壁虎活性组分促进肝癌细胞HepG2焦亡的作用

Effect of Gecko active components on promoting the pyroptosis in HepG2 cells

目的探讨壁虎活性组分(GACs)促进肝癌细胞HepG2焦亡的作用及相关机制。方法用不同质量浓度GACs(0,0. 15,0. 2,0. 25,0. 3,0. 35,0. 4,0. 45和0. 5 mg·mL-1)处理HepG2细胞24 h,用噻唑蓝(MTT)比色法检测细胞增殖情况,根据MTT结果分为空白对照组和低、中、高剂量(GACs:0. 1,0. 15,0. 225mg·mL-1)实验组。以乳酸脱氢酶(LDH)法检测HepG2细胞凋亡情况,以酶联免疫吸附(ELISA)法检测细胞上清中白细胞介素-1β(IL-1β)释放情况,以Western blot法检测细胞中活化胱天蛋白酶1 (cleaved caspase-1),消皮素D(GSDMD),IL-1β和白细胞介素-18(IL-18)的表达。结果 GACs可抑制HepG2细胞的增殖,并具有质量浓度依赖性,作用24,48,72 h后其IC50值分别为0. 24,0. 22,0. 18 mg·mL-1。空白对照组和低、中、高剂量实验组细胞培养上清液中的LDH含量分别为(63. 68±20. 54),(79. 33±14. 22),(81. 56±14. 38),(332. 96±6. 32) U·L-1;IL-1β含量分别为(1. 73±0. 05),(2. 11±0. 05),(2. 24±0. 01),(2. 59±0. 08) ng·L-1;cleaved caspase-1蛋白表达比值分别为(6. 14±0. 46)×10-2,(10. 71±1. 21)×10-2,(20. 29±2. 96)×10-2,(20. 61±2. 67)×10-2;GSDMD蛋白表达比值分别为(9. 76±0. 66)×10-2,(23. 67±4. 77)×10-2,(41. 17±3. 69)×10-2,(46. 56±6. 31)×10-2;IL-1β蛋白表达比值分别为(8. 99±0. 48)×10-2,(34. 39±5. 51)×10-2,(36. 04±6. 42)×10-2,(66. 33±5. 65)×10-2;IL-18蛋白表达比值分别为(11. 54±1. 89)×10-2,(30. 83±3. 29)×10-2,(55. 72±6. 39)×10-2,(56. 12±5. 41)×10-2;低、中、高剂量组各指标与对照组比较,差异均有统计学意义(均P <0. 01)。结论壁虎活性组分激活了caspase-1,诱导肝癌细胞HepG2焦亡,并促进IL-1β释放。

Objective To evaluate the effects of gecko active components(GACs)on the pyroptosis in HepG2 cells.Methods The HepG2 cells were treated with different concentrations of GACs(0,0.1,0.15,0.2,0.25,0.3,0.35,0.4,0.45,0.5 mg·mL-1)for 24 h,and then the corresponding indicators were detected with respective methods.3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide(MTT)assay was used to detect the viability of HepG2 cells.The concentration of blank control group and low,middle,high dose experimental groups were separately 0,0.1,0.15,0.225 mg·mL-1,according to the results of MTT.Pyroptosis was investigated by a standard lactate dehydrogenase release assay(LDH).Interleukin-1β(IL-1β)content in the cell culture supernatant was quantified by enzyme-linked immunosorbent assay(ELISA).The expression of cleaved cysteine aspartate-specific protease-1(caspase-1),gasdermin D(GSDMD),IL-1β,interleukin-18(IL-18)were detected by Western blot analysis.Results The results showed that GACs could inhibit the proliferation of HepG2 cells in dose-and time-dependent manners.After treatment for 24,48,72 h,the 50%inhibitory dose(IC50)were 0.24,0.22,0.18 mg·mL-1.The LDH contents in the cell culture supernatants of blank control group and low,middle,high dose experimental groups were(63.68±20.54),(79.33±14.22),(81.56±14.38),(332.96±6.32)U·L-1;the IL-1βcontents were(1.73±0.05),(2.11±0.05),(2.24±0.01),(2.59±0.08)ng·L-1;the cleaved caspase-1 protein expression ratios were(6.14±0.46)×10-2,(10.71±1.21)×10-2,(20.29±2.96)×10-2,(20.61±2.67)×10-2;the GSDMD protein expression ratios were(9.76±0.66)×10-2,(23.67±4.77)×10-2,(41.17±3.69)×10-2,(46.56±6.31)×10-2;the IL-1βprotein expression ratios were(8.99±0.48)×10-2,(34.39±5.51)×10-2,(36.04±6.42)×10-2,(66.33±5.65)×10-2;the IL-18 protein expression ratios were(11.54±1.89)×10-2,(30.83±3.29)×10-2,(55.72±6.39)×10-2,(56.12±5.41)×10-2;Compared with blank control group,the differences were all statistically significant(all P<0.01).Conclusion Gecko active components activated caspase-1,promoted pyroptosis and IL-1βrelease in HepG2 cells.