類別:

高鹽飲食對腎臟免疫的影響

2017-01-01T00:00:00Z

標題: 高鹽飲食對腎臟免疫的影響; The impact of high-salt intake in kidney immunity 作者: Pei-Zhen Tsai; 蔡佩蓁摘要: 鹽對地球多數生物來說是不可或缺的，適當濃度的鹽能維持細胞與器官正常的生理功能。但最近越來越多研究顯示高鹽飲食有可能是高血壓、慢性發炎以及自體免疫疾病發展的重要因子，而現代人鹽攝取量也逐年增加，因此，研究高鹽飲食與免疫疾病之間的關係是十分重要的。過去的研究指出未分化的CD4+ T細胞會受到外加10-40 mM的鹽往輔助型T細胞17(T helper 17 cells;Th17 cells)分化，而高鹽所誘導的分化在體外及體內的實驗都證實受到血清糖皮質激酶1(serum glucocorticoid kinase 1;SGK1)及介白素-23受體(IL-23R)的表現所調控，高鹽也會影響巨噬細胞的功能及極化。然而一般生理狀況下，在身體內具有如此高的鹽濃度只可能在腎臟發現，因此我們想知道腎臟是否為發生Th17 cells活化的重要場所，以及如果Th17 cells的活化真的發生在腎臟，半乳糖凝集素3(galectin-3;Gal3)是否會抑制Th17 cells的活化。我們給予8~12週大的C57BL/6公鼠餵食高鹽飼料(8% NaCl )當作實驗組，控制組則給予一般飼料(0.4% NaCl )，兩組皆可自由飲水，藉此來觀察餵食高鹽飼料組別與控制組是否有差異性。實驗結果顯示餵食高鹽飼料的組別尿液裡的鈉離子、氯離子濃度明顯高於控制組，但血壓及血漿內鈉離子、氯離子濃度並無明顯差別。腎臟萃取的RNA在檢測Th17 cells相關基因表現上兩組並沒有差別，但galetin-3的表現則在高鹽組升高。因此，我們從腎臟分離出CD45+細胞看高鹽是否會影響Th17 cells的活化，結果顯示餵食高鹽組別相對於控制組在和Th17 cells活化相關基因表現量有明顯上升的趨勢，代表餵食高鹽飼料後Th17 cells的活化可能會發生在腎臟。但進一步從腎臟分離出的CD11b+巨噬細胞及CD4+ Th cells並沒有Th17 cells活化的情形。另外，培養的巨噬細胞在高鹽環境下似乎也有促進Th17 cells活化的情形，但galectin-3的表現反而下降。巨噬細胞實驗與我們發現餵食高鹽飼料後整個腎臟與CD45+細胞的galectin-3表現量會上升剛好相反，這之間的關係及作用機制仍需更多的實驗來完成。總結來說，餵食高鹽飼料可能會導致在腎臟有Th17 cells活化的情形，細胞實驗也能互相驗證，但我們目前還未找出Th17 cells活化的情形發生在腎臟哪些細胞上，以及腎臟中的變化是否會影響身體其他部分的免疫反應仍需更多實驗來釐清。; Like the majority of other lives on earth, humans cannot survive without salt. An ideal concentration of salt, or sodium chloride (NaCl), is required for proper functioning of cells and organ systems. A high intake of dietary salt has been implicated in the development of hypertension, chronic inflammation, and autoimmune diseases. And modern people increase salt intake year by year. Therefore, it is important to study the relationship between high-salt diet and immune diseases. Recent evidence has shown that Th17 cells can be preferentially induced by treating CD4+ T cells in cell culture medium with an additional 10-40 mM NaCl. High-salt treatment enhances the differentiation of Th17 cells by upregulation of serum glucocorticoid kinase 1 (SGK1) and IL-23 receptor (IL-23R) both in vitro and in vivo. High-salt treatment also affects the function and polarization of macrophages. However, a high level of sodium or hyperosmolality can only be found in the kidneys in normal physiology. We would like to know whether the hypertonic renal medulla is the exact niche for Th17 cells activation. If the hypertonic renal medulla is the exact niche for Th17 cells activation, we would like to know whether galectin-3 inhibit the activation of Th17 cells in renal medulla. We fed 8% NaCl diets to 8~12-week-old C57BL/6 male mice as a high salt diet (HSD) group, and control mice were fed with 0.4% NaCl diets. Both groups had free access to tap water. Experimental results show that urine Na+, Cl- concentrations were higher in HSD group, but blood pressure and plasma Na+, Cl- concentration did not have the significant changes. RNA extracted from kidney did not differ in the detection of Th17 cells-associated gene expression, except for increased galectin-3 expression in HSD group. We then isolated CD45+ cell from control and HSD kidneys to analyze gene expression associated with Th17 cells, and the data showed higher expression levels of Il23/Il23r/Il17 in HSD group than control group. Then we isolated CD11b+ macrophages and CD4+ Th cells from control and HSD kidneys to repeat the same analyses, but there’s no difference between groups. In addition, cultured macrophages seemed to promote Th17 cells activation in high salt environment. Although we found that galectin-3 expression increased in the kidneys and CD45+ cells after high-salt diet, the galectin-3 expression in high salt-treated macrophages in vitro was in contrast. The role of galectin-3 in regulating high salt-induced Th17 activation still needs more experiments to verify. In conclusion, mice fed high-salt diets may result in the activation of Th17 cells in the kidney, and cell culture experiments can be used to verify each other. But we have not yet identified which cells in the kidney are responsible for activation of Th17 cells and whether the activation of Th17 cells in kidney will affect the other body parts after high salt intake. We need more experiments to clarify.

高量細菌內毒素 (LPS) 引起腸道上皮細胞凋亡及緊密連結蛋白破壞：CD14參與之機制探討

2008-01-01T00:00:00Z

標題: 高量細菌內毒素 (LPS) 引起腸道上皮細胞凋亡及緊密連結蛋白破壞：CD14參與之機制探討; High Dose of Bacterial LPS Induce Apoptosis-dependent Tight Junctional Destruction in Intestinal Epithelial Cells：Role of CD14 作者: Wei-Ting Kuo; 郭瑋庭摘要: 腸道屏障由單層上皮細胞及細胞之間的緊密連結 (tight junctions) 組成，可防止腸腔面細菌及抗原產物進入漿膜面循環至全身。緊密連結蛋白包括occludin、claudin及zonula occluden (ZO) 等。臨床上，有報導指出在細菌性腸炎及發炎性腸疾中均有腸道屏障缺損之現象。根據先前研究，利用高劑量革蘭氏陰性菌內毒素刺激腸道上皮細胞Caco-2之腸腔面，會引起細胞凋亡及增加細胞間通透性 (Yu et al., 2005 & 2006)。內毒素之受體複合物包括CD14、TLR-4及MD-2等在單核球及其分化細胞上可發現，然而，在腸道上皮細胞有不同的表現形式。因此，本篇研究目的為1) 探討內毒素引起緊密連結構造缺損是否為細胞凋亡途徑之下游反應，2) 檢測內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞的機制中，內毒素受體複合分子如CD14及TLR-4所扮演的角色，3) 尋找內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞所參與之細胞訊息傳遞途徑為何。實驗中將人類大腸直腸癌上皮細胞株Caco-2細胞培養達全滿，以50 μg/mL之大腸桿菌內毒素刺激腸腔面24小時後，測量細胞凋亡程度。利用Hoechst及TUNEL染色法觀察發現內毒素刺激會引起染色質濃縮及DNA片段化現象。利用流式細胞技術以DiOC6(3) 染劑亦可偵測到內毒素刺激引起粒線體膜電位下降。預先投予caspase-3抑制劑 (z-DEVD-FMK) 能阻斷內毒素刺激所引起之上皮細胞凋亡。Caco-2之控制組細胞其緊密連結蛋白如：ZO-1、occludin在免疫螢光染色下呈現鐵絲網圍欄狀。而內毒素刺激會導致緊密連結蛋白結構破壞，預先投予caspase-3抑制劑則可阻斷此現象。除此之外，預先投予polymyxin B可阻斷內毒素刺激引起之細胞凋亡及緊密連結蛋白結構改變，表示細胞凋亡反應乃因內毒素之脂質A作用所造成，而非由於其他細菌組成成份污染所致。本實驗更進一步瞭解受體複合物如：CD14及TLR-4在內毒素刺激引起腸道上皮細胞凋亡及緊密連結蛋白破壞中所參與之機制。以免疫螢光染色法及流式細胞技術可偵測到Caco-2上皮細胞中表現CD14，由共軛焦XZ連續堆疊影像顯示CD14表現在細胞頂面膜。利用三種TLR-4單株抗體 (clones HTA-125、76B357.1及lG11) 藉由免疫螢光染色法證實Caco-2細胞並無TLR-4表現，而陽性控制組T84細胞則可見TLR-4染色。在內毒素刺激下，CD14及TLR-4表現在上述細胞中並無明顯改變。此外，西方墨點法結果證實CD14 (分子量53∼55 kDa) 表現於Caco-2細胞。最後，結果顯示預先投予中和性抗CD14之抗體 (clone 134620) 能減少內毒素刺激引起之細胞凋亡及緊密連結蛋白結構之破壞，且呈現濃度依賴形式，然而，預先投予中和性抗TLR-4之抗體 (clone HTA-125) 則無作用。過去針對單核球/巨噬細胞之研究顯示，內毒素刺激會經由TLR-4引起MyD88依賴性途徑，使得轉錄因子NF-κB發生核轉移及MAPK磷酸化現象。為進一步確認或排除已知的TLR-4媒介訊息參與之可能性，利用西方墨點法檢測NF-κB、 MyD88及MAPK在Caco-2細胞之表現。結果顯示內毒素刺激Caco-2細胞不會發生NF-κB核轉移、MyD88總量改變及JNK、p38之磷酸化現象，而陽性控制組THP-1細胞則可見。綜合上述，這些結果顯示內毒素引起腸道上皮細胞Caco-2細胞凋亡會導致緊密連結蛋白結構之破壞及屏障缺損，且這些病理現象是經由CD14而非TLR-4所參與的。; Intestinal barrier is composed of a single layer of epithelial cells connected by tight junctions that prevents luminal bacteria and antigenic products from gaining access into the body proper. Tight junctional proteins include occludin, claudin, and zonula occluden (ZO) etc. Clinical manifestations of intestinal barrier defects were reported in bacterial enteritis and inflammatory bowel disease. Previous studies utilizing intestinal epithelial Caco-2 cell culture have demonstrated that luminal exposure to high dose of Gram (-) bacterial LPS induced cell apoptosis and increased paracellular permeability (Yu et al., 2005 & 2006). LPS receptor complexes including CD14, TLR-4 and MD-2 were identified on monocytic lineage cells. However, distinct expression patterns on intestinal epithelial cells have been reported. The aim of the current study was to 1) examine whether LPS-induced tight junctional destruction is downstream of apoptotic pathway, 2) investigate the role of LPS receptor components, i.e. CD14 and TLR-4, in the mechanism of LPS-induced intestinal epithelial apoptosis and tight junctional destruction, 3) explore the cell signaling pathway involved in the LPS-induced intestinal epithelial apoptosis and tight junctional destruction. Colorectal adenocarcinoma Caco-2 cells grown to confluence were luminally exposed to E.coli LPS at 50 μg/ml for 24 hrs and examined for cell apoptosis. Hoechst staining and TUNEL assay showed chromatin condensation and DNA fragmentation in cells following LPS exposure. Flow cytometric analysis of DiOC6(3) staining exhibited mitochondrial membrane potential collapse after LPS challenge. Pretreatment with caspase-3 inhibitor, z-DEVD-FMK, prevented the epithelial cell apoptosis induced by LPS. Immunostaining revealed chicken-wire pattern of tight junctional proteins, i.e. ZO-1 and occludin, on untreated Caco-2 cell monolayer en face. LPS caused destruction of ZO-1 and occludin in which the effect was abolished by pretreatment with caspase-3 inhibitor. In addition, polymyxin B blocked the apoptotic phenomenon and tight junctional reorganization, verifying the specificity of the lipid A moiety of LPS. The role of LPS receptor components, e.g. CD14 and TLR-4, was further assessed in the mechanism. Presence of CD14, but not TLR-4, protein expression was identified on Caco-2 epithelial cells demonstrated by immunofluorescent staining and flow cytometry. The expression of CD14 on apical membrane of cells was evidenced by confocal XZ serial imaging. The absence of TLR-4 expression on Caco-2 cells was verified using three different monoclonal antibodies (clones HTA-125, 76B357.1, and 1G11), whereas positive TLR-4 staining was showed on T84 cells. After LPS challenge, the cellular expression of CD14 and TLR-4 did not change. Moreover, western blotting results confirmed the presence of CD14 (M.W. = 53~55 kDa) on Caco-2 cells. Furthermore, our results showed that neutralizing anti-CD14 antibody (clone 134620) reduced the level of LPS-induced apoptosis and tight junctional destruction in a dose-dependent manner, whereas anti-TLR-4 (clone HTA-125) had no effect. Classical LPS/TLR-4 signaling pathway induced nuclear translocation of NF-κB and phosphorylation of MAPK by a MyD88-dependent manner. In order to confirm or rule out the involvement of TLR-4-mediated signaling pathway, western blotting was utilized to examine the expression of NF-κB, MyD88 & phosphorylation level of MAPK in intestinal epithelial Caco-2 cells. Our results showed that there are no nuclear translocation of NF-κB, phosphorylation of JNK and p38, nor augment of MyD88 in LPS challenged Caco-2 cells, in contrast to the positive control THP-1 cells. Taken together, these findings suggest that LPS-induced cell apoptosis leads to tight junctional disruption and barrier defects in intestinal epithelial Caco-2 cells. These pathological effects on enterocytes were mediated via epithelial CD14, whereas TLR-4-mediated signaling pathway did not play a role.

骨骼肌遠端缺血前置處理對心肌之保護--自由基之啟動者角色

2005-01-01T00:00:00Z

標題: 骨骼肌遠端缺血前置處理對心肌之保護--自由基之啟動者角色; Skeletal Remote Ischemic Preconditioning in Myocardial Protection--free radical as a trigger 作者: Yih-Sharng Chen; 陳益祥摘要: 缺血性前置處理(IPC)在各種動物實驗研究探討已超過10年，而且除了心臟也在以外器的官發現此現象。器官內之“遠端前置處理”(RPC)的概念也形成，可用來保護心肌。而後，器官內遠端前置處理概念也推展到器官間遠端前置處理。骨骼肌遠端前置處理似乎是一個臨床上可行之心肌保護方法，但是仍有些爭論存在，而且沒有進一步研究來探討其作用機轉。此研究之目的便是在探討骨骼肌遠端前置處理對心肌梗塞之作用與自由基所扮演的角色。首先，骨骼肌前置處理之設計是對大鼠之單側股動脈行四次十分鐘缺血與十分鐘之再灌流。實驗分成四組:第一組(I):模擬組，第二組(Ⅱ):只做骨骼肌前置處理，第三組(Ⅲ):只有心肌梗塞，第四組(IV): 骨骼肌前置處理，2小時後再行心肌梗塞。以化學冷光分析儀來分析在行骨骼肌前置處理後，自由基的變化，發現在四次缺血/再灌流所產生之自由基最明顯，2小時後最高，4小時後便下降。如果在骨骼肌前置處理期間，給予MPG之注射，則發現自由基增加的現象會消失。此結果可以幫助往後實驗釐清自由基角色上的判讀。我們首先以一種特殊染色來區別梗塞區域與存活區域，由此來做解剖學上之研究。其梗塞面積(infarct size)在第四組明顯比第三組少(24.7±8.8% in IV vs 51.4±9.1% in III, p < 0.01)。且此現象可以被MPG前處理所抑制(49.2±6.3% in MPG+III vs. 50.1±8.2% in MPG+IV, p > 0.05)。我們測定心肌酵素之釋放來校正之，發現CK-MB與TnI在第四組的釋放量比第三組來的少;同時，其保護效果也可以被MPG所拮抗。用西方漬墨法來探討熱休克蛋白質及抗氧化酵素在心肌中之變化，發現在第Ⅱ、Ⅲ、IV組可以看到熱休克蛋白質及抗氧化酵素(Mn-SOD與GPx)的增加。不過，使用MPG前處理，只看到熱休克蛋白質的下降，而抗氧化酵素則沒有變化。此外，由危險區域與非危險區域蛋白質之分佈，骨骼肌前置處理可能經由神經系統傳遞信息。大鼠骨骼肌前置處理可以在梗塞模式產生心肌保護作用，其作用會經自由基啟動，而且和熱休克蛋白質與抗氧化酵素有關。; Ischemic preconditioning (IPC) has been widely explored in various experimental models for more than 10 years and it has been also observed in several organs other than the heart. The concept of intraorgan “remote preconditioning” (RPC) has been previously advocated in heart to reduce the infarct size. The concept of intraorgan RPC had beem extended to the interorgan RPC. Skeletal RPC seemed to be a good way for myocardial protection, but some controversial data still exists and no further mechanism was investigated. The aim of this thesis was to investigate the effect of skeletal ischemia/reperfusion on myocardial infarction and the role of free radicals. First, skeletal RPC was designed in rats by repeated 4-cycle 10-min ischemia-reperfusion of femoral artery. Four experimental groups were included: I, sham group; II, RPC only; III, infarction only; IV, incorporating both RPC and infarction. Chemiluminescence study showed significant elevation of free radical in groups II and IV, and pretreated mercaptopropionyl-glycine (MPG), a free radical scavenger, could abolish the production of free radicals. The result would help to delinate the role of free radicals in RPC. The infarct size was significantly reduced in group IV (24.7±8.8%) compared to group III (51.4±9.1%) (p < 0.001), and this effect was abolished by MPG pretreatment (49.2±6.3% in MPG+III vs. 50.1±8.2% in MPG+IV, p > 0.05). Cardiac enzymes also revealed significant decrease in group IV compared to group III, and the protective effect could be abolished by MPG. Western blotting of heat shock protein (HSP) revealed that consistent elevation of HSP 25 and 70 in group II, III and IV, and the elevation can be abrogated by MPG pretreatment. The expression of the antioxidant enzymes, Mn-superoxidase dismutase and glutathione peroxidase, in the area of risk were consistently elevated in groups II, III, and IV similar to HSP. However, the MPG pretreatment could not decrease the expression of the antioxidant enzymes as HSP. The skeletal RPC in rats can produce a protective effect in an infarction model that may be triggered through free radical pathway, and this protective effect was associated with heat shock protein and antioxidant enzymes.

離子通道功能異常導致小腦性運動失調症之分子機制

2020-01-01T00:00:00Z

標題: 離子通道功能異常導致小腦性運動失調症之分子機制; Molecular Mechanism of Cerebellar Ataxias Associated with Ion Channel Dysfunction 作者: Cheng-Tsung Hsiao; 蕭丞宗摘要: 脊髓小腦性運動失調症是一群以影響小腦功能為主的遺傳性神經退化性疾病，其臨床表徵與致病基因具有高度多樣性，診斷此類疾病的要領在於了解病人之疾病史與家族病史，並由身體檢查評估病人是否具有小腦功能障礙，搭配其他特定的臨床病徵可作為診斷之線索，基因檢測是確定診斷的方式之一。多麩醯胺酸疾病是脊髓小腦性運動失調症中最常見的一群，這類疾病於臨床、病理、與疾病分子機制有許多相似之處。近年的研究發現已有將近五十種基因變異與脊髓小腦性運動失調症相關，使此疾病的病生理分子機制得以被更深入的了解。神經細胞中表現著許多種類的離子通道蛋白，各式各樣的離子通道以及其相關的分子精確的分佈在神經細胞中特定的位置並執行其專責的功能。由於基因定序技術快速的發展，有越來越多的病人被發現是由於離子通道或相關分子功能變異所致。這些研究揭示由於離子通道蛋白功能變異所致的脊髓小腦性運動失調症在臨床表現與分子基因層面上都具有高度的歧異性，凸顯診斷這類疾病的困難性。然而，針對探討這類疾病的病生理機轉的研究仍然很有限。這些離子通道蛋白功能變異如何引起本身功能的改變，進而影響與其他分子交互作用的改變，並產生對神經細胞生理功能乃至於神經突觸功能恆定的影響，甚或產生增強或衰減整個神經網絡活性的變化，最終導致神經功能退化的過程，以及對病人臨床表徵的影響…等，這些問題都是神經科學與神經醫學研究中非常有趣的議題。這份研究論文旨在探討本土脊髓小腦性運動失調症病人中是否存在由於離子通道蛋白變異所致的個案，進而分析其臨床表徵之特性並尋找是否有明確的基因型與表現型的關聯性。透過細胞電生理實驗、生物化學實驗、以及免疫螢光實驗，我們可以探討這些變異對離子通道功能的影響，進一步探索其與相關分子的交互作用以及對於神經生理恆定的影響。此外，我們期能建立與此疾病之病生理機轉相關聯的疾病模式動物，藉此研究更深入複雜的神經突觸或神經網絡之議題。透過次世代基因定序，我們發現了離子通道蛋白變異所致之脊髓小腦性運動失調症的本土個案，這些病患在臨床表現上具有高度的變異性，除了小腦性運動失調症以外，這些病人有很高的比例具有認知功能障礙的特徵，然而其基因型與表現型並不具有明顯的關聯性。我們也深入的探索由於這些變異對離子通道蛋白功能產生的影響，並成功的建立與此疾病有高度關聯性的疾病模式果蠅。未來可望能透過更清楚了解這些疾病的病生理機轉與小腦及相關細胞生理調控機制，探索相關的分子作用並藉由調控目標分子的功能作為發展新治療的方針。; Spinocerebellar ataxia (SCA) is a group of hereditary neurodegenerative diseases with mainly affecting the function of the cerebellum and its network. The clinical features and the causes of SCA are highly diverse. In combination of understanding the clinical presentations and the family histories, a comprehensive physical examination to assess the cerebellar function and the other specific clinical features can provide insights to determine the diagnosis of SCA. The genetic testing is the gold standard to establish the definite diagnosis of SCA. The group of polyglutamine disorders comprises the most frequent causes of SCA and shares the clinical, pathological and molecular similarities. To date, there are approximate fifty disease-causing genes or chromosomal loci have been mapped to be associated with SCA. The identification of disease-causing mutations has also contributed to a deeper understanding for the molecular mechanism and pathophysiology of this group of diseases. There are numerous subtypes of ion channel proteins expressing in the nervous systems. These ion channels and their related molecules are precisely distributed in the subcellular locations to be responsible for their special functions. Due to the recent advance in genetic sequencing technologies, there are more and more SCA subtypes linked to ion channel dysfunctions. Those studies have revealed that the clinical and molecular features of SCA subtypes associated with ion channel dysfunction are heterogeneous, which highlights the difficulty in diagnosing such diseases. However, studies focusing on exploring the pathogenesis of cerebellar ataxias associated with ion channel dysfunction are still sparse. Whether the sequence variants resulting in ion channel dysfunction or not is still unclear. There are still open questions that how do ion channel dysfunctions lead to alternation of molecular network, disruption of synaptic homeostasis, perturbation of neuronal activities, induction of neurodegenerative processes, and consequent clinical presentations. Numerous intriguing issues remained to be further elucidated in the neuroscience and clinical neurology fields. In this study, we aim to investigate whether there are patients with SCA associated with ion channel gene mutations in Taiwanese population. We also intend to evaluate the molecular mechanism of these disorders by using multidisciplinary experiments. Utilizing the next-generation sequencing technologies, we found that there are cases of SCA caused by mutations in ion channel genes. The clinical presentations of this group of patients are variable. Cerebellar ataxia is exclusively manifested by the patients and a notable proportion of the patients are characterized by cognitive dysfunction as. However, there is not definite genotype-phenotype correlation for the patients. Besides functional characterizations for the ion channel mutations in vitro, and we have also developed a disease-relevant model using Drosophila melanogaster as the model organism. By means of in vivo exploring the molecular mechanism underlying these disorders we will able to conduct more in-depth investigation for the complex issues such as neural synapses homeostasis or neural network in regarding to the ion channel functions in the future.