AIM:To highlight the importance of microRNA(miRNA)-21-5p in directing the phosphatase and tensin homolog(PTEN)gene to control the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)...AIM:To highlight the importance of microRNA(miRNA)-21-5p in directing the phosphatase and tensin homolog(PTEN)gene to control the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway in retinal pigment epithelial(RPE)cells in humans subjected to photodamage.METHODS:Human adult RPE cell line-19(ARPE-19)was cultured in vitro and randomly divided into control,damage,overexpression,negative,and PI3K/Akt blocker groups to establish a photodamage model of ARPE-19 cells.The models were subjected to 24h of light exposure,after which the corresponding indices were detected.The cell counting kit-8 assay quantified cell viability,while flow cytometry determined apoptosis rates.The miRNA-21 mimics and miRNA mimic NC were transfected into ARPE-19 cells using a transient transfection technique.Quantitative reverse transcription polymerase chain reaction(SYBR Green)and Western blotting analyzed expression levels of miRNA-21-5p,PTEN,p-PI3K/PI3K,p-mTOR/mTOR,and p-Akt/Akt.Statistical analyses comprised one-way analysis of variance and the Student-Newman-Keuls test for multiple group comparisons.RESULTS:The photodamage group demonstrated reduced cell survival rates than the control group(P<0.01).The overexpression group exhibited higher cell survival rates than the injury group(P<0.01).The negative group showed no difference in viability(P>0.05).The PI3K/Akt blocker group demonstrated lower cell viability,compared with the overexpression group(P<0.01).CONCLUSION:miRNA-21-5p significantly increases ARPE-19 cell survival after photodamage and inhibits lightinduced ARPE-19 cell apoptosis,suggesting that it may play a protective role in RPE by activating the PI3K/Akt/mTOR pathway while downregulating PTEN expression.展开更多
目的探讨苦参碱(Matrine)对慢性脑缺血(chronic brain ischemia,CCI)大鼠认知功能障碍及磷酸酯酶肿瘤抑制基因(gene of phosphate and tension homology deleted on chromosome ten,PTEN)/磷脂酰肌醇-3-羟激酶(phosphatidylinositol-3-h...目的探讨苦参碱(Matrine)对慢性脑缺血(chronic brain ischemia,CCI)大鼠认知功能障碍及磷酸酯酶肿瘤抑制基因(gene of phosphate and tension homology deleted on chromosome ten,PTEN)/磷脂酰肌醇-3-羟激酶(phosphatidylinositol-3-hydroxykinase,PI3K)/AKT信号通路的影响。方法选择SPF级SD大鼠,应用动脉永久结扎(Two-vessel occlusion,2-VO)法建立CCI大鼠模型。将大鼠随机分为7组:假手术组(Sham组)、脑缺血组(CCI组)、苦参碱低剂量组(Matrine-L组)、苦参碱中剂量组(Matrine-M组)、苦参碱高剂量组(Matrine-H组)、苦参碱+PTEN病毒过表达组(Matrine+AAV-PTEN组)和苦参碱+病毒空载体组(Matrine+AAV-NC组)。2-VO法术后第1天开始腹腔注射3种不同剂量苦参碱,每天1次,连续8周。用PPI分析苦参碱可能的靶点。分子对接验证苦参碱和PTEN的结合活性。Morris水迷宫实验观察苦参碱对CCI大鼠认知功能的影响。神经功能评分和尼氏染色观察神经损伤。ELISA测定海马组织中枢神经特异蛋白(s-100β)和神经元特异性烯醇化酶(neuron specific enolase,NSE)水平。免疫荧光检测PTEN和神经元(neuron,NeuN)共定位。TUNEL染色检测神经元凋亡。结果苦参碱可以通过氨基酸残基ARG-173、GLU-299、ILE-303和TYR-176稳定地结合到PTEN蛋白结构上。中剂量苦参碱对CCI大鼠治疗效果最佳。CCI组较Sham组逃避潜伏期显著缩短(P<0.01)。Matrine组较CCI组较大鼠逃避潜伏期显著延长(P<0.01)。而Matrine+AAV-PTEN组较Matrine组大鼠逃避潜伏期缩短(P<0.01)。CCI组较Sham组神经功能评分较差,细胞萎缩,s-100β和NSE表达升高,神经元凋亡增加(P<0.01)。Matrine较CCI组神经功能评分提高,细胞形态恢复,s-100β和NSE表达降低,神经元凋亡减少(P<0.01)。然而在Matrine组基础上给予AAV-PTEN后,Matrine的保护作用被逆转(P<0.01)。结论Matrine能改善CCI大鼠认知障碍,这可能与抑制PTEN/PI3K/AKT信号通路有关。展开更多
文摘AIM:To highlight the importance of microRNA(miRNA)-21-5p in directing the phosphatase and tensin homolog(PTEN)gene to control the phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)pathway in retinal pigment epithelial(RPE)cells in humans subjected to photodamage.METHODS:Human adult RPE cell line-19(ARPE-19)was cultured in vitro and randomly divided into control,damage,overexpression,negative,and PI3K/Akt blocker groups to establish a photodamage model of ARPE-19 cells.The models were subjected to 24h of light exposure,after which the corresponding indices were detected.The cell counting kit-8 assay quantified cell viability,while flow cytometry determined apoptosis rates.The miRNA-21 mimics and miRNA mimic NC were transfected into ARPE-19 cells using a transient transfection technique.Quantitative reverse transcription polymerase chain reaction(SYBR Green)and Western blotting analyzed expression levels of miRNA-21-5p,PTEN,p-PI3K/PI3K,p-mTOR/mTOR,and p-Akt/Akt.Statistical analyses comprised one-way analysis of variance and the Student-Newman-Keuls test for multiple group comparisons.RESULTS:The photodamage group demonstrated reduced cell survival rates than the control group(P<0.01).The overexpression group exhibited higher cell survival rates than the injury group(P<0.01).The negative group showed no difference in viability(P>0.05).The PI3K/Akt blocker group demonstrated lower cell viability,compared with the overexpression group(P<0.01).CONCLUSION:miRNA-21-5p significantly increases ARPE-19 cell survival after photodamage and inhibits lightinduced ARPE-19 cell apoptosis,suggesting that it may play a protective role in RPE by activating the PI3K/Akt/mTOR pathway while downregulating PTEN expression.
文摘目的探讨苦参碱(Matrine)对慢性脑缺血(chronic brain ischemia,CCI)大鼠认知功能障碍及磷酸酯酶肿瘤抑制基因(gene of phosphate and tension homology deleted on chromosome ten,PTEN)/磷脂酰肌醇-3-羟激酶(phosphatidylinositol-3-hydroxykinase,PI3K)/AKT信号通路的影响。方法选择SPF级SD大鼠,应用动脉永久结扎(Two-vessel occlusion,2-VO)法建立CCI大鼠模型。将大鼠随机分为7组:假手术组(Sham组)、脑缺血组(CCI组)、苦参碱低剂量组(Matrine-L组)、苦参碱中剂量组(Matrine-M组)、苦参碱高剂量组(Matrine-H组)、苦参碱+PTEN病毒过表达组(Matrine+AAV-PTEN组)和苦参碱+病毒空载体组(Matrine+AAV-NC组)。2-VO法术后第1天开始腹腔注射3种不同剂量苦参碱,每天1次,连续8周。用PPI分析苦参碱可能的靶点。分子对接验证苦参碱和PTEN的结合活性。Morris水迷宫实验观察苦参碱对CCI大鼠认知功能的影响。神经功能评分和尼氏染色观察神经损伤。ELISA测定海马组织中枢神经特异蛋白(s-100β)和神经元特异性烯醇化酶(neuron specific enolase,NSE)水平。免疫荧光检测PTEN和神经元(neuron,NeuN)共定位。TUNEL染色检测神经元凋亡。结果苦参碱可以通过氨基酸残基ARG-173、GLU-299、ILE-303和TYR-176稳定地结合到PTEN蛋白结构上。中剂量苦参碱对CCI大鼠治疗效果最佳。CCI组较Sham组逃避潜伏期显著缩短(P<0.01)。Matrine组较CCI组较大鼠逃避潜伏期显著延长(P<0.01)。而Matrine+AAV-PTEN组较Matrine组大鼠逃避潜伏期缩短(P<0.01)。CCI组较Sham组神经功能评分较差,细胞萎缩,s-100β和NSE表达升高,神经元凋亡增加(P<0.01)。Matrine较CCI组神经功能评分提高,细胞形态恢复,s-100β和NSE表达降低,神经元凋亡减少(P<0.01)。然而在Matrine组基础上给予AAV-PTEN后,Matrine的保护作用被逆转(P<0.01)。结论Matrine能改善CCI大鼠认知障碍,这可能与抑制PTEN/PI3K/AKT信号通路有关。
