Antioxidant, Anti-tumor Activities And Underlying Molecular Mechanisms Of Carnosic Acid

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.