氯胺酮对脂多糖诱导下人脐静脉内皮细胞活化的影响

Effects of ketamine on lipopolysaccharide-induced activation of human umbilical vein endothelial cells

目的 观察氯胺酮和N-甲基-D-天门冬氨酸(NMDA)受体非竞争性拮抗剂MK-801对脂多糖(LPS)刺激下人脐静脉内皮细胞(HUVECs)胞间粘附分子-1(ICAM-1)表达及核因子-kappa B(NF-kB)易位表达的影响。方法 采用Jaffe方法培养的HUVECs随机分为10组:对照组(C组,RPMI-1640),LPS组(L组,LPS1μg/ml),氯胺酮组(K组,依浓度不同分为KⅠ、KⅡ、KⅢ、KⅣ亚组,即氯胺酮12.5、25.0、100、300μmol/L+LPS1μg/ml),MK-801组(M组,依浓度不同分为MⅠ、MⅡ、MⅢ、MⅣ亚组,即MK-801 1.25、2.50、10、30μmol/L+LPS1μg/ml)。在37℃、5％CO_2中孵育18h后,用流式细胞仪检测ICAM-1的表达阳性率。NF-kB易位表达的测定分组处理同上,在LPS1μg/ml刺激2h后,用免疫组化(SP)方法测定内皮细胞中NF-kB p65亚基的表达。结果 KⅡ、KⅢ、KⅣ亚组可抑制LPS作用下HUVECs表面ICAM-1的表达和细胞内部NF-kB的易位表达(P<0.05),且两者的变化呈正相关(r=0.985,P<0.01)。M组各亚组对LPS作用后HUVECs表面ICAM-1的表达和NF-kB的易位表达无明显影响(P>0.05)。结论 氯胺酮对炎症反应中内皮细胞的活化具有抑制作用,但并非通过NMDA受体途径。

Objectivc To study the effects of ketamine and MK-801 on lipopolysaccharide (LPS)-induced expression of intercellular adhesion molecule-1 (ICAM-1 ) and the translocation of nuclear factor-kappa B (NF-κB) into nuclei of human umbilical vein endothelial cells (HUVECs). Methods The endothelial cells obtained from human umbilical vein by digestion with collagenase 1 were cultured at 37℃ in a 5 % CO_2-95% room air atmosphere for 7 days. The cultured human umbilical vein endothelial cells (HUVECs) wer randomly divided into (1) control group in which nothing was added to the RPMI-1640 culture medium; (2) LPS group in which us 1 μg·ml^(-1) was added to RPMI-1640; (3) ketamine group was further divided into 4 subgroups according to the concentration of ketamine added: 12.5 (KⅠ), 25 .0 (KⅡ), 100 (KⅢ) and 300μmol·L^(-1) (KⅣ); (4) MK-801 group was further divided into 4 subgroups M Ⅰ-Ⅳ, according to the different concentrations of MK-801 added (1 .25, 2.50, 10, 30μmol·L^(-1)). In one set of experiment in ketamine and MK-801 groups after addition of ketamine and MK-801 to the RPMI-1640 culture medium, the HUVECs were incubated for 30 min at 37℃, then LPS1μg·ml^(-1) was added and incubated for 18 h in a 5 % CO_2 atmosphere at 37℃. The HUVECs were then harvested for determination of ICAM-1 expression by flow cytometry. In another set of experiment the cultured HUVECs were stimulated with LPS 1μg·ml^(-1) for 2 h in the absence or presence of ketamine (12.5, 25 .0, 100 and 300μmol·L^(-1) ) or MK-801 (1.25, 2.50, 10 and 30μmol·L^(-1)). The translocation of NF-κB p65 into nuclei was detected by immunohistochemistry technique. Results In group K Ⅱ, KⅢ and KⅣ the upregulation of ICAM-1 and translocation of NF-κB into nuclei of HUVECs induced by LPS were significandy reduced (P<0. 05) There was positive correlation between ICAM-1 expression on HUVECs and translocation of NF-κB into nuclei of HUVECs (r = 0.985, P<0.01). In group MⅠ, MⅡ, MⅢ, MⅣ no effect on the expression of ICAM-1 and the translocation of MF-KB into nuclei was observed (P>0.05). Conclusion Ketamine can suppress LPS- induced HUVEC activation at the higher concentrations (≥25μmol·L^(-1)). The inhibitory effects of ketamine is most likely not mediated through NMDA receptor interactions.