千里光肝脏毒性研究

【作者】 王秀坤；

【导师】 梁爱华；

【作者基本信息】 中国中医科学院 ， 中西医结合基础， 2008， 博士

【摘要】 千里光属植物Senecio spp.在世界各地分布广泛,全球约有1500多种,我国约有160余种,其中药用品种38种。在国外有人服用后中毒死亡的报道,而我国药典只收载了一种千里光Senecio scandens Bach-Ham,这个品种在我国临床上未见导致中毒的报道。虽然在我国历史上对其毒性认识并不统一,但一直延用至今。有研究表明千里光属植物普遍含有吡咯里西啶类生物碱(PAs),不饱和吡咯里西啶类生物碱对动物和人具有明显的肝脏毒性。国外的千里光品种与我国药用品种也有差异,毒性强弱不同。千里光在我国很多地区都有广泛的分布,不同地区的千里光的PAs含量以及毒性是否有差别也没有研究数据,不同植物的PAs含量相差较大,毒性强弱也不一。另外,在我国,多数使用的是复方,而不是纯的PAs。复方中由于成分很复杂,众多成分之间相互影响,因此千柏鼻炎片的毒性是不能简单地与PAs或千里光划等号的。但是,由于目前国内外对S.scandens Bach-Ham的化学和毒理没有研究,因此该品种是否有毒、毒性强弱及其特点、其是否含有不饱和PAs、含量有多少、中药复方配伍是否能够减毒等问题均不清楚。PAs的中毒机理,尤其在分子水平上目前尚未完全阐述,应深入对其进行研究。为了搞清楚上述问题,本研究采用千里光总生物碱、千里光单味药提取物、千柏鼻炎片进行平行比较,结合化学研究和PAs含量测定,从急性毒性、蓄积毒性、遗传毒性等方面,研究了三者之间的差别,以期揭示千里光的毒性作用及其特点,生物碱含量与毒性之间的量—毒关系、毒—效关系以及复方配伍减毒作用等,为千里光的安全合理应用提供科学依据。检测千里光致大鼠肝细胞DNA损伤的方法。选用四种PAs单体化合物IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine对Hep G2细胞毒性进行研究,探讨GSH对于肝细胞的保护作用,并采用免疫荧光和WESTERN-BLOT等方法,考察细胞小分子蛋白与PAs的毒性关系,不仅对千里光的全面深入了解发挥一些作用,而且对于含PAs的其他种属植物的毒性的预防和治疗,提供参考。本论文进行了以下几个方面的研究:1.急性毒性研究1.1不同产地千里光的总生物碱含量及其急性毒性比较研究为了搞清楚我国主要产区千里光的PAs含量和比较它们的毒性差异,为临床和生产中选择安全性高的药材提供科学依据。比较了全国主要的代表性产区采集的千里光PAs含量和急性毒性。结果显示不同产地的千里光毒性有很大的差别,其毒性强弱与千里光总生物碱含量有一定关系。其中,河南产千里光的生物碱含量最高,毒性也最强,而江苏、浙江千里光也有一定的毒性,可造成少量动物死亡,但是二者的毒性较河南产千里光的毒性明显减弱,其PAs含量也相对较低。湖北和广西产千里光的生物碱含量最低,其毒性也最小。河南产千里光死亡和存活动物肝脏病理学检查显示具有明显的肝脏毒性作用,主要发现肝脏中央静脉扩张淤血、局灶性肝窦轻度扩张淤血、双核细胞和轻度钙化灶。结果提示,在临床用药和含千里光的中成药生产中,应该注意产地选择,选择PAs含量低,毒性小的千里光入药,以保证安全性。1.2千里光及其复方制剂经口用药急性毒性研究本研究选择毒性最大的河南千里光,进行了成年小鼠、大鼠以及幼年小鼠的LD50测定,以分析其急性用药的量—毒关系,在其安全剂量范围内用药,可以保证用药安全,为临床用药提供参考。结果显示成年小鼠、幼年小鼠和大鼠一次性灌胃给予千里光水提物,在剂量过高时均显示出有明显的毒性和致死性,并有明显的量—毒关系。对小鼠与大鼠的量—毒关系比较,可见大鼠对千里光的耐受较小鼠更强,表明千里光的毒性有一定的种属差异。成年小鼠一次性灌胃给予千柏鼻炎片水提物(含河南千里光),在剂量过高时显示出有明显的毒性和致死性,并有明显的量—毒关系,千柏鼻炎片的毒性略低于千里光提取物毒性。2.千里光及其复方制剂长期用药的肝脏毒性研究采用毒性最强的河南产千里光的水提物、千柏鼻炎片及总生物碱部分,以20.0、6.0、3.0、1.5 g生药/kg四个剂量水平(相当于临床剂量的100、30、15、7.5倍)给大鼠连续灌胃给药4w,观察对肝脏的毒性。结果显示千里光水提物、总生物碱提取物、千柏鼻炎片分别结果表明,受试物的20.0 g生药/kg剂量组对凝血指标APTT有一定缩短作用,千里光无论是单味药、总生物碱提取部分还是复方,对肝脏生化指标AST均有一定的影响。组织病理学显示三种受试物的20.0g生药/kg剂量组对大鼠肝脏有轻度毒性反应。3千里光致毒机理研究3.1千里光致大鼠肝脏细胞DNA损伤的研究采用反复灌胃给药4w的千里光水提取物大鼠肝脏,利用彗星试验检测千里光提取物对大鼠肝细胞DNA损伤的遗传终点即DNA断裂。结果显示千里光3个剂量均能使肝细胞脱尾率及DNA迁移率与对照组相比显著提高,且肝细胞拖尾率与剂量存在相关性,说明连续灌胃30天给予大鼠河南产的千里光能够导致大鼠肝细胞DNA断裂,造成DNA损伤,损伤的频率随剂量的增加而增大,故该种千里光可导致肝细胞DNA发生损伤及影响肝功能。3.2千里光体内致突变作用研究本实验采用反复灌胃给药4w的大鼠股骨骨髓涂片,进行骨髓细胞微核试验,以确定千里光、千柏鼻炎片和总生物碱致突变作用。结果显示三种受试物20.0g生药/kg剂量下均能显著升高大鼠骨髓细胞微核率,与对照组比较有非常显著差异(P＜0.05或P＜0.01)。结果表明千里光、千柏鼻炎片和总生物碱具有一定的升高骨髓细胞微核率的作用,但是作用较弱。3.3 PAs对Hep G2肝细胞毒性研究运用Hep G2细胞作模型,研究IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine四种PAs单体化合物的细胞毒性,考察致毒过程中生化指标的变化及对肝细胞DNA损伤的影响,预测含HPAs植物药的毒性。结果显示:选用的四种PAs单体化合物IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine在各自的高浓度时对细胞皆有高抑制率,明显抑制细胞活性,Monocrataline、Retrorsine的细胞毒性较强。对生化指标测定结果显示,当细胞受损伤时,细胞内的生物活性酶就会大量释放到上清中,IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine能够明显影响肝细胞的CK、LDH生物活性酶,增加这两种活性酶的释放率;Monocrataline和Retrorsine细胞毒性相对较强,Monocrataline和Retrorsine的适当浓度能够使肝细胞AST、ALT的释放率增加。说明Monocrataline、Retrorsine比IntegerrimineA、B对肝细胞损伤程度高,对Hep G2细胞毒性更强。单细胞凝胶电泳结果表明四种PAs单体化合物可引起DNA迁移的变化,拖尾细胞率明显高于正常细胞。导致DNA段裂,导致细胞坏死和凋亡。3.4谷胱甘肽对PAs毒性的影响通过检测PAs致HEP G2细胞毒性GSH的含量变化,研究PAs致毒与GSH相关性,选用细胞活性抑制率和PAs肝细胞毒性显著性指标ALT,通过生物合成抑制剂丁胱亚磺酰亚胺(BSO)及合成GSH的前提物质N-乙酰半胱氨酸(NAC)的作用于Hep G2细胞分别抑制和增加细胞内GSH的含量,间接探讨GSH在PAs所致肝细胞毒性过程中的作用。结果显示:用Hep G2细胞探讨PAs致肝毒性GSH对其影响,发现PAs具有明显的肝细胞毒性,GSH合成抑制剂BSO可使PAs肝细胞毒性明显增强,而GSH合成前体物半胱氨酸可使PAs肝细胞毒性减弱,间接说是GSH对PAs肝细胞毒性具有保护作用。在肝细胞培养液中加入GSH合成前体物半胱氨酸,肝细胞可摄取半胱氨酸并用来合成GSH,4 h后,细胞内GSH浓度可提高。在肝细胞培养基中加入GSH合成抑制剂BSO,肝细胞不能继续合成GSH,原有的GSH经过16 h的逐渐消耗,肝细胞GSH浓度降低。BSO和半胱氨酸对肝细胞GSH浓度有完全相反的影响,对PAS肝细胞毒性也具有完全相反的作用,因此,可以认为细胞内GSH与PAs肝细胞毒性相关。3.5 PAs对Hep G2细胞的小分子蛋白及细胞因子的影响充分利用细胞生物学、分子生物学、免疫化学等手段深入研究IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine的细胞毒性,研究其对VEGF、GM130、Cav-1的影响,进一步探讨其对肝脏的毒性机制。结果显示:PAs致Hep G2细胞毒后VEGF呈现高表达,且IntegerrimineA、Monocrataline、Retrorsine在致HEP G2细胞毒24h时,Hep G2表达VEGF呈高峰,IntegerrimineA、Monocrataline、Retrorsine诱导VEGF具有时间依赖性。Hep G2细胞分别经四种不同PAs(IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine)作用后经免疫荧光标记,四种吡咯里西啶生物碱均能降低Hep G2胞浆内GM130的含量,IntegerrimineB、Monocrataline、Retrorsine能够升高Hep G2细胞膜及胞浆内Cav-1的含量。PAs致细胞毒性的机制可能是通过增加细胞膜及胞浆内的Cav-1,物质转运发生相应变化,通透性改变,肝细胞和其他非实质性细胞处于缺血缺氧状态,并且所合成和分泌的化学物、以及代谢产物不容易进入血液循环,降低GM130,影响细胞内质网功能,加重肝脏病理性损伤,促进肝纤维化形成过程。更多还原

【Abstract】 Senecio are distributed widely and have 1500 species in the world.In other country it be reported having toxicity.There are more than 160 species which 38 medicinal species be included in our country,but it has no reported in clinic.Though it be recognise differently in toxicity in history,it is be used as yet.Previous studies showed that Senecio commonly contained PAs.Unsaturated PAs have hepatic toxicity obviously.Foreign Senecio are different in species and toxic intensity from ours’.Senecio existed in our coutry widely,it has no research in the diferent of PAs content and toxic in the different area.In addition,it is used mostly in complex preparation in our country,but not pure PAs.because complex preparation have complex components which affect and act each other,the toxic relationship between complex preparation and PAs or single herb can’t be over simplified into a sign of equivalence.It have no studies about chemistry and toxicology of S.scandens Bach-Ham at home and abroad at present,so this species is unclear in character and toxic tensity,wether it had any toxic and what about unsaturated PAs the function in detoxic about complex preparation.It is not be confirmed completely in the toxic mechanism,especially in the molecular level,so it should be reseach deeply.In order to make these clear.We take the methods of parallel comparing climbing water extract of groundsel herb with climbing groundsel herb total alkloids or Qianbaibiyanpian from the aspect of acute toxic accumulation toxic and genetic toxic combining chemistry experiment and contents of PAs. Studying the difference of these three,revealing climbing groundsel herb toxic character,the relationeship between toxic and quantity,the relationship between toxic and efficiency.All these studies can provide scientific discipline for climbing groundsel herb safty and reasonable application.To investgate DNA damages of livers of rats exposed to Climbling Groudsel Herb.We select four mono-compounds IntegerrimineA,IntegerrimineB,Monocrataline,Retrorsine to studies their toxicity to Hep G2 cell.Researching effection of GSH on pretecting the hepatic cell, using the methods of immunofluorescence and WESTERN-BLOT,studies the relationship micromolecule protein with the toxic of PAs,all these studies not only play some effect on getting the message of climbing groundsel herb,but also provide some information to prevent and treat the PA’s toxicity of other genera botanicThis thesis take the following studies:Acute toxicity studies1.1 Comparative study on total alkloids and acute toxicity of climbing groundsel herb from different producing areaIn order make clear PAs content of climbing groundsel herb of different producing area and compare the difference of their toxic to offer science basis for select safty medical herb for clinic and production.We compare the toxic and PAs content of climbing groundsel herb from representative area of our country.The results show that the toxicity of climbing groundsel herb varies with its producing area and has relationship with its conent of PAs.The climbing groundsel herb from Henan has the the most PAs and the highest toxic one among these herbs.There is some degree of toxicity in climbing groundsel herb from Jiangsu,Zhejiang,but their toxicity are weaker and the PAs contents is lower than Henan.The toxicity of climbing groundsel herb varies with its producing area.No marked toxicity was found in climbing groundsel herb from Guangxi and Hubei,content of PAs in there are the lowest than others.There is some degree of toxicity in climbing groundsel herb from Jiangsu, Zhejiang..Pathological examination of died and survival mice has obviously hepatic toxic,A microscopic examination revealed more binuclear,macronuclear,and irregular nuclear cells in the liver,inflammatory cellular infiltration,multiple calcific foci,and dilatation and steas in hepatic central veins and hepatic sinusoid.Result show that when it is be used in clinic and Chinese formulated products produing,it should be select the specis which has lower contents of PAs and toxic to ensure its safty.1.2 Studies on acute toxicity of climbing groundsel herb and its compound preparation.Climbing groundsel herb from Henan which the toxic is highest are seclected to detect LD50 of mice rats and young mice,analysis the relationship of quantity and toxicity of acute medication. It can ensure its safty and provide information of clinic medication.The results show that mice,rats and young mice are perfused to the stomach with water extracts of climbing groundsel herb of Henan.It show obviously toxic and fetal when the highest dosage be used.It also has relationship with quantity and toxicity obviously.Compared on the relationship of quantity and toxicity in mice and rats.The tolerance in rates is greater than in mice.so climbing groundsel herb’s toxicity is related to species difference.Water extacts of Qianbaibiyanpian were perfused to adult mice,it show obviously toxic and fetal when the highest dosage be used and also has the relationship with quantity and toxicity.The toxicity of Qianbaibiyanpian contained Henan climbing groundsel herb is lower than Water extacts of climbing groundsel herb.2.Studies of hepatic toxicity of long-term use climbing groundsel herb and its compound preparation.Water extracts,compound preparation and total alkloids of Henan climbing groundsel herb which the toxicity is highest are seclected four dosage of 1.5,3.0,6.0,20.0g/kg and perfused to rats 4 weeks tobserved the hepatic toxicity.Result show that 20.0g/kg dosage of this three agent can reduce APTT.This three agent can effect the biochemical indicator AST.Histopathology show that 20.0g/kg dosage of those agent had the mild heptic toxicity.3.Studies on the mechanism of toxicity of climbing groundsel herb3.1 Studies on the damages of livers of rats exposed to Climbling Groudsel Herb.Liver of Rats which was perfused to water extracts of climbing groundsel herb.DNA damages were detected by single cell gel electrophoresis(comet assay).The results show that The incidence rates of comet cells in the experimental groups were significantly higher than those of the controls.Furthermore the incidence rates of comet has the relationship with the dosage.It demonstration that water extrats of climbling Groudsel Herb could induce DNA break and damage of rats hepatic cell cells.The frequency of damage is increase with dosage.So climbing groundsel herb can induce the damage DNA of hepatic cell and change of hepatic functions.3.2 Studies on the mutagenic action of climbing groundsel herb.Rats which was perfused to water extracts of climbing groundsel herb,then femoral bone was be maken to bone marrow slides,taking the bone marrow cell micronucleus test to ensure the mutagenic ation of climbing groundsel herb,total alkloids and Qianbaibiyanpian.Results show that 20.0g/kg dosage of this three objects can increase bone marrow cell micronuclear rates obviously and the data were significantly different compared with the control group(P＜0.05orP＜0.01).It demonstrate that climbing groundsel herb,total alkloids and Qianbaibiyanpian has the function of improving the one marrow cell micronuclear rates,but the function is weak3.3 Studies of the hepatic celluar toxicity of PAs on Hep G2.Using the model of Hep G2,We research the four chemical compounds of IntegerrimineA,IntegerrimineB,Monocrataline and Retrorsine cell toxicity,studies the changance of biochemical indicator and the effect to hepatic cell DNA in the progress of induing toxicity.All this can anticipate the toxicity of botanic herb which contents PAs.The results show that this four chemical compounds IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine has the higher inhibition ratio when it be used in the highest dosage.It can inhibit cell activity.Cell toxicity of Monocrataline、Retrorsine is higher than two other.Biochemical indicator was be detected and results show that biological enzyme in the cell has be released to the cell supematant,IntegerrimineA、IntegerrimineB、Monocrataline、Retrorsine can effect CK,LDH of hepatic cell,increase the release rate of this two biological enzyme. Monocrataline and Retrorsine has higher cell toxity than the other two objects,the suitable dosage of this two objects can increase the release ratio of AST,ALT.All this demonstrate Monocrataline、Retrorsine can induce serious hepatic cellular damage,especially to the Hep G2.Single cell gel electrophoresis(comet assay)show that this four chemical compounds can cause the DNA immigration,The incidence rates of comet cells in the experimental groups were significantly higher than those of the controls.It induce breaking of DNA and cellular necrosis and apoptosis.3.4 The effect of GSH on the toxicity of PAsContents of GSH was be detected when PAs cause the Hep G2 cellular toxicity.Studies the relationship between the toxicity of PAs and GSH.Cytoactive inhibition ratio and ALT which is the obvious indicator for the heptic cellular toxicity of PAs.BSO which is the catastaltic of GSH and NAC which is precurosor of GSH effect on the Hep G2,then contents of GSH in cell be inhibited and increase respectively.Approach the function of GSH in the process of PAs effect the heptic cellular toxicity.The results show that PAs have heptic cellular toxicity when we studies the effect of GSH on the PAs’ hepatic cellular toxicity in Hep G2.BSO can increase the PAs’ heptic cellular toxicity,NAC can reduce the PAs’ heptic cellular toxicity,GSH can protect the cell indirectly. When NAC be added in the heptic cell culture fluid,cell can intake the NAC to syntheise GSH. Four hours later,concentration of GSH will increase.While,BSO be added in the heptic cell culture fluid,cell can’ t continue to syntheise GSH.Intrinsic GSH has been exhausted, concentration of GSH in cell lower.So BSO and NAC have adverse effect on concentration of GSH in cell,and PAs’ hepatic toxicity.Therefore,It can be consumed that GSH in cell has the relationship with PAs’ heptic celluar toxicity.3.5 The effect of PAs to the micromolecule protein and cell cytokine in Hep G2Utilization the method of cytobiology,molecula biological,immunochemistry to studies the cellular toxicity of IntegerrimineA,IntegerrimineB,Monocrataline,Retrorsine,studies what’s effect on the VEGF、GM130、Cav-1 and research its mechanism ofheptic toxicity.The results show that VEGF in Hep G2 has higher expression when PAs caused the cell toxicity.After IntegerrimineA,Monocrataline,Retrorsine effect cell 24 hours,VEGF in Hep G2 higher express.IntegerrimineA、Monocrataline、Retrorsine induce VEGF which have time dependence.When PALS effect Hep G2,cell be labeled with immunofluorence,this four PAs could reduce concentration of GM130 in intracytoplasm,IntegerrimineB、Monocrataline、Retrorsine could increase the concentration of Cav-1 in cytomembrance and intracytoplasm in Hep G2.The mechanism of PAs cellular toxicity may be that increasing Cav-1 can change the material transport and permeability,GM130 reducence can effect the function of endocytoplasmic reticulum,aggravate the damage of hepatic,promot the process of hepatic fibrosis.更多还原