The effects of two routes of melatonin administration on the behavior and histopathology in focal cerebral ischemic rats / 中华行为医学与脑科学杂志

Objective To explore the effects of two routes of melatonin (MT) administration including intraperitoneal and caudal vein injection on the behavior,histopathology and the expression of myelin basic protein (MBP) and active caspase-3 protein in focal cerebral ischemic rats.Methods 84 male Sprangue-Dawley rats were randomly divided into normal control group (CON,n=12),middle cerebral artery occlusion group(MCAO,n=24),MT-intraperitoneal group (n=24) and MT-intravenous injection group (n=24) by random number table.Twenty-four hours after ischemia reperfusion (IR),Morris water maze was used to observe the effects of two routes of MT administration on behavior in focal cerebral ischemic rats.7 d after IR,MBP immunohistochemical and hematoxylin eosin (HE) staining were used to examine the expression of MBP in striatum and histopathological changes in hippocampal CA1 region.24 h,72 h and 7 d after IR,the expression of active caspase-3 in hippocampal CA1 region was observed by immunohistochemical staining.Results The average escape latencies in Morris water maze in MT-intravenous injection group at different time points were all lower than those of the MT-intraperitoneal,and they were all lower than those of the MCAO group.Swimming time percentage of target quadrant in MT-intravenous injection group were higher than those of the MT-intraperitoneal,and they were all higher than those of the MCAO group (all P＜0.01);7 d after IR,the results of HE staining showed that the hippocampus cells in MCAO group were disarranged with hyperchromatic nucleus and cytoplasm.More hippocampal cells were observed in MT-intraperitoheal and MT-intravenous injection groups,and they were relatively well arranged.The optical density (OD)of MBP in MT-intravenous injection group (105.60±4.04) was significantly higher than those in MCAO group (95.60±2.07) and MT-intraperitoneal injection group (98.00±4.18) (both P＜0.01).Immunohistochemical results showed that the number of active caspase-3 positive cells in MT-intravenous injection group ((116.93± 12.58)/mm2,(130.16±21.22)/mm2,(88.25±7.80)/mm2) at each time point were significantly lower than those in MT-intraperitoneal injection group ((156.64± 32.54)/mm2,(176.49± 17.44)/mm2,(127.96±16.73)/mm2) (all P＜0.05).At the time points of 24 h and 72 h after IR,there were less active caspase-3 positive cells in MT-intraperitoneal and MT-intravenous injection group compared with those in MCAO group((273.56±32.54)/mm2,(288.63±35.17)/mm2)(all P＜0.01).Conclusion MT administration by both intraperitoneal and intravenous injection can significantly improve the behavior and attenuate the histopathology and white matter damage,and reduce the cell apoptosis in hippocampal CA1 region in focal cerebral ischemic rats,and the therapeutic effects of MT-intravenous injection are better than MT-intraperitoneal injection.

The effect of melatonin on retinal apoptosis in rats with ischemia-reperfusion injury / 中华眼底病杂志

Objective To observe the effect of melatonin (MT) on retinal apoptosis in rats with ischemia-reperfusion injury (RIRI).Methods A total of 54 male healthy Sprague-Dawley adult rats were randomly divided into the normal control (CON) group (6 rats), RIRI group (24 rats) and MT group (24 rats). The rats of RIRI and MT group were induced using suture-occluded methods to establish RIRI model. The rats of MT group were injected with MT in the left carotid artery 30 minutes after RIRI, and RIRI group was injected with the same amount of saline. On 6, 24 hours and 3, 7 days after RIRI, the morphological changes of retina were evaluated by hematoxylin and eosin (HE) staining; the effects of MT on retinal cell apoptosis and Nrf2,HO-1 proteins were examined by immunohistochemistry staining. The correlation between active Caspase-3 and Nrf2 protein, active Caspase-3 and HO-1 protein in MT group were analyzed by linear regression analysis. Results HE staining results showed that the morphology of retinal cells was regular and retinal cells were well arranged in the CON and MT group. In the RIRI group, both the thickness of inner retinal layer and the number of retinal ganglion cells (RGC) were decreased. On 6, 24 hours and 3, 7 days after RIRI, the thickness of inner retinal layer (F=16.710, 62.303, 68.389, 57.132;P<0.01) and RGC number (F=24.250, 11.624, 14.155, 32.442;P<0.05) in MT group were more than those in RIRI group. Immunohistochemistry staining results showed that less active Caspase-3+ cells were observed in MT group as compared with those in RIRI group at each time points (F=49.118, 134.173, 76.225, 18.385;P<0.01). There were more Nrf2+ (F=11.041, 31.480, 59.246, 6.740;P<0.05) and HO-1+ cells (F=128.993, 21.606, 51.349, 8.244;P<0.05) in MT group as compared with those in RIRI group at each time points. Linear regression analysis results showed that the difference of active Caspase-3+cells were all linearly correlated with the Nrf2+ cells and HO-1+cells in the MT group (r2=0.810, 0.730;P<0.01). Conclusion MT could reduce retinal cell apoptosis in RIRI rats, and its mechanism may be associated with increased Nrf2 and HO-1 expression, reduced active Caspase-3 expression.

Active caspase-3 expression levels as bioindicator of individual radiosensitivity

ABSTRACT Several molecules and events involved in cell response to radiation-induced damage have been investigated towards a personalized radiotherapy. Considering the importance of active caspase-3 in the proteolytic cascade that ensures radiation-induced apoptosis execution, this research was designed to evaluate the expression levels of this protein as a bioindicator of individual radiosensitivity. Peripheral blood samples of 10 healthy individuals were gamma-irradiated (cobalt-60 source) with 1, 2 and 4 Gy (control: non-irradiated samples), and active caspase-3 expression levels were measured in lymphocytes, by flow cytometry, ex vivo and after different times of in vitro incubation (24, 48 and 72 hours). Short-term incubation of 24 h was the most adequate condition to evidence correlations between dose radiation and active caspase-3 expression. For each radiation dose, it was observed a significant inter-individual variation in active caspase-3 expression intensity, suggesting that this parameter may be suitable for evidence individual radiosensitivity. The methodology presented and discussed in this work may help to predict healthy tissues response to radiation exposure toward the better patient outcome.

Effects of N-acetylserotonin on expression of active caspase-3, Bcl-2 and Bax protein in rat retina after ischemia-reperfusion injury / 眼科新进展

Objective To investigate the effects of N-acetylserotonin (N-AS) on the expression of active caspase-3,Bcl-2 and Bax in rat retinas induced by retinal ischemia-reperfusion injury (RIRI).Methods Adult male Sprague-Dawley rats were randomly divided into the normal control group (6 cases),RIRI group (30 cases) and NAS group (30 cases),RIRI models in NAS group were established after giving NAS,the groups were sub-divided into 6 hours,12 hours,24 hours,48 hours and 72 hours group based on the time of RIRI.Morphologic changes were evaluated by HE staining.The expression of active caspase-3,Bcl-2 and Bax protein in the retina of rats was detected by immunohistochemistry.Results HE staining showed that the retinal structure in the normal control group was clear,and the cells in each layer were tightly packed;Each layer of retina was edema in the RIRI group after 6 hours and 12 hours,the edema gradually alleviated after 24 hours,the ganglion cells decreased gradually,the distribution was in disorder,with the prolongation of time,the retinal ganglion cells were defected;drug group of as Compared with RIRI group,the cell edema in the NAS group at 6 hours and 12 hours were obvious reduced,the cells in 24 hours,48 hours,72 hours group arranged regularly,the loss number of ganglion cells were reduced.The number of active caspase-3 positive cells in RIRI group increased at 6 hours after peffusion,the number was (561.15 ±37.19) cell ·mm-2,and reached the high level at 24 hours,the number was (1522.61 ±84.36) cell · mm-2,and then decreased gradually.The number of active caspase-3 positive cells in NAS group was significantly lower than that in RIRI group,the difference was statistically significant (all P ＜ 0.05).The expression of Bcl-2 positive cells in RIRI group began to decrease after 6 hours,and decreased to a low level at 24 hours,and the number of Bcl-2 positive cells in NAS group was significantly higher than that in RIRI group at each time point,the differences were statistically significant (all P ＜ 0.05).There were almost no Bax positive cells in the retina of the control normal group,and the Bax positive cells were found to be higher of the RIRI group at the 6 hours after RIRI,and reached the higher level at 24 hours,and decreased at 48 hours.The Bax positive cells of NAS group were significantly less than those in the RIRI group at different time points,and the differences were statistically significant (all P ＜0.05).Conclusion NAS can promote the expression of Bcl-2 protein in rat retina after RIRI,inhibit the expression of Bax protein,decrease the expression of active caspase-3 protein,alleviate cell apoptosis,and have neuroprotective effects.

Anti-apoptotic effect of Icariin on renal tubular epithelial cell in SHR / 中国药理学通报

Aims To observe the effect of Icariin( Ica) on apoptosis of renal tubular epithelial cell in spontane-ously hypertensive rats( SHR) , and to explore its pos-sible mechanism. Methods 21 male SHR of 14 weeks were randomly divided into model groups, the Ica low and high dose groups (20 or 40 mg·kg-1 ,ig, bid, to 26 weeks ) , 14 week-old male homologous Kyoto rats ( WKY ) as control group, and the number of each group was 7 . WKY group and model group were ad-ministered by gavage with the same volume of double distilled water. The pathological changes were observed by using the HE staining and the apoptosis was detec-ted by TUNEL method in renal tubular epithelial cell, respectively. The mRNA levels of Bok, Bax, Bcl-2 were detected by the real time RT-PCR and the protein expressions of Bcl-2 , Bax and Active caspase-3 were detected by Western blot method in the renal tissue. Results Compared with WKY, renal capsule was nar-row and irregular, and glomerular mesangial matrix was increased with the cell arrangement disorder and the capillary dilation and congestion. Several glomeruli shrank and renal tubular epithelial cell was edema with luminal stenosis in model group. The apoptosis of renal tubular epithelial cell was evident and the mRNA levels of Bok and Bax, as well as the protein expressions of Bax and Active caspase-3 were significantly up-regula-ted in model group, while the mRNA Level and protein expression of Bcl-2 significantly down-regulated ( P <0. 05 or P<0. 01). Compared with model group, renal glomerular capsule widened, proliferation of glomerular mesangial matrix was reduced, and cell arrangement disorder and capillary dilation and renal tubular lumen stenosis were improved in Ica group. Renal tubular ep-ithelial cell apoptosis was decreased evidently, and the mRNA level of Bax, Bok and protein expression of Bax were significantly down-regulated in Ica group, and the mRNA level and protein expression of Bcl-2 in Ica-H group were significantly up-regulated while the protein expression of Active caspase-3 significantly down-regu-lated( P <0. 05 or P <0. 01 ) . Conclusion Ica can inhibit the apoptosis of renal tubular epithelial cell in spontaneously hypertensive rats and its mechanism may be related to the down-regulation of Bok, Bax, and Active caspase-3 , accompanied with the up-regulation of Bcl-2 .