摘要
目的探讨红枣色素对高糖诱导的小鼠足细胞损伤的影响及其机制。方法设正常对照组(NG)(5 mmol/L D-葡萄糖),高糖处理(HG)组(30 mmol/L D-葡萄糖),HG+红枣色素-低组(30 mmol/L D-葡萄糖+50 mg/L红枣色素)、HG+红枣色素-中组(30 mmol/L D-葡萄糖+150 mg/L红枣色素)、HG+红枣色素-高组(30 mmol/L D-葡萄糖+250 mg/L红枣色素);将miR-NC、miR-29b-3p、anti-miR-NC、anti-miR-29b-3p分别转染至未经任何处理的MPC5细胞中,记为miR-NC组,miR-29b-3p组、anti-miR-NC组、anti-miR-29b-3p组;将miR-NC、miR-29b-3p、si-NC、si-蛋白激酶1(MST1)染至单纯30 mmol/L的D-葡萄糖处理的MPC5细胞中,记为HG+miR-NC组,HG+miR-29b-3p组、HG+si-NC组、HG+si-MST1组。将anti-miR-NC、anti-miR-29b-3p转染至30 mmol/L的D-葡萄糖和250 mg/L红枣色素处理的MPC5细胞中,记为HG+红枣色素+anti-miR-NC组、HG+红枣色素+anti-miR-29b-3p组,均采用脂质体法。qRT-PCR检测miR-29b-3p和MST1 mRNA的表达水平;Western Blot检测蛋白表达;超氧化物歧化酶(SOD)和丙二醛(MDA)试剂盒检测SOD活性和MDA含量;流式细胞术检测细胞凋亡;双荧光素酶报告基因检测实验检测荧光活性。结果与NG组比较,HG组小鼠足细胞中miR-29b-3p的表达水平显著降低,MST1 mRNA的表达水平显著升高,SOD活性显著降低,MDA含量显著升高,Bcl-2、Nephrin、Podocin表达水平显著降低,Bax、MST1表达水平显著升高,细胞凋亡率显著升高,差异有统计学意义(P<0.05);与HG组比较,不同浓度红枣色素的高糖处理组小鼠足细胞中miR-29b-3p的表达水平逐渐显著升高,MST1 mRNA的表达水平显著降低,SOD活性显著升高,MDA含量显著降低,Bcl-2、Nephrin、Podocin表达水平显著升高,Bax、MST1表达水平显著降低,细胞凋亡率显著降低,差异有统计学意义(P<0.05)。Western Blot结果显示,miR-29b-3p组小鼠足细胞中MST1蛋白的表达水平(0.21±0.03)显著低于miR-NC组(0.42±0.04);anti-miR-29b-3p组小鼠足细胞中MST1蛋白的表达水平(0.87±0.08)显著高于anti-miR-NC组(0.40±0.04),差异均有统计学意义(P<0.05)。与HG+miR-NC组比较,HG+miR-29b-3p组小鼠足细胞中miR-29b-3p的表达水平显著升高,SOD活性显著升高,MDA含量显著降低,Bcl-2、Nephrin、Podocin表达水平显著升高,Bax表达水平显著降低,细胞凋亡率显著降低,差异有统计学意义(P<0.05);与HG+miR-NC组比较,HG+si-MST1组小鼠足细胞SOD活性显著升高,MDA含量显著降低,Bcl-2、Nephrin、Podocin表达水平显著升高,Bax、MST1表达水平显著降低,凋亡率显著降低,差异有统计学意义(P<0.05)。与HG+红枣色素+anti-miR-NC组比较,HG+红枣色素+anti-miR-29b-3p组小鼠足细胞中miR-29b-3p的表达水平显著降低,SOD活性显著降低,MDA含量显著升高,Bcl-2、Nephrin、Podocin表达水平显著降低,Bax、MST1表达水平显著升高,凋亡率显著升高,差异有统计学意义(P<0.05)。结论红枣色素对高糖诱导的小鼠足细胞凋亡有保护作用,其机制可能与调控miR-29b-3p/MST1及SOD和MDA活性有关。红枣色素可作为治疗肾损伤的药物,miR-29b-3p、MST1可成为肾损伤治疗的靶点。
Objective To investigate the effect of jujube pigment on high glucose-induced podocyte injury in mice and its mechanism.Methods For the glucose treatment,mice were divided into normal control group(5 mmol/L D-glucose),the high-glucose-treated(HG)group(30 mmol/L D-glucose),HG+jujube pigment-low group(30 mmol/L D-glucose+50 mg/L jujube Pigment),HG+jujube pigment-medium group(30 mmol/L D-glucose+150 mg/L jujube pigment),HG+jujube pigment-high group(30 mmol/L D-glucose+250 mg/L jujube pigment).The miR-NC,miR-29 b-3 p,anti-miR-NC,and antimiR-29 b-3 p were used to transfect into MPC5 cells without any treatment,and recorded as miR-NC group and miR-29 b-3 p group,anti-miR-NC group and anti-miR-29 b-3 p group.The miR-NC,miR-29 b-3 p,si-NC,and si-MST1 were used to transfect into MPC5 cells treated with only 30 mmol/L D-glucose,and recorded as HG+miR-NC group,HG+miR-29 b-3 p group,HG+si-NC group,HG+si-MST1 group,respectively.Anti-miR-NC and anti-miR-29 b-3 p were used to transfect into30 mmol/L D-glucose and 250 mg/L jujube pigment-treated MPC5 cells and recorded as HG+jujube pigment+anti-miR-NC group and HG+jujube pigment+anti-miR-29 b-3 p groups,respectively using the liposome method.qRT-PCR was used to detect miR-29 b-3 p and MST1 mRNA expression levels;Western Blot was used to detect protein expression;SOD and MDA kits were used to detect SOD activity and MDA content,respectively;flow cytometry was used to detect apoptosis.The double luciferase reporter assay was used to detect fluorescent activity.Results Compared with the NG group,the expression level of miR-29 b-3 p in podocytes of HG group was significantly reduced;the expression level of MST1 mRNA was significantly increased;SOD activity was significantly reduced,and MDA content was significantly increased;the expression levels of Bel-2,Nephrin and Podocin were significantly reduced,the expression levels of Bax and MST1 were significantly increased,and the apoptosis rate was significantly increased(p<0.05).Compared with the HG group,the expression levels of miR-29 b-3 p in podocytes of mice treated with different concentrations of jujube pigment were gradually increased;the expression level of MST1 mRNA decreased significantly;SOD activity increased significantly,and MDA content decreased significantly.The expression levels of Bel-2,Nephrin,and Podocin were significantly increased;the expression levels of Bax and MST1 were significantly reduced,and the apoptosis rate was significantly reduced(p<0.05).Western Blot results showed that the expression level of MST1 protein in podocytes of miR-29 b-3 p group(0.21±0.03)was significantly lower than that of miR-NC group(0.42±0.04);the expression level of MST1 protein in podocytes of anti-miR-29 b-3 p group(0.87±0.08)was significantly higher than that of anti-miR-NG group(0.40±0.04)(p<005).Gompared with the HG+miR-NC group,the expression level of miR-29 b-3 p in podocytes of mice in the HG+miR-29 b-3 p group was significantly increased;SOD activity was significantly increased,and MDA content was significantly reduced.The expression levels of Bel-2,Nephrin and Podocin were significantly increased;the expression levels of Bax were significantly decreased,and the apoptosis rate was significantly reduced(p<0.05).Compared with the HG+miR-NC group,the podocyte SOD activity in the HG+si-MST1 group was significantly increased,the MDA content was significantly reduced,the expression levels of Bel-2,Nephrin,and Podocin were significantly increased,and the expression levels of Bax and MST1 were significantly reduced,the apoptosis rate was significantly reduced,and the difference was statistically significant(P<0.05).Compared with HG+jujube pigment+anti-miR-NC group,the expression level of miR-29 b-3 p in podocytes of HG+jujube pigment+anti-miR-29 b-3 p group was significantly reduced;SOD activity was significantly reduced,and MDA content was significantly increased;the expression levels of Bel-2,Nephrin and Podocin were significantly reduced;the expression levels of Bax and MST1 were significantly increased,and the apoptosis rate was significantly increased(P<0.05).Conclusions Jujube pigment had protective effect on high glucose-induced apoptosis of mouse podocytes,and its mechanism might be related to the regulation of miR-29 b-3 p/MST1 and SOD and MDA activities.Jujube pigment could be used as a drug for the treatment of kidney damage;miR-29 b-3 p and MST1 might be the targets for the treatment of kidney injury.
作者
马媛
张大鹏
王想
MA Yuan;ZHANG Da-peng;WANG Xiang(Department of Endocrinology,Zhumadian Central Hospital,Zhumadian,Henan 463000,China)
出处
《热带医学杂志》
CAS
2020年第1期6-12,44,共8页
Journal of Tropical Medicine
基金
河南省基础研究计划项目(142300410052).
