Short Term Effects Of E64d On The Pineal Gland Of HIBD Rats In Vivo

Objective：To comprehend the changes of apoptosis, autophagy and necrosis pathways ofthe pineal gland in hypoxic ischemic brain damage (HIBD) rat models, then to discuss theshort term effects and mechanism of E64d to the pineal gland damage and repair processes.Methods：The HIBD model was established by using SD rats(7postnatal days) and ac-cording to the modified Rice-Vannucci method. Rats were randomly divided into threegroups (E64d group, DMSO group, Sham operation group), then were subdivided into fivesubgroups after2h,6h,12h,24h and48h from HIBD operation. We used Western blot tomeasure Cleaved caspase-3, Beclin-1and Spectrin αⅡprotein concentration, Hematox-ylin-Eosin staining to measure the tissue damage, while Cleaved caspase-3、IL-21wereassessed by immunohistochemistry.Results：1. Compared to Sham group, Cleaved caspase-3in DMSO group significantlyincreased in pineal gland at6h after HIBD, peaked at12h and the increase continued atleast for24h（P<0.05）, then the level returned to normal at48h (P>0.05). Compared toDMSO group, E64d engendered decline of Cleaved caspase-3at about6h and12h(P<0.05),but presented no significance at24h (P>0.05). While, in the immunohistochemistry,Cleaved caspase-3expressed both in interstitial cells and pinealocytes, it increased at2h,continued at48h(P<0.05), and E64d can decrease it(P<0.05).2. Compared to Sham group, autophagy related protein Beclin-1in DMSO group rose,and showed statistical difference at6h, peaked at24h, and still high until48h（P<0.05）.Compared to DMSO group, Beclin-1in E64d group only decreased at24h in the pinealgland(P<0.05), and had no statistical difference at the remaining time-points (P>0.05).3. Compared to Sham group, membrane skeleton protein Spectrin α II decompositionascended at2h-48h(P<0.05) except for the6h point(P>0.05). After adding E64d, comparedto DMSO group, the cell skeleton decomposition was descended in a certain period of time(at12h and24h, P<0.05), but after that, the protein was still on the rise, the48h subgrouphad no difference (P>0.05). 4. Immunohistochemistry showed that Interleukin-21existed in intercellular sub-stance of pineal gland tissue, there were no significance among the three groups at2h(p>0.05). Compared to Sham group, IL-21in DMSO group significantly increased at48h(p<0.05), while decreased in E64d group compared to DMSO group（P<0.05）Conclusions：1. The pineal gland cells are damaged in a short term after HIBD, the dam-age appears as karyolysis and karyorrhexis, parts of the cells have blurred boundary.Apoptosis, autophagy and necrosis are involved and the three processes had dynamicchanges in this model.2. In the presented research, the intraperitoneal injection of E64d has a direct effect onpineal gland in vivo after HIBD. E64d does show effects on Cleaved caspase-3and Spec-trin αⅡprotein, but Beclin-1is relatively barely influenced. In the model, E64d shows itsprotection within24h by inhibiting apoptosis and maintaining the integrity of the cyto-membrain.. E64d fails to effectively stop this trend.3、Immune mechanism maybe involve in the pineal gland damage after HIBD byupregulating Interleukin-21, E64can decrease it significantly.