微小核醣核酸miR-196a可能藉由抑制骨橋蛋白來改善第一型糖尿病小鼠模型的腎臟纖維化

Abstract

糖尿病(Diabetic mellitus, DM)屬於代謝性症候群，其病症之共同特徵為高於一般人之血糖濃度，並容易引起慢性發炎反應與相關的併發症。當腎臟功能因糖尿病而受損時，我們稱之為糖尿病腎病變(Diabetic nephropathy, DN)。近年來的研究指出，患有糖尿病的病人在骨質疏鬆方面亦有較高的風險。為了探討糖尿病與低骨質密度之間的關係，骨橋蛋白(Osteopontin, OPN)被認為是一個具潛力的指標。骨橋蛋白目前被定位為一種多功能型的蛋白質，可誘導介白素(Interleukin, IL) -1β、介白素-6 (IL-6)、腫瘤壞死因子(Tumor necrosis factor, TNF) -α、單核球趨化蛋白(Monocyte chemo-attractant protein, MCP) -1等促炎性細胞因子，並協助巨噬細胞滲入組織與聚集於特定區域。此外，骨橋蛋白亦與轉化生長因子(Transforming growth factor, TGF) -β的訊息傳導路徑相關，參與第一型膠原蛋白的生成。為了找尋可能調控骨橋蛋白的機制，我們認為微小核醣核酸miR-196a可能是一個值得探討的目標。miR-196a最早被發現跟胚胎發育與哺乳類動物的肢體發育有關，而一組科學團隊於2016年發現miR-196a的過量表現可藉由直接互補配對其標的物—轉化生長因子的第二型受體(TGF-βRII) —來抑制其表現量，並因此而減輕腎臟的纖維化。我們的目標為證明miR-196a是藉由抑制骨橋蛋白的表現量，來改善第一型糖尿病小鼠模型的腎臟纖維化。在我們的研究中，我們以鏈脲黴素(Streptozotocin, STZ)誘發小鼠的第一型糖尿病，並於miR-196a基因轉殖小鼠中發現，其過量表現可延緩腎臟功能衰退的進程，並抑制與維持白蛋白滲出至尿液中的量。在基因轉錄過程，miR-196a造成腎臟中的骨橋蛋白以及腫瘤壞死因子-α、介白素-6等發炎反應指標的表現量下降，顯示其抑制發炎反應的可能性。轉化生長因子-β在腎臟的核糖核酸表現量亦下降，表示miR-196a亦有可能抑制腎臟的纖維化。另外，利用腎臟組織切片的蘇木精-伊紅染色(Hematoxylin and eosin stain, H&E stain)和天狼星紅染色(Picro-sirius red stain)，我們發現miR-196a的過量表現在第一型糖尿病小鼠中，可以抑制其腎絲球的膨脹、白血球的浸潤與膠原蛋白的堆積。這些證據皆支持miR-196a減輕糖尿病腎病變病徵的潛力。因此，我們認為miR-196a可經由降低發炎反應與纖維化來抑制糖尿病腎病變，而且骨橋蛋白可能為連結miR-196a與這些致病機轉的中介者。在未來，我們相信隨著更多這方面的研究，miR-196a有機會成為治療糖尿病腎病變的其中一種有效方法。

Diabetes Mellitus (DM) is a kind of metabolic disorders in high blood glucose condition, leading to chronic inflammation and several complications. DM may become diabetic nephropathy (DN) when it destroys the renal function. Recent researches have mentioned that the patients who have DM would increase the risk to get osteoporosis. To focus on the relationship between DM and low bone mineral density, osteopontin (OPN) is referred as one of the candidates. OPN is recently reported as a multifunctional protein to induce proinflammatory cytokines such as interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, monocyte chemoattractant protein (MCP)-1, and recruit macrophages. It may also be involved in type I collagen synthesis of TGF-β/Smad pathway. To find the possible pathway that may regulate OPN, miR-196a was first discussed the associations with mammalian limb development and embryogenesis. A recent research showed that overexpression of miR-196a might alleviate kidney fibrosis by targeting on transforming growth factor β receptor 2 (TGFβR2). Our aim is to confirm that miR-196a improve kidney fibrosis through the regulation of OPN in type-I diabetic mice model, so as to be regarded as a new candidate for the treatment to DM. In our study, overexpression of miR-196a delayed the process of kidney dysfunction and resisted the exacerbation of urine albumin excretion in streptozotocin (STZ)-induced type I diabetic mice model. In transcription level, OPN and inflammation markers, TNF-α and IL-6, were decreased in the kidney while miR-196a was overexpressed, showing that miR-196a may regulate inflammation in the model. Fibrotic marker TGF-β was also reduced, showing that miR-196a may inhibit kidney fibrosis. By analyzing kidney sections with Hematoxylin-eosin (H&E) and picro-sirius red stain, glomerular expansion, leukocyte infiltration, and collagen accumulation appeared to be suppressed in the mice model with miR-196a overexpression, giving the evidence for miR-196a to improve DN. In conclusion, we demonstrated that miR-196a may attenuate DN symptoms including inflammation and fibrosis, and OPN may be the mediator for connecting miR-196a to those symptoms. With the further studies about miR-196a, we believe that it may be a potential treatment for DN amelioration in the near future.