Pentraxin 3激活JNK信號並調節腎纖維化中的上皮間質轉化

研究目的：腎小管間質纖維化可導致末期腎臟疾病。五聚環蛋白-3 (Pentraxin 3, PTX3)是一種由駐留型和先天性免疫細胞所產生的急性反應蛋白質。我們研究了五聚環蛋白-3對培養的人類近端腎小管上皮（human proximaltubular epithelial cell line, HK-2）細胞和大鼠單側輸尿管阻塞（unilateral ureteral obstruction, UUO）腎臟纖維化模式的影響。希望藉由本研究，能對於造成腎纖維化及上皮間質轉化的機制，有較多的了解並能找到可能的輔助治療方法。研究方法及材料：本研究利用重組人類五聚環蛋白-3 (recombinant human Pentraxin 3, Rh-PTX3)對人類近端腎小管上皮(HK-2)細胞的作用以MTT和細胞移動實驗測量細胞增殖和體外細胞遷移作用。使用免疫組織化學分析單側輸尿管阻塞(UUO)腎纖維化模式的大鼠內五聚環蛋白-3，p-JNK1/2和上皮間質轉化相關蛋白(snail、slug、N-cadherin和 vimentin)表現。使用反轉錄聚合酶鏈式反應和西方墨點法分析五聚環蛋白-3，p-JNK1/2和上皮間質轉化相關mRNA和蛋白質表現。研究結果：利用重組人類五聚環蛋白-3處理的人類近端腎小管上皮HK-2細胞不影響細胞生長，但是細胞遷移能力顯著增加。此外，重組人類五聚環蛋白-3增加了snail、slug、N-cadherin和 vimentin蛋白的表現，降低了E-cadherin蛋白的表現，增加了JNK1/2的磷酸化表現。利用SP600126（專一性JNK1/2活化抑制劑）和重組人類五聚環蛋白-3共同作用抑制JNK1/2活化、snail和slug蛋白表現，和增加E-cadherin的表現。利用單側輸尿管阻塞腎臟纖維化模式的大鼠證實隨著時間增加會造成PTX3、p-JNK1/2, and 和vimentin表現增加，且E-cadherin的表現下降。結論與建議：綜合上述結果發現，PTX3透過JNK1/2活化作用機制來調控上皮間質轉化相關蛋白表現因而誘導細胞遷移能力增加，因此本研究證實PTX3在人類腎臟纖維化的致病機轉的角色

Objective：Tubulointerstitial fibrosis can lead to end-stage renal disease. Pentraxin 3 (PTX3) is an acute phase protein produced by resident and innate immunity cells. We investigated the effect of PTX3 on cultured human proximal tubular epithelial (HK-2) cells and a rat unilateral ureteral obstruction (UUO) model of renal fibrosis.Methods and Materials：Gain-of-function experiments were used to examine the effect of recombinant human PTX3 (Rh-PTX3) on HK-2 cells. Cell proliferation (MTT assay) and in vitro cell migration were measured. The levels of PTX3, p-JNK1/2, and EMT markers were measured using immunohistochemistry, RT-PCR, and western blotting in UUO rats and HK-2 cells.Results：HK-2 cells treated with Rh PTX3 did not affect cell viability, but significantly increased cell migration. Moreover, Rh-PTX3 increased the expression of snail, slug, N-cadherin, and vimentin, decreased the expression of E-cadherin, and increased the phosphorylation of JNK1/2. SP600126 (a specific JNK1/2 inhibitor) enhanced the effects of Rh-PTX3. Rats with UUO exhibited time-dependent increased levels of PTX3, p-JNK1/2, and vimentin, and decreased expression of E-cadherin.Conclusion and Suggestion：Our results suggest that PTX3 induces cell migration via upregulation of EMT in a JNK1/2-dependent mechanism, and highlight the role of PTX3 in the pathogenesis renal fibrosis.