【作者】 董菊；

【导师】 詹瑧；

【作者基本信息】 南京中医药大学 ， 中西医结合基础， 2012， 博士

【摘要】 牛黄解毒片是中医临床清热解毒的经典方剂,用于治疗火热内盛,咽喉肿痛,牙龈肿痛,口舌生疮及目赤肿痛等症。为评价牛黄解毒片的安全性,本研究采用Ames试验、染色体畸变分析、单细胞凝胶电泳试验(SCGE).微核试验等方法,探讨了牛黄解毒片在体内、外实验条件下,有无潜在的遗传毒性作用；此外,配伍是最具中医特色的减毒方法和技术之一,为探讨牛黄解毒片配伍对雄黄是否具有减毒作用,本研究在已有研究基础上,设立牛黄解毒片全方及其不同拆方组,通过可溶性砷盐含量、砷在机体组织的分布、病理损伤、氧化损伤、遗传损伤、细胞凋亡等指标的检测,拟初步探讨牛黄解毒片方药配伍对雄黄有无减毒作用及其可能机制。1牛黄解毒片的遗传毒性研究1.1牛黄解毒片遗传毒性的体外实验研究1.1.1牛黄解毒片对鼠伤寒沙门氏菌菌株的回复突变作用采用平皿掺入法,按照Maron DM和Ames BN推荐程序,根据预试验结果,以5mg.IIII-1为最高剂量,下设1.5、0.5、0.15、0.05mg.IIII-1四个剂量组和阴、阳性对照组开展实验。结果表明,牛黄解毒片水溶液在试验剂量为0.05mg.IIII-1至5mg.IIII-1,在加与不加S9混合液两种情况下,各剂量组和阴性对照组比较,差异均无统计学意义,且各菌株的回复菌落数均未超过阴性对照组2倍,也未呈现一定剂量-反应关系。1.1.2牛黄解毒片对CHL细胞的染色体畸变作用采用细胞计数法测出牛黄解毒片水溶液对CHL细胞的半数生长抑制浓度(IC50)约为54μg.ml-1,以此浓度为高剂量组,采用倍比稀释法,设立中、低剂量组(分别为27和13.5μg.ml-1)进行染色体畸变实验。结果表明,牛黄解毒片在接触24h时和在加入活化系统S9接触6h时,高、中、低三个剂量组的染色体畸变率均小于5%,为阴性反应；而牛黄解毒片在接触48h时,中剂量组的畸变率为8.5%,大于5%为可疑阳性反应,高、低二个剂量组畸变率小于5%,为阴性反应。1.1.3牛黄解毒片对CHL细胞的DNA损伤作用应用单细胞凝胶电泳实验(SCGE)检测了牛黄解毒片对CHL细胞DNA链的断裂作用,结果显示,CHL细胞接触牛黄解毒片水溶液48h后,中、低剂量组与阴性对照组比较,差异无显著性；而高剂量组与阴性对照组比较,差异有显著性(P<0.05),表明本实验条件下,牛黄解毒片水溶液高浓度时会引起体外培养CHL细胞DNA的损伤。1.2牛黄解毒片遗传毒性的整体动物实验研究1.2.1牛黄解毒片对小鼠体细胞染色体完整性与分离改变的影响采用经口给药,进行小鼠骨髓嗜多染红细胞微核试验。设立三个剂量组(3345、1670、835mg.kg-1)和一个阳性对照组(环磷酰胺,按40mg.kg-1腹腔注射)及一个阴性对照组,按两种方案(“0”和“24”小时两次给药和连续6周给药)检测牛黄解毒片对染色体的影响。结果表明,“0”与“24”小时两次给药,各剂量组与阴性对照组比较均无显著性差异(P>0.05),提示本方案下,牛黄解毒片对小鼠体细胞染色体的完整性和染色体分离无明显影响。但给药6w后,在剂量为1672mg.kg-1及3340mg.kg-1时,小鼠骨髓嗜多染红细胞微核率与阴性对照组比较差异有统计学意义(P<0.05),且各个剂量组之间存在明显剂量反应关系(r=0.999,P=0.023),提示给药6w,牛黄解毒片可引起小鼠骨髓嗜多染红细胞微核率增加。1.2.2牛黄解毒片对小鼠生殖细胞遗传损伤的影响采用经口给药,进行小鼠精子畸形试验。设立三个剂量组(3345、1670、835mg.kg-1)和一个阳性对照组(环磷酰胺,按40mg.kg-1腹腔注射)及一个阴性对照组,按两种方案(5d给药和连续6周给药)检测牛黄解毒片对小鼠生殖细胞的遗传损伤作用。结果显示,两种给药方案,各剂量组与阴性对照组比较均无显著性差异(P>0.05),表明本实验条件下,牛黄解毒片对小鼠生殖细胞无明显遗传损伤作用。1.2.3牛黄解毒片对小鼠主要组织细胞DNA链断裂作用的检测采用单细胞凝胶电泳技术(SCGE),检测不同给药剂量(3345、1670、835mg.kg-1)及不同给药时段(1w、2w、3w、4w和5w)的牛黄解毒片对小鼠外周血细胞DNA的断裂情况,并于末次给药后检测牛黄解毒片对肺、肝、肾、脾细胞DNA的断裂作用。结果显示,给药2w、3w时,高剂量组即可引起外周血淋巴细胞DNA链发生断裂,给药4w、5w时,高、中、低三个剂量均可引起外周血淋巴细胞DNA发生断裂作用；牛黄解毒片经口给药6w后,高、中剂量组肝、肺细胞及高剂量组的肾细胞的DNA损伤与阴性对照组比较,差异有统计学意义,表明牛黄解毒片经口给药6w可能会致肝、肺及肾细胞DNA损伤作用。初步结论：以上研究结果表明,在体内、外实验条件下,牛黄解毒片无明显遗传毒性作用,但随着给药剂量及给药时间的延长,牛黄解毒片可能具有一定蓄积性的遗传毒性；2牛黄解毒片配伍减低雄黄砷毒性作用初探2.1牛黄解毒片配伍对雄黄可溶性砷含量的影响采用原子荧光法测定牛黄解毒片全方及其不同拆方组中的可溶性砷盐的含量。结果显示,不管是人工胃液还是人工肠液处理,牛黄解毒片全方配伍后,均可明显降低雄黄中可溶性砷含量,并且,牛黄解毒片去甘草、黄芩、大黄后,其可溶性砷又明显升高,这表明牛黄解毒片全方配伍对雄黄可能有减毒作用,而甘草、黄芩、大黄三味中药可能是发挥减毒作用的药物。2.2牛黄解毒片配伍减低雄黄砷毒性的动物实验研究2.2.1牛黄解毒片配伍对雄黄中砷吸收分布的影响采用原子荧光光谱法测定并分析牛黄解毒片全方配伍对雄黄中砷在机体内的吸收与分布的影响。结果显示,给药9周后,雄黄组的肝、肾、脾、心、肺组织砷浓度均高于阴性对照组,差异有统计学意义(P<0.05),进一步证实雄黄中砷在机体内可被吸收并分布。各组与全方组比较,雄黄组的肝、肾、脾、心、肺组织砷浓度均明显高于全方组(P<0.05),而全方去甘草、黄芩、大黄组的肝、脾、心组织砷浓度明显高于全方组(P<0.05),这提示牛黄解毒片全方配伍后可减少雄黄中砷在机体内的吸收与分布,并且去除甘草、黄芩、大黄三味中药后,部分机体组织对雄黄中砷的吸收可能增加。2.2.2牛黄解毒片配伍对雄黄遗传毒性的影响采用彗星技术、微核试验等测定牛黄解毒片全方及其拆方对遗传损伤的影响。研究显示,与阴性对照组比较,单味雄黄组小鼠血细胞DNA损伤、骨髓嗜多染红细胞微核形成率增加及精子畸变率均升高(P<0.05)；与雄黄组比较,牛黄解毒片全方组小鼠血细胞DNA损伤与微核形成率明显降低(P<0.05),精子畸变率无明显变化；全方去甘草、黄芩、大黄后,与全方组比较,DNA损伤作用明显增强,但微核形成率、精子畸形率未见明显改变。表明牛黄解毒片配伍可能会减低雄黄的遗传损伤作用,但具体药味尚不能明确与甘草、黄芩、大黄有关。2.2.3牛黄解毒片配伍对雄黄肝肾毒性的影响采用病理组织学观察,分析牛黄解毒片配伍对雄黄肝肾毒性的影响。结果显示,雄黄组肝组织细胞显著水肿,局部有坏死；而肾脏组织出现局部肾小管细胞重度水肿；牛黄解毒片全方组及牛黄解毒片去雄黄组的肝肾组织未见明显异常,表明牛黄解毒片配伍能减低雄黄组肝肾毒性。2.2.4牛黄解毒片配伍对肝肾组织氧化损伤的影响采用生物化学方法,测定各组肝肾组织脂质过氧化物MDA含量、抗氧化酶SOD和GSH-px的活力。结果显示,与阴性对照组及与牛黄解毒片全方组比较,雄黄组的肝、肾组织中GSH-px酶、SOD活力活力均显著下降(P<0.05),而MDA均明显升高(P<0.05),这表明雄黄的肝肾毒性可能与氧化损伤作用有关,而牛黄解毒片配伍减低雄黄肝肾毒性可能与抗氧化损伤有关。全方去甘草、黄芩、大黄组与全方组比较未见有明显差异,提示甘草、黄芩、大黄三味药在方中抗氧化尚不能明确。2.2.5牛黄解毒片配伍对细胞凋亡的影响采用TUNNEL、免疫组化法测定牛黄解毒片配伍对肝细胞凋亡和凋亡相关分子表达的影响。结果显示,与阴性对照组比较,雄黄组的TUNEL阳性细胞数明显增加,Bax表达平均光密度明显增加,Bcl2表达平均光密度明显减少(P<0.05)：牛黄解毒片配伍后,TUNEL阳性细胞数较雄黄组明显减少,Bax表达平均光密度明显减少,Bcl2表达平均光密度明显增多(P<0.05)。这表明诱导肝细胞凋亡可能是雄黄引起肝损伤的可能机制；牛黄解毒片配伍对雄黄的诱导凋亡作用产生了抑制,抑制过程可能是通过促进Bcl2表达而阻碍bax表达来实现的。全方去甘草、黄芩、大黄组凋亡细胞数较全方组增加,Bax表达明显增加,但Bcl2表达无明显变化。初步结论：牛黄解毒片配伍可以减低雄黄的砷毒性(肝肾毒性、遗传毒性),甘草、黄芩及大黄在方中可能是发挥减毒作用的重要药味,机制可能与减少可溶性砷的吸收与分布、抗氧化、抗凋亡有关。更多还原

【Abstract】 The Niuhuang jiedu tablet is a representative of Qingrejiedu prescription in TCM, usually using for filled hot, sore throat, sore gums, tongue festered, red and swelling eyes and etc. For the evaluation Niuhuangjiedu security, the Ames test, chromosome aberration analysis, single cell gel electrophoresis assay (SCGE) and micronucleus tests were used, respectively, to explore the potential genotoxic effects of Niuhuang jiedu tablet. In addition, compatibility detoxification is one of the most characteristic methods and techniques for TCM, to explore whether the compatibility for Niuhuang jiedu tablet could reduce the toxicity of realgar and what is its possible mechanisms, different groups about Niuhuangjiedu tablet and its disassembled presciption were arranged, and soluble arsenic, arsenic in the body tissue distribution, pathological damage, oxidative damage, genetic damage, apoptosis etal, were detected.1Genetoxicity evaluation for Niuhuang jiedu tablet1.1Genetoxicity studies of Niuhuang jiedu tablet in in vitro bioassays1.1.1Effects of Niuhuangjiedu tablet on gene reverse mutation Plate incorporation assay was adopted, in accordance with Maron, DM and Ames BN recommended procedures. Based on the pre-test results,5mg/dish was arranged as the highest dose group, then,1.5,0.5,0.15,0.05mg/dish, negtive and positive control groups were also arranged to carry out the experiment. The results showed no difference on gene reverse mutation of Trphoid Salmonella Strains, from0.05mg/dish to5mg/dish, both with and without S9mixture, and replies colonies number of various strains did not exceed2times the negative control group, and dose-response relationship was not showed.1.1.2Chromosomal aberrations effect of Niuhuangjiedu tablet on CHL cells The IC50of Niuhuangjiedu tablet was determined as54μg.ml-1, by cell counting method. Dose range for chromosome aberration test was1.0,1/2,and1/4IC50values, i.e,54,27.13.5μg.ml-1respectively. The results showed that chromosome aberration rate of all dose group were less than5%, at24hrs exposure, and at9hrs exposure with the activation system S9, which is a negative response, chromosome aberration rate of the middle dose group was8.5%, which is greater than5%, and is a suspicious positive, at48hrs exposure, but chromosome aberration rate of high and low dose group is less than5%.1.1.3DNA damage effect of Niuhuangjiedu tablet on CHL cells The DNA strand break effect of Niuhuangjiedu tablet on CHL cells was tested by SCGE. Dose range was also1.0,1/2,and1/4IC50values, i.e,54,27.13.5μg.ml-1respectively. The results showed DNA damage rised at the54μg.ml-1dose group after48hrs exposure in comparation with the negative control group, and the difference was significant (P<0.05), but there were no difference was found at medium and low dose group, which means Niuhuangjiedu tablet could induce DNA strand break of CHL cells at high dose.1.2Genetoxicity studies of the Niuhuangjiedu tablet in in vivo assays1.2.1The influence of Niuhuangjiedu tablet on chromosome integrity and separation changes Bone marrow PCE cell micronucleus test in mice exposed Niuhuangjiedu tablet through oral delivery with three dose groups (3345,1670,835mg.kg-1) was carried out, and two administering drugs regimens ("0" and "24" hrs administration at twice and6weeks continuous administration) were arranged. The results showed that the "0" and "24" hrs administration at twice, no significant difference (P>0.05)was found at each dose group,which suggested that under this regimen, the Niuhuangjiedu tablet had no influence on mice chromosome integrity and chromosome segregation. But under6weeks administration regimen, micronucleus rate at dose of1672mg.kg-1and3345mg.kg-1was found higher than that of negative control group (P <0.05), and the obvious dose-response relationship (r=0.999, P=0.023)was found, which means Niuhuangjiedu tablet could increase the mouse bone marrow cells micronucleus rate under long time administration.1.2.2Sperm abnormality test in mice exposed to Niuhuangjiedu tablet Sperm abnormality test in mice exposed to Niuhuangjiedu tablet was carried out with three dose groups (3345,1670,835mg.kg-1), and the two administering drugs regimens (5d days administration and6weeks administration) were arranged. The results showed each dose group and the negative control group showed no significant difference (P>0.05) under two regimens, which means Niuhuangjiedu tablet has no significant genetic damage effect on germ cells in mice under this experimental conditions.1.2.3DNA strand break of main tissues and organs cells in mice exposed to Niuhuangjiedu tablet SCGE technique was used to detect DNA strand breaks of peripheral blood cell in mice exposed to Niuhuangjiedu tablet, at different dose (3345,1670,835mg.kg-1) for different administration times (1w,2w,3w,4w and5w), and after the last administration, DNA strand breaks of mice lung, liver, kidney, spleen cell were also detected. The results showed that the DNA of peripheral blood lymphocytes began to perform strand breaks at administration2w or3w with high dose, and then significant DNA strand breaks were detected at all dose groups after administration4w or5w. Significant DNA strand breaks were also detected in cells of liver, lung cells at high and medium dose groups, and in renal cell at high dose groups, when compared with negtive control group, which indicates that Niuhuangjiedu tablet may induce DNA damage of liver, lung and renal cell after administration6w and through oral delivery.Preliminary Conclusion:Niuhuangjiedu tablet had no obvious genetoxicity in in vitro and vivo experimental conditions, but with the increase of administration dose and the extension of administration time, which may have cumulative genetoxicity. 2The preniminary exploration on Niuhuangjiedu compatibility reducing realgar arsenic toxicity2.1Determination of Soluble Arsenic in NiuhuangJiedu Tablet and its disassembled presciption Different groups about NiuhuangJiedu Tablet and its disassembled presciption were arranged, treated with artificial gastric juice or artificial intestine juice, then Atom Fluorescence Spectrophotometry(AFS) was used to detected the content of soluble Arsenic in the different groups samples. The result showed the content of soluble Arsenic in the whole presciption of NiuhuangJiedu Tablet was significant lower than that in the single relgar (P<0.01), the content of soluble Arsenic in the whole presciption with Gancao,Hangqin,Dahuang excluded was significant higher than that in the whole presciption of NiuhuangJiedu Tablet (P<0.01),which suggested the compatibility for Niuhuangjiedu Tablet could reduce the toxicity of realgar,and Gancao,Hangqin and Dahuang may be the possible herbal medicines that play the role of reducing toxicity.2.2Animal experiment studies on Niuhuangjiedu Tablet compatibility reducing realgar arsenic toxicity2.2.1The influence of Niuhuangjiedu tablet compatibility on the absorption and distribution of arsenic in realgar The AFS was used to detect and analye the influence of Niuhuangjiedu tablet on the absorption and distribution of arsenic in realgar. The results showed arsenic content in liver, kidney, spleen, heart and lung tissue in the single realgar was significantly higher than that of negtive group after administration9W (P<0.05), which confirmed that the arsenic in realgar could be absorpted and distributed in bodies. Compared with the whole presciption of NiuhuangJiedu Tablet, arsenic content in liver, kidney, spleen, heart and lung tissue in realgar was significantly higher (P<0.05), and arsenic content in liver, spleen, heart tissue in the whole presciption with Gancao,Hangqin,Dahuang excluded was also significant higher (P<0.05) which suggested the compatibility for Niuhuangjiedu Tablet could reduce the absorption and distribution of arsenic in realgar, and the absorption and distribution of arsenic in realgar may increase if Gancao, Hangqin.Dahuang in whole presciption of NiuhuangJiedu Tablet are excluded.2.2.2The influence of Niuhuangjiedu tablet compatibility on the oxidative stress The lipid peroxide product MDA and antioxidant enzymes SOD and GSH-px were determined to explore the influence of Niuhuangjiedu tablet compatibility on the oxidative damage. The results showed GSH-px enzyme activity in the liver, kidney, lung, heart tissue and SOD enzyme activity in liver kidney, spleen tissue decreased significantly (P<0.05).and MDA content in liver, kidney, heart tissue increased significantly (P<0.05),when single realgar compared with the negtive group. However, the GSH-px and SOD enzyme activity and MDA content in the whole presciption and in the whole presciption with Gancao, Hangqin, Dahuang excluded showed no significant change (P>0.05), which indicates that the realgar could cause oxidative damage, and Niuhuangjiedu compatibility could reduce oxidative damage caused by realgar, but which are the main herbs that play the role of antioxidative damage may not be determined.2.2.3The influence of Niuhuangjiedu tablet compatibility on genetic damage Comet technology, the micronucleus test, etc were used to determined the influence of Niuhuangjiedu tablet compatibility on genetic damage. The results showed the DNA damage in peripheral blood cell, micronucle formation rate in bone marrow PCE cell, and the sperm abnormality rate in single realgar increased signiffcantly (P<0.05),when compared with the negative group. DNA damage, micronucleus formation rate decreased signiffcantly (P<0.05), sperm abnormality rate had no signiffcant difference in the whole presciptio of Niuhuangjiedu tablet in comparation with single realgar; DNA damage, micronucle formation rate and sperm abnormality rate in the whole presciption with Gancao,Hangqin,Dahuang excluded showed no significant change. These results indicates that the Niuhuangjiedu compatibility could reduce the genetic injury of realgar, but the specific herbs that play role of antigenetic damage was not yet determined.2.2.4The influence of Niuhuangjiedu tablet compatibility on liver and kidney toxicity The pathological and hisological observation was used to analye the influence of Niuhuangjiedu tablet compatibility on liver and kidney toxicity of realgar. The results showed that liver tissue in the realgar was significantly edema, local necrosis; and local renal tubular cells in kidney tissues in the realgar was severe edema; The liver and kidney tissues showed no abnormal changes except local edema in the whole presciption of NiuhuangJiedu Tablet and the whole presciption with realgar excluded, which suggested Niuhuangjiedu tablet compatibility may could reduce the liver and kidney toxicity of realgar. The liver tissue showed mild edema and kidney tubular epithelial cells showed moderate edema in the whole presciption with Gancao, Hangqin, Dahuang excluded, which indicates the liver and kidney toxicity may be enhanced with Gancao, Hangqin, Dahuang removed from the whole presciption of NiuhuangJiedu.2.2.5The influence of Niuhuangjiedu tablet compatibility on liver apoptosis The TUNEL and immunohistochemical methods were used to determine the influence of Niuhuangjiedu tablet compatibility on liver apoptosis and apoptosis related molecules expression. Results showed the TUNEL positive cell numbers and mean density of Bax expression increased significantly, but mean density of Bcl2expression reduced significantly, when realgar group compared with negative group (P<0.05). However, TUNEL-positive cell numbers and mean density of Bax expression decreased significantly, and mean density of Bcl2expression increased significantly, when the whole presciption compared with the single realgar (P<0.05), indicates the Niuhuangjiedu compatibility may inhibit liver apoptosis that realgar induced, and such inhibition may be achieved by promoting bcl2expression and inhibiting bax expression. The whole presciption with Gancao, Hangqin, Dahuang excluded in compared with the whole presciption, the TUNEL positive cell numbers and mean density of Bax expression increased significantly, but mean density of Bcl2expression had no apparent changes, which indicated Gancao, Hangqin. Dahuang in the whole presciption may reduce the liver toxicity of realgar by antiapoptosis.Preliminary Conclusion:The compatibility of Niuhuangjiedu tablet may reduce the asenic toxicity of realgar, and reducing absorption and distribution of soluble arsenic, antioxidative damage, antiapoptosis, and et al maybe the possible mechanisms. The specific herbs that reduced asenic toxicity of realgar were not yet determined in this study, which needs further research.更多还原