Enhanced intestinal production of pro-inflammatory cytokines has been considered to contribute to the pathogenesis of inflammatory bowel disease (IBD) such as Crohn's disease and ulcerative colitis. In the course of inflammation, interleukin-8 (IL-8), a CXC chemokine, attracts and activates neutrophils at the site of infection, which may subsequently culminate into epithelial cell damage. Because the migration of neutrophils and their activation by IL-8 has the potential to perpetuate inflammation by producing reactive oxygen species (ROS), which are not only directly injurious but further increase IL-8 production, the down-regulation of IL-8 is vital in the prevention of inflammation.

Carnosine is a dipeptide composed of β-alanine and L-histidine, its physiological concentration in human skeletal muscle ranges from 2 to 25 mM. Carnosine has been reported to serve as a physiological buffer, divalent metal ion chelator, antioxidant and free radical scavenger. Several studies have demonstrated that carnosine inhibited the angiotensin-converting enzyme, delayed aging, and stimulated wound-healing. Since carnosine is supplied from normal meals like meat and also from dietary supplements through the intestines, the intestinal tissues are thought to be exposed to high concentration of carnosine. However, the functions of carnosine in the intestines have not previously been reported. In the present study, cell culture experiments with Caco-2 cells were therefore conducted to examine the hypothesis that carnosine regulates the secretion of inflammatory cytokines under inflammatory conditions

The regulatory effects of carnosine on IL-8 secretion

The secretion of IL-8 was increased by H2O2, whereas the secretion of IL-1β, TNF-α, or IFN-γ was not significantly affected in Caco-2 cells. We examined whether or not carnosine could suppress the H2O2-induced IL-8 secretion in Caco-2 cells. A co-treatment with carnosine and H2O2 did not affect the level of H2O2-induced IL-8 secretion. The inhibitory effect of carnosine on the H2O2-induced IL-8 secretion was marked after a pretreatment with carnosine than by a simultaneous treatment with carnosine and H2O21. However, cycloheximide, a protein synthesis inhibitor, had no effect on the inhibitory activity of carnosine, suggesting that de novo protein synthesis during the pretreatment was not required for the carnosine-mediated inhibition of IL-8 secretion. On the other hand, the increased uptake of carnosine that occurred during the preincubation did seem to be important. The competition experiment using Gly-Sar, a PepT1 substrate, indicated that a sufficient uptake and high intracellular content of carnosine were required to significantly inhibit IL-8 secretion under inflammatory conditions. In other words, the uptake of carnosine seems to have been the rate-limiting step in the inhibition of IL-8 secretion.

The inhibition of IL-8 secretion may have occurred at a various stages of protein synthesis such as transcription, translation and secretion. To test whether or not carnosine regulated the mRNA level, we determined the relative levels of IL-8 mRNA in the presence of carnosine. Interestingly, carnosine did not reduce the level of H2O2-induced IL-8 mRNA after a co-treatment or pretreatment with carnosine. We also examined whether carnosine inhibited another stimulus-induced IL-8 secretion through the same mechanism. Significant inhibition of IL-8 secretion by carnosine was observed in TNF-α-, cadmium- and humulene-treated Caco-2 cells without reducing the IL-8 mRNA level. These results suggest that carnosine did not inhibit the transcription of IL-8 gene.

Requirement of carnsoine structure for inhibition of IL-8 secretion

Using the component amino acids of carnosine and various histidine-containing dipeptides, we found that there was certain structural requirement for carnosine to show its unique inhibitory characteristic. The H2O2-induced IL-8 secretion in intestinal epithelial cell lines was markedly inhibited by a treatment with histidine while there was no inhibitory effect by β-alanine, suggesting that histidine is responsible for the carnosine action. The suppressive effect of histidine on IL-8 secretion was, however, mediated by inhibition of the transcriptional level, especially the inhibition of NF-κB activation2. These results indicate that the inhibitory mechanism of carnosine on IL-8 secretion differed from that of histidine, a component amino acid. To examine whether other histidine-containing dipeptides would also show an inhibition pattern similar to carnosine, we investigated the effects of Gly-His, Ala-His, and anserine (β-Ala-1-methyl-His) on the H2O2-induced IL-8 secretion. These three peptides inhibited the H2O2-induced IL-8 secretion in a dose-dependent manner. They also reduced the H2O2-induced IL-8 mRNA expression, indicating that the anti-inflammatory mechanism for these histidine-containing dipeptides differed from that for carnosine. By the analysis of intracellular amino acids, we observed that Gly-His and Ala-His had been degraded to the component amino acids. It is therefore reasonable to think that the inhibitory effect of Gly-His and Ala-His on IL-8 secretion and mRNA expression could be attributed to histidine. Although anserine, a methylated derivative of carnosine, remained intact in the cells, the inhibitory mechanism for anserine also differed from that of carnosine. This result suggests that the structure of intact carnosine was required to inhibit IL-8 secretion without affecting the level of mRNA. Although we do not yet know how the carnosine structure is recognized and which molecule in the cells is involved in this recognition, the inhibitory effect of carnosine on IL-8 secretion was uniquely related to its structure.

Mechanism for the inhibitory effect of carnosine on IL-8 production

Several potential mechanisms could be proposed to explain this unique mechanism of carnosine. (1) carnosine may overwhelm the H2O2-induced IL-8 mRNA stabilization, (2) carnosine may down-regulate the translation of IL-8, (3) carnosine may promote IL-8 degradation, or (4) carnosine may inhibit IL-8 secretion through the Golgi complex. IL-8 is secreted after the cleavage of a signal peptide via common secretory apparatus involving the Golgi complex and small secretory vesicles. In this respect, the fourth proposed mechanism can be excluded, because the intracellular IL-8 concentration was significantly decreased in the presence of carnosine. The data for laser scanning confocal microscopy also supports carnosine inhibiting stimulus-induced IL-8 synthesis rather than the secretion of IL-8 through the Golgi complex. The data with the treatment of actinomycin D, a transcriptional inhibitor, show that there was no difference in the degradation rate of IL-8 mRNA in the presence or absence of carnosine, demonstrating that carnosine did not inhibit the stability of IL-8 mRNA in the Caco-2 cells. The inhibitory effect of carnosine on the IL-8 secretion without altering the expression and degradation of IL-8 mRNA suggests that carnosine exerts a direct effect at the translation or post-translational level. Translation is divided into three distinct phases: initiation, elongation, and termination. Among these, the most important step in the translational process is the binding of mRNA to ribosome (i.e. initiation). eIF4E is one of the main regulatory initiation factors and the activation of eIF4E is regulated by its abundance, by its phosphorylation state, and by its binding proteins (4E-BP). We found that carnosine strongly inhibited H2O2-induced eIF4E phosphorylation. Carnosine also significantly inhibited the phosphorylation of Akt, which is the key event regulating the 4E-BP phosphorylation in the mTOR/FRAP pathway. The suppression of Akt phosphorylation by carnosine could inhibit a 4E-BP phophorylation and lead to the suppression of eIF4F complex formation, including eIF4E, eIF4A and eIF4G. These results suggest that the second proposed mechanism, in which carnosine is presumed to down-regulate the translation of IL-8, would be the most plausible.

Carnosine, a bioactive peptide found in most types of dietary meat, was tested for its ability to modulate immune reactions, specifically with respect to the IL-8 secretion in intestinal epithelial cells. Our results demonstrate that carnosine inhibited the stimulus-induced IL-8 production and secretion in the intestinal cells. This inhibition was not likely to have occurred at a transcriptional level, but down-regulation of the translation process via the decreased phosphorylation of eIF4E would have been involved in this inhibition3.

Such a unique inhibitory mechanism of carnosine toward IL-8 secretion would effectively serve to suppress inflammation in the intestinal epithelium, providing a better effect by collaborating with other anti-inflammatory substances such as anserine and histidine which would inhibit IL-8 production at the transcriptional level. Although a more detailed investigation into the translational or protranslational level is required to elucidate the mechanism involved in this phenomenon, the results of this present study provide the first evidence for carnosine acting as an effective suppressor of the inflammatory response of intestinal epithelial cells to various stimuli. These results will help to provide practical intervention for inhibiting intestinal inflammation.

近年、食生活の欧米化に伴って増加している潰瘍性大腸炎やクローン病などの炎症性腸疾患の発症には炎症性サイトカインが深く関わっており、その主な治療法としては免疫抑制剤や抗体などが使われている。しかし、漢方薬や機能性食品などによる症状の緩和・予防も最近注目されるようになってきた。申請者は、抗酸化能などの生理機能を持つことが知られている食品由来ペプチド、カルノシンの抗炎症機能を見出し、その作用機構の解明を試みた。カルノシンはβ-Ala-Hisの構造を持つジペプチドで、肉類等に多く含まれているほか、サプリメントの成分としそも利用されている。これらの食品を介して摂取されたカルノシンは高濃度で腸管に達すると考えられるが、腸管におけるその動態、機能については報告がない。本論文は、ヒト腸管上皮細胞モデル系としてCaco-2を用い、カルノシンによる炎症性サイトカイン分泌抑制効果とその作用機構について詳細に検討しているもので、序論を含む4章および総合討論から構成されている。

序論では研究の背景になる炎症性腸疾患とカルノシンを紹介するとともに、本研究の意義と目的について述べている。

第一章では炎症性サイトカインであるインダーロイキン-8(IL-8)の分泌亢進に与えるカルノシンの影響を検討している。まず、カルノシンの取込み活性がH+依存性であり、ジペプチドによってその活性が抑制されたことから、カルノシンはペプチドトランスポーターPepT1を介して腸管細胞内に輸送されることが示唆された。Caco-2細胞では、過酸化水素、TNF-α(tumor necrosis factor-α)、カドミウム、α-フムレンなどの種々の刺激によりIL-8の分泌が亢進されるが、カルノシンはいずれの刺激によるIL-8分泌亢進も濃度依存的に抑制した。しかし、PepT1の基質であるGly-Sarを共存させカルノシンの細胞内輸送を阻害すると、カルノシンのIL-8分泌抑制効果は軽減されたことから、カルノシンは細胞内に輸送されてIL-8分泌亢進を抑制すると考えられた。各種刺激によるIL-8mRNA発現誘導に及ぼすカルノシンの影響を調べた結果、mRNA発現の亢進はカルノシンによって全く抑制されなかった。このようなことから、カルノシンのIL-8分泌抑制効果は、従来報告されてきたようなIL-8の転写段階を抑制する抗炎症性物質とは異なり、転写以降の段階を抑制によるものであることが示唆された。

第二章ではカルノシンの構造と抑制効果との関係について述べている。カルノジンの構成アミノ酸がIL-8分泌亢進に与える影響を調べた結果、β-*アラニンにはIL-8分泌亢進抑制効果が見られなかったが、ヒスチジンでは強い抑制効果が見られた。また、ヒスチジンを含むカルノシン、アンセリン(β-Ala-Methyl His)、Gly-His、Ala-Hisも過酸化水素によるIL-8分泌亢進を強く抑制したことから、これらのジペプチドのIL-8分泌抑制効果にはヒスチジンが重要な役割を果たしていることが示唆された。そこでヒスチジンのIL-8分泌抑制作用について解析した結果、ヒスチジンは「IKB分解の抑制→NF-KBの核内移行阻害→IL-8の転写活性抑制」というプロセスを介して転写段階でIL-8分泌を抑制することが示唆された。これは転写以降の段階を抑制するカルノシンとは異なる機構であり、カルノシンの効果は構成アミノ酸であるヒスチジンの作用では説明できないことが明らかになった。さらに、Gly-His、Ala-His、アンセリンも転写レベルでIL-8分泌を抑制することが確認された。Gly-HisとAla-Hisは細胞内でそれぞれの構成アミノ酸に分解されるため、その効果はヒスチジンによるものと考えられた。以上の結果から、カルノシン特有の構造的要因がそのIL-8分泌抑制効果にとって重要であることが示された。

第三章ではカルノシンのIL-8分泌抑制の機構について詳細に調べている。第一章の結果から、カルノシンはIL-8の転写以降の段階を抑制することが示唆されたため、IL-8mkNAの安定性、IL-8の翻訳、細胞外への分泌、合成されたIL-8の分解といった各段階へのカルノシンの作用を一つずつ検討した。まずmRNA合成抑制剤アクチノマイシDを用いた実験の結果、カルノシンはIL-8mRNAの安定性には影響を与えないことが分かった。またレーザ共焦点顕微鏡とELISA法を用いて細胞内のIL-8量を測定した結果、カルノシン処理はIL-8のタンパク質産生自体を顕著に抑制していることが認められた。翻訳に着目して実験を進めた結果、カルノシンは翻訳開始因子の一つであるeIF4Eのリン酸化を抑制することによってIL-8産生を抑制すること、eIF4Eのリン酸化に関わるERKI/2とp38MAPKのリン酸化を抑制することを見出し、カルノシンによるユニークなIL-8産生制御機構の一端が明らかになった。

以上要するに、本研究はごニークな食品由来ペプチドであるカルノシンが抗炎症作用を持つことを明らかにするとともに、炎症性サイトカインの分泌抑制におけるその新規な機構を解明したもので、学術的にも応用的にも寄与するところが大きい。よって審査委員一同は、本研究が博士(農学)の学位論文として価値あるものと認めた。