in the medium added 200umol/L Mb+2mmol/L VitC and normal culture medium for 48h,annexinⅤ/PI double-staining and flow cytometry were employed to detect the apoptosis changes in HK2 cells. (5) HK2 cells were cultured in the medium added 200umol/L Mb+2mmol/L VitC for selected durations(0h、3h、6h、12h、24h、36h、48h),then extracted celluar proteins GRP78、Caspase9、CytC、Caspase8、4/5 were analysized by Western blot.
Results:(1) The optimal dosage ratio between Mb and VitC was determined at the point of 1:10. (2) The proliferations of HK2 cells was diminished and LDH was decreased by deoxygenized Mb. (3) The proliferations of HK2 cells was diminished and LDH was decreased by 200umol/L Mb+2mmol/L VitC. (4) Cultured by Mb (200umol/L Mb+2mmol/L VitC) for 48h the apoptotic cells were more than the cells cultured by normal culture medium. (5) GRP78 at the protein level was found to decrease with the procession of intervening duration(3h、6h、12h、24h), while CytC and Caspase9 were found to reach the peak at 48h.On the contrary, Caspase8 and Caspase 4/5 were detected to show no obvious changes.
Conclusions:(1) Vit C can deoxygenize Mb and 10 times Vit C can completely deoxygenize Mb.(2)The deoxygenized Mb can injure HK2 cells. (3) The apoptosis was induced by deoxygenized Mb (200umol/L Mb+2mmol/L VitC) when cultured 48h. (4) The apoptosis is transmitted by the mitochondria apoptosis signal pathway when HK2 cells cultured by Mb.
引文
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3. Allison RC, Bedsole DL. The other medical causes of rhabdomyolysis. Am J Med Sci 2003;326:79-88.
4. Bagley WH, Yang H, Shah KH. Rhabdomyolysis.Intern Emerg Med 2007;2:210-8.
5. Fernandez-Sola J, Grau JM, Pedro-Botet JC, et al. Nontraumatic rhabdomyolysis: a clinical and morphological analysis of 53 cases. Med Clin (Barc) 1988;90:199-202. (In Spanish.)
6. Giannoglou GD, Chatzizisis YS, Misirli G. The syndrome of rhabdomyolysis: pathophysiology and diagnosis. Eur J Intern Med 2007;18:90-100.
7. Wrogemann K, Pena SD. Mitochondrial calcium overload:a general mechanism for cell necrosis in muscle diseases.Lancet 1976;1:672-4.
8. Vanholder R, Sever MS, Erek E, et al. Rhabdomyolysis. J Am Soc Nephrol 2000; 11:1553-61.
9. Holt SG, Moore KP. Pathogenesis and treatment of renal dysfunction in rhabdomyolysis. Intensive Care Med 2001;27:803-11.
10. Melli G, Chaudhry V, Cornblath DR.Rhabdomyolysis:an evaluation of 475 hospitalized patients. Medicine (Baltimore) 2005;84:377-85.
11. Ward MM. Factors predictive of acute renal failure in rhabdomyolysis. Arch Intern Med 1988; 148:1553-7.
12. Veenstra J, Smit WM, Krediet RT, et al. Relationship between elevated creatine phosphokinase and the clinical spectrum of rhabdomyolysis. Nephrol Dial Transplant 1994;9:637-41.
13.de Meijer AR, Fikkers BG, de Keijzer MH, et al. Serum creatine kinase as predictor of clinical course in rhabdomyolysis:a 5-year intensive care survey. Intensive Care Med 2003;29:1121-5.
14. Woodrow G, Brownjohn AM, Turney JH. The clinical and biochemical features of acute renal failure due to rhabdomyolysis.Ren Fail 1995;17:467-74.
15. Knochel JP. Rhabdomyolysis and myoglobinuria.Annu Rev Med 1982;33:435-43.
16.Zager RA, Gamelin LM. Pathogenetic mechanisms in experimental hemo-globinuric acute renal failure. Am J Physiol 1989;256:446-455.
17.Zager RA. Studies of mechanisms and protective maneuvers in myoglobinuric acute renal injury. Lab Invest 1989;60:619-29.
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