海藻糖芭樂汁對第二型尿病大鼠之腎臟及胰的保護效應

Abstract

第二型糖尿病(Type 2 diabetes, T2DM)是目前廣泛流行的代謝症候群。研究預測第二型糖尿病的人口數，在西元2030年時會攀升至3.66億。芭樂 (Psidium guajava)，被發現具有抗氧化(anti-oxidation)、抗發炎(anti-inflammation)及抗糖尿病(anti-diabetes)的特性。並且也被發現在第一型糖尿病的大鼠上，可以保護胰臟的β細胞，免於受到氧化壓力損害。然而，很少研究是利用芭樂的可食部位探討其對第二型糖尿病之間的作用機制。所以我們利用芭樂可食部位製成芭樂汁(40%)，以研究此主題。為了提升芭樂汁的適口性，我們在果汁當中添加了海藻糖(Trehalose)。海藻糖是一種雙醣，目前已被應用為細胞冷凍保存的工具。先前的研究指出它可以防止阿茲海默症中，類澱粉蛋白的累積。以及亨丁頓氏舞蹈症中，多聚谷氨酰胺的形成而產生抗氧化之作用。本研究之主要目的在評估芭樂汁(40%)添加海藻糖(5%)對於第二型糖尿病大鼠在腎臟與胰臟的保護作用效應。 利用Nicotinamide (NA)及Streptozotocin (STZ)腹腔注射以誘發 Wistar品系雌性大鼠第二型糖尿病。誘發成功後，分為六組進行實驗，分別為CON, DM, T1, T2, T5和B1。每日灌餵芭樂汁，連續四周。灌餵之劑量如下: T1, T2, T5: 4, 8, 20 ml/kg BW芭樂汁含5%海藻糖。 B1: 4 ml/kg BW芭樂汁不含5% 海藻糖。紀錄葡萄糖耐受性試驗(OGTT)、血清胰島素、糖化血色素與換算之胰島素阻抗和分泌量。並測量腎臟活體自由基，而後進行犧牲，收取組織進行免疫組織染色、螢光染色、西方墨點法及離體血清自由基測試。我們亦以LC/MS的方式定量芭樂有效成份。 結果顯示，芭樂汁中含有高量的槲皮素，且槲皮素與芭樂汁可清除H2O2 and HOCl。而本研究也發現，海藻糖可清除H2O2，但無法清除HOCl。第二型糖尿病(DM組)會增加大鼠之氧化壓力及發炎反應。相較之下，T1, T2, T5組在灌餵海藻糖芭樂汁之後，表現出較低程度的氧化壓力及發炎指標，如IL-1β, Caspase 3及4-HNE。第二型糖尿病(DM組)增加胰島素阻抗和降低胰島素分泌量。相較之下，T1, T2, T5組在灌餵海藻糖芭樂汁之後，會降低第二型糖尿病(DM組)所增加之腎臟和胰臟氧化壓力及發炎反應包括降低IL-1β, Caspase 3 及4-HNE之表現。而且會降低胰島素阻抗和部份增加胰島素分泌量。此外，我們發現在離體血清自由基測試中，B1組的血清自由基較T1, T2, T5組高，其中T2及T5組統計達顯著差異(P< 0.05)。免疫組織染色及螢光染色結果也有相同趨勢。這結果表示海藻糖芭樂汁對於腎臟及胰臟的保護功效，較單獨芭樂汁佳。 總結，海藻糖可以提升芭樂汁在第二型糖尿病中的保護功效。將兩者合併攝取，可降低腎臟及胰臟的氧化壓力及發炎反應。Type II diabetes is one of the most epidemic metabolic syndrome. It was predicted that people with T2DM would rise to 366 million in 2030. Guava (Psidium guajava) has been reported to provide anti-oxidation, anti-inflammation, and anti-diabetes. Besides, it protected β cells from the damage of oxidative stress in type I DM rats. However, there were few studies which report the mechanism between the edible proportion of guava and the T2DM. Therefore, we utilized guava juice to investigate its effect on T2DM. To improve the palatability, we added one kind of sugar, trehalose. Trehalose is a disaccharide, which has been used in cryopreservation of cells. In addition, it was found to avoid β-amyloid formation and polyglutamine (polyQ) in Alzheimer disease and Huntington’s disease indicating its protective function. This study is to discover the protective mechanism of guava juice (40%) combination with trehalose (5%) on the pathophysiology of kidney and pancreas in T2DM rats. T2DM was induced in female Wistar rats by intraperitoneal administration of nicotinamide and streptozotocin and combination with high fructose diets for 8 weeks. After successful induction (> 230 mg/dL), the rats were divided into 6 groups, CON, DM, T1, T2, T5, B1, and were fed with different dosage of guava juice combination with or without trehalose for 4 weeks (Dose: T1, T2, T5: 4, 8, 20 ml/kg BW guava juice with 5% trehalose; B1: 4 ml/kg BW guava juice without trehalose).OGTT, plasma insulin, HbA1c, Homeostasis model assessment of IR (HOMA-IR, an indicator of insulin resistance) and HOMA-β (an index of the function of β cell in pancreas and insulin secretion) were determined. We also measured the kidney reactive oxygen species (ROS) in vivo. The oxidative and inflammatory degrees were measured by immunohistochemistry stain, fluorescent stain, serum free radical value and western blotting. We also measured the active component of guava juice with LC/MS analysis. We found high content of quercetin existing in the guava juice. Quercetin and guava juice could scavenge H2O2 and HOCl, whereas trehalose can selectively reduce H2O2, not HOCl in the in vitro study. The results showed that the rats in group DM had elevated the degree of oxidative stress and inflammatory levels. In contrast, rats treated with oral intake of trehalose and guava juice in group T1, T2, T5 showed less expression of oxidative and inflammatory indicators, such as IL-1β, Caspase 3 and 4-HNE compared to DM group. Consistently, in the measurement of serum free radical levels, we found that rats in T1, T2 and T5 have significantly (P< 0.05) lower free radicals counts than B1 and DM groups. The results of immunohisotchemic and fluorescent stain also showed that oral intake of guava juice with trehalose in T1, T2, T5 rats had less (P < 0.05) oxidative damage, autophagy and apoptosis in the kidney and pancreas than B1 rats. In conclusion, trehalose supplement seems to provide the additively protective effect of guava juice in T2DM. Combination with trehalose and guava juice not only increases palatability, but also protects pancreas and kidney against oxidative and proinflammatory damages in T2DM.