壁虎多肽混合物对HepG2细胞增殖及内质网应激途径的影响

Effects of Gecko peptide mixture on HepG2 cells proliferation and endoplasmic reticulum stress pathway

目的研究壁虎多肽混合物(GPM)对人肝癌HepG2细胞增殖及内质网应激途径的影响。方法用不同浓度GPM(0,0.15,0.20,0.25,0.30,0.35,0.40,0.45 mg·mL^(-1))处理HepG2细胞24 h。以噻唑蓝(MTT)比色法检测细胞增殖,根据MTT结果,将细胞分为空白组和低、中、高3个质量浓度(GPM:0.1,0.2,0.3 mg·mL^(-1))实验组,对照组(氟尿嘧啶10μg·mL^(-1))。以Western blot法检测内质网应激相关蛋白及凋亡相关蛋白的表达水平。结果 GPM可抑制HepG2细胞的增殖,并具有质量浓度依赖性,作用24,48,72h后,其IC50值分别为0.27,0.23,0.20 mg·mL^(-1)。正常组、对照组和低、中、高3个质量浓度实验组的双链RNA依赖的蛋白激酶样ER激酶(PERK)与GAPDH灰度比值分别为(4.31±0.81)×10^(-2),(8.92±0.91)×10^(-2),(20.73±0.97)×10^(-2),(24.04±0.95)×10^(-2),(11.65±1.67)×10^(-2)。这5组的GRP78与GAPDH灰度比值分别为(27.99±2.36)×10^(-2),(35.58±1.02)×10^(-2),(42.55±1.19)×10^(-2),(54.91±1.20)×10^(-2),(7.31±1.01)×10^(-2)。这5组的ATF4与GAPDH灰度比值分别为(20.82±1.42)×10^(-2),(39.60±0.56)×10^(-2),(52.02±1.83)×10^(-2),(73.39±1.83)×10^(-2),(18.13±2.28)×10^(-2);这5组的CHOP与GAPDH灰度比值分别为(8.71±0.76)×10^(-2),(11.27±1.07)×10^(-2),(41.29±1.36)×10^(-2),(48.55±1.37)×10^(-2),(33.01±3.95)×10^(-2)。这5组的多聚ADP-核糖聚合酶(PARP)与GAPDH灰度比值分别为(13.06±2.88)×10^(-2),(36.79±2.10)×10^(-2),(58.72±1.53)×10^(-2),(67.61±1.68)×10^(-2),(34.88±2.02)×10^(-2)。这5组的半胱氨酸天冬氨酸蛋白酶-3(Caspase-3)与GAPDH灰度比值分别为(5.92±0.33)×10^(-2),(14.71±1.11)×10^(-2),(17.58±1.33)×10^(-2),(35.41±2.91)×10^(-2),(5.94±1.61)×10^(-2),与正常组相比,4个给药组的以上各项指标差异均有统计学意义(均P<0.05)。提示GPM可能通过诱导HepG2细胞内质网应激从而诱导细胞凋亡。结论 GPM可抑制HepG2细胞增殖并诱导其凋亡,其机制可能与诱导HepG2细胞内质网应激有关。

Objective To investigate the effect of Gecko polypeptide mixture （GPM） on the proliferation and endoplasmic reticulum stress （ERS） pathway of human hepatocellular carcinoma HepG2 cells. Methods The HepG2 cells were treated with different concentration of GPM（0,0. 15,0.20,0.25,0.30,0.35,0.40,0.45 mg · mL-1） for 24 h, and then corresponding indicators were detected with respective methods.The 3 -（4,5 -dimethyl-2- thiazolyl） -2,5 -diphenyl-2- H- tetrazolium bromide （MrlT） assay was used to de- tect the viability of HepG2 cells. The concentration of blank group, GPM low - dose, middle - dose and high - dose experimental groups was respectively 0, 0. 1, 0. 2, 0. 3 mg · mL- 1, according to the results of MTr. 5 - Fluorouracil was chosen as the positive control drug, which concentration was 10 ixg · mL-1. Western blot analysis was applied to observe the expression of ERS - related proteins and apoptosis - related proteins in HepG2 cells. Results The GPM could inhibit the proliferation of HepG2 cells in a dose - and time - dependent manners. After treatment of GPM for 24, 48, 72 h, the 50% inhibitory dose （ICs0） values were 0. 27,0. 23,0. 20 mg · mL-1. Compared with the normal group, the proteins expression levels of double - strand RNA - activated protein kinase - like ER kinase （ PERK）, glu- cose - regulated protein78 （ GRP78 ）, activating transcription factor - 4 （ ATF4 ）, C/EBP - homologous protein （CHOP） and apoptosis -related poly -ADP -ribos polymerase （PARP）, cleaved cysteinyl aspartate specific protein- ase - 3 （ Caspase - 3 ） were significantly up - regulated after treatment of GPM in vitro （ P 〈 0. 05 or P 〈 0. 01 ）. PERK and GAPDH grayscale average ratio in normal group, control group, and three concentration experimantal groups were （4. 31 ±0. 81） xl0-2, （8.92 ±0. 91） x 10-2, （20. 73 ±0. 97） x 10-2, （24. 04 ±0. 95） x 10-2, （ 11.65 ± 1.67） x 10.2 ; GRP78 and GAPDH grayscale average ratio in the five groups were （27. 99 ±2.36） x 10 -z, （35.58 ± 1.02） x 10-2, （42. 55 ± 1.19） x 10 -2, （54. 91 ± 1.20） x 10 -2, （7.31 ± 1.01 ） x 10 -2 ;ATF4 and GAPDH grayscale average ratio in the five groups were （20.82 ± 1.42） x 10-2, （39.60 ± 0.56） x 10-2, （52.02 ± 1.83） x 10-2, （73.39 ± 1.83） x 10-2,（ 18. 13 ± 2. 28） x 10-2;CHOP and GAPDH grayscale average ratio in the five groups were （8.71 ±0.76） x10-2,（11.27±1.07） x10-2,（41.29±1.36） x10-1,（48.55±1.37） x10 -2 , （ 33. 01 ±3.95） x 10-2 ;PARP and GAPDH grayscale average ratio in the five groups were（ 13.06 ± 2. 88） x10-2, （36.79 ± 2. 10） x10 -2, （58.72 ± 1.53 ） x10 -2, （67. 61 ± 1.68 ） x 10 -2, （34. 88 ± 2. 02 ） x10 -2 ; Caspase - 3 and GAPDH grayscale average ratio in the five groups were （5.92 ± 0.33） x 10-2, （14.71 ± 1.11） x10-2, （17.58 ±1.33） x10-2, （35.41 ±2. 91 ） x10-2, （5.94 ± 1.61 ） x10-2. Compared with the normal group, the expression levels of GRP78, ATF4, CHOP in the four groups were significantly up - regulated （P 〈 0. 05 or P 〈 0. 01 ）. Conclusion GPM can inhibit proliferation and induce apoptosis of HepG2 cells, which may be associated with inducing the HepG2 cells endonlasmic reticulum stress.