药学学报

No	1	2	3	4	5	6	7	8	9	10	11
Gender	Female	Female	Female	Female	Female	Female	Female	Female	Male	Female	Female
Age (years)	65	68	45	57	57	67	40	63	64	57	60
Other possible causes induced liver injury investigation
Autoimmune antibodies	-	-	Negative	-	-	-	Negative	-	-	-	-
Hepatitis A, hepatitis C, and hepatitis E antibodies	-	-	Negative	-	Negative	-	Negative	-	-	-	-
Hepatitis B serological test	All negative	All negative	Hbcab positive	All negative	All negative	-	All negative	-	-	-	-
Drink history	-	-	-	-	-	-	-	No	No	No	No
Clinical type of liver injury	-	-	Hepatocellular	-	-	-	-	Hepatocellular	Mixed	Hepatocellular	Mixed
Medication history	Compound reserpine and triamterene tablets, compound Ginkgo leaves tablets	Cisapride	-	Omeprazole capsules, Dengzhan Shengmai capsules, calcitriol soft gelatin capsules, levothyroxine tablets	Amlodipine besylate Tablets	Alfarosalitol, levothyroxine tablets	Diclofenac sodium sustained release capsules	-	-	-	-
Duration from medication to liver injury (days)	180	60	10	60	20	194	28	30	300	30	60
Clinical characteristics
ALP/U·L^-1	-	-	129	-	-	-	-	267.65	163	88.16	579
ALT/U·L^-1	85	158	1 098	389	2 196	930	-	1 477.4	770	150	629
TBIL/μmol·L^-1	-	-	47.2	-	-	-	496	313.51	228.3	12.4	12
INR	-	-	-	-	-	-	-	-	-	-	-
RUCAM scores	-	-	-	-	-	-	-	8	6	7	6
Final outcome	Cured	Cured	Cured	Cured	Cured	Cured	Death	-	-	-	-
Integrated evidence chain evaluation	-	-	-	-	-	-	-	-	-	-	-

No	1	2	3	4	5	6	7	8	9	10	11
Gender	Female	Female	Female	Female	Female	Female	Female	Female	Male	Female	Female
Age (years)	65	68	45	57	57	67	40	63	64	57	60
Other possible causes induced liver injury investigation
Autoimmune antibodies	-	-	Negative	-	-	-	Negative	-	-	-	-
Hepatitis A, hepatitis C, and hepatitis E antibodies	-	-	Negative	-	Negative	-	Negative	-	-	-	-
Hepatitis B serological test	All negative	All negative	Hbcab positive	All negative	All negative	-	All negative	-	-	-	-
Drink history	-	-	-	-	-	-	-	No	No	No	No
Clinical type of liver injury	-	-	Hepatocellular	-	-	-	-	Hepatocellular	Mixed	Hepatocellular	Mixed
Medication history	Compound reserpine and triamterene tablets, compound Ginkgo leaves tablets	Cisapride	-	Omeprazole capsules, Dengzhan Shengmai capsules, calcitriol soft gelatin capsules, levothyroxine tablets	Amlodipine besylate Tablets	Alfarosalitol, levothyroxine tablets	Diclofenac sodium sustained release capsules	-	-	-	-
Duration from medication to liver injury (days)	180	60	10	60	20	194	28	30	300	30	60
Clinical characteristics
ALP/U·L^-1	-	-	129	-	-	-	-	267.65	163	88.16	579
ALT/U·L^-1	85	158	1 098	389	2 196	930	-	1 477.4	770	150	629
TBIL/μmol·L^-1	-	-	47.2	-	-	-	496	313.51	228.3	12.4	12
INR	-	-	-	-	-	-	-	-	-	-	-
RUCAM scores	-	-	-	-	-	-	-	8	6	7	6
Final outcome	Cured	Cured	Cured	Cured	Cured	Cured	Death	-	-	-	-
Integrated evidence chain evaluation	-	-	-	-	-	-	-	-	-	-	-

No	1	2	3	4	5	6
Gender	Female	Male	Female	Male	Female	Female
Age (years)	70	75	55	82	75	64
Medication history	Xianling Gubao capsules, tinidazole, Qianggu capsules, calcitriol soft gelatin capsules	Xianling Gubao capsules, loxoprofen sodium tablets	Xianling Gubao capsules, Thai snake medicine	Xianling Gubao capsules, olanzapine, estazolam, calcitriol, cervus and cucumis polypeptide, cobamamide	Xianling Gubao capsules, warfarin, calcitriol, Qianggu capsules, calcarea carbonica	Xianling Gubao capsules, Gushukang capsules
Duration from medication to liver injury (days)	180	30	20	40	30	25
Clinical characteristics
ALP/U·L^-1	120	113	101	168	225	165
ALT/U·L^-1	904	775	1 149	666	520	610
TBIL/μmol·L^-1	390.9	100.3	13.4	432	8.8	165.69
INR	2	1.75	1.03	3.82	1.82	1.08
RUCAM scores	5	8	9	7	7	9
Outcome	Invalid	Improved	Improved	Invalid	Improved	Improved
Final outcome	Death	Cured	Cured	Death	Cured	Cured
Integrated evidence chain evaluation	Suspected diagnosis	Suspected diagnosis	Clinical diagnosis	Suspected diagnosis	Suspected diagnosis	Clinical diagnosis

No	1	2	3	4	5	6
Gender	Female	Male	Female	Male	Female	Female
Age (years)	70	75	55	82	75	64
Medication history	Xianling Gubao capsules, tinidazole, Qianggu capsules, calcitriol soft gelatin capsules	Xianling Gubao capsules, loxoprofen sodium tablets	Xianling Gubao capsules, Thai snake medicine	Xianling Gubao capsules, olanzapine, estazolam, calcitriol, cervus and cucumis polypeptide, cobamamide	Xianling Gubao capsules, warfarin, calcitriol, Qianggu capsules, calcarea carbonica	Xianling Gubao capsules, Gushukang capsules
Duration from medication to liver injury (days)	180	30	20	40	30	25
Clinical characteristics
ALP/U·L^-1	120	113	101	168	225	165
ALT/U·L^-1	904	775	1 149	666	520	610
TBIL/μmol·L^-1	390.9	100.3	13.4	432	8.8	165.69
INR	2	1.75	1.03	3.82	1.82	1.08
RUCAM scores	5	8	9	7	7	9
Outcome	Invalid	Improved	Improved	Invalid	Improved	Improved
Final outcome	Death	Cured	Cured	Death	Cured	Cured
Integrated evidence chain evaluation	Suspected diagnosis	Suspected diagnosis	Clinical diagnosis	Suspected diagnosis	Suspected diagnosis	Clinical diagnosis

Drugs	Diagnostic cases	Medication cases	Occurrence rate/%	95% CI
Allopurinol	114	22 623	0.504	0.41%-0.60%
Leflunomide	6	3 680	0.163	0.033%-0.29%
Celecoxib	60	125 686	0.048	0.036%-0.060%
XLGB	50	149 838	0.034	0.025%-0.043%

Drugs	Diagnostic cases	Medication cases	Occurrence rate/%	95% CI
Allopurinol	114	22 623	0.504	0.41%-0.60%
Leflunomide	6	3 680	0.163	0.033%-0.29%
Celecoxib	60	125 686	0.048	0.036%-0.060%
XLGB	50	149 838	0.034	0.025%-0.043%

仙灵骨葆相关肝损伤的临床病例分析及拆方实验研究

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黄迎 ¹^,² , 刘亚蕾 ² , 马润然 ² , 李春雨 ³ , 马致洁 ⁴ , 景婧 ² , 高源 ⁵ , 沈鹏 ⁶ , 林鸿波 ⁶ , 郭玉明 ² , 柏兆方 ² , 肖小河 ^*^,² , 王伽伯 *^,²

药学学报 | 研究论文 2021,56(1): 266-370

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药学学报 | 研究论文 2021, 56(1): 266-370

仙灵骨葆相关肝损伤的临床病例分析及拆方实验研究

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黄迎¹^,², 刘亚蕾², 马润然², 李春雨³, 马致洁⁴, 景婧², 高源⁵, 沈鹏⁶, 林鸿波⁶, 郭玉明², 柏兆方², 肖小河^*^,², 王伽伯*^,²

作者信息

1.湖南中医药大学药学院, 湖南长沙 410208

2.中国人民解放军总医院第五医学中心全军中医药研究所, 北京 100039

3.中国医学科学院肿瘤医院, 北京 100039

4.首都医科大学附属北京友谊医院, 北京 100039

5.首都医科大学中医药学院, 北京 100039

6.宁波市鄞州区疾病预防控制中心, 浙江宁波 315000

通讯作者:

*肖小河, Tel/Fax: 86-10-66933322, E-mail: pharmacy302xxh@126.com

王伽伯, Tel/Fax: 86-10-66933322, E-mail: pharmacy302xxh@126.com

Clinical case analysis and disassembled prescription study of liver injury related to Xianling Gubao

Ying HUANG¹^,², Ya-lei LIU², Run-ran MA², Chun-yu LI³, Zhi-jie MA⁴, Jing JING², Yuan GAO⁵, Peng SHEN⁶, Hong-bo LIN⁶, Yu-ming GUO², Zhao-fang BO², Xiao-he XIAO^*^,², Jia-bo WANG*^,²

Affiliations

1. School of Pharmacy, Hunan University of Chinese Medicine, Changsha 410208, China

2. China Military Institute of Chinese Medicine, the Fifth Medical Centre of Chinese PLA General Hospital, Beijing 100039, China

3. Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100039, China

4. Beijing Friendship Hospital, Capital Medical University, Beijing 100039, China

5. School of Traditional Chinese Medicine, Capital Medical University, Beijing 100039, China

6. Ningbo Yinzhou District Center for Disease Control and Prevention, Ningbo 315000, China

出版时间: 2021-01-12 doi: 10.16438/j.0513-4870.2020-1477

文章导航

摘要

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仙灵骨葆为骨科常用药, 临床疗效好, 但近年来发现有致肝损伤风险。本研究通过对文献不良反应报告和重点医院病例进行分析, 发现文献检索到的仙灵骨葆肝损伤病例存在较大比例的报告信息不完整, 难以准确判断其因果关系。重点医院药物性肝损伤(DILI)病例中, 筛选出仙灵骨葆相关肝损伤患者6例, 其中2例经整合证据链法评价达到了“临床诊断”标准。进一步对区域全人群健康大数据进行分析, 估算其粗发生率为0.034%, 属于罕见水平, 具有显著的个体差异和特异质属性, 仙灵骨葆相关肝损伤总体发生水平低于其他骨病用药。基于免疫应激介导的特异质肝损伤模型, 通过拆方研究发现, 方中淫羊藿和补骨脂是引起肝损伤的相关药味, 全方的肝损伤严重程度弱于淫羊藿和补骨脂, 提示方中另外4味药(续断、知母、地黄、丹参)有减轻二者引起的肝损伤作用。进一步进行拆方研究, 发现4味药均有减轻淫羊藿和补骨脂所致肝损伤作用, 其中丹参的配伍减毒效果最好。综上, 仙灵骨葆可能在极少数易感个体引起特异质肝损伤, 但发生风险低于其他常用骨病药物。临床应用仙灵骨葆要注意避免在免疫应激患者应用。仙灵骨葆引起肝损伤的主要药味为补骨脂、淫羊藿, 且丹参在全方中起到配伍减毒作用。本研究可为仙灵骨葆临床合理用药提供参考。

关键词

仙灵骨葆 / 整合证据链 / 免疫应激 / 淫羊藿 / 补骨脂 / 配伍减毒

Abstract

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Xianling Gubao is a common and effective medicine in the treatment of orthopedic diseases. In recent years, it has been reported to be associated with liver injury. However, through the analysis of the adverse drug reaction reports and key hospital cases, we found that there is considerable incomplete information in the reports of Xianling Gubao-related liver injury cases retrieved from the literature. Thus, it is difficult to accurately judge causality between the drug and liver injury. Six cases of liver injury related to Xianling Gubao were identified in key hospitals, two of which achieved the clinical diagnosis according to the assessment of the integrated evidence chain method. We further analyzed the public health data of all residents in Yinzhou. The gross incidence rate of Xianling Gubao-related liver injury was 0.034%, which corresponds to a level of rare incidence. This revealed that Xianling Gubao-related liver injury has significant divergence in individuals and an idiosyncratic nature. The gross incidence of liver injury related to Xianling Gubao was lower than that of other medicines for the treatment of orthopedic diseases. Based on the idiosyncratic drug-induced liver injury model mediated by immune stress, it was found that Epimedii Folium and Psoraleae Fructus were the major components that lead to liver injury, and the liver injury caused by a full prescription was less serious than that encountered with only Epimedii Folium and Psoraleae Fructus. This suggests that the other 4 herbs(Dipsaci Radix, Anemarrhenae Rhizoma, Rehmanniae Radix, Salviae Miltiorrhizae Radix et Rhizoma) can prevent/alleviate the liver injury. Through disassembled prescription analysis, we found that the attenuation efficacy of Salviae Miltiorrhizae Radix et Rhizoma was the most significant.In conclusion, Xianling Gubao may cause idiosyncratic liver injury in a tiny minority of susceptible individuals, but the incidence risk is lower than that of other commonly used drugs for orthopedic disease. Xianling Gubao should be discreetly applied to patients with immune stress. The major components that induced liver injury in Xianling Gubao were Epimedii Folium and Psoraleae Fructus, and Salviae Miltiorrhizae Radix et Rhizoma appears to attenuate this toxicity. This study provides a reference for the rational clinical medication with Xianling Gubao.

Key words

Xianling Gubao / integrated evidence chain / immune stress / Epimedii Folium / Psoraleae Fructus / compatibility-mediated toxicity

引用本文

黄迎, 刘亚蕾, 马润然, 李春雨, 马致洁, 景婧, 高源, 沈鹏, 林鸿波, 郭玉明, 柏兆方, 肖小河, 王伽伯. 仙灵骨葆相关肝损伤的临床病例分析及拆方实验研究. 药学学报, 2021 , 56 (1) : 266 -370 . DOI: 10.16438/j.0513-4870.2020-1477

Ying HUANG, Ya-lei LIU, Run-ran MA, Chun-yu LI, Zhi-jie MA, Jing JING, Yuan GAO, Peng SHEN, Hong-bo LIN, Yu-ming GUO, Zhao-fang BO, Xiao-he XIAO, Jia-bo WANG. Clinical case analysis and disassembled prescription study of liver injury related to Xianling Gubao[J]. Acta Pharmaceutica Sinica, 2021 , 56 (1) : 266 -370 . DOI: 10.16438/j.0513-4870.2020-1477

正文

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仙灵骨葆制剂在临床上应用广泛, 是用于治疗骨质疏松、骨折、骨关节炎、骨无菌性坏死等骨科疾病的苗药制剂, 收载于《国家基本药物目录》。近年来, 相继有仙灵骨葆引起药物性肝损伤的报道^{[1, 2]}, 国家食品药品监督管理局也发出了相关通报^[3]。然而, 仙灵骨葆引起药物性肝损伤(drug-induced liver injury, DILI)的风险大小尚不完全清楚。

仙灵骨葆由淫羊藿、续断、知母、补骨脂、地黄和丹参六味中药组成, 从组方来看仙灵骨葆中均为传统无毒中药。课题组前期研究发现仙灵骨葆对正常动物不会引起肝损伤, 但在免疫应激模型上可引起大鼠血浆ALT和AST水平升高以及组织学损伤^[4]。仙灵骨葆方中的主要致肝损伤药味, 以及是否存在配伍减毒, 尚未充分研究。

基于此, 为进一步明确仙灵骨葆肝损伤的临床特征、风险因素以及风险大小, 本研究对仙灵骨葆进行文献不良反应报告、重点医院病例和区域全人群健康大数据分析, 评估其引起肝损伤在临床DILI中的占比及粗发生率, 为其安全性评价和临床合理用药提供参考^[5]。进一步对仙灵骨葆进行两步拆方的动物实验, 以期明确其导致肝损伤的原因和具体药味, 为降低仙灵骨葆肝损伤风险提供参考。

材料与方法

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文献回顾性分析 检索数据库中国知网(CNKI), 根据医学主题词表, 限定检索的关键词为“仙灵骨葆”并且“肝功能不全”或“肝功能异常”或“肝损伤”或“肝损害”或“肝衰竭”, 检索的截止日期为2018年12月31日, 对检索到的病例进行统计比较。相同方法检索其他骨病用药并进行病例数统计, 与仙灵骨葆病例数进行比较, 判断仙灵骨葆相关肝损伤的总体发生水平。

住院病例再评价 在解放军总医院第五医学中心(2008年1月1日~2017年12月31日)数据库中诊断为“药物性肝损伤”、“药物性肝炎”且服用骨病用药的病例, 将仙灵骨葆与其他骨病用药进行比较, 对其肝损伤发生水平进行再评价。

在首都医科大学附属友谊医院(2009年1月1日~2018年12月31日)数据库中诊断为“药物性肝损伤”、“药物性肝炎”且服用过仙灵骨葆制剂的病例。参照中华中医药学会《中草药相关肝损伤临床诊疗指南》(以下简称《指南》)推荐的基于整合证据链法的HILI诊断策略对住院病例进行客观性评价^[6]。

肝损伤粗发生率估算 在2009年1月1日~2017年12月31日期间, 基于鄞州社区全人群健康大数据检索出仙灵骨葆相关肝损伤病例进行再评价, 合并同一病例, 删除重复病例, 筛选得到服用仙灵骨葆后发生肝功能异常并被诊断为“药物性肝损伤”或“药物性肝炎”的病例。按照肝损伤发生率的计算公式估算仙灵骨葆肝损伤的粗发生率。同时基于鄞州社区全人群健康大数据筛选出几种治疗骨病化学药物的相关肝损伤病例, 计算其肝损伤粗发生率, 与仙灵骨葆进行比较, 进一步判断仙灵骨葆的相对发生风险。肝损伤粗发生率的计算公式^[7]: $仙灵骨葆肝损伤年化粗发生率=\frac{\text { 研究时间内服用过仙灵骨保且发生肝损伤的病例数 }}{\text { 研究时间内所有服用过仙灵骨保的病例数 }} \times 100\%$。采用正态近似法, 计算其95%置信区间, 公式为: $P \pm \mu_{\alpha / 2} \mathrm{Sp}, \mathrm{Sp}=\sqrt{\frac{P(1-P)}{n}}$

动物健康SPF级雄性C57小鼠80只, 6周, 体重18±2 g, 购于斯贝福实验动物科技有限公司[合格证号SYXK (京) 2017-0002]。分笼饲养于解放军总医院第五医学中心实验动物中心, 自由饮水饮食。饲养环境: 湿度50%~60%、温度(25±1)℃, 明暗周期(各12 h), 通风良好。动物实验获得中国人民解放军总医院第五医学中心动物伦理委员会批准(批准号: IACUC-2019-0019)。

仪器微量分析天平(Mettler Toledo AL204瑞士); 低温离心机(美国Thermo Fisher Scientific Sigma公司); SynergyH2全功能微孔板检测仪(美国BioTek公司); Nikon E200光学显微镜(日本Nikon公司); Leica2016石蜡切片机(上海莱卡仪器有限公司); Leica EG1150组织包埋机等。

药物与试剂 仙灵骨葆(XLGB)处方药物: 淫羊藿(批号181102, 产地甘肃)、补骨脂(批号181102, 产地云南)、丹参(批号181101, 产地山东)、地黄(批号181001, 产地河南)、知母(批号181101, 产地安徽)、续断(批号181101, 产地四川), 均由贵州同济堂制药有限公司提供。仙灵骨葆各药按原方比例称取适量, 根据要求制作水煎液。

脂多糖(lipopolysaccharide, LPS)购于Sigma公司(批号: 028M4094V); 丙氨酸氨基转移酶(ALT)试剂盒(货号: C009-2)、天冬氨酸氨基转移酶(AST)试剂盒(货号: C010-2)购于南京建成生物工程研究所。按照给药剂量(2.8 mg·kg^-1)^[8]计算所需脂多糖的量, 采用无菌生理盐水涡旋溶解, 4℃贮存备用。

仙灵骨葆拆方实验

拆方实验一 C57小鼠80只随机分为8组(每组10只), 分为正常组(N)、仙灵骨葆全方组(X)、淫羊藿+补骨脂组(A)、地黄+丹参+知母+续断组(B)、LPS模型组(L)、LPS+仙灵骨葆组(LX)、LPS+淫羊藿+补骨脂组(LA)、LPS+地黄+丹参+知母+续断组(LB)。正常组给予生理盐水, 各给药组分别灌胃给予对应药液(20 mL·kg^-1), 淫羊藿、补骨脂、地黄、丹参、知母、续断剂量分别为1.68、0.12、0.12、0.12、0.12和0.24 g·kg^-1。免疫应激模型动物尾静脉注射LPS 0.5 mg·kg^-1, 其余各组尾静脉注射等体积生理盐水, 2.5 h后给予相应药液, 6 h后摘眼球取血, 4℃、3 500 r·min^-1离心15 min, 分离血浆, 于-80℃冰箱内保存; 取小鼠肝大叶部分, 放置于4%中性多聚甲醛溶液中固定。

拆方实验二 C57小鼠60只随机分为6组(每组10只), 分为正常组(N)、LPS+淫羊藿+补骨脂组(LA)、LPS+淫羊藿+补骨脂+地黄组(DH)、LPS+淫羊藿+补骨脂+丹参组(DS)、LPS+淫羊藿+补骨脂+知母组(ZM)、LPS+淫羊藿+补骨脂+续断组(XD)。给药方式、给药剂量及操作同上。

肝功能指标检测和肝脏病理学检查 按照说明书操作测定并计算血浆ALT、AST的活力值。肝组织HE染色, 显微镜下观察病理切片。

统计分析 采用SPSS 22.0统计软件进行分析, 计量资料采用单因素方差分析(ANOVA), P < 0.05或P < 0.01表示具有显著性差异。

结果

收起

1 基于CNKI检索仙灵骨葆相关肝损伤病例回顾性分析

从CNKI数据库中检索到的仙灵骨葆相关肝损伤文献中, 剔除会议摘要、综述, 符合要求的文献一共7篇, 合计11例患者^[9-15], 基本情况见表 1, 整合信息发现很大比例的病例报告信息不完整, 联合用药多, 因果关系不清晰。同时检索到另外5种致肝损伤的骨病用药^[16-20], 构成比排序为见图 1。

2 基于整合证据链的仙灵骨葆相关肝损伤病例评价

从两家重点专科医院数据库中, 收集解放军总医院第五医学中心(2008~2017年)和首都医科大学附属友谊医院(2009~2018年)的药物性肝损伤病例共4 275例, 其中仙灵骨葆相关肝损伤的病例只有6例。收集到的6例患者中男性2例, 女性4例, 年龄在55~82岁之间, 病例的基本情况见表 2。

以上住院病例中, 患者均在服用仙灵骨葆一个月左右发生肝损伤, 多数患者经停药并积极治疗后, 病情好转出院。通过整合证据链的方法, 对这6例病例进行再评价, 2名患者达到“临床诊断”标准, 另外4名患者为“疑似诊断”。

从解放军总医院第五医学中心数据库中, 对检索的仙灵骨葆相关肝损伤病例进行统计, 同时检索到另外4种致肝损伤的骨病用药, 构成比排序: 骨康胶囊 > 丹鹿通督片 > 尼美舒利 > 仙灵骨葆制剂 > 金天格胶囊。

3 基于区域全人群健康大数据的仙灵骨葆相关肝损伤粗发生率估算

鄞州区域全人群健康大数据从2009年到2017年间, 服用过仙灵骨葆的病例总数为149 838例, 其中发生肝功能异常并被诊断为“药物性肝损伤”和“药物性肝炎”的有50例, 估算仙灵骨葆药物性肝损伤的粗发生率为: 0.034%(95%CI, 0.025%~0.043%)。参照国际医学科学组织理事会(CIOMS)对不良反应发生率的风险分级: 十分常见(≥10%), 常见(1%~10%, 含1%), 偶见(0.1%~1%, 含0.1%), 罕见(0.01%~0.1%, 含0.01%), 十分罕见(< 0.01%), 仙灵骨葆的粗发生率属于“罕见”。将仙灵骨葆与另外三种治疗骨病的化学药物进行对比, 结果显示, 仙灵骨葆肝损伤总体发生风险低于化学药(表 3)。

4 基于免疫应激模型的仙灵骨葆致肝损伤的拆方实验

课题组前期利用脂多糖(LPS)介导的免疫应激模型, 发现壮骨关节丸的组成药味淫羊藿、补骨脂均可导致免疫特异质肝损伤^[21]。基于此研究结果, 以淫羊藿和补骨脂为药对, 对仙灵骨葆进行拆方分析研究, 验证淫羊藿和补骨脂在免疫应激模型下的肝损伤作用, 并观察其余4味药是否有肝损伤作用。

仙灵骨葆全方、淫羊藿和补骨脂、其余4味药均未引起正常小鼠肝功能生化指标AST、ALT明显改变, 也未引起肝脏病理明显改变。在免疫应激状态下, 全方组及淫羊藿和补骨脂组相对其他组, 小鼠肝功能指标AST, ALT显著升高(P < 0.01)、肝脏病理发生变化, 出现明显的肝损伤表型, 淫羊藿和补骨脂组比全方组肝损伤程度更严重, 而其他4味药无明显作用(图 2)。结果提示在免疫应激状态的条件下, 仙灵骨葆方中淫羊藿和补骨脂可致肝损伤, 可能为仙灵骨葆的损肝组分。

5 基于免疫应激模型的仙灵骨葆致肝损伤的配伍减毒评价

在免疫应激动物模型上, 仙灵骨葆方中淫羊藿和补骨脂可致肝损伤, 且毒性要高于全方组, 说明仙灵骨葆方中其余四味药存在一定的减毒效果, 本部分将基于拆方实验的内容, 分别对地黄、丹参、知母、续断的减毒作用进行评价。

在免疫应激状态下, 淫羊藿和补骨脂组、淫羊藿和补骨脂分别配伍其余4味药组肝功能指标ALT、AST均显著升高, 以淫羊藿和补骨脂组最为明显(图 3)。与模型组相比, 加地黄、丹参、知母、续断的各组均有不同程度的减毒作用, 其中丹参组减毒作用最明显, 具有显著性差异(P < 0.05)。结果提示在免疫应激状态下, 方中丹参对仙灵骨葆全方具有配伍减毒作用。

讨论

收起

仙灵骨葆是临床常用的治疗骨病的中成药, 近年来有关其肝损伤的报道逐渐引起社会各界关注, 评估仙灵骨葆致肝损伤的发生风险和方中损肝药味变得尤为重要。通过文献回顾性分析, 发现仙灵骨葆临床病例中大多报告不准确, 存在因果关系不清晰的情况^[22]。仙灵骨葆临床DILI病例的比例排序低于其他骨病用药, 提示仙灵骨葆相关肝损伤总体发生水平较低。对鄞州社区全人群健康大数据记载的病例进行筛选统计, 估算仙灵骨葆肝损伤粗发生率为0.034%, 属罕见范畴, 说明仙灵骨葆相关肝损伤仅发生在极少数人身上, 绝大多数的人服用仙灵骨葆是安全的。将仙灵骨葆与用于治疗骨病的三种化药进行对比发现其肝损伤发生率低于同类型的化学药物。综合提示, 仙灵骨葆可能在极少数易感个体引起特异质肝损伤, 发生风险低于其他常用骨病药物。

拆方研究表明在免疫应激状态的条件下, 仙灵骨葆方中淫羊藿和补骨脂是引起肝损伤的相关药味, 丹参对仙灵骨葆全方具有配伍减毒作用。此研究明确了仙灵骨葆导致肝损伤的原因和具体药味, 以及仙灵骨葆全方中丹参可降低其引起肝损伤毒性, 可为仙灵骨葆安全性评价和临床安全用药提供参考。课题组针对淫羊藿的研究表明淫羊藿次苷II可通过增强NLRP3炎症小体的激活导致特征性肝损伤^{[23, 24]}。另有文献研究表明补骨脂素、补骨脂二氢黄酮、补骨脂二氢黄酮甲醚、补骨脂酚等与补骨脂潜在肝毒性相关^[25]。此外, 丹参水溶性成分丹酚酸A、丹酚酸B、丹参素和脂溶性成分丹参酮IIA均有保肝作用^{[26, 27]}。但淫羊藿、补骨脂、丹参成分均复杂, 仍需要深入研究。特别是不同产地的丹参成分含量差异很大, 选择适宜产地的丹参原料可能更好地降低仙灵骨葆全方的肝损伤风险, 有待深入研究。

作者贡献: 王伽伯、肖小河负责实验思路设计; 黄迎、马润然为本文主要撰写者; 刘亚蕾、黄迎负责完成主要的实验部分; 李春雨、马致洁和高源提供实验思路; 景婧、沈鹏和林鸿波提供病例信息; 王伽伯、郭玉明和柏兆方负责论文修改; 所有作者均对本文有所贡献。

利益冲突: 本文无任何利益冲突。

基金

收起

国家自然科学基金项目(81630100)
国家重点研发计划项目(2018YFC1707002)
解放军总医院科研专项(2019-JQPY-003)

参考文献

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文献

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2021年第56卷第1期

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文章信息

doi: 10.16438/j.0513-4870.2020-1477

接收时间：2020-09-11
首发时间：2025-12-18
出版时间：2021-01-12

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出版历史

收稿日期：2020-09-11
修回日期：2020-10-29

基金

国家自然科学基金项目(81630100)

国家重点研发计划项目(2018YFC1707002)

解放军总医院科研专项(2019-JQPY-003)

作者信息

1.湖南中医药大学药学院, 湖南长沙 410208

2.中国人民解放军总医院第五医学中心全军中医药研究所, 北京 100039

3.中国医学科学院肿瘤医院, 北京 100039

4.首都医科大学附属北京友谊医院, 北京 100039

5.首都医科大学中医药学院, 北京 100039

6.宁波市鄞州区疾病预防控制中心, 浙江宁波 315000

通讯作者:

*肖小河, Tel/Fax: 86-10-66933322, E-mail: pharmacy302xxh@126.com

王伽伯, Tel/Fax: 86-10-66933322, E-mail: pharmacy302xxh@126.com

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2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

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No	1	2	3	4	5	6	7	8	9	10	11
Gender	Female	Female	Female	Female	Female	Female	Female	Female	Male	Female	Female
Age (years)	65	68	45	57	57	67	40	63	64	57	60
Other possible causes induced liver injury investigation
Autoimmune antibodies	-	-	Negative	-	-	-	Negative	-	-	-	-
Hepatitis A, hepatitis C, and hepatitis E antibodies	-	-	Negative	-	Negative	-	Negative	-	-	-	-
Hepatitis B serological test	All negative	All negative	Hbcab positive	All negative	All negative	-	All negative	-	-	-	-
Drink history	-	-	-	-	-	-	-	No	No	No	No
Clinical type of liver injury	-	-	Hepatocellular	-	-	-	-	Hepatocellular	Mixed	Hepatocellular	Mixed
Medication history	Compound reserpine and triamterene tablets, compound Ginkgo leaves tablets	Cisapride	-	Omeprazole capsules, Dengzhan Shengmai capsules, calcitriol soft gelatin capsules, levothyroxine tablets	Amlodipine besylate Tablets	Alfarosalitol, levothyroxine tablets	Diclofenac sodium sustained release capsules	-	-	-	-
Duration from medication to liver injury (days)	180	60	10	60	20	194	28	30	300	30	60
Clinical characteristics
ALP/U·L^-1	-	-	129	-	-	-	-	267.65	163	88.16	579
ALT/U·L^-1	85	158	1 098	389	2 196	930	-	1 477.4	770	150	629
TBIL/μmol·L^-1	-	-	47.2	-	-	-	496	313.51	228.3	12.4	12
INR	-	-	-	-	-	-	-	-	-	-	-
RUCAM scores	-	-	-	-	-	-	-	8	6	7	6
Final outcome	Cured	Cured	Cured	Cured	Cured	Cured	Death	-	-	-	-
Integrated evidence chain evaluation	-	-	-	-	-	-	-	-	-	-	-

Gender

Female

Male

Female

Age (years)

Other possible causes
induced liver injury
investigation

Autoimmune antibodies

Negative

Hepatitis A, hepatitis C,
and hepatitis E antibodies

Negative

Hepatitis B serological test

All negative

Hbcab positive

All negative

Drink history

Clinical type of liver injury

Hepatocellular

Mixed

Hepatocellular

Mixed

Medication history

Compound reserpine and triamterene tablets, compound
Ginkgo leaves tablets

Cisapride

Omeprazole capsules, Dengzhan Shengmai capsules, calcitriol soft gelatin capsules, levothyroxine tablets

Amlodipine
besylate
Tablets

Alfarosalitol, levothyroxine tablets

Diclofenac
sodium sustained release capsules

Duration from medication
to liver injury (days)

180

194

300

Clinical characteristics

ALP/U·L^-1

129

267.65

163

88.16

579

ALT/U·L^-1

158

1 098

389

2 196

930

1 477.4

770

150

629

TBIL/μmol·L^-1

47.2

496

313.51

228.3

12.4

INR

RUCAM scores

Final outcome

Cured

Death

Integrated evidence chain evaluation

No	1	2	3	4	5	6
Gender	Female	Male	Female	Male	Female	Female
Age (years)	70	75	55	82	75	64
Medication history	Xianling Gubao capsules, tinidazole, Qianggu capsules, calcitriol soft gelatin capsules	Xianling Gubao capsules, loxoprofen sodium tablets	Xianling Gubao capsules, Thai snake medicine	Xianling Gubao capsules, olanzapine, estazolam, calcitriol, cervus and cucumis polypeptide, cobamamide	Xianling Gubao capsules, warfarin, calcitriol, Qianggu capsules, calcarea carbonica	Xianling Gubao capsules, Gushukang capsules
Duration from medication to liver injury (days)	180	30	20	40	30	25
Clinical characteristics
ALP/U·L^-1	120	113	101	168	225	165
ALT/U·L^-1	904	775	1 149	666	520	610
TBIL/μmol·L^-1	390.9	100.3	13.4	432	8.8	165.69
INR	2	1.75	1.03	3.82	1.82	1.08
RUCAM scores	5	8	9	7	7	9
Outcome	Invalid	Improved	Improved	Invalid	Improved	Improved
Final outcome	Death	Cured	Cured	Death	Cured	Cured
Integrated evidence chain evaluation	Suspected diagnosis	Suspected diagnosis	Clinical diagnosis	Suspected diagnosis	Suspected diagnosis	Clinical diagnosis

Gender

Female

Male

Female

Male

Female

Age (years)

Medication history

Xianling Gubao
capsules, tinidazole, Qianggu capsules,
calcitriol soft
gelatin capsules

Xianling Gubao capsules,
loxoprofen
sodium tablets

Xianling Gubao capsules, Thai snake medicine

Xianling Gubao capsules, olanzapine, estazolam, calcitriol, cervus and
cucumis polypeptide,
cobamamide

Xianling Gubao capsules, warfarin, calcitriol, Qianggu capsules, calcarea carbonica

Xianling Gubao capsules,
Gushukang
capsules

Duration from medication to liver injury (days)

180

Clinical characteristics

ALP/U·L^-1

120

113

101

168

225

165

ALT/U·L^-1

904

775

1 149

666

520

610

TBIL/μmol·L^-1

390.9

100.3

13.4

432

8.8

165.69

INR

1.75

1.03

3.82

1.82

1.08

RUCAM scores

Outcome

Invalid

Improved

Invalid

Improved

Final outcome

Death

Cured

Death

Cured

Integrated evidence chain evaluation

Suspected
diagnosis

Clinical
diagnosis

Suspected
diagnosis

Clinical
diagnosis

Drugs	Diagnostic cases	Medication cases	Occurrence rate/%	95% CI
Allopurinol	114	22 623	0.504	0.41%-0.60%
Leflunomide	6	3 680	0.163	0.033%-0.29%
Celecoxib	60	125 686	0.048	0.036%-0.060%
XLGB	50	149 838	0.034	0.025%-0.043%

Drugs

Diagnostic cases

Medication cases

Occurrence rate/%

95% CI

Allopurinol

114

22 623

0.504

0.41%-0.60%

Leflunomide

3 680

0.163

0.033%-0.29%

Celecoxib

125 686

0.048

0.036%-0.060%

XLGB

149 838

0.034

0.025%-0.043%