萝卜硫素对高糖诱导人肾小管上皮细胞增殖和凋亡的影响及机制
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篇名: 萝卜硫素对高糖诱导人肾小管上皮细胞增殖和凋亡的影响及机制
TITLE: Effects of Sulforaphane on the Proliferation and Apoptosis of Human Renal Tubular Epithelial Cells Induced by High Glucose and Its Mechanism
摘要: 目的:研究萝卜硫素对高糖诱导人肾小管上皮细胞HK-2增殖和凋亡的影响,并初步探讨其作用机制。方法:将HK-2细胞分为正常组、高糖组、厄贝沙坦组(阳性对照,1μmol/L)和萝卜硫素低、中、高浓度组(10、20、40μmol/L),正常组细胞用DMEM完全培养基培养96h,其余各组细胞均用高糖DMEM完全培养基(含40mmol/L葡萄糖)培养48h诱导细胞损伤后,再加入相应药物继续培养48h。检测各组细胞的存活率、凋亡率以及细胞中细胞周期蛋白D1(cyclinD1)、胱天蛋白酶3(caspase-3)、B细胞淋巴瘤2(Bcl-2)、B细胞淋巴瘤2相关X蛋白(Bax)mRNA和磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、磷酸化腺苷一磷酸活化蛋白激酶(p-AMPK)、磷酸化蛋白激酶B(p-Akt)、磷酸化磷脂酰肌醇3激酶(p-PI3K)蛋白的表达量。另将HK-2细胞分为正常组、高糖组、萝卜硫素高浓度组(40μmol/L)、阿卡地新组(AMPK激动剂,1mmol/L)、萝卜硫素高浓度+compoundC组(萝卜硫素40μmol/L+AMPK抑制剂compoundC40μmol/L)、哌立福辛组(Akt抑制剂,19.95μmol/L)、萝卜硫素高浓度+SC79组(萝卜硫素40μmol/L+Akt激动剂SC794μmol/L),同法培养后,检测细胞中p-mTOR、p-AMPK、p-Akt、p-PI3K蛋白的表达量。结果:与正常组比较,高糖组细胞的存活率以及细胞中cyclinD1、Bcl-2mRNA和p-AMPK蛋白的表达量均显著降低(P<0.05),凋亡率以及细胞中cas-pase-3、BaxmRNA和p-mTOR、p-Akt、p-PI3K蛋白的表达量均显著升高(P<0.05)。与高糖组比较,萝卜硫素低、中、高浓度组和厄贝沙坦组细胞上述指标均显著改善(P<0.05),其中萝卜硫素中、高浓度组细胞上述指标的改善效果均显著优于萝卜硫素低浓度组(P<0.05)。与厄贝沙坦组比较,萝卜硫素高浓度组细胞上述指标差异均无统计学意义(P>0.05)。与萝卜硫素高浓度组比较,阿卡地新组细胞中p-AMPK、p-mTOR蛋白和哌立福辛组细胞中p-mTOR、p-Akt、p-PI3K蛋白的表达量差异均无统计学意义(P>0.05);萝卜硫素高浓度+compoundC组细胞中p-AMPK蛋白的表达量显著降低(P<0.05),p-mTOR蛋白的表达量显著升高(P<0.05);萝卜硫素高浓度+SC79组细胞中p-mTOR、p-Akt、p-PI3K蛋白的表达量均显著升高(P<0.05)。结论:萝卜硫素可促进高糖诱导肾小管上皮细胞增殖,抑制其凋亡,其机制可能与上调p-AMPK表达,下调p-mTOR、p-Akt、p-PI3K表达有关。
ABSTRACT: OBJECTIVE:To study the effects of sulforaphane on the prolifera tion and apoptosis of human renal tubular epithelial cells HK- 2 induced by high glucose ,and to investigate its mechanism primarily. METHODS :HK-2 cells were divided into normal group ,high glucose group ,irbesartan group (positive control ,1 μmol/L),sulforaphane low ,medium and high concentration groups (10,20,40 μmol/L). The cells in normal group were cultured in DMEM medium for 96 hours. T he cells in other groups were cultured in high glucose DMEM medium (containing 40 mmol/L glucose )for 48 hours. After inducing cell injury,the cells were added with corresponding drugs for 48 hours. Survival rate and apoptotic rate of cells were detected. mRNA expression of cyclin D 1,caspase-3,Bcl-2 and Bax as well as protein expression of p-mTOR ,p-AMPK,p-Akt and p-PI 3K were also determined. In addition ,HK-2 cells were divided into normal group ,high glucose group ,sulforaphane high concentration group(40 μmol/L),acardicin group (AMPK agonist ,1 mmol/L),sulforaphane high concentration+compound C group (sulforaphane 40 μmol/L+AMPK inhibitor compound C 40 μmol/L),perifoxine group (Akt inhibitor ,19.95 μmol/L)、sulforaphane high concentration+SC 79 group(sulforaphane 40 μmol/L+Akt agonist SC79 4 μmol/L). After cultured with the same method , protein expression of p-mTOR ,p-AMPK,p-Akt and p-PI 3K were detected in HK- 2 cells. RESULTS :Compared with normal group,survival rate of HK- 2 cells,mRNA expression of cyclin D 1 and Bcl- 2 as well as protein expression of p-AMPK were decreased significantly in high glucose group (P<0.05);apoptotic rate ,mRNA expression of caspase- 3 and Bax ,protein expression of p-mTOR ,p-Akt and p-PI 3K in HK- 2 cells were increased significantly (P<0.05). Compared with high glucose group,above indexes of sulforaphane low ,medium and high concentration groups ,irbesartan group were all improved significantly (P<0.05);the improvement of above indexes in sulforaphane medium and high concentration groups were significantly better those of sulforaphane low concentration group (P<0.05). There was no significant difference in above indexes between sulforaphane high concentration group and irbesartan group (P>0.05). Compared with sulforaphane high concentration group,there were no significant difference in the protein expression of p-AMPK ,p-mTOR in acardicin group and p-mTOR ,p-Akt and p-PI 3K in perifoxine group (P>0.05);the protein expression of p-AMPK in sulforaphane high concentration+compound C group was decreased significantly (P<0.05),while the protein expression of p-mTOR was increased significantly (P<0.05);the protein expression of p-mTOR 、p-Akt、p-PI3K in sulforaphane high concentration+SC 79 group were increased significantly (P< 0.05). CONCLUSIONS :Sulforaphane can promote the proliferation of renal tubular epithelial cells and inhibit its apoptosis ;its mechanism may be associated with up-regulating the expression of p-AMPK and down-regulating the expression of p-mTOR ,p-Akt and p-PI 3K.
期刊: 2021年第32卷第24期
作者: 周磊,艾望,陈小娟,闻春月
AUTHORS: ZHOU Lei,AI Wang,CHEN Xiaojuan ,WEN Chunyue
关键字: 萝卜硫素;高糖;人肾小管上皮细胞;磷脂酰肌醇3激酶/蛋白激酶B信号通路;腺苷一磷酸活化蛋白激酶/哺乳动物雷帕
KEYWORDS: Sulforaphane;High glucose ;Human renal tubular epithelial cells ;PI3K/Akt signaling pathway ;AMPK/mTOR
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