| 摘要: | 微生物基因組群 (microbiome) 在人類健康和疾病發病機制中扮演重要的角色。我們現有的數據是基於皮膚微生物基因組群 (skin microbiome) 在皮膚病學的顯現例如瘙癢或發炎反應，尤其著重在尿毒性瘙癢與慢性紫外線 ((UV)-B) 引起的皮膚發炎反應之中的益生菌作用。尿毒性搔癢伴隨皮膚中磷酸鈣 (calcium phosphate; CaP) 濃度的升高是慢性腎臟病 (chronic kidney disease; CKD) 患者常見的症狀。在這項研究中我們證明，皮內注射到小鼠的磷酸鈣所引起的老鼠抓癢行為，隨著注射劑量的增加而增加，是透過正向調控在皮膚中的IL-6的表現量與背根神經節(dorsal root ganglion; DRG)中ERK的磷酸化程度。在IL-6基因剔除小鼠的背根神經節中，磷酸鈣誘導的ERK磷酸化 (p-ERK) 的正向調控程度大幅減少，因此IL-6是不可缺少的。磷酸鈣注射後，微陣列分析加上即時聚合酶鏈鎖反應分析顯示，在背根神經節中布魯頓氏酪氨酸激酶 (Bruton’s tyrosine kinase; BTK) 基因的mRNA表現量更高。透過Ibrutinib將BTK抑制後明顯地減少磷酸鈣誘導的IL-6表現量與ERK磷酸化的正向調控程度。在慢性腎臟病患者的搔癢皮膚與血液中檢測到 較高量的IL-6。經過IL-6重組蛋白處理後，DRG初代細胞中的p-BTK與p-ERK的表現量分別在第1分鐘與第10分鐘達到最高程度，並且兩者的表現量在細胞同時處理IL-6重組蛋白與Ibrutinib之後顯著地降低。磷酸鈣誘導的搔癢機制是透過IL-6/p-BTK/p-ERK訊息傳導路徑所介導的。
皮膚共生益生菌在磷酸鈣誘導的尿毒性搔癢和發炎反應中的作用尚未被鑑定與揭露。在這項研究中我們證實皮膚共生菌，痤瘡嗜酸桿菌 (Cutibacterium acnes) 透過葡萄糖發酵產生的短鏈脂肪酸 (short-chain fatty acids; SCFAs)，例如丁酸，溶解磷酸鈣。像丁酸一樣，N-[2-(2-Butyrylamino-ethoxy)-ethyl]-butyramide (BA-NH-NH-BA)，一種丁酸的衍生物，在角質細胞中顯著地誘導組織蛋白H3離胺酸 (histone H3 lysine 9; AcH3K9) 的乙醯化。在小鼠皮膚上局部施用發酵的痤瘡丙酸桿菌、丁酸或BA-NH-NH-BA可以有效地改善磷酸鈣誘導的皮膚搔癢 角質細胞中白介素IL-6的正向調控程度，以及背根神經節中p-BTK和p-ERK表現程度。在IL-6基因剔除小鼠中，磷酸鈣誘導的p-BTK和p-ERK正向調控明顯減少。在慢性腎臟病患者的搔癢皮膚中量測到的角質屬細菌含量相對較低。我們的結果確定了皮膚發酵的痤瘡丙酸桿菌能夠改善磷酸鈣誘導的IL-6/p-BTK/p-ERK訊息傳導路徑的活化並造成發炎反應的作用。此外，我們更提供BA-NH-NH-BA作為一種後抗生素治療尿毒性瘙癢症的潛在治療功效的證據。
我們還檢測到皮膚共生益生菌，表皮葡萄球菌(Staphylococcus epidermidis) 對慢性紫外線輻射的抗發炎作用是透過甘油發酵產生例如丁酸的短鏈脂肪酸來進行的。我們檢測到皮膚微生物基因組群中的表皮葡萄球菌在體外以毫摩爾範圍的濃度產生丁酸。小鼠背側皮膚暴露於UVB光引起顯著的IL-6產量增加。局部單獨施用丁酸或表皮葡萄球菌與甘油一起施用可顯著改善UVB誘導的IL-6產生。小鼠皮膚中2的短鏈脂肪酸受體2 (short-chain fatty acid receptor 2; FFAR2) 的體內剔除明顯地阻斷了表皮葡萄球菌對於抑制UVB誘導的IL-6產生的益生作用。這些結果表明，發酵中的皮膚益生菌的代謝產物中的丁酸介導FFAR2來調節UVB誘導的促炎性細胞因子的產生。總體而言，我們已經闡明了尿毒性搔癢症和UVB誘導的皮膚發炎反應的分子信號傳導路徑，並且驗證了丁酸的抗發炎作用。丁酸是痤瘡丙酸桿菌和表皮葡萄球菌皮膚益生菌透過發酵碳源產生的主要代謝產物，可提供預防和治療尿毒性瘙癢和UVB引起的皮膚慢性發炎的治療方法。;Microbiome plays a significant role in human health and disease pathogenesis. Our current data based on the probiotic role of the skin microbiome in dermatological manifestations like pruritus or inflammation with a special emphasis on uremic pruritus and chronic ultraviolet (UV)-B induced skin inflammation. Uremic pruritus with elevated levels of calcium phosphate (CaP) in skin is a common symptom in patients with chronic kidney disease (CKD). In this study, we demonstrate that intradermal injection of CaP into mice triggered scratching by up-regulating the IL-6 in skin and phosphorylation of ERK in dorsal root ganglion (DRG) in a dose-dependent manner. IL-6 is essential because the CaP-induced up-regulation of phosphorylated (p)-ERK in DRG was considerably reduced in the IL-6 knockout mice. Microarray analysis in conjunction with real-time PCR revealed higher mRNA expression of Bruton’s tyrosine kinase (BTK) gene in DRG after CaP injection. The inhibition of BTK by ibrutinib noticeably decreased the CaP-induced up-regulation of IL-6 and p-ERK in mice. A high amount of IL-6 was detected in itchy skin and blood of patients with CKD. The expressions of p-BTK and p-ERK in DRG primary cells reached maximum levels at 1 and 10 min, respectively, after treatment of recombinant IL-6 and were significantly reduced by treatment of IL-6 along with ibrutinib. The mechanism by which the CaP-induced pruritus mediated by the IL-6/p-BTK/p-ERK signaling was revealed.
The role of skin commensal microbiota in the CaP-induced uremic pruritus and inflammation remains uncharacterized. In this study, we have shown that skin commensal, Cutibacterium acnes (C. acnes) can solubilize CaP by the production of short-chain fatty acids (SCFAs), such as butyric acid, through glucose fermentation. Like butyric acid, the N-[2-(2-Butyrylamino-ethoxy)-ethyl]-butyramide (BA-NH-NH-BA), a butyric acid derivative, remarkably induced acetylation of histone H3 lysine 9 (AcH3K9) in keratinocytes. Topical application of fermenting C. acnes and its metabolite, butyric acid or BA-NH-NH-BA onto mouse skin effectively ameliorated CaP-induced skin itching, interleukin IL-6 up-regulation in keratinocytes, p-BTK and p-ERK in DRG. The upregulation of p-BTK and p-ERK by CaP was markedly reduced in IL-6 knockout (KO) mice. Genus Cutibacterium was detected in relatively low abundance in itchy skin of patients with CKD. Our results identify a role for the skin fermenting C. acnes in ameliorating CaP-induced activation of IL-6/p-BTK/p-ERK signaling and resulting skin inflammation. Moreover, we provide evidence for the potential therapeutic efficacy of BA-NH-NH-BA as a postbiotic for the treatment of uremic pruritus.
We have also detected the anti-inflammatory effect of skin commensal, Staphylococcus epidermidis (S. epidermidis) against chronic ultraviolet B (UVB) radiation by the production of SCFAs, such as butyric acid, through glycerol fermentation. We detected that S. epidermidis in the skin microbiome produced butyric acid in vitro at concentrations in the millimolar (mM) range. The exposure of the dorsal skin of mice to UVB light provoked a significant increase in IL-6 production. Topical application of S. epidermidis with glycerol or butyric acid alone remarkably ameliorated the UVB-induced IL-6 production. In vivo knockdown of short-chain fatty acid receptor 2 (FFAR2) in mouse skin considerably blocked the probiotic effect of S. epidermidis on suppression of UVB-induced IL-6 production. These results demonstrate that butyric acid in the metabolites of fermenting skin probiotic bacteria mediates FFAR2 to modulate the production of pro-inflammatory cytokines induced by UVB. Overall, we have unraveled the molecular signaling in uremic pruritus and UVB induced skin inflammation and validated the anti-inflammatory role of butyric acid, a major metabolite from the fermentation of carbon source by C. acnes and S. epidermidis skin probiotic bacteria could provide a therapeutic approach for the prevention and treatment of uremic pruritus and UVB induced chronic inflammation in skin. |
