생쥐신장에서 Glycerol 유도 급성신부전에서 TNF-α, IL-6 분비에 관한 스쿠알렌의 효과

Abstract

신장에 급성신부전이 발생되어지면 사구체여과 기능, 신장 독성, 관 손상, 이온의 흡수 및 배설 기능에 영향을 받는다. NO는 mitogenesis 억제 및 대식세포 독성, TNF-α와 LPS 자극에 의한 apoptosis 조절, 신장 근위세뇨 RDPase 방출 조절 등 면역병리 과정에 관여한다. TNF-α는 면역반응과 염증반응을 유도하는 염증유도매개 싸이토카인으로 apoptosis, necrosis 등의 기능 관여, 미생물 감염 시 발현량이 증가, 식세포의 싸이토카인 분비 증가를 유도하여 미생물에 대한 숙주세포의 항상성을 유지하는 중요한 방어기전 역할을 담당한다. IL-6는 TNF-α와 IL-1과 함께 급성기 단백반응의 유도체로서 anti-inflammatory와 pro-inflammatory cytokine으로 알려져 있다. IL-6는 CD⁴+ T-세포, cytotoxic T-세포 분화와 NK(natural killer) 세포 기능을 자극하고, IL-1ra 합성과 수용성 TNF 수용체 방출을 유도하는 작용을 가지고 있다. 스쿠알렌은 콜레스테롤의 생합성에 아주 중요한 선구물질이다. 스쿠알렌는 상처 치유, 동맥경화 억제 작용, 심근경색, 화상 치료, 카드뮴 독성 완화, 항암효과, 섬유아세포 성장인자 활성 효과 등이 있다. 본 연구에서는 생쥐에 glycerol를 이용하여 ARF를 유도한 후, 스쿠알렌 처치후 싸이토카인 중 조직내 염증 반응 및 면역에 관여하는 NO, TNF-α와 IL-6의 생성 정도와 신장 조직 회복에 관해 알아보고자 하였다. In vitro 실험의 경우 Renca 세포(2×105/well)를 이용하여 세포독성 지표인 LDH,염증반응의 지표인 NO를 측정하였고, glycerol(4 mM)-유도 싸이토카인 중 TNF-α, IL-6를 측정하였다. In vivo 실험의 경우 생쥐에 glycerol(50 %, 8 ㎖/㎏)을 근육 주사하고, 스쿠알렌(80 ㎖/㎏) 처치 한 후, glycerol에 의해 유도되어진 TNF-α, IL-6 생성량을 측정하였다. 실험 결과, in vitro의 경우 스쿠알렌 자체만으로는 세포독성이 없으며, NO생성에도 큰 영향을 주지 않았다. glycerol-유도 Renca 세포주에서 TNF-α, IL-6의 생성량도 glycerol 단독 처치 군에 비해 유의하게 감소시킴을 관찰할 수 있었다(P<0.05). In vivo 실험의 경우 glycerol 단독 처치 군에 비해 스쿠알렌을 1일 1회 처치한 군에서 48, 72 시간째에 TNF-α, IL-6 생성량을 감소시킴을 관찰할 수 있었다(P<0.05). 본 연구를 통해서 급성신부전을 유발시킬 수 있는 물질 중 하나인 glycerol에 의해 유도되어 지는 싸이토카인의 분비 억제에 스쿠알렌 처치가 효과가 있어서 면역증진 효과를 기대할 수 있을 것으로 사료된다.|Kidney had recovery function toxicants, ischemia, reperfusion -induced damage, acute-renal failure (below, called ARF). ARF is characterized by a deterioration of renal function over a period of hours to days. ARF results in failure of the kidney to excrete nitorgenous waste products and maintain fluid and electrolyte homeostasis. Nitric oxide (below, called NO) is a recently discovered mediator of cell communication involved in a variety physiological and pathophysiological processes. NO is syn- thesized from L-arginine by a family of enzymes called NO synthases (below, called NOS). This enzyme is present in a variety of tissues including smooth muscle cells and macrophages and take part in several immunopathological process. NO has a short half-life and is rapidly oxidized to the stable, inactive end-products, nitrite and nitrate. Macrophages play a major role in host defense against infection and cancer. Activation of macrophages induces the production and release of many inflammatory mediators, including cytokines (TNF-α,IL-6), arachidonic acid metabolites and NO. Pro-inflammatory cytokines are produced in response to variety of stimuli including bacterial endotoxin, pro-oxidants, and certain environmental agents. Tumor necrosis factor-α (below, called TNF-α, a 17-kDa trimeric protein) is a proinflammatory cytokine that often is overexpressed in a number of disease states such as sepsis syndrome, rheumatoid arthritis, inflammatory bowel disease and periodontitis. TNF-α is a known to be a key mediator for the induction of apoptosis in murine thymus and organization of lymphoid organs, as well as development of humoral immune response. And TNF-α can induce the production of NO. However, at high concentrations, TNF-α has dis- advantageous effects, such as inducing tissue injury and potentiating septic shock. Interleukin-6 (below, called IL-6, a 26-kDa protein) is a multifunctional cytokine. IL-6 is released by T and B lympho- cytes, macrophages, fibroblasts, endothelial cells, mesengial cells, and tubular epithelial cells. And, IL-6 release is stimulated by TNF-α. Squalene (below, called SQ), a polyunsaturated triterpene that contain six isoprene units, is a key intermediate in cholesterol biosynthesis. It received its name because of occurrence in shark liver oil (Squaluss spp.) which is considered to be the richest source of squalene. SQ has been reported to possess antioxidant, membrane stabilizing properties, detoxic effect against diverse chemicals, protect the skin from ultraviolet radiation, protect the cyclophosph- amide-induced toxicity, effect of bFGF. protect of alcohol damage. The aim of this study is to evaluate the effects squalene on the prevention of experimental ARF induced by glycerol. In vitro experiments, The cytotoxic effects of SQ (0.1%) was evaluated by the LDH. And, We were investigated to production of NO, TNF-α and IL-6 in Renca kidney cells (2×105/well) after treatment glycerol(4mM) or SQ (0.1%). In vivo experiments, We were investigated to production of TNF-α and IL-6 after treatment with glycerol. The experimental groups were divided into three groups. Group 1 was normal mouse. Group 2 was not treatment with squalene after intramuscular injection of glycerol (50 %, 8 ml/kg). And, Group 3 was treated with squalene (180 ㎎/㎏) after intramuscular injection of glycerol (50 %, 8 ml/kg). All groups were used to 10 mice. The supernatants or tissue were analyzed for TNF-α and IL-6 by a using a commercial ELISA kit (Minneapolis, USA) according th the manufacturer′s guidelines. In vitro results, SQ did not affect on the LDH and NO production by itself. At 6 hour, the production of TNF-α was affected compared with only glycerol group (p<0.05). But, at 24 hour, the production of TNF-α did not affected. On the other hand, IL-6 was affected compared with glycerol only at 24 hour (p<0.05). In vivo results, SQ was decreased the glycerol-induced TNF-α and IL-6 production compared with glycerol only at 48 and 72 hours (p<0.05). It was concluded that SQ will increase the immune response on the glycerol induced-renal damage.