ObjectiveTo 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.MethodsBy 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. One-way 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.ResultsOptical 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 (r2=0.923, 0.936; P＜0.01).ConclusionsNAS 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.
Objective To investigate the effect of N-acetylserotonin (NAS) on the retinal microglia polarization in retinal ischemia-reperfusion injury (RIRI) rats and explore its mechanism via nucleotide-bound oligomeric domain 1 (NOD1)/receptor interacting protein 2 (Rip2) pathway. MethodsHealthy male Sprague Dawley rats were randomly divided into Sham (n=21), RIRI (n=21) and NAS (injected intraperitoneally 30 min before and after modeling with NAS, 10 mg/kg, n=18) groups, using random number table. And the right eye was used experimental eye. The RIRI model of rats in RIRI group and NAS group was established by anterior chamber high intraocular pressure method. Rats in NAS group were intraperitoneally injected with 10 mg/kg NAS before and 30 min after modeling, respectively. The retinal morphology and the number of retinal ganglion cell (RGC) in each group were detected by hematoxylin-eosin staining and immunohistochemical staining. The effect of NAS on polarization of retinal microglia was detected by immunofluorescence staining. Transcriptome sequencing technology was used to screen out the differentially expressed genes between Sham and RIRI groups. Western blot and real-time quantitative polymerase chain reaction (RT-PCR) were used to examine the differentially expressed genes. Immunohistochemical staining, Western blot and RT-PCR were used to investigate the effect of NAS on the expression of NOD1 and Rip2 protein and mRNA in retinal tissue and microglia of rats. General linear regression analysis was performed to determine the correlation between the number difference of NOD1+ cells and the number difference of M1 and M2 microglia in retinal tissues of rats in NAS group and RIRI group. ResultsA large number of RGC were observed in the retina of rats in Sham group. 24 h after modeling, compared with Sham group, the inner retinal thickness of rats in RIRI group was significantly increased and the number of RGC was significantly decreased. The thickness of inner retina in NAS group was significantly thinner and the number of RGC was significantly increased. Compared with Sham group, the number of retinal microglia of M1 and M2 in RIRI group was significantly increased. Compared with RIRI group, the number of M1 microglia decreased significantly and the number of M2 microglia increased significantly in NAS group. There was statistical significance in the number of M1 and M2 microglia in the retina of the three groups (P＜0.05). Transcriptome sequencing results showed that retinal NOD1 and Rip2 were important differential genes 24 h after modeling. The mRNA and protein relative expressions of NOD1 and Rip2 in retina of RIRI group were significantly higher than those of Sham group, with statistical significance (P＜0.05). The number of NOD1+ and Rip2+ cells and the relative expression of mRNA and protein in retinal microglia in RIRI group were significantly higher than those in Sham group, and NAS group was also significantly higher than that in Sham group, but lower than that in RIRI group, with statistical significance (P＜0.05). The number of Iba-1+/NOD1+ and Iba-1+/Rip2+ cells in retinal microglia in RIRI group was significantly increased compared with that in Sham group, and the number of Iba-1+/Rip2+ cells in NAS group was significantly decreased compared with that in RIRI group, but still significantly higher than that in Sham group, with statistical significance (P＜0.05). Correlation analysis results showed that the difference of retinal NOD1+ and Rip2+ cells in NAS group and RIRI group was positively correlated with that of M1 microglia (r=0.851, 0.895), and negatively correlated with that of M2 microglia (r=−0.797, −0.819). The differences were statistically significant (P＜0.05). ConclusionNAS can regulate the microglial polarization from M1 to M2 phenotype, the mechanism is correlated with the NOD1/Rip2 pathway.