PLD2通过Nrf2-NFKB途径缓解胰腺细胞损伤过程中泛凋亡的机制研究
Mechanism by which PLD2 alleviates panapoptosis during pancreatic cell injury via Nrf2-NFκB pathway
张荣 1王朝海 1杜超1
作者信息
- 1. 山西省儿童医院儿童重症医学科,太原 030000
- 折叠
摘要
目的 探讨磷脂酰胆碱特异性磷脂酶D2(phosphatidylcholine specific phospholipase D2,PLD2)通过Nrf2-NFKB途径缓解胰腺细胞损伤过程中泛凋亡的机制研究.方法 将大鼠胰腺AR42J细胞用于实验研究,采用随机数字法分为CON组(常规条件下培养的AR42J细胞)、CER组(建立体外胰腺炎模型,在AR42J细胞中加入10nM cerulein)、CER+pcDNA组(在体外胰腺炎模型的基础上,建立非靶质粒阴性对照PcDNA)、CER+PLD2-OE组(在体外胰腺炎模型的基础上,建立了 PcDNA的PLD2过表达质粒PLD2-OE).通过RT-qPCR检测PLD2的表达.通过RT-qPCR检测细胞上清液炎症因子的水平.通过蛋白印迹分析Nrf2-NFKB信号通路的表达.通过蛋白印迹分析凋亡相关、焦亡相关和坏死相关蛋白的表达.结果 CER组(0.54±0.01)和 CER+pcDNA 组(0.62±0.01)PLD2 mRNA 表达较 CON 组(1.02±0.03)降低,CER+PLD2-OE 组(1.79±0.12)PLD2 mRNA 表达较 CON 组升高.CER+PLD2-OE 组的 TNF-α mRNA(2.95±0.21)、IL-6 mRNA(2.35±0.18)和 IL-10 mRNA(3.22±0.20)表达较CER+pcDNA组(TNF-α mRNA:4.25±0.25、IL-6 mRNA:3.64±0.21、IL-10 mRNA:3.22±0.20)降低.CER 组 Nrf2 蛋白(0.49±0.01)表达较 CON 组(1.02±0.01)降低,CER 组NFκB 蛋白(2.52±0.21)表达较 CON 组(1.01±0.01)升高.CER+PLD2-OE 组 Nrf2 蛋白(1.24±0.03)表达较 CER+pcDNA 组(0.50±0.01)升高,CER+PLD2-OE 组 NFκB 蛋白(1.68±0.14)表达较 CER+pcDNA 组(2.46±0.22)降低.CER 组 Bax 蛋白(1.83±0.14)表达较 CON 组(1.04±0.02)升高,CER 组 Bcl-2 蛋白(0.31±0.01)表达较 CON组(1.02±0.01)降低.CER+PLD2-OE组 Bcl-2蛋白(0.75±0.02)表达较CER±pcDNA 组(0.30±0.01)升高,CER+PLD2-OE 组 Bax 蛋白(1.42±0.11)表达较 CER+pcDNA 组(1.85±0.13)降低.CER 组 Gasdermins 蛋白(1.72±0.13)、Caspase-1 蛋白(1.88±0.15)和 NLRP3 蛋白(1.77±0.13)表达较 CON组(Gasdermins:1.13±0.04、Caspase-1:1.08±0.02、NLRP3:1.05±0.03)升高,CER+PLD2-OE 组 Gasdermins 蛋白(1.24±0.05)、Caspase-1 蛋白(1.16±0.04)和 NLRP3 蛋白(1.17±0.05)表达较 CER+pcDNA 组(Gasdermins:1.69±0.12、Caspase-1:1.75±0.13、NLRP3:1.80±0.14)降低.CER组RIPK1/RIPK3蛋白(0.52±0.01)、MLKL蛋白(0.48±0.01)和TNFR1 蛋白(0.51±0.01)表达较 CON 组(RIPK1/RIPK3:1.04±0.02、MLKL:1.03±0.01、TNFR1:1.01±0.01)降低,CER+PLD2-OE RIPK1/RIPK3 蛋白(0.65±0.02)、MLKL 蛋白(0.54±0.01)和 TNFR1 蛋白(0.63±0.01)表达较 CER+pcDNA 组(RIPK1/RIPK3:1.72±0.15、MLKL:1.65±0.13、TNFR 1:1.81±0.16)降低.结论 PLD2在调节泛凋亡过程(凋亡、焦亡和坏死)中发挥关键作用,通过激活Nrf2抗氧化途径和抑制NFκB炎症途径,有效减轻胰腺细胞损伤.
Abstract
Objective To investigate the mechanism of PLD2 alleviating panapoptosis during pancreatic cell injury through Nrf2-NFκB pathway.Methods Rat pancreatic AR42J cells purchased from ATCC were used for experimental study.The cultured and treated cells were divided into the following groups:CON group(AR42J cells cultured under conventional conditions),CER group(AR42J cells were added with 10 nM cerulein in order to establish the in vitro pancreatitis model),CER+pcDNA group(non-target plasmid negative control PcDNA was established on the basis of the in vitro pancreatitis model),and CER±PLD2-OE group(based on in vitro pancreati-tis model,PLD2-OE of PcDNA PLD2-overexpression plasmid was established).PLD2 expression was detected by RT-qPCR.The levels of inflammatory factors in cell supernatant were detected by RT-qPCR.The expression of Nrf2-NFκB signaling pathway was analyzed by protein imprinting.The expressions of apoptosis-related,pyrodeath relat-ed and necrotic proteins were analyzed by protein imprinting.Results The expression of PLD2 mRNA in CER+PLD2-OE group(1.79±0.12)was higher than that in CON group(0.54±0.01)and CER+pcDNA group(0.62±0.01).The expressions of TNF-α mRNA(2.95±0.21),IL-6 mRNA(2.35±0.18)and IL-10 mRNA(3.22±0.20)in CER+PLD2-OE group were higher than those in CER+pcDNA group(4.25±0.25;IL-6 mRNA:3.64±0.21;IL-10 mRNA:3.22±0.20).The expression of Nrf2 protein(0.49±0.01)in CER group was lower than that in CON group(1.02±0.01),and the expression of NFκB protein(2.52±0.21)in CER group was higher than that in CON group(1.01±0.01).The expression of Nrf2 protein(1.24±0.03)in CER+PLD2-OE group was higher than that in CER+pcDNA group(0.50±0.01),and the expression of NFκB protein(1.68±0.14)in CER+PLD2-OE group was lower than that in CER+pcDNA group(2.46±0.22).The expression of Bax protein in CER group(1.83±0.14)was high-er than that in CON group(1.04±0.02),and the expression of Bcl-2 protein in CER group(0.31±0.01)was lower than that in CON group(1.02±0.01).The expression of Bcl-2 protein(0.75±0.02)in CER+PLD2-OE group was higher than that in CER+pcDNA group(0.30±0.01),and the expression of Bax protein(1.42±0.11)in CER+PLD2-OE group was lower than that in CER+pcDNA group(1.85±0.13).The expression of Gasdermins protein(1.72±0.13),Caspase-1 protein(1.88±0.15)and NLRP3 protein(1.77±0.13)in CER group was higher than that in CON group(Gasdermins:1.13±0.04;Caspase-1:1.08±0.02;NLRP3:1.05±0.03).The expression of Gasdermins protein(1.24±0.05),Caspase-1 protein(1.16±0.04)and NLRP3 protein(1.17±0.05)in CER+PLD2-OE group was higher than that in CER+pcDNA group(Gasdermins:1.69±0.12;Caspase-1:1.75±0.13;NLRP3:1.80±0.14).The expressions of RIPK1/RIPK3 protein(0.52±0.01),MLKL protein(0.48±0.01)and TNFR1 protein(0.51±0.01)in CER group were higher than those in CON group(RIPK1/RIPK3:1.04±0.02;MLKL:1.03±0.01;TNFR1:1.01±0.01),and CER+PLD2-OE RIPK1/RIPK3 proteins(0.65±0.02),MLKL proteins(0.54±0.01)and TNFR1 proteins(0.63±0.01)were more expressed than CER+pcDNA group(RIPK1/RIPK3:1.72±0.15;MLKL:1.65±0.13;TNFR1:1.81±0.16).Conclusion PLD2 plays a key role in the regulation of panapoptosis(apoptosis,pyro-death and necrosis),and effectively alleviates pancreatic cell damage by activating Nrf2 antioxidant pathway and inhibiting NFκB inflammatory pathway.
关键词
磷脂酰胆碱特异性磷脂酶D2/核因子红细胞相关因子2/胰腺细胞损伤/泛凋亡Key words
PLD2/Nrf2/Pancreatic cell injury/Panapoptosis引用本文复制引用
出版年
2024
NETL
NSTL
万方数据