氯喹对脂多糖诱导的Ⅱ型肺泡细胞损伤的影响  

Effect of chloroquine on typeⅡalveolar cell injury caused by lipopolysaccharide

作　　者：朱莹莹[1] 张姣姣 孙俊楠 王海嵘[1] ZHU Yingying;ZHANG Jiaojiao;SUN Junnan;WANG Hairong(Department of Emergency,Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine,Shanghai 200092,China)

机构地区：[1]上海交通大学医学院附属新华医院急诊医学科,上海200092

出　　处：《内科理论与实践》2021年第3期197-201,共5页Journal of Internal Medicine Concepts & Practice

摘　　要：目的:探究氯喹(chloroquine,CQ)对脂多糖(lipopolysaccharide,LPS)所致Ⅱ型肺泡(alveolar typeⅡ,AT2)细胞株损伤的影响。方法:用不同浓度LPS和CQ分别单独处理AT2细胞,用细胞计数试剂盒-8(cell counting kit-8,CCK-8)检测细胞活力以筛选LPS和CQ的作用浓度。根据细胞处理情况将实验分为对照组(不做任何处理的细胞)、LPS组、CQ组和CQ+LPS组4组。用蛋白质印迹法检测各组裂解的胱天蛋白酶3、Bcl-2、Bax的表达和Akt的磷酸化水平。之后增加磷脂酰肌醇-3-激酶(phosphatidylinositol-3-kinase,PI3K)/Akt信号通路抑制剂LY294002与LPS和CQ共同处理细胞,即LPS+CQ+LY294002组,通过蛋白质印迹法检测裂解的胱天蛋白酶3、Bcl-2和Bax的蛋白表达。结果:用终浓度为1、10和50 mg/L的LPS作用24 h,当LPS浓度为50 mg/L时,AT2活力下降显著(P<0.05),故选择该浓度的LPS构建脓毒症细胞损伤模型。用终浓度0.5μmol/L和5μmol/L CQ作用4 h时,AT2细胞活力无明显改变(P>0.05),当CQ浓度达10μmol/L,AT2细胞活力开始下降,浓度越高,细胞活力下降越明显,故选择5μmol/L的CQ作为治疗浓度。用50 mg/L的LPS刺激后,细胞内裂解的胱天蛋白酶3表达增高,Bcl-2/Bax比值及Akt蛋白磷酸化减少;在加入5μmol/L的CQ后,裂解的胱天蛋白酶3含量下降,Bcl-2/Bax比值和Akt蛋白磷酸化显著增加;当加入LY294002后,CQ的作用被消除,Bcl-2/Bax比值减少,裂解的胱天蛋白酶3表达增高。结论:5μmol/L的CQ可以通过激活PI3K/Akt信号通路,抑制细胞线粒体凋亡,减轻LPS诱导的AT2细胞损伤。Objective To explore the effect of chloroquine(CQ)on lipopolysaccharide(LPS)-induced damage to alveolar typeⅡ(AT2)cell.Methods Different concentrations of LPS and CQ were used to treat AT2 cells separately,and cell counting kit-8(CCK-8)was used to detect cell viability to screen the concentration of LPS and CQ.After that,the AT2 cells were divided into 4 groups based on different treatment,including control(without treatment),LPS,CQ and LPS+CQ groups.The expression of cleaved caspase 3,Bcl-2,Bax and the phosphorylation level of Akt were detected by Western blotting after each group of cells were processed.After that,LY294002,the signaling pathway inhibitor of phosphatidylinositol-3-kinase(PI3K)/Akt,was added in the cells which were treated by LPS and CQ successively(LPS+CQ+LY294002 group),and the protein expression of cleaved caspase 3,Bcl-2 and Bax was detected by Western blotting in this group.Results The LPS in the concentration of 1,10 and 50 mg/L was kept for 24 h.The activity of AT2 decreased significantly(P<0.05)when the LPS concentration was increased to 50 mg/L,so LPS in this concentration was used to stimulate AT2.AT2 cell viability did not change significantly(P>0.05)when they were treated with 0.5μmol/L and 5μmol/L CQ for 4 h.However,AT2 cell viability began to decrease and showed a concentration-dependent manner when the CQ concentration increased to 10μmol/L.According to the cell response to CQ in different concentration,5μmol/L CQ was chosen to treat the cells.After stimulation with 50 mg/L LPS,the expression of cleaved caspase 3 in the cell increased,and the Bcl-2/Bax ratio and the phosphorylation of Akt decreased.After adding 5μmol/L CQ,the cleaved caspase 3 dropped,and Bcl-2/Bax ratio and Akt protein phosphorylation rise significantly.After the PI3K inhibitor LY294002 was added,the effect of CQ was eliminated,which showed the Bcl-2/Bax ratio decreased,and the expression of cleaved caspase 3 increased.Conclusions CQ(5μmol/L)could inhibit cell mitochondrial apoptosis by activating the PI3K

关 键 词：氯喹 Ⅱ型肺泡细胞 细胞凋亡 PI3K/AKT 

分 类 号：R631[医药卫生—外科学]