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Objective@# To explore whether Lycium barbarum polysaccharide (LBP) can reduce the apoptosis of retinal photoreceptor cells in retinitis pigmentosa (RP) mice by inhibiting nuclear factor-kappa B (NF-κB)/NOD-like receptor thermal protein domain-associated protein 3 (NLRP3) signaling pathway. @*Methods@# (i) In vitro experiments, mouse retinal ganglion cells (661W cells) were divided into normal, model, LBP low-dose (LBP-L, 40 mg/L), LBP middle-dose (LBP-M, 80 mg/L), LBP high-dose (LBP-H, 160 mg/L), and positive drug control (NLRP3 inhibitor, 160 mg/L) groups. And the 661W cells were exposed to varying concentrations of H2O2 ranging from 50 to 400 μmol/L to determine the optimal concentration for inducing apoptosis (200 μmol/L). Then the cell viability was assessed using Cell Counting Kit-8 (CCK-8), while the apoptosis rate was detected by flow cytometry; the expression of NLRP3 was detected by immunofluorescence; and the expression of apoptosis markers was detected by enzyme-linked immunosorbent assay (ELISA) and Western blot (WB). (ii) In vivo assays were carried out with the use of C57/BL6 and Rd10 mice. The animal experimental groups were divided into normal, model, LBP-L, LBP-M, LBP-H, and NLRP3 inhibitor groups, in which the normal group was C57/BL6 mice and the other groups were Rd10 mice. Ten mice were included in each group, and the corresponding drugs were administered intragastrically for a duration of four weeks. NF-κB/NLRP3 pathway and the expression of apoptosis markers were observed by electroretinogram, histopathological examination, and WB to assess the effects of LBP on retinal photoreceptor cell apoptosis.@*Results@#(i) In vitro experiments, compared with the normal group, the apoptosis rate of 661W cells in model group was significantly increased (P < 0.01), and the expression levels of key proteins of NF-κB/NLRP pathway, such as NLRP3, NF-κB, p-NF-κB, and pro-apoptotic protein caspase-3, were up-regulated (P < 0.01). The rate of Bax/Bcl-2 was increased (P < 0.01), and the concentrations of interleukin (IL)-1β and tumor necrosis factor (TNF)-α were significantly increased (P < 0.01). Compared with the model group, high dose of LBP decreased the apoptosis rate of 661W cells (P < 0.01), and down-regulated the expression levelsof the key proteins of NF-κB/NLRP3 pathway, including NF-κB, NLRP3, p-NF-κB, and caspase-3 (P < 0.01). The rate of Bax/Bcl-2 was decreased (P < 0.01), and the concentrations of IL-1β and TNF-α were decreased (P < 0.01). (ii) In vivo experiments, high dose of LBP significantly increased morphological changes in the outer nuclear layer (ONL) thickness of Rd10 mice, as well as functional changes in the amplitudes of the a-wave and b-wave (P < 0.01), which also down-regulated the expression levels of NF-κB (P < 0.05), NLRP3, p-NF-κB, and caspase-3 (P < 0.01), reduced the Bax/Bcl-2 rate (P < 0.01), and decreased the concentrations of IL-1β (P < 0.01) and TNF-α (P < 0.05). @*Conclusion@#LBP could improve both retinal morphology and function, providing protection to photoreceptors from apoptosis through the inhibition of the NF-κB/NLRP3 pathway.
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Objective@#To understand the epidemiological characteristics of Human coronavirus (HCoV), the patterns of emergence and circulation, and the genotype distribution of human coronavirus OC43 (HCoV-OC43) from November, 2009 to April, 2016 in Shanghai.@*Methods@#A total of 6 059 respiratory specimens, including pharyngeal swab, sputum, nasopharyngeal aspirates and alveolar lavage fluid, as well as relative clinical data were collected from patients with acute respiratory infections from seven sentinel hospitals during November, 2009 to April, 2016 in Shanghai. Respiratory specimens were tested by RT-PCR with HCoV-conserved primers and subsequently genotyped by DNA sequencing. Using specific primers to amplify and sequence full-length Spike (S), RNA-dependent RNA polymerase (RDRP) and nucleocapsid (N) gene from HCoV-OC43 positive samples. Further genotype and phylogenetic analysis of HCoV-OC43 were performed by conducting phylogenetic trees.@*Results@#Among 6 059 patients, the total frequency of HCoV was 63 (1.04%), in which HCoV-OC43 was the most frequently detected species with 34 positive samples, followed by human coronavirus 229E (HCoV-229E) and human coronavirus HKU1 (HCoV-HKU1) with 18 and 10 positive sample respectively. However, other HCoV like human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle-East Respiratory Syndrome Coronavirus (MERS-CoV), were not been detected, which illustrated that HCoV-OC43 was the dominant subtype. The full-length of S, RDRP and N gene were obtained from 29 HCoV-OC43 positive samples. According to the sequence-analysis, 27 of which was genotype D, 2 of which was genotype B and others genotype, including genotype E, F and G, were not detected. The result indicated that the genotype D may be the dominant genotype. Further analysis of S protein that help HCoV-OC43 to entry host cell and stimulate the host immune system to produce neutralizing antibody found that two important functional domains in S protein, N-terminal domain (NTD) and receptor-binding domain (RBD) contained more amino acid substitution and positive selection sites, accompanied with amino acid insertion/deletion. 13 positive selection sites were all located in the NTD or RBD, 10 of which were located in the NTD and 3 in the RBD.@*Conclusion@#Human coronavirus OC43 was the major circulation human coronaviurs in Shanghai from 2009 to 2016, in which genotype D was the dominant genotype. NTD and RBD regions of the S protein were hypervariable region during HCoV-OC43 evolution, and had amino acid substitutions as well as amino acid insertion/deletion.