Advanced Oxidation Protein Products Induce Oxidative Stress In THP-1Macrophages And Drug Interference

1. Background:Advanced oxidation protein products (AOPP) was initially found to be elevated as an important macromolecule uremic toxin in the plasma of CRF patients by Witko-Sarsat in1996, which is the dityrosine-containing and cross-linking protein products formed during oxidative stress by reaction of plasma protein with chlorinated oxidants through neutrophil myeloperoxidase-catalyzing reaction.The level of AOPP in plasma is positively correlated with the levels of hallmarks of oxidized protein such as dityrosine, pentose prime and neopterin, meanwhile negatively correlated with antioxidants such as glutathione peroxidase (GSH-PX). Therefore, AOPP has been considered as a novel marker of oxidative stress in CRF, which has certain biological activity as a pro-inflammatory mediator and uremia toxin molecule participating in CKD, pathololgic and physiological process of clinical complications in dialysis patient.AOPP is able to trigger the oxidative burst of monocytes and their release of inflammatory cytokines such as IL-1β, IL-6and TNF-a, inflammatory effect, similarly, stimulate the respiratory burst of neutrophil, which result in stronger oxidative stress. The positive feedback increases its own production and enhances the ability of monocytes, contributing to systemic micro-inflammatory reaction. It has been confirmed that AOPP is a pro-inflammatory and oxidative stress media which selectively targets to monocytes/macrophages as the major source of inflammatory cytokines and oxidant.Niaoduqing granule is a medicine treating renal failure NanFang Hospital developed, which can effectively reduce the patients’clinical symptoms, improve renal function and attenuate renal dysfunction. Compound Huanggan Granule (FHG) is second-generation product of Niaoduqing granule. Our previous animal studies in vivo proved that compound huanggan extract could decrease apparently the production of AOPP in the circulation of rats with CRF, which indicated that compound huanggan extract could attenuate the injury by oxidative stress, but the underlying mechanism remains unclear.Paeonol (Pae), a major active component extracted from the herb Pycnostelma paniculatum (Bunge) K.S., and the root of the plant Paeonia Sufrruticosa Andrew, Mounting evidences have been demonstrated that paeonol possess extensive pharmacological activities such asanti-inflammatory, anti-allergic and have significant immunomodulatory effects on immune organs such as spleen, thymus and immune molecules such as lymphocytes, monocytes. In addition, paeonol have a wide range of pharmacological effects of anti-oxidation, anti-microbial, anti-tumor, cardiovascular protection and sedation, hypnosis, anti-pyresis, analgesic. However, it is not clear whether paeonol can affect the injury by oxidative stresson macrophages.The purpose of this experiment is to investigate their effects on injury by oxidative stress and the possible mechanism by compound huanggan extract and paeonol in AOPP-stimulated THP-1macrophages. 2. Methods:2.1Preparation of AOPPAOPP was prepared according to the method described by Witko-Sarsat. Briefly, endotoxin free BSA was incubated with HC10at molar ratio of1/70,1/140and1/280at room temperature for30min. AOPP concentrations were determined by measuring absorbance at340nm in acidic condition and were calibrated with chloramines-T in the presence of potassium iodide. Their protein content was measured at595nm by Bradford method. The endotoxin content was detected qualitativly with tachypleus amebocyte lysate by the gel method (sensitivity0.25EU/ml).2.2Effect of Compound Huanggan Extract and paeonol on oxidative stress in AOPP-induced THP-1macrophages2.2.1Differentiation of THP-1macrophagesTHP-1cells were stimulated with phorbol-12-myristate-13-acetate (PMA) uncontained FBS at a final concentration of160nM for24h.2.2.2Effect of Compound Huanggan Extract and paeonol on cell viability in AOPP-induced THP-1macrophagesCell viability was determined by the MTT assay. After incubating the cells with different concentrations and modified degree of AOPP alone or AOPP plus various concentrations of paeonol and compound huangan extract respectively for24h, absorbance was calculated spectrophotometrically at570nm by using a microplate reader.2.2.3Effect of Compound Huanggan Extract on NO production in AOPP-induced THP-1macrophagesAfter incubating the cells with different concentrations and modified degree of AOPP alone or AOPP plus various concentrations of paeonol and compound huangan extract respectively for24h, the supernatant was removed from the cultures. The nitrite accumulated in culture medium was measured as an indicator of nitric oxide (NO) production according to the Griess reaction. The mRNA level of iNOS was calculated by RT-PCR.2.2.4Effect of Compound Huanggan Extract, paeonol and inhibitors on ROS production and Cytokine secretion in AOPP-induced THP-1macrophagesTHP-1macrophages were incubated with various concentrations and different modified degree AOPP for24h and then added into an oxidant-sensitive2’7’-dichlorefluorescin (DCFH). Fluorescence intensity were quantified with multi-function microplate reader in485nm excitation and535nm emission filters, representing directly the production of active oxygen. Moreover, THP-1macrophages were pre-incubated for0.5h,1h,2h with compound huanggan extract and paeonol prior to incubation with AOPP (200μg/ml) for24h. Meanwhile, to verify the sources of ROS generation, THP-1macrophages were pre-incubated for0.5h,1h,2h with a free radical scavenger N-acetylcysteine (NAC,10μM), NADPH oxidase inhibitor (apocynin,1mM; DPI,100μM), a specific inhibitor of nuclear factor kappa-B (NF-кB) pyrrolidine dithiocarbamate (PDTC,10μM) and paeonol respectively prior to incubation with AOPP (200μg/ml) for24h, then intracellular ROS production was measured. Quantification of tumor necrosis factor-a (TNF-a), interleukin-1β (IL-1(3) and interleukin-6(IL-6) at protein level in cell culture supernatants were carried out with Enzyme Immuno Assay (ELISA) kit. Quantification of TNF-a, IL-1β, IL-6and MCP-1in mRNA levels were determined by RT-PCR.2.2.5Effect of inhibitors PDTC on iNOS mRNA level in AOPP-induced THP-1macrophagesTHP-1macrophages were pre-incubated for24h with AOPP prior to incubation with inhibitor PDTC for24h. Then the gene expression of iNOS was detected by RT-PCR. 2.2.6Effect of paeonol on RAGE, CD36, SR-A, SR-B1mRNA level in AOPP-inducedTHP-1macrophagesTHP-1macrophages were pre-incubated for24h with AOPP prior to incubation with different concentrations of paeonol for24h. Then the gene expression of RAGE, CD36, SR-A, SR-B1was detected by RT-PCR.3. Results:3.1Characterization of AOPPThree kinds of AOPP with different degree of oxidation were prepared according to the method described by Witko-Sarsat. AOPP (1/70,1/140,1/280) concentrations were168.5μM,1047.8μM and2340.6μM respectively, which all were much higher than native BSA with0.58μM. Furthermore, endotoxin levels in the preparation were tested with limulus amebocyte lysate kit and were found to be less than0.25endotoxin units/ml.3.2Effect of Compound Huanggan Extract and paeonol in AOPP-induced THP-1macrophages3.2.1Effect of Compound Huanggan Extract and paeonol on cell viability in AOPP-induced THP-1macrophagesAfter incubating the cells with different concentrations and modified degree of AOPP alone or AOPP plus various concentrations of paeonol and compound huangan extract respectively for24h, cell viability was calculated by the MTT assay. The results proved that AOPP, paeonol and compound huangan extract have no effects on macrophages viability compared to the control group.3.2.2Effect of AOPP modification on NO production in THP-1macrophagesThe native BSA induced significant amounts of NO production in THP-1macrophages. Low modified degree of AOPP(1/70) also induced remarkly NO production. However, High modified degree of AOPP (1/140) had not ability of inducing NO production. Meanwhile, AOPP (1/140) could notably inhibit NO production in a dose-dependent manner in THP-1macrophages as the concentration was up to200μg/ml.3.2.3Effect of Compound Huanggan Extract and paeonol on NO production in AOPP-induced THP-1macrophagesCompound huanggan extract increased apparently NO production induced by AOPP in a concentration-dependent way in THP-1macrophages at100-400ug/ml. On the other hand, paeonol also enhances obviously NO production in time-dependent manner at100-400μM.3.2.4Effect of AOPP on ROS production in THP-1macrophagesGenerally when cells were exposed to1/140and1/280AOPP, a significant increase in the intracellular reactive oxygen species (ROS) level could be provoked; in contrast, BSA could not induce ROS production. In addition, ROS production reached the peak when1/140AOPP was at a concentration of200μg/ml. The THP-1macrophages were pretreated with apocynin, DPI, NAC, PDTC, paeonol for0.5h,1h and2h respectively before AOPP was added and cultured for24h. The results showed that different inhibitors and paeonol decreased ROS production apparently in a time-and dose-dependent manner. PDTC showed the strongest effect above all the inhibitors including paeonol.3.2.5Effect of Compound Huanggan Extract and paeonol on ROS production in THP-1macrophagesCompound huanggan extract exhibited time-dependent inhibition of ROS production induced by AOPP in THP-1macrophages when pretreated before AOPP stimulation. Moreover, the ability of inhibiting ROS production reached the peak (25.75%) when the pretreat time was1h and the concentration was400μg/ml, which was weaker than that of PDTC, but stronger than others.Paeonol also decreased significantly ROS production in a dose-dependent way in AOPP-induced THP-1macrophages. When the cell was exposed to400μM paeonol for1h, the inhibition rate was19.57%, which was slightly higher than the inhibitor apocynin, DPI and NAC, but lower than PDTC.3.2.6Effect of Compound Huanggan Extract and paeonol on inflammation cytokines in AOPP-induced THP-1macrophagesAOPP could up-regulate the pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in mRNA and protein level. However, different antioxidants remarkably suppressed their secretion. In parallel, there was a tendency that paeonol and compound huanggan extract inhibited strongly AOPP-stimulated pro-inflammatory cytokines expression in THP-1macrophages. Nevertheless, the inhibitory of paeonol and compound huanggan extract were weaker than DPI and PDTC.3.2.7Effect of Compound Huanggan Extract and paeonol on inflammation cytokines in AOPP-induced THP-1macrophagesAOPP could significantly up-regulate RAGE. Intervention of paeonol inhibited remarkly RAGE gene expression by AOPP in THP-1macrophages at100-200μM. Meanwhile, intervention of paeonol at100-400μM significantly reduced the upregulation of CD36induced by AOPP. In contrast, AOPP decreased SR-A in mRNA level, intervention of paeonol (up to200μM) accelerated SR-A gene expression in a dose-dependent manner and increased the downregulation of SR-B1at100-400μM induced by AOPP.4. Conclusions:4.1Compound huanggan extract was effective in delaying renal failure in that it could alleviate inflammation response by inducing NO and oxidative stress injury by reducing ROS, TNF-a, IL-1β, IL-6and MCP-1.4.2Paeonol could decrease inflammation cytokines (TNF-a, IL-1β, IL-6and MCP-1) in THP-1macrophages likely through a RAGE, CD36, SR-A and SR-B1-mediated signals involving NADPH oxidase dependent ROS generation and activator NF-кB. In conclusion, paeonol could attenuate the injury by oxidative stress.