灯盏花素对人肝细胞缺氧再给氧损伤的保护作用及机制

Protective effect of breviscapine on human hepatocytes under hypoxia/re-oxygenation and its possible mechanism

目的观察中药灯盏花素对人肝细胞（L-02）缺氧再给氧（H／R）损伤的保护作用，并探讨其可能的保护机制。方法体外建立L-02细胞H／R损伤模型。L-02细胞随机分为正常对照组、缺氧组和灯盏花素组（灯盏花素预处理＋缺氧）OL-02细胞缺氧处理10h后，分别再给氧1、2、4、8、16、24h后检测各组细胞MICA分子和蛋白水平的表达；利用乳酸脱氢酶释放法测定NK细胞对L-02细胞的杀伤能力。结果H／R1h后，缺氧组MICA分子和蛋白水平显著高于正常对照组（P〈0．05）；且以再给氧8h达到峰值，于24h恢复正常。NK细胞对H／RL-02细胞的杀伤活性与MICA的表达水平呈现相同的变化规律。H／R处理的L-02细胞经MICA抗体封闭后，NK细胞对其的杀伤活性显著性降低（P〉0．05）。灯盏花素组MICA分子和蛋白表达水平及NK细胞对其的杀伤作用在再给氧1h内达峰后逐渐下降；灯盏花素组MICA的表达水平和NK细胞对其的杀伤作用在各时间点均明显低于缺氧组（均P〈0．05）。结论H／R损伤可能是通过上调L-02细胞MICA的表达而诱导NK细胞对其的杀伤作用；灯盏花素对L-02细胞H／R损伤的保护作用可能是通过抑制MICA的上调，从而抑制NK细胞对其的杀伤作用。

Objective To explore the protective effect of breviscapine on human hepatocytes （L- 02） under hypoxia/re-oxygenation （H/R） and elucidate its possible mechanism. Methods A in vitro model of H/R was employed to mimic H/R injury of graft organ. L-02 cells were randomly divided into 3 groups： control, I-I/R and breviscapine treatment （pre-treated with breviscapine and H/R）. After a 10 h hypoxic culturing under 1% 02, 94% N2 and 5% COs, L-02 cells received oxygen for 1, 2, 4, 8, 16 and 24 h respectively. MICA mRNA and protein levels were detected by quantitative real-time polymerase chain reaction （PCR） and Western blot. And the activity of natural killer （NK） cell cytotoxicity for L-02 was measured by lactate dehydrogeuase （LDH） release assay. After blocking I/R treatment with NKG2D antibody, the activity of NK cell cytotoxicity for L-02 was detected. Results After 10 h hypoxia, MICA mRNA and protein levels significantly increased from 1 h, stayed up-regulated until 8 h and then went back to normal level after reoxygenation versus the control group. The activity of NK cell cytotoxicity for L-02 under the treatment of H/R increased markedly from 1 h post-reoxygenation and stayed up-regulated from 1 h to 8 h versus the control group. After hypoxia, L-02 cells were blocked with NKG2D antibody and the NK cell cytotoxicity for L-02 significantly decreased versus the I/R group. The administration of breviscapine significantly lowered the mRNA and protein levels of MICA in L-02 under I/R and then significantly decreased the NK cell cytotoxicity. Conclusion The process of I/R may mediate the NK cell cytotoxicity activity toward to L-02 by inducing a strong increase of MICA at mRNA and protein levels in L-02 cells. And the dministration of Breviscapine significantly reduces the NK cell cytotoxicity for L-02 under I/R through the down-regulated expression of MICA.