嘉寶果多酚萃取物(MCPE)可抑制高糖誘導人類足細胞生長、發炎與相關機制之研究

Study on the effect inhibitory of Myrciaria cauliflora extract the growth, inflammation and related mechanisms of human podocytes induced by high glucose.

蔡書瑜(Shu-Yu Cai)

指導教授 ： 黃惠珮

共同指導教授 ： 陳霈霓(Pei-Ni Chen)

中山醫學大學/醫學院/醫學研究所/碩士(2022年)

https://doi.org/10.6834/csmu202200228

摘要

糖尿病腎病變 (Diabetic nephropathy, DN)為導致腎病末期的主要原因之一，糖尿病患者長期處於高血糖狀態，血糖控制不佳，進而導致血管病變，增加了腎臟負擔、改變組織結構並影響細胞激素分泌。其致病機轉分為代謝及血液動力學異常(高血糖、angiotensin II)，促使DN之病程進展。嘉寶果 (Myrciana cauliflora) 又名樹葡萄，其成份具有許多功效，如抗氧化與抗發炎等，本實驗室先前研究利用高脂飲食誘導糖尿病鼠腎病變，再給予嘉寶果水萃物，可改善蛋白尿與腎臟纖維化相關因子、降低糖尿病鼠的Reactive oxygen species (ROS)與發炎因子，並在細胞實驗中，確認嘉寶果水萃物透過Ras路徑，抑制高糖對系膜細胞的損傷。近年來，許多實驗顯示足細胞(podocyte)受損對DN的發展至關重要，因足細胞在DN早期最易遭受破壞，進而影響Cytokine與粒線體功能。本研究觀察嘉寶果多酚萃取物(MCPE)對足細胞之保護作用，並探討相關機制。細胞實驗分為五組，控制組、高糖組(33 mM)、高糖(33 mM) + MCPE (0.025、0.05、0.1 mg/mL)，處理96小時後，分析其存活現象與細胞凋亡變化，證實MCPE能夠改善足細胞因高糖誘導而凋亡之情形。接著利用流式細胞儀分析高糖誘導後氧化壓力及粒線體膜電位之情形，發現MCPE處理組能夠降低氧化壓力及避免粒線體膜電位下降。此外利用ELISA進一步對粒線體功能進行分析；另外透過西方墨點法分析發炎相關因子(TNF-α、IL-6)之表達。結果證實MCPE能夠抑制高糖所引起的氧化壓力，改善粒線體Complex I、III功能損傷進而透過內源性凋亡途徑減緩細胞凋亡，並且能夠透過NFκB的途徑減緩發炎作用，藉以延緩足細胞受損脫落而導致糖尿病腎病變之過程。

關鍵字

糖尿病腎病變 ； 足細胞 ； 嘉寶果 ； 粒線體 ； 發炎反應

並列摘要

Diabetic nephropathy (DN) is one of the main causes of end-stage renal disease. Diabetic patients suffer from the poor glycemia control, which leads to vascular disease, increases the burden on the kidneys, changes tissue structure and affects the release of cytokines. The pathogenesis of DN is divided into metabolic and hemodynamic abnormalities (hyperglycemia and angiotensin II), which promotes the progression of DN. Myrciana cauliflora, also known as tree grape, contains many phytochemicals with antioxidant and anti-inflammatory effects. Our previous studies indicated that Myrciana cauliflora extract can improve proteinuria, reduce oxidative stress and inflammation in streptozotocin/nicotinamide mice on a high fat diet. We also confirmed that Myrciana cauliflora extract can inhibit the damage of mesasngial cells induced by high glucose through Ras/ PI3K/Akt pathway. Recent studies demonstrated that the destruction of podocytes is crucial to the development of DN. Podocytes are most vulnerable to damage in the early stage of DN, which further affects the cytokine secretion and mitochondria function. The purpose of this study was to determine the protective effect of Myrciaria cauliflora polyphenol extract (MCPE) on podocytes. First, podocytes were divided to five groups, control group, high glucose (33 mM) group, and high glucose + MCPE (0.025, 0.05, 0.1 mg/mL)groups. After treatment with 96 hours, cell viability and cell apoptosis were analyzed. The results show that MCPE can improve podocyte apoptosis induced by high glucose treatment. Then, the oxidative stress and mitochondria membrane potential are analyzed by flow cytometry. MCPE can reduce the oxidative stress and avoid the decrease of mitochondria membrane potential. Moreover, we measure the mitochondria function by ELISA method and the inflammatory cytokines (TNF-α, IL-6) by western blotting. Our results indicate that MCPE can suppress the oxidative stress caused by high glucose, then reduce mitochondrial complex I and III dysfunction, attenuate cell apoptosis via intrinsic apoptotic pathway, and inhibit inflammation via the NFκB pathway, thereby delaying or inhibiting the process of DN.

並列關鍵字

Diabetic nephropathy ； podocytes ； Myrciana cauliflora ； mitochondrial ； inflammation

參考文獻

1. Tervaert, T.W Pathologic classification of diabetic nephropathy. J Am Soc Nephrol, 2010.21(4): p. 556-63.

2. Chenyang Qi., et al., Classification and Differential Diagnosis of Diabetic Nephropathy. J Diabetes Res.2017;2017:8637138

3. S M Kobrin., et al., Diabetic nephropathy. Dis Mon.1998 May;44(5):214-34.

4. Puneet Garg., et al., A Review of Podocyte Biology. Am J Nephrol . 2018;47 Suppl 1:3-13

5. Jia Fu., et al., Glomerular endothelial cell injury and cross talk in diabetic kidney disease. Am J Physiol Renal Physiol. 2015 Feb 15;308(4):F287-97.