乙酰苯肼致血虚证模型小鼠的证候物质基础初探
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摘要
目的研究乙酰苯肼引起的血虚证模型小鼠证候可能的物质基础。方法以雄性ICR小鼠为对象,采用乙酰苯肼170 mg/kg皮下注射,第1天和第4天各1次制作血虚证模型,并设立正常对照组。动态观察小鼠体重变化,显微镜下观察血液细胞与股骨骨髓细胞,取肾脏、肾上腺和骨髓分别抽提RNA,以实时荧光定量PCR技术检测相关基因表达、以ELISA方法检测血清皮质酮含量。结果与正常对照组比较,乙酰苯肼小鼠造模3天后体重显著下降(P<0.05),小鼠呈现虚弱征象,小鼠脾脏代偿性增大而胸腺却明显萎缩(P<0.01)。乙酰苯肼组小鼠血液红细胞形态异常、正常红细胞数量减少、白细胞代偿性增多;骨髓成熟的环状核细胞减少。与正常对照组比较,乙酰苯肼组小鼠骨髓IL-5和IL-6基因表达显著升高(P<0.05),而巨噬细胞集落刺激因子(M-CSF)、IL-1b和IL-7基因表达显著下降(P<0.05);肾脏促红细胞生成素(Epo)、粒细胞集落刺激因子(G-CSF)和M-CSF基因表达也显著升高(P<0.05,P<0.01),而粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因表达则被乙酰苯肼抑制(P<0.05);肾上腺Cyp21a1与Cyp11b1基因表达显著升高(P<0.05);小鼠血清皮质酮含量明显升高(P<0.01)。结论乙酰苯肼所复制的证候以血虚证为主,脾脏肿大、肾脏Epo基因代偿性高表达以及继发肾上腺皮质功能虚性亢奋是该证候物质与功能变化最突出特征。
Objective To study the material base on acetylphenylhydrazine induced blood deficiency syndrome(BDS) model mice. Methods Male ICR mice were subcutaneously injected with acetylphenylhydrazine(170 mg/kg). BDS model was prepared on the 1~(st) day and the 4~(th) day. A normal control group was also set up by injecting normal saline at the same dosage. Body weight was dynamically observed. Changes of each organ were detected after sacrificed. Blood and femur bone marrow smears were made and observed under a microscope. RNAs were extracted from kidney, adrenal gland, and bone marrow using TRIzol kit. Related gene expressions were detected by real-time quantitative PCR, and serum corticosterone content was tested by ELISA. Results Compared with the normal control group, body weight of mice in the acetylphenylhydrazine group significantly decreased after 3 days modeling(P<0.05). The mice showed weak signs. Their spleens were compensatively enlarged, but the thymus was severely atrophic(P<0.01). Their erythrocytes′ morphologies were abnormal and the numbers of normal red blood cells were reduced, and leucocyte were compensatively increased. Mature ring neutrophils in bone marrow were reduced. Compared with the normal control group, gene expressions of bone marrow IL-5 and IL-6 increased significantly(P<0.05), while gene expressions of M-CSF, IL-1b, and IL-7 decreased significantly(P<0.05). Gene expressions of renal Epo, G-CSF, and M-CSF significantly increased(P<0.05, P<0.01), but the gene expression of renal GM-CSF were inhibited by acetylphenylhydrazine(P<0.05). Gene expressions of adrenal gland Cyp21a1 and Cyp11b1 increased significantly(P<0.05). Moreover, serum corticosterone content increased obviously in the acetylphenylhydrazine group(P<0.01). Conclusions Acetylphenylhydrazine could cause typical BDS. The most prominent features included splenomegaly, compensatory high expression of renal Epo gene, and secondary adrenal hyperactivity.
Objective To study the material base on acetylphenylhydrazine induced blood deficiency syndrome(BDS) model mice. Methods Male ICR mice were subcutaneously injected with acetylphenylhydrazine(170 mg/kg). BDS model was prepared on the 1~(st) day and the 4~(th) day. A normal control group was also set up by injecting normal saline at the same dosage. Body weight was dynamically observed. Changes of each organ were detected after sacrificed. Blood and femur bone marrow smears were made and observed under a microscope. RNAs were extracted from kidney, adrenal gland, and bone marrow using TRIzol kit. Related gene expressions were detected by real-time quantitative PCR, and serum corticosterone content was tested by ELISA. Results Compared with the normal control group, body weight of mice in the acetylphenylhydrazine group significantly decreased after 3 days modeling(P<0.05). The mice showed weak signs. Their spleens were compensatively enlarged, but the thymus was severely atrophic(P<0.01). Their erythrocytes′ morphologies were abnormal and the numbers of normal red blood cells were reduced, and leucocyte were compensatively increased. Mature ring neutrophils in bone marrow were reduced. Compared with the normal control group, gene expressions of bone marrow IL-5 and IL-6 increased significantly(P<0.05), while gene expressions of M-CSF, IL-1b, and IL-7 decreased significantly(P<0.05). Gene expressions of renal Epo, G-CSF, and M-CSF significantly increased(P<0.05, P<0.01), but the gene expression of renal GM-CSF were inhibited by acetylphenylhydrazine(P<0.05). Gene expressions of adrenal gland Cyp21a1 and Cyp11b1 increased significantly(P<0.05). Moreover, serum corticosterone content increased obviously in the acetylphenylhydrazine group(P<0.01). Conclusions Acetylphenylhydrazine could cause typical BDS. The most prominent features included splenomegaly, compensatory high expression of renal Epo gene, and secondary adrenal hyperactivity.
引文
[1] 贲长恩, 叶百宽. 血虚动物模型初探[J]. 上海中医药杂志, 1981, 27(6): 38-39.
[2] 金若敏, 宁炼, 陈长勋, 等. 血虚模型动物制备及当归补血汤的作用研究[J]. 中成药, 2001, 24(4): 36-39.
[3] 卢文丽, 方肇勤, 潘志强, 等. 四种不同品系小鼠气虚血虚证候的比较与评价[J]. 辽宁中医杂志, 2007, 34(4): 519-522.
[4] 钱宏梁, 潘志强. 中医血虚证及其动物模型制备方法评析[J]. 广州中医药大学学报, 2018, 35(1): 176-181.
[5] French JK, Winterbourn CC, Carrell RW. Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine[J]. Biochem J, 1978, 173(1): 19-26.
[6] Winterbourn CC. Protection by ascorbate against acetylphenylhydrazine-induced Heinz body formation in glucose-6-phosphate dehydrogenase deficient erythrocytes[J]. Br J Haematol, 1979, 41(2): 245-252.
[7] Sathyanarayana P, Dev A, Fang J, et al. EPO receptor circuits for primary erythroblast survival[J]. Blood, 2008, 111(11): 5390-5399.
[8] McNiece IK, Langley KE, Zsebo KM. Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages[J]. Exp Hematol, 1991, 19(3): 226-231.
[9] 李程程, 孟爱民. G-CSF动员造血干细胞机制研究进展[J]. 生命科学, 2016, 28(4): 480-485.
[10] 陈智松, 吴志奎, 蔡辉国, 等. 从补肾生血药对衰老小鼠骨髓GM-CSF 基因转录水平的影响探讨肾生髓的分子基础[J]. 中国中医基础医学杂志, 2001, 7(4): 28-32.
[11] 陈笛, 王莎莉, 王亚平, 等. 人参总皂甙诱导人骨髓基质细胞表达GM-CSF 的实验研究[J]. 重庆医科大学学报, 2003, 28(4): 405-408.
[12] 郭平, 梁乾德, 胡剑江, 等. 四物汤对放射线致血虚证小鼠骨髓Epo 和G-CSF 基因表达的影响[J]. 中国中药杂志, 2005, 30(15): 1173-1176.
[2] 金若敏, 宁炼, 陈长勋, 等. 血虚模型动物制备及当归补血汤的作用研究[J]. 中成药, 2001, 24(4): 36-39.
[3] 卢文丽, 方肇勤, 潘志强, 等. 四种不同品系小鼠气虚血虚证候的比较与评价[J]. 辽宁中医杂志, 2007, 34(4): 519-522.
[4] 钱宏梁, 潘志强. 中医血虚证及其动物模型制备方法评析[J]. 广州中医药大学学报, 2018, 35(1): 176-181.
[5] French JK, Winterbourn CC, Carrell RW. Mechanism of oxyhaemoglobin breakdown on reaction with acetylphenylhydrazine[J]. Biochem J, 1978, 173(1): 19-26.
[6] Winterbourn CC. Protection by ascorbate against acetylphenylhydrazine-induced Heinz body formation in glucose-6-phosphate dehydrogenase deficient erythrocytes[J]. Br J Haematol, 1979, 41(2): 245-252.
[7] Sathyanarayana P, Dev A, Fang J, et al. EPO receptor circuits for primary erythroblast survival[J]. Blood, 2008, 111(11): 5390-5399.
[8] McNiece IK, Langley KE, Zsebo KM. Recombinant human stem cell factor synergises with GM-CSF, G-CSF, IL-3 and epo to stimulate human progenitor cells of the myeloid and erythroid lineages[J]. Exp Hematol, 1991, 19(3): 226-231.
[9] 李程程, 孟爱民. G-CSF动员造血干细胞机制研究进展[J]. 生命科学, 2016, 28(4): 480-485.
[10] 陈智松, 吴志奎, 蔡辉国, 等. 从补肾生血药对衰老小鼠骨髓GM-CSF 基因转录水平的影响探讨肾生髓的分子基础[J]. 中国中医基础医学杂志, 2001, 7(4): 28-32.
[11] 陈笛, 王莎莉, 王亚平, 等. 人参总皂甙诱导人骨髓基质细胞表达GM-CSF 的实验研究[J]. 重庆医科大学学报, 2003, 28(4): 405-408.
[12] 郭平, 梁乾德, 胡剑江, 等. 四物汤对放射线致血虚证小鼠骨髓Epo 和G-CSF 基因表达的影响[J]. 中国中药杂志, 2005, 30(15): 1173-1176.