京尼平对脂多糖介导的血管通透性增加的影响及其可能机制

Effect of genipin on lipopolysaccharide-mediated vascular hyperpermeability and its underlying mechanism in vitro and in vivo

摘要:

目的 探讨京尼平对脂多糖(LPS)介导的血管通透性增加的影响及可能机制。方法 体外实验:将人脐静脉内皮细胞(HUVECs)分为4组,对照组仅接受0.1%的二甲基亚砜(DMSO)处理24.5h(DMSO)；模型组接受同等浓度DMSO预处理30min后与500ng/ml LPS共孵育24h(DMSO+LPS)；治疗组接受京尼平 50μmol预处理30min后与500ng/ml LPS共孵育(京尼平+LPS)；抑制剂组接受10μmol EX527及京尼平50μmol预处理30min后与500ng/ml LPS共孵育(京尼平+EX527+LPS)。检测各组细胞内皮通透性Pa值(Transwell法)、天冬氨酸特异性半胱氨酸蛋白酶3(caspase-3)活性(酶标仪检测荧光强度)、Sirtuin-1蛋白(SIRT1)表达(Western blotting)、线粒体膜电位变化(JC-1探针法)及细胞凋亡情况(TUNEL染色法)。体内实验:另将雌性大鼠24只按随机数表法分为4组,对照组大鼠注射0.5ml DMSO 30min后注射生理盐水0.5ml(DMSO+生理盐水)；模型组大鼠注射0.5ml DMSO 30min后注射LPS 10mg/kg(DMSO+LPS)；治疗组大鼠注射京尼平 5mg/kg 30min后注射LPS 10mg/kg(京尼平+LPS)；抑制剂组大鼠接受5mg/kg京尼平及5mg/kg EX527 30min后注射LPS 10mg/kg(京尼平+EX527+LPS)。生理盐水或LPS注射60min后检测大鼠血管通透性ΔI(FITC-白蛋白荧光法)及SIRT1表达(Western blotting)。应用SPSS 20.0软件进行统计分析。根据数据类型,组间比较采用单因素方差分析、LSD两两比较或Tamhane′s T2检验；组内比较采用单因素重复测量的方差分析。结果 体外实验:与对照组[Pa:1.00±0.04；caspase-3活性:100.0±4.4；JC-1绿色/红色荧光比例:(100.0±4.8)%；细胞凋亡率:(2.3±1.4)%；SIRT1:(100.0±8.9)%]比较,模型组[1.68±0.09、216.0±23.5、(343.0±28.3)%、(40.8±8.9)%、(61.0±8.5)%],治疗组[1.35±0.07、165.0±15.0、(220.0±13.3)%、(28.2±6.6)%、(86.7±7.6)%]及抑制剂组[1.60±0.10、202.0±16.5、(309.0±18.5)%、(38.0±9.5)%、(64.3±4.8)%]细胞Pa值、caspase-3活性、JC-1绿色/红色荧光比例及细胞凋亡率显著增加,SIRT1水平显著降低；与模型组比较,治疗组Pa、caspase-3、绿色/红色荧光比例及细胞凋亡率显著下降,SIRT1表达显著升高；与治疗组比较,抑制剂组Pa、caspase-3、JC-1绿色/红色荧光比例及细胞凋亡率显著增加,SIRT1表达显著降低,差异均有统计学意义(P<0.05)。体内实验:模型组、治疗组及抑制剂组大鼠在LPS注射30min及60min后,与处理前及对照组比较血压明显下降(P<0.05),但3组大鼠间血压比较差异无统计学意义(P>0.05)。与对照组[SIRT1:(100.0±4.0)%,ΔI:(0.12±0.03)]比较,模型组[(49.3±8.3)%、0.54±0.07],治疗组[(87.3±4.7)%、0.32±0.05]及抑制剂组[(55.3±4.9)%、0.53±0.06]SIRT1表达量显著降低,ΔI显著增加；与模型组比较,治疗组SIRT1表达显著上升,ΔI显著减弱；与治疗组比较,抑制剂组SIRT1表达显著降低,ΔI显著增强,差异均有统计学意义(P＜0.05)。结论 京尼平通过抑制线粒体凋亡信号激活最终抑制LPS介导的血管通透性增加,其作用可能与上调SIRT1有关。

Abstract:

Objective To investigate the effect of genipin on lipopolysaccharide (LPS)-mediated hyperpermeability and its possible mechanism. Methods In vitro experiment:human umbilical vein endothelial cells (HUVECs) were divided into 4 groups, that is, control group (DMSO, 0.1% DMSO for 24.5h), model group (DMSO+LPS, same concentration of DMSO pretreatment for 0.5h followed by 500ng/ml LPS for 24h), treatment group (genipin+LPS, 50μmol pretreatment with genipin for 0.5h followed by same LPS treatment), and inhibitor group (genipin+EX527+LPS, 0.5h pretreatment of 10μmol EX527 and 50μmol genipin and then same dose of LPS). The Pa value (representing endothelial permeability) was tested by Transwell chamber test, activity of cysteine-containing aspartate-specific proteases 3 (caspase-3) was measured by fluorescence intensity with microplate reader, Sirtuin-1 (SIRT1) level was detected by Western blotting, the change of mitochondrial membrane potential was measured by MitoProbeTM JC-1 assay kit, and apoptosis rate was tested by TUNEL. In vivo experiment:24 female rats were randomly divided into 4 groups. The rats of control, model, treatment and inhibitor groups were respectively injected with 0.5ml DMSO pretreated and 0.5ml normal saline (DMSO+normal saline), 0.5ml DMSO pretreatment followed by 10mg/kg LPS treatment (DMSO+LPS), 5mg/kg of genipin pretreatment and then LPS treatment (genipin+LPS), and pretreatment of 5mg/kg genipin and 5mg/kg EX527 and LPS treatment (genipin+EX527+LPS). The pretreatment time lasted for 30min in all the groups. The vascular permeability ΔI was measured by FITC-albumin fluorescence assay, and expression level of SIRT1 was detected with Western blotting in 60min after LPS or normal saline injection. SPSS statistics 20.0 was used for statistical analysis. According to the data type, single factor analysis of variance, LSD multiple comparison or Tamhane′s T2 test was used for intergroup comparison, and single factor repeated measurement analysis of variance was used for intragroup comparison. Results In vitro experiment:compared with control group [Pa:1.00±0.04; caspase-3 activity:100.0±4.4; JC-1 green/red fluorescence ratio:(100.0±4.8)%; apoptosis rate:(2.3±1.4)%; SIRT1:(100.0±8.9)%], Pa value, caspase-3 activity, JC-1 green/red fluorescence ratio and apoptosis rate were significantly increased in the model group [1.68±0.09, 216.0±23.5, (343.0±28.3)%, (40.8±8.9)%], treatment group [1.35±0.07,5.0±15.0, (220.0±13.3)%, (28.2±6.6)%] and inhibitor group [1.60±0.10,2.0±16.5, (309.0±18.5)%, (38.0±9.5)%], and the expression level of SIRT1 level was decreased[(61.0±8.5)%, (86.7±7.6)%, and (64.3±4.8)% respectively]. Compared with the model group, the former indices in treatment group were reduced obviously, while SIRT1 level was increased (P<0.05). Compared with the treatment group, the Pa, caspase-3, JC-1 green/red ratio and apoptosis rate were elevated significantly in the inhibitor group, while the expression of SIRT1 was decreased significantly (P<0.05). In vivo experiment:the blood pressure of rats in the model, treatment and inhibitor groups were decreased significantly (P<0.05) after LPS injection for 30 and 60min when compared with the values before LPS pretreatment and those of the control group, but there was no significant difference in blood pressure among the 3 groups (P<0.05). Compared with the control group [SIRT1:(100.0±4.0)%, ΔI:(0.12±0.03)], the expression level of SIRT1 was decreased while ΔI was increased in the model group [(49.3±8.3)%, 0.54±0.07], the treatment group [(87.3±4.7)%, 0.32±0.05], and the inhibitor group [(55.3±4.9)%, (0.53±0.06)]. When compared with the model group, the expression of SIRT1 was increased, and ΔI was decreased significantly in the treatment group, but the level was significantly decreased and ΔI was significantly increased in the inhibitor group. Statistical significances were seen in above indicators (P<0.05). Conclusion Genipine inhibits LPS-mediated hyperpermeability by inhibiting the activation of mitochondrial apoptotic signal, and this effect may be related to the up-regulation of SIRT1.