天然植化素槲皮素與蘿蔔硫素對糖尿病大鼠的泌尿系統保護機轉

Abstract

本論文主要在探討天然植化素（phytochemicals）對於糖尿病大鼠的泌尿系統保護作用，並研究有關細胞凋亡（apoptosis）、細胞自噬（autophagy）、發炎性細胞凋亡（pyroptosis），和粒線體功能的作用機轉。我們建立了兩種不同誘發糖尿病的動物模型，第II型糖尿病（Type 2 diabetes mellitus, T2DM）模型，與第I型糖尿病（Type I diabetes mellitus, T1DM）模型。T2DM模型主要研究對象是第II型糖尿病（Type 2 diabetes mellitus, T2DM）之腎臟細胞損傷與保護，而T1DM模型則是用於研究糖尿病的排尿功能障礙，這通常會發生在較嚴重的T1DM高血糖狀態，因為T1DM模型可以快速誘導糖尿病膀胱（diabetic bladder）損傷。

我們萃取富含槲皮素（quercetin）的番石榴汁，並混合不同比例的海藻糖（trehalose），來研究其對於T2DM大鼠腎臟和胰臟損傷的保護作用，並採用高效液相色譜分析法以測定番石榴汁的有效成分。通過腹腔注射菸鹼醯胺（nicotinamide）和鏈脲佐菌素（streptozocin），結合高果糖飲食誘導Wistar大鼠T2DM模型，持續8周。用不同劑量的番石榴汁混和海藻糖餵養大鼠4周，檢測口服葡萄糖耐量試驗（Oral Glucose Tolerance Test, OGTT）、血漿胰島素（insulin）、糖化血色素（glycated hemoglobin, HbA1c）、胰島素抗性指數（Homeostasis Model Assessment-Insulin Resistance index, HOMA-IR）、β細胞功能和胰島素分泌指數（Homeostasis Model Assessment of β-cell function, HOMA-β）。我們也使用了免疫組織化學染色法、螢光染色法和西方墨點法來測定氧化和發炎程度，用化學發光分析儀測定了血清和腎組織活性氧類（Reactive Oxygen Species, ROS）濃度。

結果發現，番石榴汁中高含量的槲皮素對過氧化氫（Hydrogen Peroxide, H2O2）和次氯酸（hypochlorous acid, HOCl）有清除作用，而海藻糖對H2O2有選擇性清除作用，而對HOCl無清除作用。對於T2DM的OGTT、insulin、HbA1c、HOMA-IR和HOMA-β水平均有影響，而番石榴混和海藻糖對T2DM改變的參數，除HbA1c外均有顯著改善。番石榴汁混和海藻糖能顯著降低T2DM所增強的腎臟ROS、4-hydroxynonenal、caspase-3/apoptosis、LC3-B/autophagy，以及 IL-1β/pyroptosis的水平。研究結果顯示：番石榴汁混和海藻糖的攝取，對於因T2DM而損傷的胰臟和腎臟細胞，具有顯著的保護作用。

嚴重的高血糖能誘發氧化壓力，造成糖尿病膀胱（diabetic bladder）,進而引發排尿功能障礙。我們在論文中探討了蘿蔔硫素（sulforaphane），一種具有抗氧化力的轉錄因子Nuclear factor erythroid 2-related factor 2（Nrf2）激活劑，是否具有預防糖尿病因高血糖而併發膀胱功能障礙的功用。糖尿病誘導前給予鏈脲佐菌素和蘿蔔硫素，用化學發光分析儀測定膀胱活性氧類，另用西方墨點法檢測粒線體功能、粒線體Bcl-2-associated X protein（Bax）和胞漿細胞色素cytochrome c、抗氧化防禦能力Nuclear factor erythroid 2-related factor 2／heme oxygenase-1（Nrf2/HO-1）、內質網壓力標誌物Activating transcription factor 6／C/EBP Homologous Protein（ATF-6/CHOP）和Caspase 3／poly ADP-ribose polymerase （Caspase 3/PARP）。糖尿病增加膀胱組織中Keap1的表現，並降低Nrf2的表現，與膀胱活性氧增加、粒線體Bax轉位、胞漿細胞色素（cytochrome c）釋放、ATF-6/CHOP、Caspase 3/PARP/apoptosis增加有關，通過增加排尿間隔時間和排尿時間導致排尿功能障礙。蘿蔔硫素能顯著活化Nrf2/HO-1軸的表現，減少膀胱活性氧、粒線體Bax轉位、細胞色素C釋放、ATF-6/CHOP和caspase 3/PARP/apoptosis，從而通過縮短排尿間期和排尿時間來改善排尿功能。根據研究結果，我們認為蘿蔔硫素通過激活Nrf2/HO-1信號通路保護了粒線體功能，並抑制糖尿病誘導的ROS、內質網壓力、細胞凋亡和排尿功能障礙。

研究顯示，天然植化素槲皮素與蘿蔔硫素，的確具有保護糖尿病大鼠泌尿系統之效益。In this study, we investigated the protective effects of natural phytochemicals on urinary system in diabetic rats, and investigated the mechanisms of apoptosis, autophagy, pyroptosis, and mitochondrial function. We have established two different animal models of diabetes, T2DM (Type II diabetes) model and T1DM (Type I diabetes) model. T2DM model is used to research the protection of T2DM rats on kidney cell damage by hyperglycemia. However, T1DM model is used to study the urination disorder occurred in the more serious hyperglycemia condition in T1DM because of diabetes mellitus bladder rapidly induced by T2DM model method.

We extracted quercetin from psidium guajava, combined with trehalose to study the protective effect on kidney and pancreas injury in T2DM rats. Use high-performance liquid chromatography (HPLC) analysis to determine the active components of guava juice. T2DM was induced in Wistar rats by intraperitoneal injection of nicotinamide and streptozocin combined with a high fructose diet for 8 weeks. Oral glucose tolerance test (OGTT), plasma insuin, glycated hemoglobin (HbA1c), homeostasis model assessment of insulin resistance (HOMA-IR) , homeostasis model assessment of the function of β cell in pancreas and insulin secretion index (HOMA-β) were observed in rats fed with different doses of guava juice and trehalose for 4 weeks. The degree of oxidation and inflammation was determined by immunohistochemical staining, fluorescence staining and western blotting, and the serum and renal reactive oxygen species (ROS) were determined by chemiluminescence analysis.

The results showed that quercetin in guava juice could scavenge H2O2 and HOCl, while trehalose could selectively scavenge H2O2, not for HOCl. T2DM had effects on OGTT, plasma insulin, HbA1c, HOMA-IR and HOMA-β, while guava and trehalose had significant effects on T2DM except HbA1c. Guava juice combined with trehalose significantly decreased T2DM-enhanced renal ROS, 3-nitrotyrosine, Caspase-3 / Apoptosis, LC3-B / autophagy and IL-1β / pyroptosis. We found that guava juice combined with trehalose had protective effects on cells of pancreas and kidney damaged by T2DM.

Hyperglycemia evoked oxidative stress to induce diabetes voiding dysfunction. We explored whether antioxidant sulforaphane, a nuclear factor erythroid 2-related factor 2 (Nrf2) activator, may ameliorate DM-induced bladder dysfunction. Streptozotocin and sulforaphane was administered before DM induction. Bladder ROS was determined by an ultrasensitive chemiluminescence analyzer. Mitochondrial function index, mitochondrial bax, cytosolic cytochrome c, antioxidant defense Nrf2/HO-1, endoplasmic reticulum stress marker ATF-6/CHOP, and Caspase 3/PARP were evaluated by western blotting. DM increased Keap1 and reduced Nrf2 expression, associated with increase of bladder ROS, mitochondrial Bax translocation, cytosolic cytochrome c release, ATF-6/CHOP, Caspase-3/PARP in bladders which resulted in voiding dysfunction by increased intercontraction intervals and micturition duration. However, sulforaphane significantly increased nuclear Nrf2/HO-1 axis expression, decreased bladder ROS amount, mitochondrial Bax translocation, cytochrome c release, ATF-6/CHOP and Caspase 3/PARP/apoptosis, thereby improved the voiding function by the shortened intercontraction intervals and micturition duration. We suggested that sulforaphane via activating Nrf2/HO-1 signaling preserved mitochondrial function and suppressed DM-induced ROS, endoplasmic reticulum stress, apoptosis and voiding dysfunction.

In conclusion, these studies have shown that natural phytochemicals quercetin and sulforaphane have the benefit of protecting the urinary system in diabetic rats.