摘要
近年来,自由基相关疾病(炎症、衰老、心血管病、肿瘤等)日益引起人们的关注。同时,关于天然抗氧化剂清除自由基作用的研究也日益深入。寻找并确定抗氧化作用较强的天然抗氧化剂,确认其抗氧化作用与防治自由基损伤相关疾病的关系,研究其有效成分,对研发保健食品或药品,用于疾病的预防以及延缓衰老等都有重要意义。
本研究分两部分、4个层次(包括基因和蛋白水平、亚细胞水平、细胞水平和动物整体水平)研究了多种中药醇提物抑制自由基生成、抗氧化应激损伤的反应特点和作用规律,发现玉麦须醇提物具有多层次的抗氧化应激损伤作用,并存在一定的剂量效应关系,具有很大的开发利用潜能。
第一部分为体外和亚细胞水平实验:实验利用化学发光和微粒体脂质过氧化模型有效的筛选了11种传统中药类抗氧化剂。体外建立了羟自由基和超氧阴离子发光体系,亚细胞水平制备了微粒体CHP、Vc/Fe~(2+)、CCl_4╱NADP~+氧化损伤模型。通过建立的抗氧化剂筛选技术平台,检测了11种中药醇提物对平台各体系激发生成的自由基及丙二醛含量的影响及其剂量效应关系。实验结果表明:虽然所选药物醇提物都有一定的抗氧化作用,但是,首次发现玉麦须醇提物具有很强的抗氧化作用。玉麦须醇提物(≥3.55μg/ml)可以显著清除化学发光体系产生的~·OH和O_2~(·-),具有明显的量效关系,并且活性远强于所选的其它中药提取物。在终浓度为6.25～2000μg/ml内,玉麦须醇提物能抑制微粒体脂质过氧化作用,也存在剂量—效应关系(r≥0.79,P＜0.05)。表明玉麦须醇提物具有比较全面、有效、系统的抗氧化作用。
第二部分分别以L929细胞(小鼠肺成纤维细胞)和BABL/c小鼠作为研究对象,分为正常对照组、单纯照射组、照射+低剂量给药组、照射+中剂量给药组、照射+高剂量给药组。利用电离辐射产生大量自由基和活性氧,造成实验细胞和动物氧化应激损伤。
(1)细胞水平实验:~(60)Co-γ射线20Gy单次照射L929细胞,照射后立即给药,给药终浓度分别为10、50、250μg/ml MSE,并于照射后4h收取细胞样品,测定氧化应激相关指标的变化。结果表明~(60)Co-γ射线20Gy单次照射能诱发显著的自由基介导的氧化应激损伤反应,表现为TBARS升高,GSH含量降低、GSH/GSSH比值降低、CAT、SOD活性明显降低。给予不同剂量MSE保护性干预后,氧化应激水平明显降低,表现为TBARS水平降低,抗氧化酶活性增强,GSH/GSSG比值升高。其中以中剂量MSE组各项指标改善明显,效果最为显著,同时发现高剂量MSE有一定的消耗GSH作用。
(2)基因和蛋白表达水平实验:提取氧化损伤的L929细胞的总RNA,通过RT-PCR检测CAT、SOD、GGS、Nrf2和TGFβmRNA表达水平,同时通过细胞免疫化学检测TGFβ蛋白水平变化。实验结果表明:照射后细胞Nrf2 mRNA表达水平下降,TGFβmRNA和蛋白表达水平升高,而CAT、SOD、GGS mRNA水平均无明显变化。给予MSE干预后,中剂量组SOD、CAT、TGFβmRNA表达水平降低,Nrf2、GCS mRNA表达水平升高,TGFβ蛋白表达水平降低。低、高剂量组Nrf2、TGFβmRNA表达水平无显著变化,SOD mRNA水平升高,CAT mRNA水平降低,低剂量组GCS mRNA水平升高,高剂量组降低。
(3)动物水平的实验:~(60)Co-γ射线5Gy单次照射BABL/c小鼠,照射前一天灌胃给药,每天一次,一直持续到观察终点。给药浓度分别为75、150、300mg/kg/d MSE。于照射后10天宰杀动物,取血清、肝肾匀浆,测MDA、GSH、GSSH含量,CAT、SOD活性。提取肝脏总蛋白,测TNFα、SOD、CAT蛋白表达水平。取全血检测WBC、RBC、HGB、PLT变化。做肝肾病理切片,H&E染色。实验结果表明,低剂量~(60)Co-γ射线照射可以使小鼠全身氧化水平增高,其中以肝脏最为敏感,肾脏最不敏感。给予不同剂量MSE保护后,肝脏和血清的TBARS含量降低,抗氧化酶活性增高,并呈现出量效关系。然而,MSE对肝、血清的GSH、GSSG含量和肝CAT活性无显著影响,同时发现MSE对肾组织不仅未表现出保护作用,低、高剂量MSE还有促TBARS生成作用。肝脏蛋白表达检测结果显示,照射组CAT、TNFα表达均无变化,SOD表达下降。给予MSE后,CAT表达无改变,中剂量组SOD、TNFα表达增强,低、高剂量组SOD表达反而降低。血常规检测结果显示,照射组各项指标均显著下降。除WBC外,给予不同剂量MSE后,指标数值均显著回升,且量效关系明显。
本研究结果提示:化学发光和微粒体脂质过氧化模型是筛选抗氧化剂的高效、可靠技术平台。经该平台测定充分证明,玉麦须具有多层次的抗氧化应激损伤作用,并存在剂量效应关系。作为一种有效的中药抗氧化剂,玉麦须在体内体外模型中,都表现出有效的氧化应激损伤保护作用,具有显著的研究、开发、应用前景。本研究为进一步研究玉麦须在自由基相关疾病中的治疗作用提供了基础,为其进一步开发利用提供了生物学信息。
Diseases such as aging,angiocardiopathy,inflammation,tumor,etc related to free radical injury have attracted more and more public concern.At the same time,further studies on roles of natural antioxidants and free radical scavengers are being conducted.Theoretically,searching efficient natural antioxidants can further confirm the relation of their antioxidative abilities and preventive abilities of diseases related to free radical injury.Practically,searching efficient natural antioxidants will contribute to further studies on their effective constituents,developing health food and medicine to prevent or cure diseases.
Our study was divided into 2 parts and 4 levels(including gene and protein, deuto-cell,cell and animal) to research the reaction characteristics and action ways of many traditional Chinese herbal medicine extracts on their scavenging free radicals and anti-oxidative stress injury activities.Maydis stigma extract (MSE) was found to have multi-level anti-oxidative stress function and exhibited a concentration-dependent relation,so it deserves a furher development and utilization.
In the part of in vitro test,we effectively screened 11 ethanol extracts from traditional Chinese herbal medicines employing chemiluminescence and microsomal lipid peroxidation models.We have investigated their direct scavenging effects on hydroxyl radicals(~·OH) and superoxide anions(O2~(·-)),as well as their anti-lipoperoxidative capacity on rat liver microsomal membrane peroxidation induced in vitro by three different sources of stimulators: Fe~(2+)/ascorbate,cumene hydroperoxide(CHP),and CCl_4/reduced form of nicotinamide-adenine dinucleotide phosphate(NADPH).The result indicated that all the tested extracts had antioxidant effect at some extent,but Maydis stigma extract was firstly found to have much stronger and broader,more efficient and systematic antioxidant ability compared with the others.It could scavenge free radicals obviously at concentrations as low as 3.55μg/ml.And between the final concentration of 6.25 and 2000μg/ml,it could effectively inhibit lipid peroxidation with concentration-dependent relations(r≥0.79, P＜0.05).
In the part of ex vivo and in vivo test,we took L929 cells and BABL/c mices as research subjects respectively.The cells and animals are divided into 5 groups: control group,radiation group,radiation+low dose MSE group, radiation+middle dose MSE group,and radiation+high dose MSE group.We made use of ~(60)Co-γray irradiation which produces a large mount of free radicals and reactive oxygen species to cause oxidative stress injury on the experimental subjects.
(1) Ex vivo studies:L929 cell was irradiated by ~(60)Co-γray with a single dose of 20Gy.MSE was added to the cells immediately after irradiation with a final concentration of 10,50,250μg/ml respectively.4 hours after irradiation,cell samples were collected and oxidative stress related indicators were tested.We found that a single dose of 20Gy irradiation could induce free radical-mediated oxidative stress injury reaction noticeably,which was marked by the increase of TBARS,decrease of GSH and GSH/GSSH ratio and reduction of CAT,SOD.Giving different dose of MSE could prevent L929 cell from oxidative injury by lowering oxidative stress level,which was showed as decrease of TBARS,increase of GSH/GSSH ratio and CAT,SOD activities.Especially,the middle dose of MSE showed the most dictinct effect on improving various indicators. Meanwhile,the high dose of MSE was found to play some GSH consumption roles.
(2) Gene and protein level studes:the total RNA was extracted from the oxidative injury L929 cells.The mRNA level changes of Nrf2,TGFβand antioxidant enzymes including CAT,SOD and GCS were observed by RT-PCR.The protein level change of TGFβwas observed by cell immunofluorescence.The results are as follows.①Down-regulation of Nrf2 and up-regulation of TGFβwere observed after irradiation.However, CAT,SOD,GGS mRNA level didn't change obviously.②Middle dose of MSE could down-regulate SOD,CAT and TGFβwhile up-regulate Nrf2 and GCS.③Low and high dose of MSE had no obvious effect on Nrf2 and TGFβmRNA level,but they could up-regulate SOD while down-regulate CAT.In addition,the effect of low-dose MSE on GCS mRNA level was up-regulation,while the effect of high-dose MSE on it was down-regulation.
(3) In vivo studies:BABL/c mice were irradiated with 20Gy single dose of ~(60)Co-γray.The mice were oral administrated with MSE(75,150 and 300mg/kg/d) 1 day before irradiation and once a day for 10 days after irradiation.All mice were sacrificed at a predetermined time of ten days postradiation.The livers and kidneys homogenates and blood serum were prepared for testing MDA,GSH,GSSH and CAT,SOD activity.The livers protein level changes of TNFαand antioxidant enzymes including CAT,SOD were observed by Western Blot.Pathological changes of livers and kidneys were observed by H&E staining.WBC,RBC,HGB and PLT were also tested in whole blood.The results showed that low dose irradiation could increase oxidative level in the mice's whole body.Liver seemed to be the most sensitive tissue to oxidative stress while kidneys to be the less sensitive.After MSE oral administration,decrease of TBARS and increase of antioxidant enzymes activity were observed in livers and serum.However,no obvious changes of GSH and GSSG were observed in livers and serum.Meanwhile,MSE had no obvious protective effect on kidneys,low and high dose of MSE even induced the generation of TBARS.The result of Western Blot in livers showed that expressions of SOD and TNFαwere altered but that of CAT was not altered.In addition, the decrease of RBC,HGB and PLT caused by irradiation could be significantly reversed by MSE in a dose-react relationship,but that of WBC could not be reversed by MSE.
These results indicated that chemiluminesence systems and microsomal lipid peroxidation models are efficient and reliable technique platform for screening antioxidants.On this platform,Maydis stigma's extract was demonstrated to possess multi-level anti-oxidative stress property and have dose-effect relation in both in vitro and in vivo experiments.However,as a byproduct of agriculture, Maydis stigma's health care and medical value has not been fully exploited,the research progress on its pharmacodynamics is limited.So Maydis stigma enjoys a great potential for research and development.Our results will provide important experimental evidence for its therapeutic action on free radical-related diseases as well as useful bio-information for its further development and utilization
引文
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