【作者】 相启森；

【导师】 刘学波；

【作者基本信息】 西北农林科技大学 ， 食品科学， 2013， 博士

【摘要】 活性氧（ROS）和活性氮（RNS）是细胞正常代谢过程产生的一类副产物，参与了宿主防御、细胞信号传导、基因表达调控等正常生理活动。但活性氧和活性氮的过量产生会对DNA、蛋白质、脂质和糖类等生物功能分子造成损伤。生物功能分子损伤的积累将导致动脉粥样硬化、肿瘤、慢性炎症、糖尿病等疾病和衰老的发生。研究证实，膳食补充抗氧化剂可预防或减轻自由基对机体造成的损伤。因被认为比合成抗氧化剂更安全和健康，植物来源的天然抗氧化剂已广泛用于功能食品的开发。因此，研究天然抗氧化剂的作用机制对功能食品的研究开发具有重要意义。鼠尾草酸（Carnosic acid, CA）是一种从迷迭香（Rosmarinus officinalis）、鼠尾草（Salvia officinalis）等唇形科植物分离得到的多酚双萜化合物。已有研究初步证实鼠尾草酸具有抗氧化、抗菌、抗肿瘤、神经保护、抑制脂肪细胞分化等多种生理活性，但关于其活性功能的研究尚不够深入。本文采用体外模型、细胞模型和动物模型等方法系统研究了鼠尾草酸对自由基诱导生物分子损伤的抑制作用、对脂多糖（LPS）诱导SD大鼠慢性炎症和肝损伤的保护作用及对人肝癌HepG2细胞凋亡诱导作用并探讨了鼠尾草酸作用的分子机制；此外，采用荧光光谱、圆二色光谱等方法研究了鼠尾草酸与人血清白蛋白（HSA）的相互作用，为揭示鼠尾草酸在体内的转运和代谢机制提供了理论依据。本文的主要研究内容和结果如下：(1)研究了CA对自由基诱导蛋白质、脂质和DNA损伤的抑制作用。试验结果表明，CA能显著抑制Cu2+/H2O2和AAPH诱导的牛血清白蛋白（BSA）氧化降解和羰基化损伤，并显著抑制H2O2和AAPH诱导的RAW264.7细胞内活性氧的产生及总蛋白羰基化损伤；CA抑制Hemin/nitrite/H2O2诱导BSA中3-硝基酪氨酸的生成并显著抑制LPS刺激的RAW264.7细胞分泌NO。同时，CA能显著抑制Fe2+/VitC和AMVN诱导亚油酸脂质过氧化过程中硫代巴比妥酸反应产物（TBARS）和共轭二烯的生成，同时显著抑制亚油酸脂质过氧化产物造成的RAW264.7细胞毒性和胞内活性氧过量产生。此外，CA显著抑制AAPH诱导DNA氧化损伤过程中TBARS的生成和pBR322质粒DNA链断裂。CA能够以浓度依赖和时间依赖的方式上调RAW264.7细胞中血红素加氧酶-1（HO-1）蛋白的表达，这可能是CA在细胞内发挥抗氧化作用的机制之一。(2)研究了CA对脂多糖（LPS）诱导Sprague-Dawley（SD）大鼠氧化/硝化应激、慢性炎症和肝损伤的保护作用。在腹腔注射LPS（1mg/kg体重）之前，对大鼠予以CA灌胃处理（15,30和60mg/kg体重）。CA可显著抑制LPS引发的脂质过氧化、蛋白羰基化、血清一氧化氮含量和一氧化氮合成酶活力升高。CA显著降低促炎性细胞因子肿瘤坏死因子-α（TNF-α）和白介素-6（IL-6）的分泌并增强抗炎性细胞因子白介素-10（IL-10）分泌。同时，CA显著抑制LPS诱导的天冬氨酸氨基转移酶（AST）、丙氨酸氨基转移酶（ALT）、碱性磷酸酶（ALP）等活力升高。此外，CA显著降低血清中总固醇和甘油三酯的含量，有效改善LPS造成的肝脏脂质代谢紊乱。CA能够显著增强机体抗氧化防御能力，主要是增加血清和肝脏中超氧化物歧化酶、谷胱甘肽过氧化物酶等抗氧化酶活力和谷胱甘肽含量，这可能是CA在体内发挥生理活性功能的机制之一。(3)研究了CA对人肝癌HepG2细胞的生长抑制作用及其机制。MTT试验结果表明，CA能够显著抑制HepG2细胞的增殖，并呈浓度依赖性。DAPI和吖啶橙/溴化乙锭（AO/EB）染色结果显示，CA处理的HepG2细胞呈现细胞核皱缩、染色质凝集和碎片化等典型的凋亡形态学特征。CA同时诱导了HepG2细胞中Caspase-3激活及PARP片段化，二者均是细胞凋亡的主要标志。CA显著降低了HepG2细胞的线粒体膜电位并诱导了细胞色素C从线粒体内膜释放到细胞质。此外，CA造成细胞中抗凋亡蛋白Bcl-2和促凋亡蛋白Bax比值的降低，而Bcl-2/Bax比值降低导致细胞发生凋亡。CA还能抑制Akt和ERK的磷酸化并促进p38和JNK的磷酸化，表明PI3K/Akt和MAPK信号通路参与了CA诱导HepG2细胞凋亡过程。(4)采用荧光光谱、圆二色光谱、分子对接、循环氧化还原染色等方法研究了CA与HSA的相互作用。荧光光谱结果表明，CA可使HSA内源荧光有规律地发生静态猝灭；该静态猝灭过程中，焓变(ΔH)>0而熵变(ΔS)<0，表明静电作用和疏水作用在CA-HSA相互作用过程中其起重要作用。荧光猝灭和分子对接结果表明，CA结合于HSA的亚结构域IIA，结合位点数码（n）为1。循环氧化还原染色结果表明，CA可通过自氧化作用与HSA的Cys-34残基发生共价结合作用。此外，同步荧光光谱和圆二色光谱结果表明，CA-HSA相互作用造成HSA微环境和蛋白构象发生变化。更多还原

【Abstract】 Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the by-productsof normal cellular metabolism, which are involved in a wide variety of physiologicalprocesses, such as host defense, cellular signaling and regulation of gene expression.However, the overproduction of ROS and RNS can cause severe damage to a broad range ofessential biomolecules, including DNA, proteins, lipids, and carbohydrates. The accumulationof damaged biomolecules eventually leads to many chronic diseases, such as atherosclerosis,cancer, chronic inflammation, diabetics, and aging in humans. Dietary supplements containinghigh levels of antioxidants are used to prevent or reduce the damage caused by free radicals inhuman body. As regarded be safer and healthier than synthetic antioxidants, naturalantioxidants from plants are widely used in functional foods. As a consequence, there hasbeen an increasing interest in exploring the molecular basis of working mechanism of naturalantioxidants.Carnosic acid (CA) is a major phenolic diterpene derived from several labiate herbs likerosemary (Rosmarinus officinalis) and sage (Salvia officinalis). Previous studies have beenconfirmed CA owns diverse biological activities including antioxidant, antimicrobialpotential, anticancer, neuroprotection, and inhibition of adipocyte differentiation. However,the studies on the biological activities CA are not enough. In this study, the protective effectsof CA on free radicals-induced damages to biomolecules, the anti-inflammatory and liverprotective effect of CA on lipopolysaccharide (LPS)-treated Sprague-Dawley (SD) rats aswell as the apoptosis-inducing effects of CA on human hepatoma HepG2cells wereinvestigated by using in vitro cell culture model and experimental animal model. In addition,the interaction of CA with human serum albumin (HSA) was also evaluated by multipleapproaches, such as fluorescence quenching spectra and far-UV circular dichroism spectra,which provided theoretical basis for the mechanisms of transport and metabolism of CA in thebody. The main research contents and primary results of this study were presented asfollowing:(1) The inhibitory effect of CA on free radical-mediated damages to proteins, lipids and DNA was investigated. The results showed that CA significantly inhibited the oxidativefragmentation and carbonylation of bovine serum albumin (BSA) initiated by Cu2+/H2O2orAAPH. CA effectively attenuated protein carbonylation and intracellular ROS production inH2O2or AAPH-treated RAW264.7cells. Additionally, CA dose-dependently inhibited theformation of3-nitrotyrosine in Hemin/nitrite/H2O2-treated BSA and significantly suppressedLPS-induced nitric oxide production in RAW264.7cells. CA also efficiently inhibited theformation of thiobarbituric acid reactive substances (TBARS) and conjugated dienes duringperoxidation of linoleic acid initiated by Fe2+/VitC or thermal decomposition of AMVN. CAinhibited the cytotoxic effects and ROS overproduction induced by oxidized linoleic acid onRAW264.7cells. Moreover, CA protected DNA against AAPH-induced oxidative damage anddecreased AAPH-induced single-strand breaks in plasmid pBR322DNA. Furthermore, CAdose-and time-dependently upregulated the protein expression of heme oxygenase-1(HO-1)in RAW264.7cells, which was proposed as one of the mechanisms for antioxidant activity ofCA.(2) The protective effects of CA on LPS-induced oxidative/nitrosative stress, chronicinflammation, and hepatic injury in Sprague-Dawley (SD) rats were studied. CA wasadministered orally to rats at doses of15,30and60mg/kg body weight before LPS challenge(single intraperitoneal injection,1mg/kg body weight). The results revealed that CAeffectively attenuated LPS-induced oxidative/nitrosative stress by decreasing lipidperoxidation, protein carbonylation, serum levels of nitric oxide and activities of nitric oxidesynthase (NOS). CA also potently inhibited secretion of proinflammatory cytokines such astumor necrosis factor-α and interleukin-6and increased serum levels of anti-inflammatorycytokine interleukin-10. The activities of aspartate aminotransferase (AST), alanineaminotransferase (ALT), and alkaline phosphatase (ALP) were raised after LPS challenge,which were significantly reversed by the pretreatment with CA in a dose-dependent manner.CA also efficiently attenuated the LPS-induced disorder of lipid metabolism in liver bydecreasing the serum levels of total cholesterol and triacylglycerol. CA supplementationmarkedly enhanced the body’s cellular antioxidant defense system by restoring the levels ofsuperoxide dismutase, glutathione peroxidase, and glutathione in serum and liver after theLPS challenge, which was proposed as one of the molecular biological mechanisms of CA.(3) The apoptosis-inducing effects of CA on human hepatoma HepG2cells wereinvestigated. The MTT assay results showed that CA significantly inhibited proliferation ofHepG2cells in a dose-dependent manner. Based on DAPI and acridine orange/ethidiumbromide (AO/EB) staining, CA-treated cells manifested typical morphologic changes ofapoptosis characterized by nuclear shrinkage, chromatin condensation, and fragmentation. Moreover, the treatment of HepG2cells with CA caused activation of Caspase-3andsubsequent PARP cleavage, which were characteristic indicators of apoptosis. CA alsodecreased the mitochondrial membrane potential (ΔΨm) and elevated the release ofcytochrome c from mitochondria to cytoplasm. Additionally, CA decreased the ratio ofantiapoptotic proteins Bcl-2and proapoptotic protein Bax, which increased the susceptibilityof HepG2cells to apoptosis. CA significantly inhibited the phosphorylation of Akt and ERKas well as promoted the phosphorylation of p38and JNK, indicating that PI3K/Akt andMAPK singing pathways were involved in CA-induced apoptosis of HepG2cells.(4) The interaction of CA with HSA was investigated by using fluorescence quenchingspectra, molecular modelling, far-UV circular dichroism spectra, and electrophoresis/blottingwith redox-cycling staining. Fluorescence data revealed that CA strongly quenched theintrinsic fluorescence of HSA through a static quenching procedure. The negative value ofenthalpy change (ΔH) and positive value of entropy change (ΔS) indicated that electrostaticinteraction and hydrophobic interaction played major roles in the interaction. From the resultsof fluorescence titration and molecular docking, the binding of CA to HSA mainly took placein subdomain IIA and the number of binding sites (n) was about1. Redox-cycling stainingindicated that CA could covalently bind to HSA and form HSA-CA quinone complexes.Moreover, synchronous fluorescence and circular dichroism showed that the binding of CA toHSA induced the micro-environmental and conformational changes of HSA.更多还原