天山堇菜通过调控内质网应激和 NF-κB 信号通路发挥对肝损伤的保护作用。
Gentianella turkestanerum Showed Protective Effects on Hepatic Injury by Modulating the Endoplasmic Reticulum Stress and NF-κB Signaling Pathway.
机构信息
Department of Pharmacy, The First Affiliated Hospital, Xinjiang Medical University, Urumqi 830011, China.
College of Pharmacy, Xinjiang Medical University, Urumqi 830011, China.
出版信息
Curr Mol Med. 2019;19(6):452-460. doi: 10.2174/1566524019666190415124838.
OBJECTIVE
To investigate the protective effects of Gentianella turkestanerum extraction by butanol (designated as GBA) on hepatic cell line L02 injury induced by carbon tetrachloride (CCl4) and hydrogen peroxide (H2O2).
METHODS
L02 cells were incubated with 5 µg/mL, 10 µg/mL, 20 µg/mL, 40 µg/mL, 60 µg/mL, 80 µg/mL and 100 µg/mL GBA for 24 hours, and then MTT assay was used to screen the cytotoxicity for GBA. Cells were divided into blank control group, CCl4/H2O2 model group, treated by CCl4 (20 mmol/L) or H2O2 (100 µmol/L); silymarin+CCl4/H2O2 group, treated by CCl4 (20 mmol/L) or H2O2 (100 µmol/L) and 5 µg/mL silymarin; GBA+CCl4/H2O2 group, treated by CCl4 (20 mmol/L) or H2O2 (100 µmol/L) and GBA (5 µg/mL, 10 µg/mL and 20 µg/mL). MTT assay was performed to determine the cellular activity. Malondialdehyde (MDA) content was determined using a commercial kit. The alanine transaminase (ALT), aspartate transaminase (AST) in the supernatant was determined. PE-Annexin V/7-ADD method was utilized to determine the apoptosis of cells. RT-PCR was used to evaluate the expression of endoplasmic reticulum stressrelated genes (CHOP, PERK, IRE1 and ATF6) mRNA. Western blot analysis was performed to determine the expression of CHOP, Caspase 12 and NF-κB protein.
RESULTS
Cellular survival after GBA (5 µg/mL, 10 µg/mL and 20 µg/mL) incubation was ≥ 75%. After GBA incubation, levels of ALT and AST showed a significant decrease (P < 0.05), while that of the MDA showed a significant decrease (P < 0.05). The apoptosis in the CCl4 or H2O2 group showed a significant increase compared to the control group (P < 0.05). In contrast, GBA-preincubation could attenuate the cellular apoptosis compared to the CCl4 or H2O2 group, which displayed a dose-dependent manner (P < 0.05). The expression of CHOP, PERK, IRE1 and ATF6 mRNA was significantly up-regulated in the presence of CCl4 or H2O2 (P < 0.05). Whereas, GBA induced a significant decrease in these mRNA thereafter (P < 0.05), together with a decrease in CHOP and Caspase 12 proteins (P < 0.05). Besides, it could attenuate the expression of NF-κB p65 in nuclear protein.
CONCLUSION
G. turkestanerum could inhibit the lipid peroxidation and increase the antioxidant activity. Also, it could inhibit the cellular apoptosis through down-regulating the transcriptional level of ERS related genes and proteins. This process was associated with the nuclear translocation of NF-κB p65 protein.
目的
研究正丁醇提取的獐芽菜提取物（命名为 GBA）对四氯化碳（CCl4）和过氧化氢（H2O2）诱导的 L02 肝细胞损伤的保护作用。
方法
将 L02 细胞分别用 5µg/mL、10µg/mL、20µg/mL、40µg/mL、60µg/mL、80µg/mL 和 100µg/mL 的 GBA 孵育 24 小时，然后采用 MTT 法筛选 GBA 的细胞毒性。将细胞分为空白对照组、CCl4/H2O2 模型组、用 20mmol/L CCl4 或 100µmol/L H2O2 处理；水飞蓟素+CCl4/H2O2 组，用 20mmol/L CCl4 或 100µmol/L H2O2 和 5µg/mL 水飞蓟素处理；GBA+CCl4/H2O2 组，用 20mmol/L CCl4 或 100µmol/L H2O2 和 5µg/mL、10µg/mL 和 20µg/mL 的 GBA 处理。采用 MTT 法测定细胞活力。采用商业试剂盒测定丙二醛（MDA）含量。测定上清液中丙氨酸转氨酶（ALT）和天门冬氨酸转氨酶（AST）的含量。采用 PE-Annexin V/7-ADD 法检测细胞凋亡情况。采用 RT-PCR 法检测内质网应激相关基因（CHOP、PERK、IRE1 和 ATF6）mRNA 的表达。采用 Western blot 法检测 CHOP、Caspase 12 和 NF-κB 蛋白的表达。
结果
GBA（5µg/mL、10µg/mL 和 20µg/mL）孵育后细胞存活率≥75%。GBA 孵育后，ALT 和 AST 水平显著降低（P<0.05），MDA 水平显著降低（P<0.05）。与对照组相比，CCl4 或 H2O2 组的细胞凋亡明显增加（P<0.05）。相反，与 CCl4 或 H2O2 组相比，GBA 预处理可减轻细胞凋亡，呈剂量依赖性（P<0.05）。与对照组相比，CCl4 或 H2O2 存在时 CHOP、PERK、IRE1 和 ATF6 mRNA 的表达显著上调（P<0.05）。然而，GBA 处理后这些 mRNA 的表达显著下调（P<0.05），同时 CHOP 和 Caspase 12 蛋白的表达也下调（P<0.05）。此外，它还可以抑制核蛋白中 NF-κB p65 蛋白的表达。
结论
獐芽菜提取物可抑制脂质过氧化，提高抗氧化活性。此外，它还可以通过下调 ERS 相关基因和蛋白的转录水平来抑制细胞凋亡。这一过程与 NF-κB p65 蛋白的核转位有关。
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