慢性肾功能不全大鼠胫骨生长板软骨细胞自噬功能下降对细胞凋亡的影响

Malfunction of autophagy in tibial growth plate chondrocytes causes increased apoptosis rate in chronic renal insufficiency rats

目的观察慢性肾功能不全(CRI)大鼠胫骨生长板软骨细胞自噬功能改变对细胞凋亡的影响。方法雄性4周龄SD幼鼠20只,分为假手术组(暴露左侧输尿管,10只)和CRI组(结扎左侧输尿管,10只)。术后6周处死大鼠前收集24 h尿液并检测总蛋白,处死大鼠后心腔取血检测血肌酐、血尿素氮浓度;取双侧胫骨近端固定脱钙制作组织学切片,番红固绿染色观测胫骨生长板增殖区软骨细胞柱细胞数量,免疫荧光检测软骨细胞自噬指标轻链蛋白3(LC-3)的细胞表达率,Tunel技术检测软骨细胞凋亡率,免疫组化检测软骨细胞糖原指标糖原蛋白1的表达水平。结果与假手术组相比,CRI组24 h尿蛋白[(163.5±11.3)mg比(38.6±9.8)mg,t=25.620,P<0.001],血肌酐[(67.3±16.2)μmol/L比(28.4±11.5)μmol/L,t=5.974,P<0.001],血尿素氮[(16.4±6.4)mmol/L比(4.8±2.0)mmol/L,t=5.198,P<0.001]均增高;CRI组胫骨生长板增殖区软骨细胞柱细胞数量减少[(4.2±2.1)个比(9.1±3.8)个,t=3.109,P=0.006],软骨细胞LC-3蛋白阳性表达率降低[(27.2±12.6)%比(51.4±18.2)%,t=3.457,P=0.003],糖原蛋白1累积增多[(6.1±2.5)分比(3.5±1.8)分,t=2.669,P=0.016],凋亡率增高[(17.2±4.8)%比(5.1±3.4)%,t=6.505,P<0.001]。结论肾功能不全大鼠胫骨生长板软骨细胞自噬功能下降,糖原累积增多,凋亡率增高,软骨细胞数量减少。

Objective To observe the effect of autophagy of tibial growth plate chondrocyte on apoptosis in chronic renal insufficiency(CRI)rats.Methods Male 4-week-old SD rats were randomly divided into two groups:(1)Sham group:only the left ureter was exposed(n=10);(2)CRI group:the left ureter was ligated to cause CRI(n=10).The urine from all the rats was collected 6 weeks after the operation and the total protein content was measured.Then all the rats were sacrificed and the concentrations of creatinine and urea nitrogen in intracardiac blood were detected.The proximal tibia were fixed and decalcified to prepare histological sections,and the number of chondrocytes of column cells in the proliferative area of tibia growth plate was observed by saffron O staining.The expression rate of protein Light Chain-3,an autophagy marker of chondrocytes,was detected by immunofluorescence.The apoptosis rate of chondrocytes was detected by the method of TUNEL assay.The level of glycogenin-1,a glycogen formation marker of chondrocyte was detected by immunohistochemistry in chondrocytes.Results The 24 h urine total protein was higher in CRI group[(163.5±11.3)mg vs(38.6±9.8)mg,t=25.620,P<0.001].The levels of blood creatinine[(67.3±16.2)μmol/L vs(28.4±11.5)μmol/L,t=5.974,P<0.001]and urea nitrogen[(16.4±6.4)mmol/L vs(4.8±2.0)mmol/L,t=5.198,P<0.001]were higher in CRI group.The number of chondrocytes of column cells in the proliferating area of tibia growth plate was lower in CRI group(4.2±2.1 vs 9.1±3.8,t=3.109,P=0.006).The expression rate of LC-3 protein in chondrocytes of CRI group was lower[(27.2±12.6)%vs(51.4±18.2)%,t=3.457,P=0.003].The level of glycogenin-1 of chondrocytes in CRI group increased significantly(6.1±2.5 vs 3.5±1.8,t=2.669,P=0.016).The apoptosis rate of chondrocytes in CRI group also increased[(17.2±4.8)%vs(5.1±3.4)%,t=6.505,P<0.001].Conclusion Malfunction of autophagy in tibial growth plate chondrocytes causes increased apoptosis rate in CRI rats,which might be caused by the failure of glycogen degradation in chondrocytes.