評估巨噬細胞中C1GALT1的抗纖維化作用

Abstract

纖維化主要發生於組織修復的異常。先前研究指出巨噬細胞會分泌細胞因子誘使成纖維細胞轉為肌纖維母細胞，其中TGFβ可促進膠原蛋白等細胞外基質堆積，在發炎及抗炎反覆作用中造成纖維化。另外細胞表面醣類結構改變會影響巨噬細胞的分化及功能，而Core 1 β1,3-galactosyltransferase（C1GALT1）為延長醣類結構的必需酶，可參與氧型醣基化修飾。目前已知C1GALT1在許多疾病中過表達，但在纖維化及巨噬細胞中的影響仍不清楚。因此本研究主要想了解巨噬細胞中C1GALT1於纖維化扮演的角色。首先，在體內實驗透過lysM-Cre系統敲除C57BL/6小鼠巨噬細胞中C1GALT1表達，並以博來黴素建立纖維化模型。結果顯示C1GALT1 f/f LysM Cre相較控制組小鼠肺及皮膚纖維化情形獲得改善，由免疫螢光染色及西方墨點法共同得到TGFβ在C1GALT1敲除後表現量下降，且在IHC及IF中TGFβ下游表現亦降低。體內實驗以THP1單核細胞進行，透過凝集素驗證巨噬細胞於極化過程中醣類結構有所改變。藉由慢病毒shC1GALT1感染再進行分化，以qPCR得到C1GALT1的下調會促進M1並抑制M2分化。另外，以市售藥物Itraconazole調降C1GALT1後，TGFβ及Smad2/3 蛋白表現下降，而M2的極化也受到限制。實驗也以條件培養基作用於NIH/3T3成纖維細胞，結果得到在調降C1GALT1的M2組別， NIH/3T3的爬行及增殖能力皆顯著下降。最後藉BALB/c小鼠建立纖維化模型並以Itraconazole治療，結果顯示施打Itraconazole的組別纖維化情形改善，TGFβ及下游表現皆受到抑制。綜合以上結果，調降巨噬細胞中C1GALT1可抑制M2分化並具抗纖維化潛力，而Itraconazole亦能有更多應用。未來也致力於更詳細的作用機制。

Fibrosis mainly occurs in the process of abnormal tissue repair. Previous studies have found that macrophages secrete cytokines to promote the conversion of fibroblasts into myofibroblasts which contain TGFβ can promote collagen in extracellular matrix. However, the repeating of inflammation and anti-inflammatory induced the impaired accumulation of collagen that caused the fibrosis. In addition, changes in the carbohydrate structure on the cell surface can affect the differentiation and function of macrophages, and Core 1 β1,3-galactosyltransferase (C1GALT1) is an essential enzyme for extending carbohydrate structures and can participate in O-glycosylation modification. It is currently known that C1GALT1 is overexpressed in many diseases, but its role of C1GALT1 in fibrosis and macrophage is still unclear. Thus, the aim of this study to understand the role of C1GALT1 in macrophages and fibrosis. In vivo, the expression of monocyte in C1GALT1 on C57BL/6 mouse was specific knocked out (KO) through the lysM-Cre system then used bleomycin to establish fibrosis model. The results showed that the lung and skin fibrotic area is less in C1GALT1f/f LysM Cre mice compared with the control group effected by bleomycin. Immunofluorescence staining and Western blotting showed the expression and the downstream expression of TGFβ was decreased in C1GALT1 KO mice. In vitro experiment was performed on the THP1 cell line, Lectins used to verify the change of carbohydrate structure on macrophages surface during the polarization process. We infected shC1GALT1 cell line by lentivirus then initiate differentiation, via qPCR showed the down-regulation of C1GALT1 promotes M1 and inhibits M2 differentiation. In addition, after the down-regulation of C1GALT1 affected with the commercial drug Itraconazole, the expression of TGFβ and Smad2/3 proteins decreased. And the polarization of M2 was also restricted. The experiment also used conditioned medium to act on NIH/3T3 fibroblasts, and the results showed that the crawling and proliferation abilities of NIH/3T3 were significantly decreased in the M2 group in which C1GALT1 was downregulated. Finally, a fibrosis model was established in BALB/c mice and treated with itraconazole. The results showed that the fibrosis in the group treated with itraconazole was improved. The expression of TGFβ and their downstream expressions were also inhibited. Taken together, down-regulation of C1GALT1 in macrophages can inhibit M2 differentiation and has anti-fibrotic potential. The mechanism of C1GALT1 maybe a potential novel protein for fibrosis disease and the application of itraconazole can further investigate.