Anti-Inflammatory Effects of Egg White Combined with Chalcanthite In
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154 INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 31: 154-162, 2013 Anti-inflammatory effects of egg white combined with chalcanthite in lipopolysaccharide-stimulated BV2 microglia through the inhibition of NF-κB, MAPK and PI3K/Akt signaling pathways EUN-A CHOI1*, HYE YOUNG PARK2*, HWA-SEUNG YOO3 and YUNG HYUN CHOI4,5 1Insan Bamboo Salt Inc. and Insan Korean Medical Cancer Clinic, Hamyang 676-805; 2Department of Pharmacy, Busan National University, Busan 609-735; 3Department of East-West Cancer Center, College of Oriental Medicine, Daejeon University, Daejeon 301-724; 4Department of Biochemistry, Dongeui University College of Oriental Medicine, Busan 614-052; 5Department of Biomaterial Control (BK21 Program), Anti-Aging Research Center and Blue-Bio Industry RIC, Dongeui University, Busan 614-714, Republic of Korea Received August 10, 2012; Accepted October 12, 2012 DOI: 10.3892/ijmm.2012.1169 Abstract. Egg white-chalcanthite (EWCC) is a mixture of c-Jun N-terminal kinase, p38 mitogen-activated protein egg white and chalcanthite prepared by roasting chalcanthite kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/Akt (which is a natural mineral mainly composed of CuSO4•5H2O) pathways. Our results indicate that the inhibitory effects of to the point of dehydration, pulverizing the dehydrated EWCC on LPS-stimulated inflammatory mediator production chalcanthite and then mixing the pulverized chalcanthite in BV2 microglia are associated with the suppression of the to react with egg white to trigger a reaction. When egg NF-κB, MAPK and PI3K/Akt signaling pathways. These find- white-chalcanthite is prepared in this manner, the toxicity ings suggest that EWCC may offer a substantial therapeutic of chalcanthite is neutralized by the egg white, so that the potential for the treatment of neurodegenerative diseases that toxicity is reduced or removed and the pharmaceutical are accompanied by microglial activation. properties are increased. However, the cellular and molecular mechanisms underlying the pharmacological activity of Introduction EWCC remain poorly understood. In this study, we inves- tigated the inhibitory effects of EWCC on the production Minerals have been traditionally used in Oriental herbal of lipopolysaccharide (LPS)-induced pro-inflammatory medicine treatment, particularly for immune-related diseases mediators in BV2 microglia. Our data indicated that the in Korea, China and other Asian countries (1-3). Among them, EWCC treatment significantly inhibited the excessive produc- chalcanthite is a richly colored blue water-soluble sulfate tion of nitric oxide and prostaglandin E2 in LPS-stimulated mineral, commonly found in the late-stage oxidation zones BV2 microglia in a concentration-dependent manner of copper deposits (4). The main ingredient of chalcanthite without causing cytotoxicity. It also attenuated the expres- is CuSO4. CuSO4 exists as a series of compounds that differ sion of inducible nitric oxide synthase, cyclooxygenase-2 in their degrees of hydration. The anhydrous form occurs as and pro-inflammatory cytokines, including interleukin-1β a rare mineral known as chalcocyanite. The hydrated copper and tumor necrosis factor-α. Moreover, EWCC exhibited sulfate occurs in nature as chalcanthite (CuSO4•5H2O) and anti-inflammatory properties by the suppression of nuclear two of the rarer ones include bonattite (CuSO4•3H2O) and factor-κB (NF-κB) activation by blocking IκB-α degradation, boothite (CuSO4•7H2O) (4). According to Oriental medicine, downregulation of extracellular signal-regulated kinase, chalcanthite has sour, acrid, cold and toxic properties, and is affiliated with the liver meridian. Its main function is to elimi- nate toxins. Internally, it functions as a purgative and helps the body get rid of excess phlegm and toxins. Chalcanthite can also be applied externally to treat skin abscesses, reduce Correspondence to: Dr Yung Hyun Choi, Department of inflammation and remove necrotic tissue. The tradition- Biochemistry, Dongeui University College of Oriental Medicine, ally used dose of chalcanthite is between 0.3 and 0.6 g of Busan 614-052, Republic of Korea powdered mineral, decocted in water for oral administration. E-mail: [email protected] Larger amounts can be used when chalcanthite is applied *Contributed equally externally. However, chalcanthite is poisonous and should be used only with extreme caution. To date, due to safety Key words: egg white-chalcanthite, anti-inflammation, nuclear issues concerning their cytotoxic mechanisms, many mineral factor-κB, mitogen-activated protein kinase, phosphoinositide medicines are rarely used. 3-kinase/Akt In addition to being a quality protein source, egg whites contain many essential minerals including potassium, magne- CHOI et al: ANTI-INFLAMMATORY EFFECTS OF EGG WHITE COMBINED WITH CHALCANTHITE 155 sium, calcium, phosphorus, copper, zinc and iron. They are its association with the inhibitory protein, IκB (24-26). Once also a good source of riboflavin and selenium along with activated by inflammatory stimulants such as LPS, κI Bs are essential vitamins such as folate, B12, niacin betaine and rapidly phosphorylated and degraded by IκB kinase (IKK). choline (5-9). In Medieval times in the West, egg whites were IκB degradation liberates NF-κB to move into the nucleus, used for medicinal purposes such as treating wounds, skin resulting in transcriptional induction of inflammation-associ- disorders and mending broken bones. At present, egg whites ated genes (27,28). are considered one of the most nutrition-rich foods and are Based on these previous observations, the present study widely recommended for children, the elderly, athletes and was designed to evaluate the inhibitory effects of EWCC patients with a variety of health conditions. on the LPS-stimulated inflammatory mediator production Egg white-chalcanthite (EWCC) is a mixture of egg white in murine BV2 microglia and the related anti-inflammatory and chalcanthite and may be prepared by roasting the chal- mechanisms. As a result of our findings, we suggest that canthite in order to dehydrate it. The dehydrated chalcanthite EWCC may be a candidate for the treatment of various is then pulverized and the pulverized chalcanthite is mixed to neurodegenerative disorders. react with the egg white in order to trigger a chemical reac- tion. In the egg white-chalcanthite prepared as noted above, Materials and methods the toxicity of the chalcanthite is neutralized by egg white, so that the toxicity is reduced or removed and the pharmaceutical Preparation of EWCC. For the preparation of EWCC, chal- properties are increased. Recently, it has been reported that canthite was heated and dehydrated. During the heating EWCC inhibits the growth of human lung cancer NCI-H460 process, chalcanthite was uniformly heated while carefully cells by inducing apoptotic cell death through caspase-3 being watched for a color change every 3-5 h until it turned activation (10). However, the precise biochemical mechanisms completely grey. The heating duration (~10-24 h) varied with underlying EWCC-induced anti-inflammatory effects have the amount and quality of the mineral used. Once the chal- not yet been clarified. canthite was dehydrated, it was cooled until the remaining Microglia have conventionally been regarded as brain heat was completely released. The moisture content of the macrophages and have been proposed to play a major role in chalcanthite ranged from 0 to 5%. After cooling, chalcanthite host defense and tissue repair in the central nervous system was finely pulverized. The pulverized chalcanthite was stored (CNS) (11). They are activated in response to environmental in a concealed airtight container in a dry place to prevent stress and exposure to lipopolysaccharide (LPS), interferon moisture from accumulating. The prepared chalcanthite (IFN)-γ and β-amyloid (12,13). Activation of microglia results powder was mixed with egg whites at ratios of 30:13 to 30:30 in the release of various bioactive molecules, including nitric for 10 min. The egg whites needed to be homogeneously oxide (NO), prostaglandin E2 (PGE2), reactive oxygen species mixed with a wooden spatula in a ceramic vessel so the (ROS), monocyte chemoattractant protein-1 (MCP-1) and utensils would not interfere or cause any chemical reactions pro-inflammatory cytokines, such as interleukin (IL)-1β, IL-6 during the mixing process. After the chalcanthite and egg and tumor necrosis factor-α (TNF-α) (14,15). Overproduction whites were sufficiently mixed, the mixture was cooled until of these inflammatory mediators causes various severe the reaction heat was completely released. One hundred milli- neurodegenerative diseases, such as Alzheimer's disease, grams of the obtained substance was then dissolved in 1 liter Parkinson's disease, cerebral ischemia, multiple sclerosis of deionized water and centrifuged at 6,000 rpm for 10 min. A and trauma (16-18). Therefore, activated microglia have been portion of the upper level concentration (0.80 µm) was filtered shown to be a major cellular source of pro-inflammatory and/ by syringe filter after the centrifugation. or cytotoxic factors that cause neuronal damage in the CNS. Previous studies have also demonstrated that a decrease in the Cell culture. The murine BV2 cell line was maintained in number of pro-inflammatory mediators in microglia attenu- DMEM supplemented with 10% fetal bovine serum (FBS), ates the severity of these disorders (19-21). Thus, control of 100 U/ml penicillin and 100 µg/ml streptomycin at 37˚C in a microglial