枇杷葉懸浮細胞培養物在高脂飼餵鼠對胰島素阻抗及高血脂的影響

Effects of suspension cell culture of Eriobotrya japonica on insulin resistance and hyperlipidemia in mice fed a high-fat diet

邱俊霖(Jiun-Lin Ciou)

指導教授 ： 施純青

中臺科技大學/健康科學院/藥物科技研究所/碩士(2012年)

摘要

在二型糖尿病(Type 2 diabetes mellitus)中，胰島素阻抗(insulin resistance) 是一個很重要的病理特徵，高脂肪的飲食造成過量的游離脂肪酸，這些游離脂肪酸會導致週邊組織對胰島素不敏性，產生胰島素阻抗，並刺激胰臟分泌更多胰島素，造成高胰島素血症，游離脂肪酸更增加三酸甘油脂以及高密度脂蛋白在血液中，進而導致高血脂症。

枇杷葉，最早記錄於《名醫別錄》。為薔薇科(Rosaceae)植物枇杷 Eriobotrya japonica Lindl.乾燥葉。傳統中醫藥用於止咳、抑制嘔吐、皮膚疾病、和控制糖尿病。研究顯示11β-hydroxysteroid dehydrogenase（11β-HSD），具有改善肝臟胰島素阻抗現象。phosphoenolpyruvate carboxykinase ( PEPCK ) 為糖質新生作用一個速率決定步驟的關鍵酶，在動物基因顯現量減少時，會降低肝臟葡萄糖的產生。

預試驗採用 ICR 小鼠測試葡萄糖耐性試驗。實驗採用高脂飲食誘導C57B/6J雄性小鼠，被誘導之糖尿病鼠會產生血糖異常以及高血脂症。小鼠被隨機分為兩組，對照組 ( CON ) 餵食低脂肪的飲食，實驗組 ( HF ) 餵食6週的45%高脂肪飼料。誘導6週之後，實驗組細分成五組，高脂肪組 ( HF ) 給予純水，口服給予枇杷懸浮培養物 ( TA ) 劑量為 T1：0.2；T2：0.5；T3：1.0 ( g/kg/day )，與對照藥品Rosiglitazone。使用三酸甘油脂檢測試劑盒，根據製造廠商的使用方式。胰島素ELISA利用檢測試劑盒測定胰島素濃度。由葡萄糖氧化酶測定法檢測血液中葡萄糖的濃度。組織利用Trizol試劑分離出mRNA,再藉由反轉錄 PCR (reverse transcription-PCR, RT-PCR)測定基因的mRNA相對表現量。

初步結果顯示，ICR 小鼠葡萄糖耐性試驗，低、中、及高劑量枇杷葉懸浮細胞培養物在具有顯著降低血糖的作用，中、高劑量於30分鐘開始降血糖，作用持續至180分鐘。枇杷葉懸浮細胞培養物顯著降低內臟脂肪重量。且具有顯著降低血糖與胰島素的濃度，而在枇杷葉懸浮培養物高劑量組具有降低三酸甘油酯濃度的作用。枇杷葉懸浮細胞培養物低中高劑量具有顯著降低小鼠體內11β-HSD與PEPCK的mRNA表現量。

我們的研究顯示，給予枇杷細胞懸浮培物後會降低血糖濃度、及降低三酸甘油酯和胰島素濃度。再者，枇杷細胞懸浮培養降低副睾脂肪量和內臟肥胖。因為內臟肥胖在代謝症候群的發病機轉是很重要的指標，我們相信枇杷細胞懸浮培養物在治療血脂異常和2型糖尿方面是非常有效的。

關鍵字

胰島素阻抗 ； 枇杷葉 ； 二型糖尿病

並列摘要

Type 2 diabetes is characterized by two metabolic defects: including the majority of condition known as insulin resistance. Excess free fatty acids caused by high-fat diet, and these free fatty acids lead to insulin insensitivity resistance, thus resulting in insulin resistance; moreover, stimulate the pancreas to secrete more insulin, thus leading to hyperinsulinemia. Free fatty acids increased triglyceride and high density lipoprotein in the blood, resulting in hyperlipidemia. Eriobotrya japonica is Traditional Chinese medicine for relieving cough and regulating the stomach to restrain vomiting. The leaves of loquat are used in the treatment of skin diseases and diabetes mellitus. However, mechanism of action of Suspension cell culture of Eriobotrya japonica (TA) on insulin resistance and hyperlipidemia in mice remain obscure.

It was examined the effects and the mechanism of action of anti-hyperglycemia and anti-hyperlipidemia of T in high-fat (HF)-fed mice, an animal model of diabetes and dyslipidemia. Firstly, mice were divided randomly into two groups; the control (CON) group was fed with a low-fat diet, whereas the experimental group was fed a 45% HF fat for 6 weeks. After 6 weeks of induction, the CON group was treated with vehicle, whereas the HF group was subdivided into five groups and was given orally TA (including T1: 0.2, T2: 0.5, T3: 1.0 g/kg/day) or rosiglitazone or not for 6 weeks afterward. The concentrations of triglyceride and free fatty acid were measured using commercial assay kits according to the manufacturer’s directions. The levels of insulin were measured by ELISA using a commercial assay kit. The level of glucose was measured by the glucose oxidase method. Total mRNA from tissue was isolated with a Trizol reagent. It was measured by relative quantitation of mRNA indicating gene expression.

Following treatment with TA, visceral fat mass was decreased. Since visceral obesity is thought to play a major role in the pathogenesis of metabolic syndrome, TA is likely to be useful in treating diseases associated with visceral obesity, such as hyperlipidemia and type 2 diabetes. TA lowered serum triglycerides and free fatty acid. Glucose lowering was comparable in rosiglitazone treated group. Following treatment with TA showed decreased PEPCK and 11β-HSD expression, which contributed in attenuating diabetic state. Moreover, TA decreased apoCIII expression leading to reduction in triglyceride levels. Thus, amelioration of diabetic and dyslipidemic state by TA in HF-fed mice occurred through regulation of apo CIII, PEPCK and 11β-HSD.

並列關鍵字

Eriobotrya japonica ； Type 2 diabetes ； insulin resistance

參考文獻

Agarwal AK, Monder C, Eckstein B, White PC, 1989. Cloning and expression of rat cDNA encoding corticosteroid 11 beta-dehydrogenase. J Biol Chem 264(32) : 18939-18943.

Aguirre MC, Delporte C, Backhouse N, Erazo S, Letelier ME, Cassels BK, Silva X, Alegria S, Negrete R, 2006. Topical anti-inflammatory activity of 2alpha-hydroxy pentacyclic triterpene acids from the leaves of Ugni molinae. Bioorg Med Chem 14(16) : 5673-5677.

Ahren B, Simonsson E, Scheurink AJ, Mulder H, Myrse U, Sundler F, 1997. Dissociated insulinotropic sensitivity to glucose and carbachol in high-fat diet-induced insulin resistance in C57BL/6J mice. Metabolism 46(1) : 97-106.

An HJ, Kim IT, Park HJ, Kim HM, Choi JH, Lee KT, 2011.Tormentic acid, a triterpenoid saponin, isolated from Rosa rugosa, inhibited LPS-induced iNOS, COX-2, and TNF-alpha expression through inactivation of the nuclear factor-kappab pathway in RAW 264.7 macrophages. Int Immunopharmacol 11(4) : 504-510.

Armoni M, Kritz N, Harel C, Bar-Yoseph F, Chen H, Quon MJ, Karnieli E, 2003. Peroxisome proliferator-activated receptor-gamma represses GLUT4 promoter activity in primary adipocytes, and rosiglitazone alleviates this effect. J Biol Chem 278(33) : 30614-30623.