ABSTRACT
Objective:To dynamically observe the effect of N-acetylserotonin (NAS) on the expression of tumor necrosis factor-α (TNF-α) protein in retina of retinal ischemia reperfusion injury (RIRI) rats, and to explore the mechanism.Methods:By using random number table method, 90 healthy male Sprague-Dawley rats were divided into sham operation group ( n=10), RIRI group ( n=40), and NAS group ( n=40). The right eye was as the experimental eye. In the RIRI group and NAS group, the anterior chamber high intraocular pressure method was used to establish the RIRI model. In the NAS group, 10 mg/kg NAS was injected intraperitoneally before modeling and 30 minutes after modeling. At 6, 12, 24, 72 h after modeling, hematoxylin-eosin staining was used to observe the pathological changes of the retina, and the retinal ganglion cells (RGC) were counted. Each group was detected by immunohistochemical staining and Western blot about the relative expression of TNF-α, nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1 (HO-1) protein in the rat retina. Oneway analysis of variance was used for differences between groups. The general linear regression method was used to analyze the correlation between the relative expression changes of TNF-α protein and the changes of Nrf2 and HO-1 protein expression after NAS intervention. Results:Optical microscope observation revealed that the retinal edema of rats in the RIRI group was observed at 6, 12, and 24 h after modeling; the thickness of the retina in the NAS group was significantly thinner than that in the RIRI group, and the difference was statistically significant ( F=9.645, 477.150, 2.432; P<0.01). At 6, 12, 24, and 72 h after modeling, the retinal RGC counts in the NAS group were significantly higher than those in the RIRI group, and the difference was statistically significant ( F=12.225, 12.848, 117.655, 306.394; P<0.05). The results of immunohistochemical staining and Western blot showed that 6 h after modeling, the relative expression of TNF-α protein in the retina of the RIRI group increased significantly compared with that in the sham operation group, reaching a higher level at 12 h, and decreased at 24 and 72 h. But all were significantly higher than the sham operation group, the difference was statistically significant (immunohistochemical staining: F=105.893, 1 356.076, 434.026, 337.351; P<0.01; Western blot: F=92.906, 534.948, 327.600, 385.324; P<0.01). At different time points after modeling, the relative expression of TNF-α protein in the retina of the NAS group was significantly lower than that of the RIRI group (immunohistochemical staining: F=15.408, 570.482, 21.070, 13.767; P<0.05; Western blot: F=12.618, 115.735, 13.176, 111.108; P<0.05), but still higher than the sham operation group (immunohistochemical staining: F=40.709, 151.032, 156.321, 216.035; P<0.01; Western blot: F=33.943, 79.729, 74.057, 64.488; P<0.01), the difference was statistically significant; 12 h after modeling, Nrf2 in the retina of the NAS group (immunohistochemical staining: F=51.122, P<0.05; Western blot: F=33.972, P<0.05), HO-1 (immunohistochemical staining: F=30.750, P<0.05; Western blot: F=18.283, P<0.05) protein relative expression was significantly higher than that of RIRI group, and the differences were statistically significant. The results of linear regression analysis showed that the difference in the number of TNF-α + cells in the RIRI group and the NAS group was negatively correlated with the difference in the number of Nrf2 + and HO-1 + cells ( r 2=0.923, 0.936; P<0.01). Conclusions:NAS can inhibit the expression of TNF-α protein in the retina of RIRI rats and reduce RIRI. The mechanism may be related to the Nrf2/HO-1 pathway.
ABSTRACT
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.
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PURPOSE: The aim of this research was to study the effects of treatment with melatonin and N-acetilserotonin in the development of pilocarpina model of epilepsy in adult male rats. METHODS: Part I - The animals were divided in 4 groups: SALINE - animals that received only saline; SE - animals submitted to status epilepticus (SE); NAS + SE - animals that received pre-treatment with N-acetylserotonin and were submitted to SE and MEL + SE - animals that received pre-treatment with melatonin and were submitted to SE. Part II - The animals were divided in 6 groups: SALINE - animals that received only saline; SE - animals submitted to status epilepticus (SE); PX + SE - animals submitted to pinealectomy and to SE 7 days later; SH + SE - animals submitted to sham-surgery and to SE 7 days later; SE + NAS - animals submitted to SE and treated with N-acetylserotonin (2,5 mg/kg), 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h and 48 h after the SE and SE + MEL - animals submitted to SE and treated with melatonin (2,5 mg/kg), 30 min, 1 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h and 48 h after the SE. Following the treatment the animals were continuously video-recorded for 60 days. The behavioral parameters were observed: latency for the SE in minutes, latency for the first spontaneous seizures (ie, duration of the silent period), number of spontaneous seizures during the chronic period and mortality. Five animals per group were perfused for neo-Timm assay. RESULTS: Part I - The animals treated with melatonin and N-acetylserotonin presented an increased of latency for the status epilepticus and decreased number of spontaneous seizures during the chronic period when compared to SE group. The mortality was reduced 100 percent in animals treated with melatonin and theses animals presented a minor mossy fibers sprouting. Part II - The latency for the first spontaneous seizures and mortality were similar in all groups. The animals treated with melatonin presented...