沒食子酸調節微核糖核酸抑制糖脂毒性誘導之小鼠肝腎病變

研究背景與目的：第2型糖尿病的特徵包括高血糖、血脂異常、胰島素阻抗、肥胖與代謝症候群，其併發症如視網膜病變、周邊與自主神經病變、腎臟病變、非酒精性脂肪肝疾病、冠狀動脈疾病、腦血管疾病等等對人體的影響及危害甚鉅。糖脂毒性為長期高血糖與過多的游離脂肪酸對胰臟β細胞造成的傷害，導致β細胞功能異常、改變了胰島素基因的表現、降低β細胞分泌胰島素的能力，最終造成β細胞衰亡。許多研究顯示氧化壓力在長期高血糖造成糖脂毒性、導致β細胞功能異常的過程中扮演重要角色。微核糖核酸(microRNA)是在真核生物中發現的一種內源性且具有調控功能的RNA，在肝臟部分，微核糖核酸34a在肝臟脂質代謝中扮演重要角色，特別是在糖解作用、脂肪酸β氧化作用、內質網壓力、脂質運送等方面，有研究顯示微核糖核酸34a可調節非酒精性脂肪肝病；在腎臟部分，微核糖核酸709在近端腎小管細胞中會有大量表現的情形，並透過抑制轉錄因子的表現，造成腎臟粒線體功能障礙和腎臟細胞凋亡。多酚類物質可藉由調控微核糖核酸的表現達到抗氧化、抗發炎、抗癌、調解脂質代謝的功能。沒食子酸為酚酸的一種且同樣具有抗氧化作用，在高脂飼料餵食小鼠產生脂肪肝炎方面，沒食子酸可以調節血糖與脂質恆定、減緩肝臟脂質生成、改善氧化壓力。研究也顯示沒食子酸對腎臟有保護效果，可減少氧化壓力、降低發炎反應。因此我們藉由系統性分析第2型糖尿病小鼠中微核糖核酸基因表達和功能，進一步建立和確認微核糖核酸在糖脂毒性誘導之糖尿病脂肪肝炎與腎病變中所扮演的角色， 並探討多酚類物質調控微核糖核酸之作用機轉。研究方法：利用微陣列技術分析，比較db/db小鼠與配合高脂飼料餵食db/db小鼠所誘導糖尿病脂肪肝炎與腎病變組織中微核糖核酸的變化差異。接著針對挑選出來的微核糖核酸對於調節脂肪肝炎或腎病變作用進行分析。以肝細胞及腎絲球細胞在高糖環境下，加以油酸與(或)棕櫚酸誘導糖尿病動物模式，配合微核糖核酸模擬劑與抑制劑，分析並驗證微核糖核酸對於脂質代謝與氧化壓力之影響。另外，在肝臟細胞過量脂肪堆積現象與高糖高油脂誘導腎臟細胞受損之模式，分析沒食子酸對於微核糖核酸的相關機轉變化，並找尋下游的目標基因。研究結果：在肝臟部份，餵食高脂飼料會誘發db/db小鼠肝臟脂質過量堆積、脂質過度氧化、增加肝臟氧化壓力、增加肝臟脂質代謝蛋白表現；在此模式下，再餵食沒食子酸達12週後，可以顯著改善肝臟脂質堆積與脂質氧化、減少氧化壓力、降低肝臟脂質代謝蛋白表現。微陣列技術分析顯示，微核糖核酸34a-5p在高脂飼料餵食db/db小鼠中呈現高度表現。在肝細胞觀察到沒食子酸經由抑制微核糖核酸34a-5p的表現可減緩上述不利條件，達到改善糖尿病脂肪肝炎的功效。在腎臟部份，高脂飼料加速db/db小鼠腎臟脂質過度氧化、抑制抗氧化酵素活性，進一步造成腎絲球氧化壓力上升與腎損傷；在此模式下，同樣餵食沒食子酸12週後，可以顯著改善腎臟脂質氧化與降低氧化壓力。微陣列技術分析顯示，微核糖核酸709在高脂飼料餵食db/db小鼠中呈現高度表現。在腎絲球細胞觀察到沒食子酸經由抑制微核糖核酸709的表現可減緩腎臟氧化壓力並改善糖尿病腎臟病變。因此沒食子酸可經由調節不同的微核糖核酸，抑制糖脂毒素誘導肝臟和腎臟產生的病變。結論： 在高脂飼料誘導db/db小鼠模式，氧化壓力異常在疾病發展過程中扮演相當重要的角色。多酚成分的沒食子酸，透過調節微核糖核酸34a-5p及微核糖核酸709的表現，改善氧化壓力、提升抗氧化酵素活性，分別減緩肝臟與腎臟因高脂飼料與糖脂毒素引起的糖尿病併發症。未來我們將繼續探討由微陣列技術分析出與糖尿病併發症相關的微核糖核酸的標的及相關機轉，並發掘其他多酚類物質，透過釐清與探討其中的相關機制，以達到預防及治療糖尿病併發症之目的。

Background and Objective: Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia, dyslipidemia, insulin resistance, obesity and metabolic syndrome. It is associated with various complications such as retinopathy, diabetic neuropathy, diabetic kidney disease, nonalcoholic fatty liver disease (NAFLD), coronary artery disease, and cerebrovascular accident. Glucolipotoxicity refers to the detrimental effects of chronic exposure to hyperglycemia and excessive free fatty acid that can lead to cellular dysfunction and irreversible pancreatic β-cell damage. Oxidative stress is involved in the mechanisms of glucolipotoxicity–induced diabetic complications. MicroRNAs (miRNAs or miRs) are endogenous noncoding single-stranded RNAs that downregulate gene expression through posttranscriptional regulation. miR-34a is a crucial regulator of insulin secretion, glucose metabolism, and hepatic lipid metabolism; its upregulation caused steatosis in NAFLD. miR-709 was significantly upregulated in the proximal tubular cells and it mediated acute tubular injury through effects on mitochondrial function. Polyphenols can regulate miRNAs during inflammation, apoptosis, carcinogenesis, lipid metabolism, and cell migration. Among various types of polyphenols, gallic acid (GA) can modulate various human diseases through its antioxidative, anti-inflammatory, and antiproliferative activities. A recent study reported that GA alleviated impaired glucose and lipid homeostasis in high-fat diet (HFD)–induced NAFLD model. In addition, the favorable effects of GA on renal function in type 2 diabetic rat have been reported. However, the molecular mechanisms of miRNAs in the development of diabetic steatosis and nephropathy and their therapies have not yet been investigated. This study examined the role of microRNA and GA in regulating glucolipotoxicity–induced steatohepatits and nephropathy in HFD-fed db/db miceMethods and Materials: We performed microRNA microarray validation to analyze microRNA gene expression in db/db mice fed with HFD, a mouse model of T2DM. In vitro, hepatocyte and glomerular mesangial cells were treated with high glucose and palmitic acid and (or) oleic acid to induce steatosis and inflammation of diabetes model. Transfected with a microRNA mimic or inhibitor disclosed the effects of microRNA on lipid metabolism and oxidative stress. The potential role of GA and its regulating microRNAs and target genes on steatohepatits and nephropathy were explored. Results: GA improved the activities of antioxidant enzymes and suppressed lipid accumulation in the HFD–induced steatotic liver of db/db mice. miR-34a-5p was one of the top 10 upregulated genes in our miRNA microarray validation. In vitro, the silencing of miR-34a-5p in hepatocyte cells ameliorated high glucose + oleic acid/palmitic acid mixture–induced accumulation of cellular triglycerides. Reduction in intracellular triglyceride and enhanced hepatic antioxidant enzymatic activities by GA were mediated by the inhibition of miR-34a-5p expression. HFD exacerbated renal oxidative injury in the glomeruli and reduced renal antioxidant enzymatic activities in the diabetic mice whereas GA partially reversed the deleterious effects of HFD. miR-709 was one of the top 10 upregulated genes in our miRNA microarray validation. In vitro, the silencing of miR-709 in glomerular mesangial cells attenuated high glucose + palmitic acid–induced renal peroxidation and oxidative stress. Enhancement of renal antioxidant enzymatic activities by GA were mediated by the inhibition of miR-709 expression. Conclusions: MicroRNA plays a pivotal role in the regulation of lipid metabolism and oxidative responses in liver and kidney. HFD aggravated oxidative injury in steatohepatits and nephropathy in diabetic mice. GA ameliorated glucolipotoxicity–induced hepatic lipogenesis and renal oxidative stress by downregulating microRNAs, indicating the potential role of GA and some polyphenolic nutraceuticals on diabetic fatty liver disease and diabetic kidney disease. Detailed future studies elucidating molecular mechanisms at the microRNA level should be performed to determine the efficacy of GA or other potential substances in the prevention and treatment of diabetic complications.