基于单抗敲除技术的葛根素与葛根改善SH-SY5Y细胞糖氧剥夺损伤炎症因子作用的量效关系研究

【作者】 吴婷婷；

【导师】 王庆国；

【作者基本信息】 北京中医药大学 ， 中医临床基础， 2014， 博士

【摘要】 中药药效物质基础是中医药现代化研究的热点和难点。中药方剂配伍理论现代机制研究、中医辨证论治现代内涵研究和中药新药研发都离不开中药药效物质的指引和证实。因此揭示中药药效物质基础一直是中医药现代化研究的关键。然而由于中药内成分繁多,又常以复方制剂应用,使得成分间交互作用极为复杂,因此确认有效成分非常困难。即便是《中国药典》记载的药物中,已明确有效成分的中药品种亦不到5%。而当前绝大部分中药用于质量控制的指标性成分仅根据含量多少而设定,不一定是为药材贡献主要药效的成分。这不仅制约着中医药理论现代化研究难以取得实质性的进展,而且容易引发药物安全性问题,因此中药药效物质基础研究又是中医现代化研究中亟待解决的难点问题。导师团队致力于中药药效物质基础研究多年,为解决这一问题,导师提出采用不同于以往“还原论”研究模式的,基于中药小分子单克隆抗体技术免疫“敲除”法的研究思路,通过特异性敲除中药中某一成分,比较敲除前后和单一成分三者之间的药效异同,不仅能够反映该成分单独作用的效果,更能反映该成分在原中药中发挥的作用与单独使用的差异,以此揭示该成分可能的交互作用和对该中药功效的真实贡献度。《中国药典》上以葛根素作为葛根质控的指标性成分,脑缺血是葛根及葛根素在临床上确有疗效且机制较为明确的适应症,经过多年的研究,虽然葛根素对脑缺血的作用机制已经被大量揭示,但是葛根素对葛根抗脑缺血功效的贡献度研究,尚未见报道。本研究以葛根为模式药物,以脑缺血的体外模型——SH-SY5Y细胞糖氧剥夺模型和为载体,通过建立基于葛根素单克隆抗体的葛根素免疫亲和色谱柱,特异性敲除葛根素后,比较葛根特异性敲除葛根素前后对该模型药效及炎症因子调节功能的量效改变,揭示葛根素对葛根功效的贡献度。第一章、葛根素免疫亲和色谱柱制备和敲除液制备和葛根提取液中葛根素的敲除目的：基于葛根素单克隆抗体,制备葛根素免疫亲和色谱柱,并对葛根提取液中的葛根素进行敲除,以获得后续研究所需的药物。方法：采用辛酸-硫酸铵法对葛根素单克隆抗体腹水进行纯化,采用SDS-PAGE法检测抗体纯度,Bradford法检测抗体浓度,ELISA法检测抗体效价和竞争,确认抗体具有制备免疫亲和色谱柱的效能；将纯化后的葛根素单抗与CNBr-activated SepharoseTM4B偶联,检测偶联比和偶联率,掌握亲和柱的性能；对葛根提取液进行上样敲除,采用HPLC法检测葛根提取液和敲除液中葛根素含量,计算敲除率,获得定量敲除葛根素的葛根敲除液。结果：SDS-PAGE结果显示纯化后葛根素单抗杂蛋白减少,纯度达92%；纯化前葛根素单抗腹水蛋白浓度为4.28mg/mL,纯化后的浓度为2.16mg/mL; ELISA检测显示纯化前后葛根素单抗的效价均约为稀释度1：51200,均与葛根素呈现良好的线性竞争,线性范围未发生明显变化；经计算,纯化后的葛根素单抗与CNBr-activated SepharoseTM4B凝胶的偶联率为99.3%,偶联比为3.4mg/mL;经HPLC法检测,原上样葛根溶液中约90%的葛根素已经被剔除。结论：辛酸-硫酸铵法纯化的葛根素单抗,去除了大量杂蛋白提高了抗体纯度,而抗体效价和竞争不受明显影响,能够满足制备免疫亲和色谱柱的需要；纯化后的葛根素单抗与CNBr-activated Sepharose TM4B凝胶偶联效果非常好,敲除率较高,能够满足药效实验的需要。第二章葛根提取液,葛根敲除液和葛根素对SH-SY5Y细胞糖氧剥夺损伤药效比较研究目的：考察葛根素SH-SY5Y细胞内的药代动力学过程揭示药效产生过程,建立稳定的SH-SY5Y细胞糖氧剥夺损伤模型,考察葛根提取液、葛根敲除液和葛根素对该模型诱导的细胞损伤的保护作用药效差异。方法：(1)考察3.9、7.8、15.6、31.3、62.5、125、250、500μg/mL8个不同浓度2h,8h,16h和24h四个不同时间葛根素对正常SH-SY5Y细胞的影响；选用31.25μg/mL、62.5μg/mL、125μg/mL三种浓度葛根素孵育细胞,于5、15、30、45、60、90、120、150、180、210、240、270、300、330、360、390、420、480、540、600min,共20个时间点取上清,间接竞争ELISA法检测上清中葛根素含量。给药浓度减去上清浓度即为细胞内葛根素浓度。(2)利用无糖细胞培养液和Na2S2O4诱导缺氧缺糖损伤模型,考察0.4、0.8、1.6,3.2mmol/L四种造模浓度,2、8、16、24h四种孵育时间的造模效果,在倒置相差显微镜下观察造模后的细胞形态,以MTT分析法测定细胞存活率和乳酸-丙酮酸法检测细胞上清中LDH水平。；(3)按葛根素的浓度和敲除前应含葛根素的浓度,将葛根提取液、葛根素和葛根敲除液分别设为3.9,7.8,15.6,31.3,62.5,125,200,250,500μg/mL九个浓度干预模型,以MTT分析法测定细胞存活率和乳酸-丙酮酸法检测细胞上清中LDH水平。结果：(1)3.9、7.8、15.6、31.3、62.5、125、250μg/mL葛根素在8小时内对细胞存活率和LDH水平均无显著影响；高、中、低三个剂量组的Cmax和AUClast呈剂量依赖性减小,中剂量组的Tmax和MRTlast高于低剂量组和高剂量组,提示该剂量在细胞内吸收过程的特殊性。(2)倒置相差显微镜下进行一般形态学动态观察,SH-SY5Y细胞经糖氧剥夺损伤后,严重变形,受损程度呈剂量和时间依赖性,16h和24h组各组细胞均损伤严重,多数死亡；通过测定细胞存活率和细胞上清中的LDH水平发现,造模8小时,0.8、1.6、3.2mmol/L剂量模型组的细胞存活率比正常组显著降低(P<0.01),造模8小时,0.4、0.8、1.6mmol/L剂量模型组细胞上清的LDH水平比正常组显著升高(P<0.01)。(3)葛根素以15.6,31.3,62.5,125、200μg/mL剂量干预SH-SY5Y细胞糖氧剥夺模型,细胞存活率和上清中LDH水平与M组相比具有显著差异(P<0.01或P<0.05),提示模型所造成的损伤能够被葛根素抑制,说明造模成功,62.5μg/mL效果最好可能与其在细胞内的特殊吸收过程有关。葛根敲除液按敲除前应含葛根素浓度15.6,31.3,62.5,125,200,250,500μg/mL剂量干预SH-SY5Y细胞糖氧剥夺模型,细胞存活率和上清中LDH水平与M组相比具有显著差异(P<0.01或P<0.05)。葛根提取液按葛根素浓度3.9,7.8,15.6,31.3,62.5,125,200,250,500μg/mL分别干预时SH-SY5Y细胞糖氧剥夺模型,细胞存活率和上清中LDH水平与M组相比具有显著差异(P均<0.01)。在15.6-250μg/mL范围内葛根提取液效果优于葛根敲除液。结论：在本实验所取剂量范围内,葛根提取液效果优于葛根素说明多种成分综合作用的效果优于单一成分单独使用；葛根提取液效果优于葛根敲除液说明敲除90%葛根素会减弱葛根整体的药效,葛根素与葛根整体药效具有关联关系；葛根提取液效果优于二者单独使用共同说明葛根素与葛根中其它成分可能具有协同作用。第三章葛根素与葛根改善SH-SY5Y细胞糖氧剥夺损伤炎症因子作用的量效关系研究目的：通过在葛根提取液、葛根敲除液和葛根素各自最佳剂量上的比较,以及含不同比例葛根素的葛根之间比较,揭示葛根素与葛根改善SH-SY5Y细胞糖氧剥夺损伤炎症因子作用的量效关系方法：(1)分别取葛根提取液、葛根敲除液和葛根素三者最佳浓度与该浓度上其它两种药物干预SH-SY5Y细胞糖氧剥夺损伤,进行敲除前后的药效对比研究；(2)取葛根提取液最佳剂量和相应剂量的葛根敲除液,向葛根敲除液中定量添加葛根素至葛根素含量为原提取液的25%,比较添加前后及与葛根提取液药效的差异；采用MTT检测细胞存活率,乳酸-丙酮酸法检测LDH水平；Westernblot检测炎症因子NF-κB、TNF-α和IL-6表达水平；采用黄嘌呤氧化酶检验法检测SOD,硫代巴比妥酸法测定MDA,硝酸还原酶法检测NO。结果：(1)在葛根素的最佳剂量上,葛根敲除液改善SH-SY5Y细胞糖氧剥夺损伤所致的细胞存活率、LDH水平、NF-κB、TNF-α、SOD、MDA、NO异常的效果低于葛根提取液和葛根素,对IL-6的效果优于葛根素,与葛根提取液明显差别；在葛根敲除液和葛根提取液各自的最佳剂量上,葛根素改善细胞存活率、LDH水平、NF-κB、TNF-α、SOD、 MDA、NO、IL-6效果低于葛根敲除液和葛根提取液,葛根敲除液对除IL-6以外的其它因子的改善效果亦明显低于葛根提取液。(2)向葛根素含量为10%的葛根敲除液中定量添加葛根素至含25%葛根素后,对细胞存活率、LDH、NF-κB、TNF-α、SOD、MDA、NO改善优于单独使用原葛根敲除液和单独使用添加剂量的葛根素,但仍差于含100%葛根素的葛根提取液。结论：(1)在本实验选取的葛根素三个不同浓度上,敲除90%葛根素对葛根改善SH-SY5Y细胞糖氧剥夺损伤药效都具有不同程度的削弱作用,说明葛根素与葛根这一药效具有关联关系；葛根素对葛根改善SH-SY5Y细胞糖氧剥夺损伤药效的影响具体包括下调NF-κB、 TNF-α水平,改善SOD含量、减少MDA、NO等炎症因子,而对IL-6的影响则较弱。本实验为葛根素与葛根功效的关联关系提供了直接证据。(2)向葛根敲除液中定量添加15%葛根素之后的葛根敲除液的药效得到增强,优于单独使用葛根敲除液和葛根素,意味着葛根敲除液中的成分和所添加的葛根素之间存在协同作用；含100%葛根素的提取液对SH-SY5Y细胞糖氧剥夺损伤所致的不同炎症因子异常表达的改善效果,不同程度地优于含25%葛根素的敲除液组和含10%葛根素的敲除液组,说明葛根素对葛根改善SH-SY5Y细胞糖氧剥夺损伤的作用受葛根素含量的影响,而且与不同炎症因子有不同的量效趋势；葛根素含量对葛根改善SH-SY5Y细胞糖氧剥夺损伤功效的影响主要涉及对NF-κB、TNF-α, SOD、MDA, NO的调节作用。更多还原

【Abstract】 The material foundation of efficacy of Traditional Chinese Medicine (TCM)is the hot and difficult spot of modern research of the theory of compound compatibility, development of new drug and clarifying the modern connotations for syndrome differentiation The biological effects of Chinese medicinal formulae and the effectiveness of syndrome differentiation and treatment all can hardly be revealed without the guidance and confirmation of medicinal material base Therefore, revealing the the material foundation of efficacy of Chinese herbs is always key issue for modernization of TCM. However, due to the complexity of the chemical constituents of Chinese herbs and the use of compound preparation, the interactions between each element could be extremely complex and the identification of active compounds could be rather difficult. Even if the Chinese herbs documented in Chinese Pharmacopeia(2010edition), the varieties of traditional Chinese medicine with definite functional components makes up less than5%。At the present, the index components of the most Chinese herbs for quality control were set depended on the content of the components. So they were not necessarily the the main efficacy components。This problem is not only restricting the modernization of TCM from achieving substantial progress, it also can easily lead to drug safety. Therefore, the research of material foundation of efficacy of TCM is also the difficult and crucial part in modernization of TCM, which should be solved urgently.The team of my tutor have striven to the research of material foundation of efficacy of TCM for years, To deal with this problem, my tutor had put forward the idea of utilizing method of "knockouts" basing on the monoclonal antibody of small molecules of TCM, which differed from the method of reductionism. Through knocking out some component of a TCM specifically, we can compare the differences and similarities of efficacy before and after knocked out and the single component. The method could not only reflect the effect of the single compoent isolated from the mix, but also could show the role the component played within the herb. Therefore, the possible connections between regulators and other circuit components and the true contribution to efficacy of herb could be revealed-PRR (PRR) is the documented index components of PRA (PRA) for quality control. PRA and PRR have definite effect in the treatment of ischemic stroke and the mechanism is relatively clear.On the basis of many years of research, although lots of mechanism of PRR for ischemic stroke have been set out, the contribution of PRR to PRA has not been reported。 So we chose PRR as pattern drug, the vitro mode of ischemic stroke—oxygen glucose deprivation (OGD)in SH-SY5Y cells as carrier, intended to reveal the contribution of PRR to PRA in regulation of the Inflammatory factors in the model by comparing the function of PRA before and after the PRR was knocked out specially.Chapter1Preparation of immunoaffinity column and the knocking out of PRR in PRA exactObjective:To Prepare immunoaffinity column basing of the PRR monoclonal antibody. Then the PRR in PR exact should be knocked out and the drug for follow-up study should be prepared.Method:The ascites containing PRR monoclonal antibody(Mab) was purified by method of octanoic acid-ammonium sulfate; purity of antibodys before and after were tested by SDS-PAGE; concentrations of antibodys were measured by Bradford quantitative determination;the titer and competition of antibodys were detected by ELISA; the purified PRR Mab was coupled with the CNBr-activated SepharoseTM4B and the conjugation efficiency and conjugation rate were computed; PRA-exact(PRA-EXT) was loaded in the immunoaffinity column and the concentrations of PRR in PRA-EXT and the knock out couple (PRA-KO)were tested by HPLC, the rate of knock out was calculated.Result:SDS-PAGE showed that the other protein in PRR Mab were reduced, the purity of PRR Mab was92%; the concentration of PRR Mab ascites is3.67mg/mL, the concentration of the purified PRR Mab is2.29mg/mL; the ELISA showed that the titer of PRR Mab are both1:51200before and after purified, no apparent changes of linearity rangewere observed; the conjugation efficiency is99.3%, conjugation rate is3.1mg/mL; the90%of PRR was knocked out form PRA-EXT.Conclution:The PRR Mab purified by by method of octanoic acid-ammonium sulfate with less Miscellaneous protein and without influence to the titer and competition, met the requirement for preparation of immunoaffinity column, the conjugation between PRR Mab and the CNBr-activated SepharoseTM4B was well and the rate of knock out could meet the demands of follow-up study.Chapter2Comparative study on the pharmacodynamics of PRA-EXT, PRA-KO and PRR on the OGD injury of SH-SY5Y cellsObjective:Studying the pharmacokinetics of PRR in SH-SY5Y cells to reveal the efficacy generation process. To establish a stable oxygen glucose deprivation-reperfusion (OGD) in SH-SY5Y cells. To compare the differences between pharmacological activities of PRA-EXT, PRA-KO and PRR for this model.Method:(1)The effect of PRR on normal SH-SY5Y cells was studied with8different concentrations(3.9,7.8,15.6,31.3,62.5,125,250,500μg/mL) and4different incubation time(2h,8h,16h,24h); Cells were incubated with3different concentrations of PRR (31.25μg/mL,62.5μg/mL,125μg/mL) respectively, the supernatant was collected at20timepoints sequentially (0.08,0.25,0.5,0.75,1,1.5,2,2.5,3,3.5,4,4.5,5,5.5,6,6.5,7,8,9,10h) and tested by icELISA for the concentration of PRR. The intracellular concentration of PRR was obtained by subtracting the concentration of PRR in supernatant from the total dosage.(2) The OGD model in SH-SY5Y cells were reduced by sugar-free cell-culture medium and Na2S2O4, the condithons of Na2S2O4with4different concentrations (0.4,0.8,1.6,3.2mmol/L) and4different incubation time (2,8,16,24h) was investigated. Cytomorphology was observed under optic microscope; Cell survival rate was tested by MTT assay, Activities of lactate dehydrogenase (LDH) also be detected.(3) The PRA-EXT, PRA-KO and PRR was used to intervene the model with nine different concentrations of PRR (3.9,7.8,15.6,31.3,62.5,125,200,250,500μg/mL) individually. Cell survival rate was tested by MTT assay, Activities of lactate dehydrogenase (LDH) also be detected.Results:(1)PRR of3.9,7.8,15.6,31.3,62.5,125,250μg/mL had no significant effect on cell viability and LDH levels within8hours; The AUCiast and Cmax of high, medium and low dose groups decreased in a dose-dependent manner, The Tmax and MRTiast of medium dose group was higher than that of low-dose group and high-dose group, which showed the he particularity of the medium dose absorbed process in the cell.(2)The result of MTT and LDH test showed that the cell survival rates of cells in groups with dose of Na2S2O4is0.8,1.6and3.2mmol/L were decreased remarkablythan that in the control groups(P<0.01); the level of LDH in supernatant of cells in groups of0.4,0.8,1.6and3.2mmol/L were significantly higher than that in the control groups.(3)The result of MTT and LDH test showed that the cell survival rates and the level of LDH in supernatant of cells in PRR groups with dose of PRR is15.6,31.3,62.5,125and200μg/mL were improved remarkably(P<0.01or P<0.05) than that in the model groups; which means the damage caused by the model can be inhibited by PRR, indicating successful model. The best dose of PRR is62.5μg/mL, which may be related to the specific intracellular absorption process..The cell survival rates and the level of LDH in supernatant of cells in PRA-KO groups with dose of PRR(Before kocked out) is15.6,31.3,62.5,12,200,250,500μg/mL were improved remarkably (P<0.01or P<0.05) than that in the model groups; The cell survival rates and the level of LDH in supernatant of cells in PRA-EXT groups with dose ofPRR is3.9,7.8,15.6,31.3,62.5,125,200,250,500μg/mL were improved remarkably(P<0.01) than that in the model groups; The best dose was250μg/mL. Within15.6-250μg/mL range the efficacy of PRA-EXT groups was better than that of PRA-KO groups.Conclusion:Taken within the dose range of this experiment, the effect of PRA-EXT was better than the effect of PRR showed the combined effects of a variety of ingredients was better than single ingredient used alone; PRA-EXT performed better than PRA-KO indicated that knockout90%PRR from EXT will weaken the overall efficacy, PRR had a relationship with the overall efficacy of PRA-EXT; the effect of PRA-EXT was better than that of both PRA-KO and PRR used alone illustrated PRR may have a synergistic effect with other components of Pueraria.Chapter3Research on the dose-effect relationship between PRR and the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injuryObjective:To reveal the dose-effect relationship between PRR and the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury by the comparison amang the PRA-EXT, PRA-KO and PRR on each optimal dose, as well as the comparison amang the PRA ontaining different proportions of PRR.Method:(1)According to the best dosage of the three drugs elected in the Chapter l,the OGD model of SH-SY5Y cells were treated by the PRA-EXT, PRA-KO and PRR on3different concentration of PRR for comparing the efficacy of them;(2)At the best dose of PRA-EXT and the corresponding dose of PRA-KO, PRR was quantitatively added into PRA-KO (the content of PRR is10%) to the content of25%for the PRA-EXT, then comparing the efficacy of PRA-KO(the content of PRR is10%), PRA-KO (the content of PRR is25%) and PRA-EXT (the content of PRR is100%);Tested the cell survival rate and LDH level; SOD activities were measured by xanthine oxidase method; the content of MDA were measured by thiobarbituric acid method;the content of NO was detected with Griess method;the expression of NF-κB、TNF-αand IL-6were was investigated by Western blot.Result:(1)At the best dose of PRR, the improving effect of PRA-KO on abnormal change of cell survival rate,LDH level, NF-κB, TNF-α, SOD, MDA, NO caused by OGD were worse than these of PRA-EXT, the improving effect of PRA-KO on IL-6is better than PRR, but non difference with PRA-EXT; at the best doses of PRA-EXT and PRA-KO, the improving effect of PRR on cell survival rate,LDH level, NF-κB, TNF-α, SOD, MDA, NO, IL-6were worse than these of PRA-EXT and PRA-KO; the improving effect of PRA-KO on cell survival rate,LDH level, NF-Kb, TNF-α SOD, MDA, NO were worse than these of PRA-EXT;(2)The improving effect of PRA-KO(the content of PRR is25%) on cell survival rate, LDH level, NF-κB, TNF-α, SOD, MDA and NO were better than these of PRA-KO(the content of PRR is10%) and PRR (which equal to the dose added in PRA-KO), but still worse than PRA-EXT (the content of PRR is100%).Conclusion:(1) In three different concentrations of PRR selected in the present study, the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury was weaken by knocking out90%PRR from PRA, indicating there was a relationship between the efficacy of PRA and PRR; the effect of PRR on the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury included reducing NF-κB, TNF-α levels, improving SOD levels, reducing MDA, NO, but the effect on IL-6is relatively weak. This experiment provided the direct evidence for the relationship between PRA and PRR.(2) After PRR was quantitatively added into PRA-KO (the content of PRR is10%) to the content of25%, the improving effect of PRA-KO(the content of PRR is25%) was enhanced and better than PRR and PRA-KO use alone, which means synergies existed between PRR and the components of PRA-KO; The improving effect of PRA-EXT (the content of PRR is100%) is better than that of PRA-KO (the content of PRR is25%) and PRA-KO(the content of PRR is10%), indicating the influence of PRR on the efficacy of PRA in improving the inflammatory cytokines of SH-SY5Y cells caused by the OGD injury worked in a dose-dependent manner; This effect mainly related to the regulation of NF-κB, TNF-α. SOD, MDA and NO.更多还原