去氢骆驼蓬碱衍生物1-(2-氯)苯基-9-丁基-β-咔啉的体内外安全性评价

Safety evaluation of harmine derivative 1-(2-chlorine)-phenyl-9-butyl-β-carboline in vivo and in vitro

目的评价去氢骆驼蓬碱衍生物1-(2-氯)苯基-9-丁基-β-咔啉(DH-330)的体外和体内安全性。方法将小鼠神经PC12细胞和肝NCTC1469细胞分别分为正常对照组和低、中、高剂量实验组。正常对照组给予等量基础培养液干预24 h;低、中、高剂量实验组分别给予10,20,40μg·mL^(-1)DH-330干预24 h。用噻唑蓝法检测细胞的抑制率,用流式细胞术检测细胞凋亡率。将140只健康成年小鼠随机分为7组,分别为空白组,低、中、高剂量对照组和低、中、高剂量实验组,每组20只。空白组给予0.9%Na Cl;低、中、高剂量对照组分别给予50,100和200 mg·kg^(-1)去氢骆驼蓬碱;低、中、高剂量实验组分别给予50,100和200 mg·kg^(-1)DH-330。7组小鼠每天灌胃给药1次,连续给药90 d。用全自动生化分析仪测定血液学指标,用苏木精-伊红染色法观察病理组织改变。结果干预24 h,DH-330对PC12及NCTC1469细胞增殖抑制率呈剂量依赖性,半抑制浓度分别为(47.74±1.24)和(73.56±2.05)μg·mL^(-1)。低、中、高剂量实验组和空白对照组对PC12细胞的总凋亡率分别为(11.51±0.13)%,(16.32±0.16)%,(43.63±0.08)%和(2.00±0.03)%,对NCTC1469细胞的总凋亡率分别为(11.41±0.14)%,(18.31±0.10)%和(45.06±0.09)%和(2.00±0.12)%;低、中、高剂量实验组的总凋亡率均显著高于空白对照组,差异均有统计学意义(均P <0.05)。高剂量对照组小鼠的谷草转氨酶、谷丙转氨酶、碱性磷酸酶分别为(223.20±19.47),(69.68±12.50)和(128.90±15.39) U·L^(-1),均明显高于空白组的(133.50±24.67),(39.40±3.93)和(76.50±15.13) U·L^(-1),差异均有统计学意义(均P <0.05)。中、低剂量对照组和低、中、高剂量实验组的血生化结果与空白组比较,差异均无统计学意义(均P> 0.05)。小鼠病理组织学结果显示,高剂量对照组的小鼠心、肝、脾、肺、肾及脑组织均有器质性病变,低、中、高剂量实验组均无器质性病变。结论 DH-330在体外和体内均具有较高的安全性。

Objective To evaluate the safety of harmine derivative 1-(2-chlorine)-phenyl-9-butyl-β-carboline(DH-330) in vivo and in vitro.Methods Mouse nerve PC 12 cells and liver NCTC1469 cells were divided into normal control group and experimental-L,-M,-H groups.The normal control group was given the same amount of basic culture medium for 24 h.The experimental-L,-M,-H groups were given 10,20,40 μg·mL^(-1) DH-330 for 24 h.Thiazole blue method was used to detect cell inhibition rate,and flow cytometry was used to detect cell apoptosis rate.A total of 140 healthy adult mice were randomly divided into 7 groups:blank group,control-L,-M,-H groups and experimental-L,-M,-H groups,with 20 mice per group.The blank group was given 0.9% NaCl.The control-L,-M,-H groups were given 50,100,and 200 mg·kg^(-1) harmine,respectively.The experimental-L,-M,-H groups were given 50,100,and 200 mg·kg^(-1) DH-330,respectively.Seven groups were administered intragastrically once a day for 90 days.An automatic biochemical analyzer was used to determine the hematological indicators,and the hematoxylin-eosin staining method was used to observe the changes of pathological tissue.Results After 24 hours of intervention,the inhibition rate of DH-330 on PC 12 and NCTC1469 cell proliferation was dose-dependent,and the half-inhibitory concentrations were(47.74±1.24)and(73.56±2.05) μg·mL^(-1),respectively.The total apoptotic rate of PC 12 cells in the experimental-L,-M,-H groups and blank control group were(11.51±0.13)%,(16.32±0.16) %,(43.63±0.08) % and(2.00±0.03) %,respectively.The total apoptosis rates of NCTC1469 cells were(11.41±0.14) %,(28.31±0.10) %,(45.06±0.09) % and(2.00±0.12) %,respectively.The total apoptosis rates of the experimental-L,-M,-H groups were significantly higher than that of blank control group,and the differences were statistically significant(all P <0.05).The glutamic-oxalacetic transaminase,glutamic-pyruvic transaminase and alkaline phosphatase of the control-H group were(223.20±19.47),(69.68±12.50) and(128.90±15.39)U·L^(-1),respectively.All were significantly higher than those of blank group [(133.50±24.67),(39.40±3.93)and(76.50±15.13) U·L^(-1),all P <0.05].Compared with the blank group,the blood biochemical results of the medium and low dose control group and the experimental-L,-M,-H groups were not statistically different(all P>0.05).The histopathological results of mice showed that the heart,liver,spleen,lung,kidney and brain tissues of the mice in the control-H group had organic lesions,while the experimental-L,-M,-H groups had no organic lesions.Conclusion The DH-330 has highly safety in vitro and in vivo.