基于体征半定量指标评价的利血平致脾气虚大鼠模型的研究
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摘要
目的基于体征半定量评价标准,构建适用于利血平致脾气虚大鼠模型生理病理及药理研究的评价方法。方法以外观体征(眼神、毛色、大便和形体神)作为脾气虚造模大鼠半定量评价指标并制订其评分标准形成外观体征评分(R);以利血平(0.6、0.8 mg·kg~(-1))皮下注射致脾气虚大鼠模型评价该方法的可行性:54只SD雄性大鼠随机分为正常对照组12只,利血平0.8 mg·kg~(-1)组22只,利血平0.6 mg·kg~(-1)组20只;测定大鼠每日外观体征评分(R)、体质量、摄食量及饮水量;测定大鼠造模前及造模5 d、9 d、25 d、32 d、39 d肛温;分别于利血平注射结束和自愈观察结束后,苏木精-伊红(HE)染色法观察各组大鼠腮腺组织学形态改变。通过观察利血平2个剂量组R≥8和R≤1时大鼠脾气虚模型相关指标(体质量、摄食量、饮水量、肛温、腮腺病理)的变化以反映脾气虚大鼠造模成功及自愈情况。结果利血平0.8 mg·kg~(-1)组注射9 d或利血平0.6 mg·kg~(-1)组注射11 d,造模大鼠可达到R≥8分,与脾气虚模型评判密切相关的宏观指标(体质量、摄食量、饮水量、肛温)明显下降,腮腺组织切片也可见明显病理改变;造模结束到大鼠自愈恢复使评分降至R≤1分时,利血平0.8 mg·kg~(-1)组需时33 d[总周期(9+33)d]、利血平0.6 mg·kg~(-1)组需时31 d[总周期(11+31)d],此时宏观指标及腮腺组织形态学改变接近正常对照组;Pearson相关分析显示,大鼠外观体征评分与利血平造模组体质量(0.8 mg·kg~(-1)组,r=-0.793,P <0.01;0.6 mg·kg~(-1)组,r=-0.823,P <0.01)、肛温(0.8 mg·kg~(-1)组,r=-0.324,P <0.05;0.6 mg·kg~(-1)组,r=-0.749,P <0.01)呈负相关。结论外观体征半定量评分标准能较准确、客观、半定量地评价利血平致脾气虚大鼠的造模结果及自身恢复情况,可根据需要选择合适的利血平造模剂量和时间,本实验结果为脾气虚大鼠模型的病理生理及药理研究提供了适宜的动物模型评价方法。
Objective To establish semiquantitative evaluation criteria for reserpine induced spleen-qi-deficiencyrat model suitable for physiological,pathological and pharmacological studies. Methods External signs(eyes,haircolor,stool,and physique mind) were used as the semiquantitative indicators of spleen-qi-deficiency rat model,and the score of external signs(R) was formulated. The feasibility of evaluation criteria was evaluated in rat modelsof spleen-qi-deficiency induced by hypodermic injection of reserpine(0.6 mg·kg~(-1)and 0.8 mg·kg~(-1)). Fifty four maleSD rats were randomly divided into 0.8 mg·kg~(-1)reserpine group(n=22),0.6 mg·kg~(-1)reserpine group(n=20),normal group(n=12). The R scores,body weights,food and water intake were measured once daily. The anal temperatures were measured prior to induction of rat models and 5 d, 9 d, 25 d, 32 d, 39 d after modelingstarted. Hematoxylin-eosin(HE) staining was performed to observe the morphological changes in parotid glandsafter the last injection of reserpine or at the end of self-healing. Results The R≥ 8 was recorded, which wasconsistent with decreased macroscopic indexes(weight,food and water intake,and anal temperature) as well asobvious pathological parotid tissue sections in the injection of reserpine(0.8 mg·kg~(-1)) for 9 d or reserpine(0.6mg·kg~(-1)) for 11 d. During self-healing period,the duration from the end of modeling to R≤ 1 was 33 d in 0.8mg · kg~(-1)reserpine group,and 31 d in 0.6 mg·kg~(-1)reserpine group. Meanwhile,macroscopic indexes and parotidhistomorphological changes were almost normal. The Pearson correlation analysis showed that the semiquantitativescore of the rat appearance was negatively correlated with both body weight(r=-0.793 in 0.8 mg·kg~(-1)reserpinegroup,P < 0.01;r=-0.823 in 0.6 mg·kg~(-1)reserpine group,P < 0.01) and anal temperature(r=-0.324 in 0.8mg · kg~(-1)reserpine group, P < 0.05; r=-0.749 in 0.6 mg · kg~(-1)reserpine group, P < 0.01). Conclusion Thesemiquantitative evaluation criteria of external signs can accurately,objectively and semiquantitatively evaluate theoutcomes after modeling and self-healing in rats with spleen-qi-deficiency induced by reserpine. It is a valuablereference for selecting suitable dose and duration of reserpine in spleen-qi-deficiency rat model. This researchprovides an appropriate assessment method for the study of pathophysiology and pharmacology in spleen-qi-deficiency rats model evaluation criteria of external sign.
Objective To establish semiquantitative evaluation criteria for reserpine induced spleen-qi-deficiencyrat model suitable for physiological,pathological and pharmacological studies. Methods External signs(eyes,haircolor,stool,and physique mind) were used as the semiquantitative indicators of spleen-qi-deficiency rat model,and the score of external signs(R) was formulated. The feasibility of evaluation criteria was evaluated in rat modelsof spleen-qi-deficiency induced by hypodermic injection of reserpine(0.6 mg·kg~(-1)and 0.8 mg·kg~(-1)). Fifty four maleSD rats were randomly divided into 0.8 mg·kg~(-1)reserpine group(n=22),0.6 mg·kg~(-1)reserpine group(n=20),normal group(n=12). The R scores,body weights,food and water intake were measured once daily. The anal temperatures were measured prior to induction of rat models and 5 d, 9 d, 25 d, 32 d, 39 d after modelingstarted. Hematoxylin-eosin(HE) staining was performed to observe the morphological changes in parotid glandsafter the last injection of reserpine or at the end of self-healing. Results The R≥ 8 was recorded, which wasconsistent with decreased macroscopic indexes(weight,food and water intake,and anal temperature) as well asobvious pathological parotid tissue sections in the injection of reserpine(0.8 mg·kg~(-1)) for 9 d or reserpine(0.6mg·kg~(-1)) for 11 d. During self-healing period,the duration from the end of modeling to R≤ 1 was 33 d in 0.8mg · kg~(-1)reserpine group,and 31 d in 0.6 mg·kg~(-1)reserpine group. Meanwhile,macroscopic indexes and parotidhistomorphological changes were almost normal. The Pearson correlation analysis showed that the semiquantitativescore of the rat appearance was negatively correlated with both body weight(r=-0.793 in 0.8 mg·kg~(-1)reserpinegroup,P < 0.01;r=-0.823 in 0.6 mg·kg~(-1)reserpine group,P < 0.01) and anal temperature(r=-0.324 in 0.8mg · kg~(-1)reserpine group, P < 0.05; r=-0.749 in 0.6 mg · kg~(-1)reserpine group, P < 0.01). Conclusion Thesemiquantitative evaluation criteria of external signs can accurately,objectively and semiquantitatively evaluate theoutcomes after modeling and self-healing in rats with spleen-qi-deficiency induced by reserpine. It is a valuablereference for selecting suitable dose and duration of reserpine in spleen-qi-deficiency rat model. This researchprovides an appropriate assessment method for the study of pathophysiology and pharmacology in spleen-qi-deficiency rats model evaluation criteria of external sign.
引文
[1]陈奇.中药药理研究方法学[M].北京:人民卫生出版社,2011:1071.
[2]李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,2006:280.
[3]陈玉龙,张海艇,李茹柳,等.四君子汤对脾虚大鼠腮腺唾液淀粉酶分泌障碍及VIP-cAMP信号通路的影响[J].中药材,2012,35(1):83-87.
[4]林传权,陈玉龙,李茹柳,等.利血平致脾虚大鼠唾液蛋白分泌改变及其机制的探讨[J].中国中西医结合杂志,2010,30(5):509-512.
[5]高小玲,郭文峰,李茹柳,等.四君子汤对脾虚大鼠尿木糖排泄率及肠黏膜ATP的影响[J].中药材,2009,32(8):1242-1245.
[6]施旭光,王闽予,吴美音,等.脾气虚证4种造模方法的比较研究[J].广州中医药大学学报,2013,(2):196-199.
[7]屈小虎,陈慧,黄玲,等.脾气虚证和脾不统血证模型大鼠脾脏能量代谢的比较研究[J].中华中医药学刊,2018,36(6):1355-1358.
[8]ZHENG X F,TIAN J S,LIU P,et al.Analysis of the restorative effect of Bu-zhong-yi-qi-tang in the spleen-qi deficiency rat model using(1)H-NMR-based metabonomics[J].J Ethnopharmacol,2014,151(2):912-920.
[9]尹玫,窦智勇,张茹玉,等.四君子汤与四君子丸干预脾气虚大鼠宏观表征的比较研究[J].当代医学,2017,23(26):30-32.
[10]TIAN R,GONG Z H,YANG X Y,et al.[Study on interference effect of Sijunzi decoction on brain-gut CaM/CaMK II of spleen Qi deficiency syndrome rats[J].Zhongguo Zhong Yao Za Zhi,2015,40(20):4075-4079.
[11]尚冰,丛培玮,王蕊芳,等.对脾虚大鼠模型建立模糊数学评价体系的研究[J].辽宁中医药大学学报,2013,15(3):64-66.
[12]方肇勤,潘志强,卢文丽,等.大鼠和小鼠辨证论治标准的建立和用途[J].中西医结合学报,2009,7(10):907-912.
[13]方肇勤,潘志强,卢文丽,等.小鼠常见证候的辨证标准[J].中国中医基础医学杂志,2006,(7):508-510.
[14]中华人民共和国卫生部.中药新药临床研究指导原则:第1辑[S].1993:93-95.
[15]方肇勤,潘志强,汤伟昌,等.小鼠“四诊工作站”构建与操作标准探讨[J].上海中医药大学学报,2006,(1):42-46.
[16]李绍华,张启元.大黄致脾虚大鼠腮腺超微结构的变化[J].中国中西医结合杂志,1995,(S1):112-113.
[17]林传权,陈玉龙,李茹柳,等.利血平对大鼠唾液蛋白分泌的影响[J].世界华人消化杂志,2009,17(17):1702-1706.
[2]李仪奎.中药药理实验方法学[M].上海:上海科学技术出版社,2006:280.
[3]陈玉龙,张海艇,李茹柳,等.四君子汤对脾虚大鼠腮腺唾液淀粉酶分泌障碍及VIP-cAMP信号通路的影响[J].中药材,2012,35(1):83-87.
[4]林传权,陈玉龙,李茹柳,等.利血平致脾虚大鼠唾液蛋白分泌改变及其机制的探讨[J].中国中西医结合杂志,2010,30(5):509-512.
[5]高小玲,郭文峰,李茹柳,等.四君子汤对脾虚大鼠尿木糖排泄率及肠黏膜ATP的影响[J].中药材,2009,32(8):1242-1245.
[6]施旭光,王闽予,吴美音,等.脾气虚证4种造模方法的比较研究[J].广州中医药大学学报,2013,(2):196-199.
[7]屈小虎,陈慧,黄玲,等.脾气虚证和脾不统血证模型大鼠脾脏能量代谢的比较研究[J].中华中医药学刊,2018,36(6):1355-1358.
[8]ZHENG X F,TIAN J S,LIU P,et al.Analysis of the restorative effect of Bu-zhong-yi-qi-tang in the spleen-qi deficiency rat model using(1)H-NMR-based metabonomics[J].J Ethnopharmacol,2014,151(2):912-920.
[9]尹玫,窦智勇,张茹玉,等.四君子汤与四君子丸干预脾气虚大鼠宏观表征的比较研究[J].当代医学,2017,23(26):30-32.
[10]TIAN R,GONG Z H,YANG X Y,et al.[Study on interference effect of Sijunzi decoction on brain-gut CaM/CaMK II of spleen Qi deficiency syndrome rats[J].Zhongguo Zhong Yao Za Zhi,2015,40(20):4075-4079.
[11]尚冰,丛培玮,王蕊芳,等.对脾虚大鼠模型建立模糊数学评价体系的研究[J].辽宁中医药大学学报,2013,15(3):64-66.
[12]方肇勤,潘志强,卢文丽,等.大鼠和小鼠辨证论治标准的建立和用途[J].中西医结合学报,2009,7(10):907-912.
[13]方肇勤,潘志强,卢文丽,等.小鼠常见证候的辨证标准[J].中国中医基础医学杂志,2006,(7):508-510.
[14]中华人民共和国卫生部.中药新药临床研究指导原则:第1辑[S].1993:93-95.
[15]方肇勤,潘志强,汤伟昌,等.小鼠“四诊工作站”构建与操作标准探讨[J].上海中医药大学学报,2006,(1):42-46.
[16]李绍华,张启元.大黄致脾虚大鼠腮腺超微结构的变化[J].中国中西医结合杂志,1995,(S1):112-113.
[17]林传权,陈玉龙,李茹柳,等.利血平对大鼠唾液蛋白分泌的影响[J].世界华人消化杂志,2009,17(17):1702-1706.