| 摘要: | 研究目的
抵抗素 (Resistin) 是一種脂肪激素，同時它也是一種與食物攝取和能量平衡有關的蛋白質，稱為ingestive peptides，最初它被認為與第二型糖尿病有關，在老鼠主要由白色脂肪組織產生，但在人類的主要來源是巨噬細胞。一般來說脂肪激素都與能量平衡有關，但resistin在人體的功能目前還不是很清楚。其他ingestive peptides，例如ghrelin，adiponectin及leptin，已被證明具神經保護作用，但resistin是否一樣有神經保護的作用，至今仍無相關的研究提出證明。本實驗的目的主要是觀察resistin是否也具有神經保護的作用。
研究方法
我們選擇dopaminergic神經元細胞株(MES23.5)作為本實驗的材料，以神經毒素6-hydroxydopamine（6-OHDA）用來引發細胞毒性。我們用MTT，SRB和LDH的細胞存活率分析法來確認6-OHDA的細胞毒性，然後利用Hoechst染色法觀察細胞的形態變化，接著利用流式細胞儀雙染分析法（PI及annexin V）檢測細胞死亡的模式。下一步我們利用DCFH- DA分析法檢測活性氧化物（ROS）的產生量，並利用Rhodamine 123(Rh123)染色觀察線粒體膜電位的變化。接著我們測量細胞在加入6-OHDA後，參與細胞凋亡作用的蛋白質之表現量。最後我們觀察細胞保護蛋白質HSP73，HO-1和Bcl-2在加入resistin之後的表現量，並對其與細胞存活的關係進行了研究。
研究結果
MTT，SRB和LDH分析法證實了6-OHDA的細胞毒性，而Hoechst染色法及流式細胞儀雙染分析法皆顯示出，加入6-OHDA後會引發細胞凋亡，而resistin的前處理則會抑制這一現象。DCFH-DA分析法觀察到，細胞在加入6-OHDA後ROS的生成量會增加，而resistin的前處理則會抑制這一現象。Rh123染色法則顯示，細胞在加入6-OHDA後粒線體膜電位會下降，而resistin的前處理則會抑制這一現象。在與細胞凋亡相關的蛋白質表現量方面，加入6-OHDA後Bcl-2會下降，而resistin的前處理則會抑制這一現象。另一方面，Bax和PARP-1/2和cleaved caspase-3則顯示相反的結果。HSP73，HO-1和Bcl-2的表現量在加入resistin呈現增加的結果，但如果經過HSP73和HO-1抑製劑的處理，細胞存活率在加入resistin後並沒有增加。
研究結論
根據上述結果，我們可以得到實驗的結論。Resistin對於6-OHDA所引起的細胞毒性有神經保護的作用，而且是經由對抗細胞凋亡的機制。將細胞經過resistin前處理，能降低ROS的產生和恢復粒線體膜電位。本實驗結果也發現，HSP73和HO-1為resistin的神經保護作用所必需的蛋白質，由此推論此兩種內生性蛋白質具有神經保護的作用，而其潛在的作用機制和臨床應用則需要進一步的研究。
Introduction
Resistin is one of the adipokines families, and it is also one of the peptides responsible for food intake and energy balance called ingestive peptides. Initially it was thought to be associated with type 2 DM. In rodent it was produced mainly by white adipose tissue, but in human the major source is from macrophage. Generally adipokines are related to energy balance, but the function of resistin in human is not fully elucidated. Other ingestive peptides, e.g. ghrelin, adiponectin and leptin, had been proved to be neuroprotective, but so far there is no convincible evidence about resistin being neuroprotective. This study was set to investigate whether resistin also exert neuroprotective effect.
Methods
A dopaminergic neuron cell line called MES 23.5 was used for this in vitro study. The neurotoxin 6-hydroxydopamine (6-OHDA) was used to induce cytotoxicity. We used MTT, SRB and LDH assays to confirm the cytotoxicity of 6-OHDA, then Hoechst stain of nuclear extract was applied to observe the morphological change. Flow cytometry with propidium iodide (PI) and annexin V was performed to evaluate the pattern of cell death. Next reactive oxygen species (ROS) production was measured by 2’,7’-dichlorfluorescein-diacetate (DCFH-DA) assay, and mitochondrial membrane potential was observed by rhodamine 123 (Rh123) staining. Various proteins involved in apoptosis were checked for their expression after addition of 6-OHDA to neuron cell. Finally HSP73, HO-1 and Bcl-2 expression and their relationship to cell surviveal were examined.
Results
MTT, SRB and LDH assays confirmed the cytotoxicity of 6-OHDA and found that it had a dose-dependent cytotoxic effect. Under phase contrast microscopy, most of the 6-OHDA-treated neurons cell lost their neurites, but the number decreased after resistin treatment. Hoechst stain of nuclear extract revealed that 6-OHDA-treated nucleus became condensed and fragmented, which are features compatible with apoptosis. Flow cytometry with propidium iodide (PI) and annexin V revealed that after 6-OHDA treatment the distribution of cells shifted to late apoptosis. Pretreatment of resistin reversed this phenomenon. DCFH-DA assay disclosed that addition of 6-OHDA to neuron cell increased ROS production, but resistin pretreatment reversed this result. Rh123 staining revealed treatment of 6-OHDA decreased mitochondrial membrane potential, but resistin pretreatment reversed this phenomenon. The amount of Bcl-2 decreased after 6-OHDA treatment, but resistin treatment reversed this phenomenon. On the other hand, Bax, PARP-1/2 and cleaved caspase-3 showed opposite results compared with Bcl-2. HSP73, HO-1 and Bcl-2 actually increased in amount after addition of resistin, but after addition of HSP73 and HO-1 inhibitors, the level of cell viability did not return in the condition of resistin treatment after 6-OHDA addition.
Conclusion
According to above findings, some preliminary conclusions can be made. Resistin exerts neuroprotective effect on dopaminergic neuronal cell via anti-apoptosis mechanism. Pretreatment of resistin decreases ROS production and restored mitochondrial membrane potential, which were resulted from 6-OHDA addition. HSP73 and HO-1(HSP32), two well known neuroprotective proteins, are necessary for this neuroprotective effect of resistin. Further investigation is needed to fully discover the underlying mechanism and its potential in clinical practice |
