新疆阿魏根提取物对白细胞介素-22诱导的角质形成细胞异常增殖的作用研究

Study on the effect of Ferula Sinkiang root extract on the abnormal proliferation of keratinocytes induced by interleukin-22

目的探讨阿魏根不同提取物对白细胞介素-22(IL-22)诱导的HaCaT细胞异常增殖的影响,为评估阿魏根提取物对银屑病HaCaT细胞作用的机制研究提供依据。方法实验分组:空白对照组,IL-22实验组,阿魏酸低、中、高剂量组(0.5、1、2 mmol/L),倍半萜类化合物低、中、高剂量组(5、10、15 mg/ml),除空白对照组外其余各组采用100 ng/ml的IL-22干预24 h后,分别加入100μl不同浓度受试物继续培养24 h,观察细胞形态。CCK8法检测各组细胞增殖。流式细胞术检测各组细胞周期。酶联免疫吸附试验法检测培养体系细胞上清液中肿瘤坏死因子-α(TNF-α)、γ干扰素(IFN-γ)、IL-23、IL-4的含量。结果细胞形态学显示阿魏酸对HaCaT细胞的生长状态无明显影响,而倍半萜类化合物能抑制细胞的过度增殖。阿魏酸各剂量组细胞增殖率与IL-22实验组比较,差异均无统计学意义(P>0.05)。而倍半萜化合物各剂量组细胞增殖率与IL-22实验组比较,差异均有统计学意义(P<0.05)。阿魏酸各剂量组细胞周期与IL-22组比较,差异无统计学意义(P>0.05)。倍半萜类化合物不同剂量组G0/G1期、S期、G2/M期百分比与IL-22实验组比较,差异有统计学意义(P<0.05)。IL-22实验组各细胞因子水平与空白对照组比较,差异有统计学意义(P<0.05)。阿魏酸及倍半萜类化合物各剂量组TNF-α含量低于IL-22实验组,差异有统计学意义(P<0.05),阿魏酸各剂量组组间比较,差异无统计学意义(P>0.05),倍半萜化合物高剂量组TNF-α含量低于倍半萜化合物低剂量组(P<0.05);阿魏酸及倍半萜类化合物各剂量组IFN-γ含量与IL-22实验组比较,差异有统计学意义(P<0.05),阿魏酸及倍半萜类化合物高剂量组IFN-γ含量低于中剂量组、低剂量组,中剂量组IFN-γ含量低于低剂量组,差异有统计学意义(P<0.05);倍半萜类化合物各剂量组IL-23的含量与IL-22实验组比较,差异有统计学意义(P<0.05),倍半萜类化合物高剂量组IL-23含量低于倍半萜类化合物中剂量组、低剂量组;各处理组组间IL-4含量比较,差异无统计学意义(P>0.05)。结论倍半萜类化合物可以抑制IL-22诱导的HaCaT细胞过度增殖,调节细胞周期和分化,阿魏酸对HaCaT细胞过度增殖无明显作用。

Objective To investigate the effects of different Ferula Sinkiang root extract on the abnormal proliferation of HaCaT cells,induced by interleukin-22(IL-22)and to provide evidence for evaluating the mechanism of action of Radix Aprofunda extract on HaCaT cells in psoriasis.Methods Experimental group:blank control group,IL-22 experimental group,ferulic acid low-dose,medium-dose and high-dose groups(0.5,1,2 mmol/L),sesquiterpenoid low-dose,medium-dose and high-dose groups(5,10,15 mg/ml),except blank control group,after 24 h intervention with 100 ng/ml IL-22,the cells were cultured for 24 h by adding 100μl of different concentrations to observe cell morphology.Cell proliferation was detected by CCK8 assay.The cell cycle of each group was detected by flow cytometry.The contents of tumor necrosis factor-α(TNF-α),interferon-γ(IFN-γ),IL-23 and IL-4 in the supernatant of cultured cells were determined by enzyme-linked immunosorbent assay.Results Ferulic acid had no significant effect on the growth state of HaCaT cells,but sesquiterpenoids could inhibit the excessive proliferation of Hacat cells.There was no significant difference in cell proliferation rate between ferulic acid dose groups and IL-22 group(P>0.05).The proliferation rate of HaCaT cells in sesquiterpene dose groups was significantly different compared with that that in IL-22 experimental group(P<0.05).There was no significant difference in cell cycle between ferulic acid groups and IL-22 experimental group(P>0.05).The percentages of G0/G1 phase,and G2/M phase in different sesquiterpenoid dose groups were significantly different compared with that those in IL-22 experimental groups(P<0.05).The cytokine levels of IL-22 experimental group were significantly different compared with that those in blank control group(P<0.05).The content of TNF-αin ferulic acid and sesquiterpene groups was lower than that in IL-22 experimental group,and the difference was statistically significant(P<0.05),but there was no significant difference between ferulic acid groups(P>0.05),and the content of TNF-αin high-dose sesquiterpene group was lower than that in low-dose sesquiterpene dose group(P<0.05).The IFN-γcontent in ferulic acid and sesquiterpene groups was significantly different compared with that that in IL-22 group(P<0.05).The IFN-γcontent in ferulic acid and sesquiterpene high-dose groups was lower than that in medium-dose and low-dose groups(P<0.05).The IFN-γcontent in medium-dose group was lower than that in low-dose group(P<0.05).The content of IL-23 in sesquiterpenoid dose groups were significantly different compared with that in the IL-22 experimental group(P<0.05).The content of IL-23 in sesquiterpenoid high-dose group was lower than that in sesquiterpenoid medium-dose group and low-dose group(P<0.05).There was no significant difference in IL-4 content among treatment groups(P>0.05).Conclusion Sesquiterpenoids can inhibit IL-22-induced abnormal proliferation of HaCaT cells and regulate cell cycle,which is the important component in the treatment of psoriasis.