泼尼松龙致不同品系小鼠药源性证候的比较
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摘要
目的研究糖皮质激素药物作用于不同品系小鼠后肾上腺皮质功能抑制程度及其诱导药源性证候的差异性。方法选用ICR、BALB/c、C57BL/6J、KM和裸小鼠5种小鼠为实验对象,采用泼尼松龙进行干预。每种小鼠16只,其中正常对照组8只,泼尼松龙组8只。以0.5 mg/(kg·d)泼尼松龙连续灌胃14 d,每日观测小鼠体重,在给药第14天,运用小鼠辨证论治实验方法学检测小鼠中医四诊信息,次日处死小鼠,取脾、胸腺称重并计算脏器指数;运用ELISA检测血清皮质酮和ACTH含量;采用实时荧光定量PCR技术检测肾上腺Star、Cyp11a1、Cyp21a1、Cyp11b1、Cyp11b2基因表达;运用Western blot方法检测肾上腺LDLR、SRBI、StAR蛋白表达。结果与各自对照组比较:1)0.5 mg/(kg·d)泼尼松龙给药后第7天起ICR小鼠体重显著下降(P<0.01);给药后第13天起BALB/c小鼠体重显著下降(P<0.01);给药后第5天起C57BL/6J小鼠体重显著下降(P<0.01);给药后第13天裸鼠体重显著下降(P<0.05)。2)泼尼松龙导致ICR小鼠抓力显著下降以及躯干平均温度显著降低(P<0.05)。3)泼尼松龙导致ICR小鼠和裸鼠脾重量显著下降(P<0.01)以及裸鼠脾指数下降(P<0.05),泼尼松龙导致ICR、C57BL/6J和KM小鼠胸腺重量和胸腺指数均显著下降(P<0.01)。4)泼尼松龙显著导致BALB/c与KM小鼠血清皮质酮下降(P<0.05),也显著引起BALB/c小鼠血清促肾上腺皮质激素(ACTH)含量下降(P<0.01)。5)泼尼松龙显著下调ICR小鼠肾上腺Cyp21a1基因表达(P<0.05),下调C57BL/6J小鼠Star基因表达(P<0.01),下调KM小鼠Cyp11a1、Cyp21a1基因表达(P<0.01),下调裸鼠Star与Cyp21a1基因表达(P<0.05);且泼尼松龙抑制ICR小鼠肾上腺LDLR蛋白表达以及KM小鼠肾上腺StAR蛋白表达。结论泼尼松龙造成的小鼠药源性虚证主要表现为"气虚",涉及中医"肾藏象"与"脾藏象",其物质基础以肾上腺皮质类固醇激素合成酶分子表达被抑制以及垂体-肾上腺皮质轴功能被抑制为主;开展糖皮质激素药源性虚证研究建议首选ICR小鼠。
Objective To study the degrees of adrenocortical inhibition after the action of glucocorticoid drugs, and the differences in its drug-induced syndromes in five mouse strains. Methods ICR, BALB/c, C57 BL/6 J, KM and nude mice were selected. Prednisolone was used as the drug for intervention. There were 16 mice of each strain, eight-each in the normal control group and prednisolone group. Prednisolone(0.5 mg/kg·d) was administered for 14 consecutive days in the prednisolone group. The body weight of mice was recorded every day, and TCM diagnostic information were detected by the experimental methodology of syndrome differentiation and treatment in mice on the 14 th day. Mice was sacrificed and their spleen and thymus were weighed, and the organ indexes were calculated. Serum levels of corticosterone and adrenocorticotropic hormone(ACTH) were measured by ELISA. Gene expression of Star(steroidogenic acute regulatory protein), Cyp11a1(cytochromes P450 11 A1), Cyp21a1(cytochromes P450 21 A1), Cyp11b1(cytochromes P450 11 B1)and Cyp11b2(cytochromes P450 11 B2)in the adrenal glands were detected by real-time fluorescent quantitative PCR. Protein expression of LDLR(low-density lipoprotein receptor), SRBI(scavenger receptor class B member 1)and StAR(steroidogenic acute regulatory protein)was detected by Western blot. Results Compared with the control group of the respective strains, the body weight of ICR mice decreased significantly 7 days after prednisolone administration(P< 0.01), BALB/c mice decreased significantly 13 days after prednisolone administration(P< 0.01), C57 BL/6 J mice decreased significantly 5 days after prednisolone administration(P< 0.01), and nude mice decreased significantly 13 days after prednisolone administration(P< 0.05). Prednisolone administration resulted in a significant decrease in the grip strength and a significant decrease in the mean temperature of the body trunk of ICR mice(P< 0.05). Prednisolone resulted in a significant decrease in the spleen weight of ICR mice and nude mice(P< 0.01) and a decrease in the splenic index of nude mice(P< 0.05), whereas prednisolone administration resulted in a significant decrease in the thymic weight and thymic index of ICR, C57 BL/6 J and KM mice(P< 0.01). Prednisolone administration significantly decreased the serum corticosterone level in BALB/c and KM mice(P< 0.05), and serum level of ACTH in BALB/c mice(P< 0.01). Prednisolone significantly downregulated the Cyp21a1 expression in ICR mice(P< 0.05),Star expression in C57 BL/6 J mice(P< 0.01), Cyp11a1 and Cyp21a1 expression in KM mice(P< 0.01), and Star and Cyp21a1 expression in nude mice(P< 0.05). In addition, prednisolone inhibited expression of LDLR and StAR in the ICR and KM mice. Conclusions The prednisolone-induced deficiency syndrome in mice is mainly characterized by "qi deficiency", which is involved in the "kidney state" and "spleen state". The basis of this deficiency syndrome is primarily inhibition of expression of adrenocortical steroid synthase and function of the pituitary-adrenocortical axis. ICR mice is suggested to be used for studies of glucocorticoid-induced asthenia.
Objective To study the degrees of adrenocortical inhibition after the action of glucocorticoid drugs, and the differences in its drug-induced syndromes in five mouse strains. Methods ICR, BALB/c, C57 BL/6 J, KM and nude mice were selected. Prednisolone was used as the drug for intervention. There were 16 mice of each strain, eight-each in the normal control group and prednisolone group. Prednisolone(0.5 mg/kg·d) was administered for 14 consecutive days in the prednisolone group. The body weight of mice was recorded every day, and TCM diagnostic information were detected by the experimental methodology of syndrome differentiation and treatment in mice on the 14 th day. Mice was sacrificed and their spleen and thymus were weighed, and the organ indexes were calculated. Serum levels of corticosterone and adrenocorticotropic hormone(ACTH) were measured by ELISA. Gene expression of Star(steroidogenic acute regulatory protein), Cyp11a1(cytochromes P450 11 A1), Cyp21a1(cytochromes P450 21 A1), Cyp11b1(cytochromes P450 11 B1)and Cyp11b2(cytochromes P450 11 B2)in the adrenal glands were detected by real-time fluorescent quantitative PCR. Protein expression of LDLR(low-density lipoprotein receptor), SRBI(scavenger receptor class B member 1)and StAR(steroidogenic acute regulatory protein)was detected by Western blot. Results Compared with the control group of the respective strains, the body weight of ICR mice decreased significantly 7 days after prednisolone administration(P< 0.01), BALB/c mice decreased significantly 13 days after prednisolone administration(P< 0.01), C57 BL/6 J mice decreased significantly 5 days after prednisolone administration(P< 0.01), and nude mice decreased significantly 13 days after prednisolone administration(P< 0.05). Prednisolone administration resulted in a significant decrease in the grip strength and a significant decrease in the mean temperature of the body trunk of ICR mice(P< 0.05). Prednisolone resulted in a significant decrease in the spleen weight of ICR mice and nude mice(P< 0.01) and a decrease in the splenic index of nude mice(P< 0.05), whereas prednisolone administration resulted in a significant decrease in the thymic weight and thymic index of ICR, C57 BL/6 J and KM mice(P< 0.01). Prednisolone administration significantly decreased the serum corticosterone level in BALB/c and KM mice(P< 0.05), and serum level of ACTH in BALB/c mice(P< 0.01). Prednisolone significantly downregulated the Cyp21a1 expression in ICR mice(P< 0.05),Star expression in C57 BL/6 J mice(P< 0.01), Cyp11a1 and Cyp21a1 expression in KM mice(P< 0.01), and Star and Cyp21a1 expression in nude mice(P< 0.05). In addition, prednisolone inhibited expression of LDLR and StAR in the ICR and KM mice. Conclusions The prednisolone-induced deficiency syndrome in mice is mainly characterized by "qi deficiency", which is involved in the "kidney state" and "spleen state". The basis of this deficiency syndrome is primarily inhibition of expression of adrenocortical steroid synthase and function of the pituitary-adrenocortical axis. ICR mice is suggested to be used for studies of glucocorticoid-induced asthenia.
引文
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[9] 许雅丽,殷莲华.类固醇激素合成急性调节蛋白(StAR)与胆固醇代谢[J].国际病理科学与临床杂志,2008,28(4):358-361.Xu YL,Yin LH.Steroidogenic acute regulatory protein (StAR) and cholesterol metabolism [J].Int J Pathol Clin Med,2008,28(4):358-361.
[10] 王火,苏彬,李玉明,等.大剂量甲基泼尼松龙对严重急性呼吸综合征患者肾上腺皮质功能的影响[J].中国实用内科杂志,2006,26(15):1161-1163.Wang H,Su B,Li YM,et al.Dynamic changes of adrenocorticotropic hormone (ACTH) and cortisol (Cr) in SARS patients who received large dose of methylprednisolone therapy [J].Chin J Pract Intern Med,2006,26(15):1161-1163.
[11] 梅长林,吉程程.糖皮质激素在内科临床的应用及思考[J].中国实用内科杂志,2006,26(19):1468-1470.Mei CL,Ji CC.Application and thinking of glucocorticoid in internal medicine [J].Chin J Pract Intern Med,2006,26(19):1468-1470.
[12] 林涛,傅德皓,杨述华,等.糖皮质激素性骨质疏松模型研究进展[J].中国骨肿瘤骨病,2011,10(2):192-195.Lin T,Fu DH,Yang SH,et al.Advances in the study of glucocorticoid osteoporosis models [J].Chin J Bone Tumor Bone Dis,2011,10(2):192-195.
[2] 潘志强,方肇勤,卢文丽,等.不同品系H22肝癌小鼠证候特征的比较研究[J].上海中医药大学学报,2011,25(1):56-59.Pan ZQ,Fang ZQ,Lu WL,et al.Comparative study on syndrome characteristics of H22 tumor mice from different strains [J] J Shanghai Univ Tradit Chin Med,2011,25(1):56-59.
[3] 潘志强.“药源性证候”的新生学术问题与思考[J].上海中医药杂志,2018,52(6):5-8.Pan ZQ.New academic problems and thinking of drug-induced syndromes [J].Shanghai J Tradit Chin Med,2018,52(6):5-8.
[4] 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,2006,20(1):42-46.Fang ZQ,Pan ZQ,Tang WC,et al.Construction and operational standard of four diagnostic methods workstation for mice [J].J Shanghai Univ Tradit Chin Med,2006,20(1):42-46.
[5] 方肇勤,潘志强,卢文丽,等.大鼠、小鼠辨证的思路与方法[J].中国比较医学杂志,2009,19(10):53-59,85.Fang ZQ,Pan ZQ,Lu WL,et al.Thoughts and methods of syndrome differentiation on rats and mice [J].Chin J Comparat Med,2009,19(10):53-59,85.
[6] 方肇勤,潘志强,卢文丽,等.大鼠、小鼠常见证候计量化辨证方法的建立及其评价[J].中国中医基础医学杂志,2007,13(7):502-505.Fang ZQ,Pan ZQ,Lu WL,et al.Establishment and evaluation of quantitative syndrome differentiation method of common witness in rats and mice [J].Chin J Basic Chin Tradit Med,2007,13(7):502-505.
[7] 陈芬,王绿娅,尹卫东.低密度脂蛋白受体功能及其影响因素研究进展[J].中国动脉硬化杂志,2006,14(5):448-450.Chen F,Wang LY,Yin WD.Research progress on receptor function of low density lipoprotein and its influencing factors [J].Chin J Arteriosclerosis,2006,14(5):448-450.
[8] 秦媛媛,凌文华.B族Ⅰ型清道夫受体与高密度脂蛋白代谢[J].国际内科学杂志,2009,36(5):286-289.Qin YY,Ling WH.Scavenger receptor class B type Ⅰ and high density lipoprotein,metabolism [J].Int J Intern Med,2009,36(5):286-289.
[9] 许雅丽,殷莲华.类固醇激素合成急性调节蛋白(StAR)与胆固醇代谢[J].国际病理科学与临床杂志,2008,28(4):358-361.Xu YL,Yin LH.Steroidogenic acute regulatory protein (StAR) and cholesterol metabolism [J].Int J Pathol Clin Med,2008,28(4):358-361.
[10] 王火,苏彬,李玉明,等.大剂量甲基泼尼松龙对严重急性呼吸综合征患者肾上腺皮质功能的影响[J].中国实用内科杂志,2006,26(15):1161-1163.Wang H,Su B,Li YM,et al.Dynamic changes of adrenocorticotropic hormone (ACTH) and cortisol (Cr) in SARS patients who received large dose of methylprednisolone therapy [J].Chin J Pract Intern Med,2006,26(15):1161-1163.
[11] 梅长林,吉程程.糖皮质激素在内科临床的应用及思考[J].中国实用内科杂志,2006,26(19):1468-1470.Mei CL,Ji CC.Application and thinking of glucocorticoid in internal medicine [J].Chin J Pract Intern Med,2006,26(19):1468-1470.
[12] 林涛,傅德皓,杨述华,等.糖皮质激素性骨质疏松模型研究进展[J].中国骨肿瘤骨病,2011,10(2):192-195.Lin T,Fu DH,Yang SH,et al.Advances in the study of glucocorticoid osteoporosis models [J].Chin J Bone Tumor Bone Dis,2011,10(2):192-195.
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