补髓生血丸对再生障碍性贫血小鼠造血功能及骨髓VEGF、TPO、VCAM-1水平的影响

Effects of Busui Shengxue Pills on Hematopoietic Function and the Contents of VEGF, TPO and VCAM-1 in Bone Marrow of Aplastic Anemia Model Mice

目的 观察补髓生血丸(熟地黄、鹿角胶、黄芪、当归等)对再生障碍性贫血(AA)小鼠的防治作用,并从改善骨髓造血微环境角度分析相关作用机制。方法 将昆明种雄性小鼠随机分为正常组、模型组、阳性药组(再造生血片,2.70 g·kg^(-1))及补髓生血丸低、中、高剂量组(2.25,4.50,9.00 g·kg^(-1)),每组15只。采用白消安(10 mg·kg^(-1))及环磷酰胺(20 mg·kg^(-1))交替腹腔注射给药12 d,制备AA小鼠模型。造模同时灌胃给药,每日1次,连续30 d。检测小鼠外周血象;采用瑞士-姬姆萨染色法观察小鼠骨髓涂片;进行骨髓有核细胞计数;HE染色法观察骨髓病理变化;ELISA法检测小鼠血清及骨髓中血管内皮生长因子(VEGF)、血小板生成素(TPO)、血管细胞黏附因子1(VCAM-1)的水平。结果 各组间的血清及骨髓TPO水平无明显差异(P>0.05)。与正常组比较,模型组小鼠的红细胞(RBC)、白细胞(WBC)、血红蛋白(HGB)和血小板(PLT)水平均明显下降(P<0.05);血清及骨髓中VCAM-1含量明显下降(P<0.05),血清中VEGF含量明显下降(P<0.05);小鼠的骨髓有核细胞数量显著减少(P<0.01),骨髓脂肪化明显,出现较大和特大的脂滴空泡,骨髓象损伤评分显著升高(P<0.01);小鼠骨髓腔明显空虚,造血细胞被大量的脂肪细胞代替,造血细胞数量明显减少,淤血、出血和间质水肿等现象显著,巨核细胞未见,骨髓病理损伤评分显著升高(P<0.01)。与模型组比较,各给药组小鼠的RBC、HGB水平均明显升高(P<0.05),补髓生血丸高剂量组小鼠的WBC、PLT水平明显升高(P<0.05);各给药组小鼠的血清及骨髓中VCAM-1含量均明显上升(P<0.05,P<0.01),补髓生血丸高剂量组小鼠血清VEGF含量明显上升(P<0.05);各给药组小鼠的骨髓有核细胞数量显著上升(P<0.01),骨髓增生活跃,有核细胞密布,脂滴明显减少、变小,补髓生血丸中、高剂量组小鼠的骨髓象损伤评分明显降低(P<0.05,P<0.01);各给药组小鼠骨髓可见有核细胞、巨核细胞逐渐增多,淤血、出血减轻,脂肪细胞减少,补髓生血丸中、高剂量组小鼠骨髓病理损伤评分明显降低(P<0.05,P<0.01)。结论 补髓生血丸能够改善AA小鼠的造血功能,可能与提高VCAM-1、VEGF水平,改善骨髓造血微环境有关。

Objective To observe the preventive and curative effects of the Busui Shengxue Pills(Rehmanniae Radix Praeparata,Cervi Cornus Colla,Astragali Radix and Angelicae Sinensis Radix)on mice with aplastic anemia(AA)and to analyze the related mechanism of action from the perspective of improving the hematopoietic microenvironment of bone marrow.Methods Male mice of Kunming species were randomly divided into normal group,model group,positive drug group(Zaizaoshengxue Tablets,2.70 g·kg^(-1))and high-,medium-and low-dose groups of Busui Shengxue Pills(2.25, 4.50, 9.00 g · kg^(-1)), with 15 mice in each group. Mice models of AA were prepared by alternate intraperitoneal administration of busulfan(10 mg·kg^(-1))and cyclophosphamide(20 mg·kg^(-1))for 12 days.Peripheral blood was examined by gavage once daily for consecutive 30 days. Bone marrow smears and bone marrow nucleated cell counts were performed by Wright-Giemsa stain,and bone marrow pathological changes were observed by HE staining. The levels of vascular endothelial growth factor(VEGF),thrombopoietin(TPO)and vascular cell adhesion molecule 1(VCAM-1)were measured by ELISA.Results There was no significant difference in serum and bone marrow TPO levels among the groups(P>0.05). Compared with the normal group,the levels of red blood cells(RBC),white blood cells(WBC),haemoglobin(HGB)and platelets(PLT)in the model group were significantly decreased(P<0.05);the contents of VCAM-1 in serum and bone marrow were significantly decreased(P<0.05),and the levels of VEGF in serum were significantly decreased(P<0.05);the number of nucleated cells in bone marrow of mice was significantly decreased;the bone marrow was significantly fatty,with large and extra-large lipid droplet vacuoles,and the myelogram damage score was significantly increased(P<0.01);the bone marrow cavity of mice was significantly empty,hematopoietic cells were replaced by a large number of adipocytes,the number of hematopoietic cells was significantly reduced, bruising, hemorrhage and interstitial edema were significant,megakaryocytes were not seen, and the bone marrow pathological damage score was significantly increased(P<0.01);the number of bone marrow nucleated cells in the mice was significantly reduced(P<0.01). Compared with the model group,the levels of RBC and HGB were significantly increased in all administered mice(P<0.05),and the levels of WBC and PLT were significantly increased in the Busui Shengxue Pills high-dose group(P<0.05);the levels of VCAM-1 in serum and bone marrow were significantly increased in all administered groups(P<0.05,P<0.01),and the levels of VEGF in serum of mice in the high-dose group(P<0.05). The quantity of bone marrow cells increased,the bone marrow proliferation was active,nucleated cells were dense,and the lipid droplets were significantly reduced and getting smaller in each group,and the bone marrow myelogram pathological injury score was significantly reduced in the medium-and high-dose of Busui Shengxue Pills groups(P<0.05, P<0.01);nucleated cells and megakaryocytes were seen to increase gradually in the bone marrow of mice in each administered group, and bruising and bleeding reduced adipocytes, and the bone marrow pathological injury score was significantly reduced in the medium-and high-dose of Busui Shengxue Pills groups(P<0.05, P<0.01);the number of nucleated cells in the bone marrow of mice in each administered group was significantly increased(P<0.01).Conclusion The ability of Busui Shengxue Pills to improve the haematopoietic function of AA mice may be related to the increase of VCAM-1 and VEGF levels and the improvement of the hematopoietic microenvironment of bone marrow.