HN242 藉由上調Nrf2 訊息傳遞路徑保護胰臟中β 細胞免受STZ引致之傷害

Abstract

第一型糖尿病是一種代謝異常的疾病，會使體內胰島素分泌量不足，進而讓血糖高居不下。目前治療第一型糖尿病的方法包括注射胰島素、幹細胞治療、胰島移植及藥物治療等，但往往因其副作用及併發症使病患存活率未得改善。先前研究得知，分泌胰島素的β 細胞損傷或死亡可能源自於氧化壓力的上升，使細胞產生發炎、細胞自噬以及粒線體的損傷。若可抑制其胞內氧化壓力的上升，則可保護β 細胞，維持其胰島素分泌功能。 HN242是一種天然萃取物，取自一種南美洲植物的樹幹，在先前的研究中已知其功用包括抗氧化以及抗發炎等，因此我們想知道HN242是否能藉由降低氧化壓力及發炎來達到保護 β 細胞的存活與胰島素的功能，最終維持血糖平衡。 當細胞內氧化壓力增加時，會啟動一連串機制，製造解毒及抗氧化酵素，而細胞內抗氧化機轉的主要調控者即是Nrf2(Nuclear factor erythroid 2-related factor 2, NFE2L2)，因此本研究目標為探討HN242是否能藉由Nrf2調整細胞內氧化壓力進而保護β 細胞，維持胰島素的分泌，最終可以穩定血糖。 實驗結果發現，在動物實驗上，我們成功的以STZ模擬第一型糖尿病，並且同時以口服的方式餵食HN242五天，不但顯著降低老鼠血糖，在GTT(葡萄糖耐受性測試)實驗中也得到改善。並以免疫染色觀察到胰島素分泌量的差異，也從TUNEL實驗證實這是由於減少細胞凋亡所造成。組織染色也發現HN242藉由增加Nrf2下游抗氧化因子NQO1的表現來對抗STZ產生的氧化壓力傷害，最終減少細胞的凋亡。接著，利用INS-1細胞(大鼠胰臟β 細胞株)實驗證明加入HN242組不論在STZ直接處理或是高糖環境下，都可增加細胞存活率、減少細胞週期sub G0/G1凋亡的細胞及細胞凋亡指標蛋白cleaved caspase3/7。也證明HN242是藉由提升Nrf2的表現及活性，一併活化其下游抗氧化因子，像是SOD1, NQO1, GCLC 及SIRT1/3來對抗過高的氧化壓力產生。我們還觀察到HN242可以在COX4, JC-1, 麩醯胺酸吸收這些與粒線體相關實驗中保護粒線體不受損傷。最後也利用過氧化物的清除劑(DTT)及NQO1 抑制劑(Dicumarol) 證明HN242的確是藉由提高抗氧化的能力來對抗第一型糖尿病所產生的過高氧化壓力，進而保護β細胞，維持胰島素的分泌。 總結上述結果，加入HN242不僅可以活化NRF2，也提升其下游抗氧化因子NQO1和GCLC的表現量，還有SIRT家族蛋白也受到調控，最終可保護 β細胞對抗氧化壓力，使細胞正常運作，穩定血糖。因此，HN242有做為治療第一型糖尿病藥物之潛力。

Type I diabetes mellitus(insulin-dependent diabetes mellitus, DM), a kind of metabolic disorder, results in β-cell damage and high blood glucose level. There are many treatments for type Ⅰ diabetes including insulin injection, stem cell therapy, islet transplantation, and drug treatment, however, most are not efficient enough due to the side effects and complications.Previous studies indicate that insulin secreting islet β-cells loss their function and be damaged by the oxidative stress-induced cell inflammation, autophagy, mitochondrial damage, and apoptosis. Therefore, decreasing the oxidative stress level may decelerate the progression of type I diabetes. HN242, a nature compound extracted from the bark of a plant grown in South America, is well known for its anti-oxidative and anti-inflammatory activities. The major regulator of cellular antioxidant mechanism is Nrf2(Nuclear factor erythroid 2-related factor 2, NFE2L2) and the activation of Nrf2 increases the production of downstream anti-oxidant enzymes, such as NQO1 and GCLC. Since the effect of HN242 on β-cell is not understood, we want to know whether HN242 can protect the function of β-cells through upregulating the Nrf2 pathway, and stabilize the blood glucose through increasing the insulin secretion. In the in vivo mice model, our results showed that oral administration of HN242, for 5 days, decreased blood glucose and increased the insulin secretion level in STZ-induced type Ⅰ diabetes mice in GTT(glucose tolerance test) assay. Morover the immunohistochemical studies showed that the insulin production and the expression levels of the antioxidant enzyme NQO1 were increased by HN242.The apoptosis of β-cell leading by STZ was also decreased in TUNEL staining. In the in vitro cell model, the expression of p-Nrf2 was increased in STZ+HN242 group, so as the following anti-oxidant enzymes, including SOD1, NQO1, GCLC and SIRT1/3, by HN242 in STZ- or high-glucose-treated β-cells. The mitochondrial activity indicators, COX4 and JC-1, the level of glutamine uptake, and the cell apoptosis marker, cleaved caspase3/7, were also reversed by HN242 under treated-STZ or -high glucose treatment. ROS scavenger, DTT, and NQO1 inhibitor, dicumarol, were also used to monitor cells with HN242 in high glucose or STZ conditions, results showed that HN242 protected β-cells from STZ-induced cell apoptosis and functional damage mainly through inhibiting the ROS production, by upregulating the Nrf2 signaling pathway. In conclusion, HN242 can protect β-cell from damage, improve the insulin secretion, and stabilize the blood glucose in type I DM through upregulating the Nrf2, the down-stream anti-oxidant enzymes, and SIRT pathways. HN242 may become a potential treatment for type I diabetes mellitus.