Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass p...Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2(LMP2)in the pathophysiology of ischemia stroke.However,the relationship between the immunoproteasome LMP2 and the BBB remains unclear.Methods:Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion(MCAO/R).Three days before MCAO,the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region.The rat brain microvascular endothelial cell(RBMVEC)line was exposed to oxygen–glucose deprivation/reperfusion(OGD/R)to mimic ischemic conditions in vitro.The RNA interference-mediated knockdown of LMP2 orβ-catenin was analysed in vivo and in vitro.Analysis of the quantity of extravasated Evans blue(EB)and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB.Immunofluorescence and Western blotting were employed to detect the expression of target proteins.Cell migration was evaluated using a scratch migration assay.The results of immunofluorescence,Western blotting and cell migration were quantified using the software ImageJ(Version 1.53).Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference(LSD)test.Results:Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins[occludin,claudin-1 and zonula occludens(ZO-1)]in the MCAO/R group compared with the sham group(P<0.001).However,inhibition of the immunoproteasome LMP2 restored the expression of these proteins,resulting in higher levels of occludin,claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group(P<0.001).In addition,inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability[e.g.,the quantity of extravasated EB:LMP2-shRNA group(58.54±7.37)μg/g vs.control-shRNA group(103.74±4.32)μg/g,P<0.001],and promoted the upregulation of Wnt-3a andβ-catenin proteins in rats following MCAO/R.In vitro experiments,OGD/R induced marked upregulation of LMP2,proapoptotic protein Bax and cleaved caspase-3,and downregulation of occludin,claudin-1,ZO-1 and Bcl-2,as well as inhibition of the Wnt/β-catenin pathway Wnt-3a andβ-catenin proteins in RBMVECs,compared with the control group under normal culture conditions(P<0.001).However,silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R.Silencing ofβ-catenin by transfection of RBMVECs withβ-catenin-si RNA aggravated the downregulation of tight junction proteins,and reduced the proliferation and migration of RBMVECs following OGD/R,compared with the control-siRNA group(P<0.001).LMP2-si RNA andβ-catenin-si RNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.Conclusions:This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury,and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.展开更多
目的探究阿司匹林通过调节铁死亡对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导的小鼠神经元HT22细胞损伤的作用。方法体外培养小鼠海马神经元HT22细胞,选取HT22细胞分为对照组、模型组、低剂量组、中剂量组、...目的探究阿司匹林通过调节铁死亡对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导的小鼠神经元HT22细胞损伤的作用。方法体外培养小鼠海马神经元HT22细胞,选取HT22细胞分为对照组、模型组、低剂量组、中剂量组、高剂量组(n=3),除对照组外,其余4组建立OGD/R神经元细胞损伤模型,低、中、高剂量组分别给予阿司匹林100、200、400μg/ml处理。检测各组细胞活力及炎性因子肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素(interleukin,IL)1β、IL-6水平;试剂盒检测超氧化物歧化酶、过氧化氢酶、谷胱甘肽、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛水平;Western blot检测铁死亡相关蛋白溶质载体家族7成员11(solute carrier family 7 members 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)以及酰基辅酶A合成酶长链家族成员4(acyl-coa synthase long chain family member 4,ACSL4)水平。结果模型组细胞活力明显低于对照组,差异有统计学意义(0.49±0.07 vs 1.00±0.12,P<0.01),低、中、高剂量组细胞活力明显高于模型组,差异有统计学意义(0.72±0.10 vs 0.49±0.07,P<0.05;0.87±0.10 vs 0.49±0.07,P<0.01;0.93±0.07 vs 0.49±0.07,P<0.01)。与对照组比较,模型组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛、ACSL4蛋白表达明显升高,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显降低,差异有统计学意义(P<0.01)。与模型组比较,低、中、高剂量组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶Fe^(2+)、丙二醛、ACSL4蛋白表达明显降低,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显升高,差异有统计学意义(P<0.05,P<0.01)。结论阿司匹林可以通过调节铁死亡,减轻OGD/R诱导的小鼠神经元HT22细胞损伤,且呈剂量依赖性。展开更多
基金supported by the National Natural Science Foundation of China(81771250)the Natural Science Foundation of Fujian Province,China(2020J011059,2020R1011004)+1 种基金the Joint Funds for the Innovation of Science and Technology of Fujian Province,China(2017Y9065)the High-level hospital foster grants from Fujian Provincial Hospital,Fujian Province,China(2020HSJJ07)。
文摘Background:Disruption of the blood–brain barrier(BBB)after a stroke can lead to brain injury and neurological impairment.Previous work confirmed the involvement of the immunoproteasome subunit of low molecular mass peptide 2(LMP2)in the pathophysiology of ischemia stroke.However,the relationship between the immunoproteasome LMP2 and the BBB remains unclear.Methods:Adult male Sprague–Dawley rats were subjected to transient middle cerebral artery occlusion/reperfusion(MCAO/R).Three days before MCAO,the rats were treated with lentivirus-mediated LMP2 shRNA preparations by stereotactical injection into the ipsilateral hemispheric region.The rat brain microvascular endothelial cell(RBMVEC)line was exposed to oxygen–glucose deprivation/reperfusion(OGD/R)to mimic ischemic conditions in vitro.The RNA interference-mediated knockdown of LMP2 orβ-catenin was analysed in vivo and in vitro.Analysis of the quantity of extravasated Evans blue(EB)and cerebral fluorescent angiography were performed to evaluate the integrity of the BBB.Immunofluorescence and Western blotting were employed to detect the expression of target proteins.Cell migration was evaluated using a scratch migration assay.The results of immunofluorescence,Western blotting and cell migration were quantified using the software ImageJ(Version 1.53).Parametric data from different groups were compared using one-way ANOVA followed by the least significant difference(LSD)test.Results:Cerebral ischemia led to lower levels of structural components of the BBB such as tight junction proteins[occludin,claudin-1 and zonula occludens(ZO-1)]in the MCAO/R group compared with the sham group(P<0.001).However,inhibition of the immunoproteasome LMP2 restored the expression of these proteins,resulting in higher levels of occludin,claudin-1 and ZO-1 in the LMP2-shRNA group compared with the control-shRNA group(P<0.001).In addition,inhibition of the immunoproteasome LMP2 contributed to higher microvascular density and decreased BBB permeability[e.g.,the quantity of extravasated EB:LMP2-shRNA group(58.54±7.37)μg/g vs.control-shRNA group(103.74±4.32)μg/g,P<0.001],and promoted the upregulation of Wnt-3a andβ-catenin proteins in rats following MCAO/R.In vitro experiments,OGD/R induced marked upregulation of LMP2,proapoptotic protein Bax and cleaved caspase-3,and downregulation of occludin,claudin-1,ZO-1 and Bcl-2,as well as inhibition of the Wnt/β-catenin pathway Wnt-3a andβ-catenin proteins in RBMVECs,compared with the control group under normal culture conditions(P<0.001).However,silencing of LMP2 gene expression reversed these protein changes and promoted proliferation and migration of RBMVECs following OGD/R.Silencing ofβ-catenin by transfection of RBMVECs withβ-catenin-si RNA aggravated the downregulation of tight junction proteins,and reduced the proliferation and migration of RBMVECs following OGD/R,compared with the control-siRNA group(P<0.001).LMP2-si RNA andβ-catenin-si RNA co-transfection partly counteracted the beneficial effects of silencing LMP2-siRNA on the levels of tight junction proteins in RBMVECs exposed to OGD/R.Conclusions:This study suggests that inhibition of the immunoproteasome LMP2 ameliorates ischemia/hypoxia induced BBB injury,and that the molecular mechanism involves the immunoproteasome-regulated activation of the Wnt/β-catenin signalling pathway under ischemic conditions.
文摘目的探究阿司匹林通过调节铁死亡对氧糖剥夺/复氧(oxygen-glucose deprivation/reoxygenation,OGD/R)诱导的小鼠神经元HT22细胞损伤的作用。方法体外培养小鼠海马神经元HT22细胞,选取HT22细胞分为对照组、模型组、低剂量组、中剂量组、高剂量组(n=3),除对照组外,其余4组建立OGD/R神经元细胞损伤模型,低、中、高剂量组分别给予阿司匹林100、200、400μg/ml处理。检测各组细胞活力及炎性因子肿瘤坏死因子α(tumor necrosis factor alpha,TNF-α)、白细胞介素(interleukin,IL)1β、IL-6水平;试剂盒检测超氧化物歧化酶、过氧化氢酶、谷胱甘肽、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛水平;Western blot检测铁死亡相关蛋白溶质载体家族7成员11(solute carrier family 7 members 11,SLC7A11)、谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)以及酰基辅酶A合成酶长链家族成员4(acyl-coa synthase long chain family member 4,ACSL4)水平。结果模型组细胞活力明显低于对照组,差异有统计学意义(0.49±0.07 vs 1.00±0.12,P<0.01),低、中、高剂量组细胞活力明显高于模型组,差异有统计学意义(0.72±0.10 vs 0.49±0.07,P<0.05;0.87±0.10 vs 0.49±0.07,P<0.01;0.93±0.07 vs 0.49±0.07,P<0.01)。与对照组比较,模型组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶、Fe^(2+)、丙二醛、ACSL4蛋白表达明显升高,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显降低,差异有统计学意义(P<0.01)。与模型组比较,低、中、高剂量组TNF-α、IL-1β、IL-6、活性氧、乳酸脱氢酶Fe^(2+)、丙二醛、ACSL4蛋白表达明显降低,超氧化物歧化酶、抗氧化酶、谷胱甘肽、SLC7A11、GPX4蛋白表达明显升高,差异有统计学意义(P<0.05,P<0.01)。结论阿司匹林可以通过调节铁死亡,减轻OGD/R诱导的小鼠神经元HT22细胞损伤,且呈剂量依赖性。
