黄杞叶提取物对小鼠造血系统辐射损伤的防护作用

Protective effects of Engelhardia roxburghiana Wall.leaf extract on radiation injury of the hematopoietic system in mice

目的研究黄杞叶提取物对一次性全身4 Gy137Cs γ射线照射导致的小鼠造血系统辐射损伤的防护作用。方法 (1)体外实验。采用1,1-二苯基-2-苦基肼(DPPH)和2,2′-联氮-双-3-乙基苯并噻唑啉-6-磺酸(ABTS)自由基体外清除实验检测黄杞叶提取物的体外抗氧化能力。(2)体内实验。①存活实验。将40只无特定病原体(SPF)级雄性C57BL/6小鼠按照区组随机法平均分为4组:照射组、照射+黄杞叶提取物低剂量(20 mg/kg)组、照射+黄杞叶提取物中剂量(40 mg/kg)组、照射+黄杞叶提取物高剂量(80 mg/kg)组, 4组均进行全身一次性7.2 Gy(致死剂量)137Cs γ射线照射, 记录照射后30 d小鼠的存活情况。②造血系统辐射损伤防护实验。将60只SPF级雄性C57BL/6小鼠按照区组随机法平均分为6组:对照组、照射组、黄杞叶提取物高剂量组(80 mg/kg)、照射+黄杞叶提取物低剂量(20 mg/kg)组、照射+黄杞叶提取物中剂量(40 mg/kg)组、照射+黄杞叶提取物高剂量(80 mg/kg)组, 其中对照组和黄杞叶提取物高剂量组不照射, 其余4组均进行一次性全身4 Gy137Cs γ射线照射。照射后第7天处死小鼠, 取外周血进行血常规检测, 取单侧股骨骨髓细胞进行骨髓有核细胞计数, 取另一侧股骨骨髓细胞进行骨髓DNA含量检测, 取胸腺和脾脏计算脏器指数, 取肝脏检测还原型谷胱甘肽(GSH)含量。体内实验中各组小鼠均于照射前7 d和照射后6 d连续灌胃给药, 其中对照组和照射组给予0.5%羧甲基纤维素钠, 其余各组给予相应剂量的黄杞叶提取物。组间两两比较采用Studentt检验, 存活率采用Kaplan-Meier生存分析。结果 (1)在体外实验中, 当浓度分别为100 μg/ml和200 μg/ml时, 与Trolox相比, 黄杞叶提取物对DPPH自由基的清除率更高(89.83%对69.37%, 90.94%对68.53%);对ABTS自由基的清除率也更高(94.81%对71.35%, 94.46%对71.93%), 且差异均有统计学意义(t=19.58~33.26, 均P<0.001)。(2)在体内实验中, 与照射组相比, 照射+黄杞叶提取物低、中、高剂量组小鼠30 d存活率均明显升高, 且差异均有统计学意义(Kaplan-Meier生存分析, 均P<0.05), 存活天数亦均明显增加[(19.40±7.70)d对(12.50±3.59)d、(20.20±8.48)d对(12.50±3.59)d、(20.90±7.96)d对(12.50±3.59)d ], 且差异均有统计学意义(t=2.57、2.79、3.04, 均P<0.05);照射+黄杞叶提取物高剂量组小鼠脾脏和胸腺的脏器指数明显升高[(2.13±0.43)mg/g对(1.67±0.20)mg/g、(1.87±0.39)mg/g对(1.39±0.31)mg/g] , 且差异均有统计学意义(t=3.00、3.03, 均P<0.05);照射+黄杞叶中剂量组小鼠红细胞数量增加最为明显[(10.12± 1.71)×1012/L对(8.26±0.87)×1012/L], 且差异有统计学意义(t=2.89, P<0.05);照射+黄杞叶提取物高剂量组小鼠白细胞、红细胞数量明显增加[(1.76±0.45)×109/L对(1.17±0.23)×109/L、(9.59±0.85)×1012/L对(8.26±0.87)×1012/L], 血红蛋白含量亦明显升高[(144.40±14.61)g/L对(126.20±13.16)g/L] , 且差异均有统计学意义(t= 3.62、3.23、2.93, 均P<0.05);此外, 照射+黄杞叶提取物高剂量组小鼠骨髓有核细胞数量、骨髓DNA含量和肝脏还原型GSH含量均明显升高, 且差异均有统计学意义(t=3.28、3.93、3.07, 均P<0.05)。结论黄杞叶提取物对小鼠造血系统辐射损伤有一定的防护作用。

Objective To study the protective effects of Engelhardia roxburghiana Wall.leaf extract on mice hematopoietic system injury caused by disposable systemic 4 Gy 137Csγ-rays irradiation.Methods(1)In vitro experiment.1,1-Diphenyl-2-picrylhydrazyl(DPPH)and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)(ABTS)free radical scavenging tests were used to detect the antioxidant activity of Engelhardia roxburghiana Wall.leaf extract in vitro.(2)In vivo experiment.①Survival experiment.Forty specific pathogen-free male C57BL/6 mice were divided into four groups following a randomized block design:irradiation group,irradiation+low dose,irradiation+middle dose,and irradiation+high dose groups of Engelhardia roxburghiana Wall.leaf extract(20,40,and 80 mg/kg,respectively).All four groups were irradiated with whole-body 7.2 Gy(lethal dose)137Csγ-rays at one time.The survival condition of mice 30 days after irradiation was recorded.②Radiation protection experiment of the hematopoietic system.Sixty specific pathogen-free male C57BL/6 mice were divided into six groups following a randomized block design:control group,irradiation group,Engelhardia roxburghiana Wall.leaf extract high dose group(80 mg/kg),irradiation+low dose,irradiation+middle dose,and irradiation+high dose groups of Engelhardia roxburghiana Wall.leaf extract(20,40,and 80 mg/kg,respectively).The control group and the Engelhardia roxburghiana Wall.leaf extract high dose group(80 mg/kg)were not irradiated,while the four other groups were irradiated with whole-body 4 Gy 137Csγ-rays at one time.On the 7th day after irradiation,the mice were killed,the peripheral blood was obtained for blood routine detection,and unilateral femoral bone marrow cells were obtained for bone marrow nucleated cells count.The bone marrow cells of the other femur were used for bone marrow DNA content detection,the thymus and spleen were taken to calculate organ index,and liver was sampled to detect the content of reduced glutathione(GSH).In the in vitro experiment,the mice in each group underwent continuous intragastric administration 7 days before and 6 days after irradiation,in which the control group and irradiation group were given 0.5%carboxymethylcellulose sodium,and the other groups were given corresponding doses of Engelhardia roxburghiana Wall.leaf extract.Student's t test was applied to compare differences between two groups,and Kaplan-Meier survival analysis was used to determine the survival rate.Results(1)In the in vitro experiment,compared with Trolox,the scavenging rate of DPPH free radical by the Engelhardia roxburghiana Wall.leaf extract were higher(89.83%vs.69.37%,90.94%vs.68.53%)when the concentration was 100μg/ml and 200μg/ml;and the scavenging rate of ABTS free radical was also higher(94.81%vs.71.35%,94.46%vs.71.93%),and the differences were statistically significant(t=19.58-33.26,all P<0.001).(2)In the in vivo experiment,compared with the irradiation group,the 30-day survival rates of mice in the low,middle,and high dose groups of irradiation+Engelhardia roxburghiana Wall.leaf extract significantly increased after receiving lethal dose irradiation,and the differences were statistically significant(Kaplan-Meier survival analysis,all P<0.05);while the survival days also significantly improved((19.40±7.70)d vs.(12.50±3.59)d,(20.20±8.48)d vs.(12.50±3.59)d,(20.90±7.96)d vs.(12.50±3.59)d),and the differences were statistically significant(t=2.57,2.79,3.04;all P<0.05).The spleen and thymus organ index increased significantly in the irradiation+high dose group of Engelhardia roxburghiana Wall.leaf extract((2.13±0.43)mg/g vs.(1.67±0.20)mg/g,(1.87±0.39)mg/g vs.(1.39±0.31)mg/g),and the differences were statistically significant(t=3.00,3.03;both P<0.05).The number of red blood cells increased most significantly in the irradiation+middle dose group of Engelhardia roxburghiana Wall.leaf((10.12±1.71)×1012/L vs.(8.26±0.87)×1012/L),and the difference was statistically significant(t=2.89,P<0.05).The number of white blood cells and red blood cells and the hemoglobin content increased significantly in the irradiation+high dose group of Engelhardia roxburghiana Wall.leaf extract((1.76±0.45)×109/L vs.(1.17±0.23)×109/L,(9.59±0.85)×1012/L vs.(8.26±0.87)×1012/L,(144.40±14.61)g/L vs.(126.20±13.16)g/L),and the differences were statistically significant(t=3.62,3.23,2.93;all P<0.05).In addition,the number of bone marrow nucleated cells,the DNA content in the bone marrow,and GSH content in the liver were significantly increased in the irradiation+high dose group of Engelhardia roxburghiana Wall.leaf extract,and the differences were statistically significant(t=3.28,3.93,3.07;all P<0.05).Conclusion The Engelhardia roxburghiana Wall.leaf extract has a certain protective effect on radiation injury of the hematopoietic system in mice.