丝胶对高糖所致足细胞损伤和JNK信号通路的影响

目的 探讨丝胶对高糖所致足细胞损伤时c-Jun氨基末端激酶（JNK）信号通路相关蛋白的表达及足细胞凋亡的影响，阐明丝胶的保护作用及其可能机制。 方法 分化成熟的条件永生化小鼠足细胞随机分为正常对照组（含5.5 mmol·L^－1葡萄糖的培养基）、高渗对照组（含5.5 mmol·L^－1葡萄糖+24.5 mmol·L^－1甘露醇的培养基）、高糖组（含30.0 mmol·L^－1葡萄糖的培养基）、低浓度丝胶组（含30.0 mmol·L^－1葡萄糖+150.0 mg·L^－1丝胶的培养基）、中浓度丝胶组（含30.0 mmol·L^－1葡萄糖+300.0 mg·L^－1丝胶的培养基）和高浓度丝胶组（含30.0 mmol·L^－1葡萄糖+600.0 mg·L^－1丝胶的培养基）。采用光学倒置显微镜观察各组足细胞形态表现，实时荧光定量PCR（RT-qPCR）法检测各组足细胞中裂孔膜蛋白（Nephrin）、丝裂原活化蛋白激酶激酶1（MEKK1）、丝裂原活化蛋白激酶激酶4（MKK4）、c-Jun氨基末端激酶1（JNK1）和c-Jun mRNA表达水平，Western blotting法检测各组足细胞中Nephrin、MEKK1、MKK4、JNK1和c-Jun蛋白表达水平，AnnexinⅤ/PI双染法检测各组足细胞凋亡率。 结果 正常对照组足细胞胞体较大，并延伸生出树枝状突起；与正常对照组比较，高糖组足细胞胞体回缩变小，细胞间隔增大，脱落和悬浮的足细胞数目增多；与高糖组比较，不同浓度丝胶组足细胞形态逐渐趋于正常。高糖组足细胞中Nephrin mRNA和蛋白表达水平较正常对照组均明显降低（P<0.05），表明高糖致足细胞损伤模型建立成功。高糖组足细胞中MEKK1、MKK4、JNK1、c-Jun mRNA和蛋白表达水平较正常对照组明显升高（P<0.05）；不同浓度丝胶组足细胞中MEKK1、MKK4、JNK1和c-Jun mRNA及蛋白表达水平较高糖组明显降低（P<0.05），且高浓度丝胶组足细胞中MEKK1、MKK4、JNK1和c-Jun mRNA及蛋白表达水平较中浓度丝胶组明显降低（P<0.05），中浓度丝胶组足细胞中MEKK1、MKK4、JNK1和c-Jun mRNA及蛋白表达水平较低浓度丝胶组明显降低（P<0.05）。高糖组和高渗对照组足细胞凋亡率较正常对照组明显升高（P<0.05）；不同浓度丝胶组足细胞凋亡率较高糖组明显降低（P<0.05），且高浓度丝胶组足细胞凋亡率较中浓度丝胶组明显降低（P<0.05），中浓度丝胶组足细胞凋亡率较低浓度丝胶组明显降低（P<0.05）。 结论 丝胶对高糖所致足细胞损伤发挥保护作用，其机制可能与抑制JNK信号通路中MEKK1、MKK4、JNK1和c-Jun的表达及抑制足细胞凋亡有关联。

Effects of sericin on injury of podocytes induced by high glucose and JNK signaling pathway

Objective： To investigate the effects of sericin on the expressions of c-Jun N-terminal kinase（JNK） signaling pathway-related proteins and of apoptosis of podocytes in the podocytes injury inuced by high glucose，and to clarify the protective effect of sericin and its possible mechanism. Methods The podocytes of immortalized mice with mature differentiation were randomly divided into normal control group（medium containing 5.5 mmol·L^－1glucose）， hypertonic control group（medium containing 30.0 mmol·L^－1 glucose and 24.5 mmol·L^－1mannitol）， high glucose group（medium containing 30.0 mmol·L^－1 glucose）， low concentration of sericin group（medium containing 30.0 mmol·L^－1glucose and 150.0 mg·L^－1sericin）， medium concentration of sericin group（medium containing 30.0 mmol·L^－1 glucose and 300.0 mg·L^－1 sericin），and high concentration of sericin group（medium containing 30.0 mmol·L^－1 glucose and 600.0 mg·L^－1 sericin）.The morphology of podocytes in various groups was observed under optical inverted microscope， real-time fluorescence quantitative PCR（RT-qPCR） method was used to detect the expression levels of podocyte membrane protein（Nephrin），mitogen-activated protein kinase kinase 1（MEKK1）， mitogen-activated protein kinase kinase 4（MKK4），c-Jun N-terminal kinase 1（JNK1） and c-Jun mRNA in the podocytes in various groups，Western blotting method was used to detect the expression levels of Nephrin， MEKK1， MKK4， JNK1， and c-Jun proteins in the podocytes in various groups，and AnnexinⅤ/PI double staining method was used to detect the apoptotic rates of the podocytes in various groups. Results The podocytes in normal control group had larger cell bodies and extended to produce dendritic foot process；compared with normal control group， the body of the podocytes in high glucose group was significantly smaller， the cell spacing was increased， and the number of detached and suspended podocytes was increased；compared with high glucose group， the morphology of the podocytes in different concentrations of sericin groups were gradually tended to be normal.Compared with normal control group，the expression levels of Nephrin mRNA and protein in the podocytes in high glucose group were obviously decreased（P<0.05）， indicating that the podocyte injury model induced by high glucose was successfully established.Compared with normal control group，the expression levels of MEKK1，MKK4，JNK1， and c-Jun mRNA and proteins in the podocytes in high glucose group were significantly increased（P<0.05）.compared with high glucose group，the expression levels of MEKK1， MKK4， JNK1， and c-Jun mRNA and proteins in the podocytes in different concentrations of sericin groups were significantly decreased（P<0.05）；at the same time， the expression levels of MEKK1， MKK4， JNK1， and c-Jun mRNA and proteins in the podocytes in high concentration of sericin group were significantly lower than those in medium concentration of sericin group （P<0.05）， and the expression levels of MEKK1， MKK4， JNK1， and c-Jun mRNA and proteins in the podocytes in medium concentration of sericin group were significantly lower than those in low concentration of sericin group （P<0.05）.Compared with normal control group，the apoptotic rate of the podocytes in high glucose group and hypertonic control group were signifiantly increased（P<0.05）；the apoptotic rates of the podocytes in different concentrations of sericin groups were significantly lower than that in high concentration of sericin group （P<0.05）， and the apoptotic rate of the podocytes in high concentration of sericin group was significantly lower than that in medium concentration of sericin group （P<0.05）， while the apoptotic rate of the podocytes in medium concentration of sericin group was significantly lower than that in low concentration of sericin group （P<0.05）. Conclusion Sericin has protective effect on the podocyte injury induced by high glucose，and its mechanism may be related to its inhibition of the expressions of MEKK1， MKK4， JNK1， and c-Jun in the JNK signaling pathway and its inhibition of the apoptosis of the podocytes.