辛伐他汀對大鼠視網膜缺血/再灌流後神經節細胞的保護作用

Abstract

Simvastatin，為statins類藥物中的一類，其作用為3-hydrixy-3-methylglutaryl Co-enzyme A的競爭型抑制劑。臨床上已廣泛應用於高血中膽固醇的病人。 本實驗的目的為觀察simvastatin是否可以提升大鼠中視網膜中HO-1的表現，並且更重要的是否也可保護視網膜，使其神經節細胞免於遭受缺血/再灌流的傷害。 首先，我們使用雌性之Wistar大鼠，以玻璃體內注射的方法注射simvastatin，如此在24小時後觀察到HO-1的表現呈現劑量依存性。能引發最大量 HO-1表現的為1.6μg，而使用此劑量可使HO-1的表現由藥物注射後6小時候開始增加，並在3天時達到尖峰，因此後續實驗接使用此劑量。 接著，我們再觀察simvastatin是否可以挽救因視網膜缺血/再灌流(I/R)而喪失的神經節細胞。先以5%的Fluorogold由上丘 (superior colliculus) 注射來對視網膜神經節細胞進行標示。視網膜的缺血則是以30號針頭刺入眼球前房，並將針頭連接至一裝有生理食鹽水之儲水袋，將此儲水袋提高至150mmHg，如此維持60分鐘，使前房內壓力高於大鼠之收縮血壓以達到視網膜缺血之目的。之後再使之再灌流不同的時間。大鼠再隨機分為兩組:I/R後3天及7天。在I/R後3天組中，在再灌流後15分鐘內由玻璃體內注射simvastatin或溶劑，結果發現到神經節細胞的存活率由溶劑組的80.32±1.62%提升至 simvastatin組的88.06±2.49% (p=0.038)，而在I/R後7天組中溶劑組及simvastatin組的存活率分別為72.48±4.55%及78.16±2.95%，而兩組之間沒有達到統計上顯著的差異(p=0.297)。 雖然在I/R後3天我們觀察到simvastatin可以增加神經節細胞的存活率，然而我們卻也同時發現到視網膜在單純遭受缺血/再灌流後HO-1即有增加的現象，自6小時侯開始增加並於24小時達到尖峰。更進一步的我們發現到無論在缺血/再灌流的1天或3天後，simvastatin皆無法更進一步的使HO-1的增加有加成的作用。因此我們認為simvastatin可能是經由HO-1以外的路徑達到神經保護的作用。 除了提升HO-1的作用外，我們也觀察視網膜缺血/再灌流後，一種促進細胞凋亡的分子-Bax的變化，發現到Bax的表現在缺血/再灌流後的8至24小時有增加的現象，而在缺血/再灌流後並未發現simvastatin有降低Bax表現的現象。 相反的，我們卻發現到simvastatin可以提升一重要抗細胞凋亡的分子Bcl-2在缺血/再灌流後的表現。 綜合以上，我們認為simvastatin可以在視網膜缺血/再灌流的早期對視網膜神經節細胞扮演ㄧ保護的角色，雖然其真正的作用機轉還未完全明白，但可能是透過HO-1之外的路徑來達成，而Bcl-2表現的提昇有可能是simvastatin造成神經保護的原因之一。

Simvastatin, one of the family of statins, is known as competitive inhibitors of 3-hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA), are widely to treat hypercholesterolemic patients. The aim of present study is to investigate whether simvastatin can up-regulate HO-1 in the rat retina and protect retina ganglion cells from I/R injury. In using female Wistar rats, Intravitreal injection of simvastatin induces retinal HO-1 protein up-regulation in dose dependent manner after 24h of simvastatin injection, the maximum increase in HO-1 was 1.6 μg, thus 1.6 μg was chosen for further experiment.By using this dose, simvastatin also increase HO-1 in a time dependent manner, start from 6h and peaked at 3 days after treatment. RGCs was labeled by injections 5% Fluorogold (FG) injected into superior colliculus. Retinal ischemia/reperfusion (I/R) was done by high intraocular pressure (HIOP)procedure, The procedure was performed by inserting with a 30-gauge needle connected to a saline reservoir, the reservoir was lifted to a height of 150 mmHg for 60 minutes, and reperfusion was allowed to different duration of time. Animals were further divided into two major groups: 3 days after I/R and 7 days after I/R. For 3 days after ischemia/reperfusion, simvastatin or solvent was administered intravitreally 15 minutes after reperfusion. RGCs survival rate was significantly increased from 80.32±1.62 % (solvent) to 88.06±2.49 % (p=0.038) , For 7days after I/R, simvastatin was administered intravitreally immediately , 2 days and 5 days after ischemia. And RGCs survival rate were 72.48±4.55 % (solvent) and 78.16±2.95%(simvastatin)respectively, but there is no statistical significant between two groups (p=0.297). Although simvastatin promotes RGCs survival 3 days after retinal I/R and promotes HO-1 expression in the retina, however, we also find that HO-1 expression after I/R alone was dramatically increase, start from 6h and peaked at 24h after retinal I/R. However, in Western blot analysis, we find that simvastatin did not further increase HO-1 both 24h and 72h after retinal I/R. Therefore, we suggest that simvastatin may protect RGCs from I/R injury not via HO-1 induction. In the present results, Bax expression was increased from 8h to 24hr after retinalI/R, but simvastatin did not reduce Bax expression 24h after retinal I/R,Interestingly, simvastatin can up-regulate Bcl-2, an important anti-apoptotic molecule in rat retina after I/R. Conclusively, these data shown that simvastatin may play a protective role in early stage of retinal I/R, although the exact mechanisms underlying the neuroprotective effects of simvastatin against retina I/R is not clear, HO-1 induction may be ruled out and our results imply that at least in part,Bcl-2, may be one of the mechanisms in simvastatin – induced neuroprotective effects in retinal I/R injury.