基质金属蛋白酶9对星形胶质细胞缺氧缺糖后水通道蛋白4表达的影响及调节机制

Effect of matrix metalloproteinase-9 on aquaporin-4 expression in astrocytes after hypoxia and glucose deprivation and its regulatory mechanism

目的探讨基质金属蛋白酶9(matrix metalloproteinase 9,MMP-9)沉默对星形胶质细胞缺氧缺糖后水通道蛋白4(aquaporin,AQP4)表达的影响及其调节机制。方法取出生后2 d的SD大鼠大脑皮层进行星形胶质细胞纯化培养,通过缺氧缺糖处理建立缺血细胞模型,实验分为正常组、阴性对照组和MMP-9沉默组。比色法测定细胞和上清液的乳酸脱氢酶(LDH)漏出率,利用慢病毒转染沉默MMP-9,并通过实时荧光定量RT-PCR和Western blot法分别检测星形胶质细胞AQP4和MMP-9的表达变化,Western blot法检测细胞PKA、PKC、PKG、CaMKⅡ蛋白含量。结果细胞经缺氧缺糖处理后,LDH漏出率与正常组相比明显增高(t=13.35,P<0.01);MMP-9沉默组星形胶质细胞MMP-9 mRNA(0.412±0.297)表达较阴性对照组(1.118±0.240)显著下调(t=-4.964,P<0.05);伴随MMP-9表达的降低,MMP-9沉默组AQP4 mRNA(1.002±0.082)较阴性对照组(1.442±0.066 )明显下调(t=-9.886,P<0.01);MMP-9沉默组AQP4蛋白(0.643±0.036)较阴性对照组(1.000±0.069)也明显下调(t=-11.073,P<0.01);MMP-9沉默组的星形胶质细胞PKC蛋白表达(0.198±0.110)较阴性对照组(0.980±0.232)明显减低(t=-7.218,P<0.01),但蛋白激酶PKA、PKG、CaMKⅡ表达均差异无统计学意义(t分别为0.875、0.818、0.933,均P>0.05)。结论缺氧缺糖导致星形胶质细胞通透性增高,基因MMP-9沉默后可介导AQP4表达下调,MMP-9可能是通过调控PKC的活性来调节AQP4的表达。

Objective To investigate the effects and regulating mechanisms of matrix metalloproteinase 9 (MMP-9) gene silencing on aquaporin 4(AQP4) expression in astrocytes induced by oxygen-glucose deprivation. Methods Cerebral cortical astrocytes from 2 days newborn SD rats were undergone the primary culture. The ischemic cell model was established by oxygen-glucose deprivation. This experiment were divided into control group, negative control group and MMP-9 gene silencing group. The leakage rate of lactated dehydrogenase(LDH)was detected by chromatoptometry. The MMP-9 gene silencing was carried out by Lentivirus transfection. Real-time fluorescence quantitative PCR and Western blot were used to detect the expression levels of AQP4 and MMP-9. The expressions of PKA, PKC, PKG and CaMKⅡ were determined by Western blot. Results Compared with control group, LDH leakage rate was significantly higher in astrocytes induced by oxygen-glucose deprivation(t=13.35, P<0.01). The expression of MMP-9 mRNA in astrocytes in MMP-9 gene silencing group(0.412±0.297) decreased significantly compared with that in negative control group(1.118±0.240)(t=-4.964, P<0.05). The expression of AQP4 mRNA in astrocytes in MMP-9 gene silencing group(1.002±0.082) decreased significantly compared with that in negative control group(1.442±0.066 )(t=-9.886, P<0.01). The expression of AQP4 protein in astrocytes in MMP-9 gene silencing group(0.643±0.036)decreased significantly compared with that in negative control group(1.000±0.069)(t=-11.073, P<0.01). The expression of PKC protein in astrocytes in MMP-9 gene silencing group(0.198±0.110)decreased significantly compared with that in negative control group(0.980±0.232)(t=-7.218, P<0.01). The expressions of PKA(t=0.875), PKG(t=0.818) and CaMKⅡ(t=0.933) protein had no statistically significant difference between negative control group and MMP-9 gene silencing group(all P>0.05). Conclusion The permeability of astrocytes is increased by oxygen-glucose deprivation. Gene silencing MMP-9 could induce expression of AQP4 mRNA and protein decreased, and MMP-9 may regulate AQP4 expression by regulating PKC activity.