摘要
目的：本研究以传统外用制剂狗皮膏为研究对象,对其长期使用的安全性及其所含高剂量的重金属铅在重要脏器的蓄积行为进行实验研究,以期揭示长期使用狗皮膏的潜在毒性、毒性靶器官及造成毒性的可能机制,为临床安全合理用药提供科学依据；
方法：(1)通过皮肤急性毒性、皮肤刺激性(单次给药和多次给药)、皮肤过敏性试验,考察狗皮膏外用的安全性；
(2)通过长期外敷给予大鼠狗皮膏及其基质,观察90天连续外敷给药后的毒性特征,预测其可能引起的临床不良反应,包括不良反应的性质、程度、剂量-反应和时间-反应关系、可逆性等,推测其重复给药的临床毒性靶器官或靶组织；预测其重复用药的安全剂量范围；提示临床应用中需重点监测的指标,为临床应用提供参考信息；
(3)选用大鼠离体完整皮肤,以Franz扩散池为透皮吸收实验装置,采用微波消解-石墨炉原子吸收光谱法,研究狗皮膏中所含铅的体外释放及透皮吸收行为,了解其透皮吸收特性；
(4)通过火焰原子吸收光谱法,测定长期毒性试验中各组大鼠的血铅水平,考察狗皮膏长期毒性试验大鼠血铅浓度动态变化规律；
(5)通过火焰原子吸收光谱法,测定狗皮膏长期毒性试验中,各组大鼠主要脏器中铅的蓄积量,考察狗皮膏主要毒性成分铅的潜在毒性靶器官；
(6)通过ELISA法,考察狗皮膏连续给药90天对大鼠尿液中β2微球蛋白(β2-MG)和N-乙酰-β-D-氨基葡萄糖苷酶(NAG)等尿蛋白及尿酶水平的影响,探索狗皮膏长期使用对肾功能早期影响指标,以期揭示狗皮膏主要毒性成分铅的潜在毒性靶器官。
结果：(1)狗皮膏没有明显的皮肤急性毒性,单次及多次给药对家兔皮肤均无刺激性,对豚鼠亦无致敏作用,表明其外用安全性较高；
(2)狗皮膏及其基质以7.00g膏药/kg(相当于临床用量的14倍)连续给药90天,动物体重、饲料消耗量、容态、被毛、给药局部皮肤、粘膜、分泌物等均无明显改变；血液学和血清生化学检测发现狗皮膏可致白细胞减少、平均血小板体积及分布宽度降低、血小板减少、肌酐降低、碱性磷酸酶升高,但上述改变均在正常范围内波动,无特定生物学意义；此外,发现狗皮膏可使肾脏系数增高,但恢复期其肾脏指数可恢复正常；其余均未见与药物相关的毒性反应；
(3)在狗皮膏所含铅元素的体外透皮吸收试验中,狗皮膏中所含铅的透皮吸收行为较为符合零级动力学方程,提示狗皮膏在用药过程中,透过皮肤的铅元素量是随着时间的增长而增加的；
(4)所建立火焰原子吸收光谱法,线性关系良好,精密度、重复性良好,方法可靠、准确,可用于测定血液中的铅含量；大鼠给予狗皮膏后,血铅浓度逐渐升高,并与给药量呈正相关,停药后血铅浓度降低,但仍维持较高水平；
(5)狗皮膏大鼠给药三个月后,心脏的铅蓄积量仅在给药后第45天有明显升高,给药90天及停药28天时,心脏铅蓄积量均无明显升高；而肾脏、肝脏和脑的铅含量则有显著的升高,提示狗皮膏所含的大量铅元素可在大鼠肾脏、肝脏、脑组织中蓄积；
(6)给药后第45天,给药组大鼠尿液B2-MG、NAG水平均无明显升高；给药90天及停药28天,狗皮膏大剂量及基质组大鼠尿液NAG水平明显升高；而其他剂量组的β32-MG、NAG水平均无明显变化；
结论：狗皮膏外用给药没有明显的急性毒性、皮肤刺激性及致敏作用；高剂量(7.0g/kg)狗皮膏连续外敷90天,大鼠体重、饲料消耗量等一般体征参数及血液学、血清生化学、骨髓学、病理组织学等指标无明显改变,但可使肾脏系数明显升高,提示器对肾脏功能可能有一定影响；结合透皮吸收试验结果,提示狗皮膏中高含量的重金属铅可透过皮肤而被吸收,其透皮吸收行为符合零级动力学方程,其透过量随着剂量和时间的增长而增长的；因此,高剂量狗皮膏连续外敷90天,铅可透过大鼠皮肤而进入血液循环,使得大鼠血铅明显升高,并可在重要脏器,特别是肾脏中蓄积,使反映肾小管功能的尿NAG活力升高,但未影响到肾脏的排泄能力和发生器质性改变,另外在临床试验研究中,给药3周后血铅、尿铅水平也有一定程度增高,但未达铅中毒标准。综合上述结果,狗皮膏连续外用的潜在毒性靶器官为肾脏,可能引起早期肾小管损伤；因此,在狗皮膏的临床使用中,不仅要控制狗皮膏使用的时间和剂量,也应密切监测早期肾功能相关指标的变化。
Objective:To evaluate the the safety of long-term external use of the traditional external preparation Gou-pi plaster and the accumulation behavior of lead in rats, then reveal the potential toxicity, toxic target organs and underlying mechanism of long-term external use of Gou-pi plaster, and provided scientific evidence to the clinical use of Gou-pi plaster.
Method:Firstly, the safety of external use of Gou-pi plaster was observed by utilizing the following methods:acute dermal toxicity test, skin irritation test and skin sensitization test.
Secondly, Gou-pi plaster was administered externally at dosages of7.0,3.5and1.75g/kg once every other day for a total of90days. Then the possible toxic characteristics of Gou-pi plaster were observed, and then predicted the possible clinical adverse reactions, including the its nature and degree; Furthermore, speculated the toxicity target organs and safe dosage of Gou-pi plaster, and prompted the index which should be greatly concerned in clinical use, as well as provided reference information for the use of Gou-pi plaster.
Thirdly, the percutaneous absorption of lead contained in Gou-pi plaster was investigated in vitro skin via microwave digestion/graphite furnace atomic absorption spectrometric method.
Fourthly, the blood lead levels were evaluated by flame atomic absorption spectrometric method, then the change regularity of blood lead was investigated in long-term toxicity study of Gou-pi plaster in rats.
Fifthly, the accumulation of lead was determined by flame atomic absorption spectrometric method in long-term toxicity study of Gou-pi plaster in rats.
Finally, the levels of urinary enzymes and protein, such as B2-microglobulin and N-acetyl-β-D-glucosaminidase, were used to be evaluated the renal function of rats in long-term toxicity study of Gou-pi plaster.
Results:Firstly, Gou-pi plaster did not cause skin acute toxicity, irritation and sensitization, which indicated that Gou-pi plaster possessed a higher external use safety.
Secondly, the general conditions of all animals had not abnormal changes in chronic toxicity study, including body weight, food intake, mentality, physique, activity, clothing hair, local treated skin, mucosal, and secretions. Hematological and biochemical results indicated that Gou-pi plaster could decrease the levels of leukocyte, platelet, mean platelet volume, mean platelet distribution width, and creatinine, increase the level of alkaline phosphatase. However, the changes of these parameters were within the fluctuation of normal physiological value. Furthermore, Gou-pi plaster could increase the kidney-body weight index. Moreover, no other significant differences were observed among the groups.
Thirdly, the study suggested that the lead contained in the Gou-pi plaster through the skin in zero-level release equation so as the prompt that the amount of the lead the permeating through the skin is increasing time goes on when the Gou-pi plaster is being used.
Fourthly, the linear relationship, accuracy, and reproducibility of flame atomic absorption spectrometric method were conformed the requirements, and which could be used to determine the blood lead level. After treatment of Gou-pi plaster, the blood lead level of rats significantly increased, and maintained in a higher level in the recovery period.
Fifthly, during the administration and recovery period, the elevated lead accumulations in kidney, liver and brain were observed in Gou-pi plaster treated rats. However, the elevated lead accumulation in heart was only observed after45days of treatment.
Finally, high dosages of Gou-pi plaster and matrix could increase the levels of N-acetyl-B-D-glucosaminidase after90days of treatment and recovery period. And the levels of B2-microglobulin had no obvious changes. Conclusion:External use of Gou-pi plaster did not cause obvious skin acute toxicity, irritation and sensitization. After90days of external use of Gou-pi plaster (7.0g/kg), there were no significant changes of the general body parameters, hematological parameters, biochemical parameters, bone marrow parameters, and histopathological parameters. However, Gou-pi plaster could increase the kidney-body weight index, which indicated that treatment of Gou-pi plaster might have effect on the renal function. Furthermore, the results obtained from absorption test, we found that the lead contained in the Gou-pi plaster through the skin in zero-level release equation so as the prompt that the amount of the lead the permeating through the skin is increasing time goes on when the Gou-pi plaster is being used. Therefore, the lead contained in Gou-pi plaster could be absorbed via skin, and then caused the elevated level of blood lead. Subsequently, the lead would be accumulated in some important organs, especially in kidney, which would cause renal tubular dysfunction. Furthermore, a clinical study indicated that the lead levels in blood and urine were elevated after treatment of Gou-pi plaster for three weeks, but the lead levels were lower than its poisoning limit. In summary, the toxicity target organ of Gou-pi plaster might be kidney, and long-term use of Gou-pi plaster would cause early renal tubular injury. Hence, the use of time and dosage of Gou-pi plaster should be controlled in the clinical usage of Gou-pi plaster; Furthermore, the early indicators of renal injury also should be greatly and closely concerned.
引文
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