Objective:To study the inhibitory effects and mechanism of Armeniacae Semen Amarum alcohol extract in melanogenesis in zebrafish embryos.Methods:48 HPF healthy zebrafish embryos were randomly divided into blank control group, arbutin group, high-, medium- and low-dosage groups. The blank control group was added with 6 ml embryo culture medium, the arbutin group was added with 8 000 μg/ml arbutin solution 6 ml, and the high-, medium- and low-dosage groups were added with 2 925, 1 950 and 1 300 μg/ml Armeniacae Semen Amarum alcohol extract, respectively. After 24 hours of intervention, the melanin area of zebrafish embryos was observed by stereomicroscope, and the inhibition rate of ethanol extract of bitter almond on tyrosinase (TYR) activity and melanin content in zebrafish embryos were detected by spectrophotometry; the antioxidant activity was evaluated by DPPH radical scavenging capacity, ABTS radical scavenging capacity and CAT activity. RT-qPCR was used to detect the mRNA levels of MITF, Tyr and TYRP2 in zebrafish embryos.Results:Compared with the arbutin group, the low-, medium-, and high-dosage groups showed an increase in melanin area ( P<0.01 or P<0.05), a decrease in TYR activity and melanin content inhibition rate ( P<0.01), and IC50 values for DPPH and ABTS free radical scavenging rates of 23.636 and 3.195 mg/ml, respectively. The CAT activity increased in the medium- and high-dosage groups ( P<0.05 or P<0.01), while the mRNA levels of TYR and MITF decreased in the low-, medium-, and high-dosage groups ( P<0.01). Conclusion:Armeniacae Semen Amarum alcohol extract can effectively inhibit the production of melanin in zebrafish embryos, and its mechanism may be closely related to TYR activity and MITF related signaling pathway.

The extraordinary advantages associated with mRNA vaccines, including their high efficiency, relatively low severity of side effects, and ease of manufacture, have enabled them to be a promising immunotherapy approach against various infectious diseases and cancers. Nevertheless, most mRNA delivery carriers have many disadvantages, such as high toxicity, poor biocompatibility, and low efficiency in vivo, which have hindered the widespread use of mRNA vaccines. To further characterize and solve these problems and develop a new type of safe and efficient mRNA delivery carrier, a negatively charged SA@DOTAP-mRNA nanovaccine was prepared in this study by coating DOTAP-mRNA with the natural anionic polymer sodium alginate (SA). Intriguingly, the transfection efficiency of SA@DOTAP-mRNA was significantly higher than that of DOTAP-mRNA, which was not due to the increase in cellular uptake but was associated with changes in the endocytosis pathway and the strong lysosome escape ability of SA@DOTAP-mRNA. In addition, we found that SA significantly increased the expression of LUC-mRNA in mice and achieved certain spleen targeting. Finally, we confirmed that SA@DOTAP-mRNA had a stronger antigen-presenting ability in E. G7-OVA tumor-bearing mice, dramatically inducing the proliferation of OVA-specific CLTs and ameliorating the antitumor effect. Therefore, we firmly believe that the coating strategy applied to cationic liposome/mRNA complexes is of potential research value in the field of mRNA delivery and has promising clinical application prospects.

Objective:To investigate the effect of ionizing radiation on the N 6-methyladenine (m 6A) modification profile of circular RNA (circRNA) in mouse bone marrow cells and provide scientific basis for revealing the relationship between RNA epigenetic modification and hematopoietic radiation injury. Methods:A total of twenty four C57BL/6 J mice were randomly divided into two groups: the healthy control group ( n=12), and ionizing radiation group ( n=12) irradiated in total body with 4 Gy of 137Cs γ-rays. At 5 min after irradiation, mice were killed and bone marrow cells were collected from the femur. Total RNAs were extracted and the changes in circRNA m6A modification profiles were investigated by RNA immunoprecipitation-high-throughput sequencing (MeRIP-Seq) technology and bioinformatics analysis. The representative alterations of m 6A peaks were validated by MeRIP-PCR assay. Results:325 and 455 m 6A sites were identified on circRNAs in the healthy control group and ionizing radiation group (178 common sites, 147 specific sites in the healthy control group and 277 specific sites in ionizing radiation group), respectively. 1 275 and 1 017 deriving genes of m 6A-circRNAs were identified in the healthy control group and ionizing radiation group (767 common genes, 508 specific genes in the healthy control group and 250 specific genes in ionizing radiation group), respectively. Compared with the control healthy group, 414 (178) m 6A peaks was significantly up- (down-) regulated in the ionizing radiation group( P < 10 -10; fold-change cut-off > 5). Moreover, Gene Ontology (GO) assay revealed that the deriving genes of circRNAs with differentially methylated m 6A sites between two groups involves various functions including chromatin regulation, ciliary transition fiber and poly (A)-specific ribonuclease activity. Kyoto Encyclopedia of Genes and Genomes (KEGG) assay revealed that the deriving genes of circRNAs with differentially methylated m 6A sites between two groups included numerous pathways such as platelet activation, Fc γ R-mediated phagocytosis and B cell receptor signaling pathway. Conclusions:Ionizing radiation triggers rapid alterations in the m 6A modification profile of circRNA in mouse bone marrow cells. The deriving genes of differentially methylated circRNAs are associated with a variety of functions and signaling pathways of hematopoietic radiobiology.

Objective To detect the protein expression change in the proliferation of human retinal microvascular endothelial cells (hRMECs) stimulated with 4-Hydroxynonenal (4-HNE).Methods hRMECs were in a logarithmic growth phase, and then were separated into 4-HNE-stimulated group and negative control group. The concentration of 4-HNE included 5, 10, 20 and 50 μmol/L in 4-HNE-stimulated group, while the negative control group was added in the same volume of ethanol (the solvent of 4-HNE). Then the cells were stimulated with 4-HNE for 24 hours following by CCK-8 kits incubating for 4 hours to detect absorbance. It was found that 10 μmol/L 4-HNE had the most obvious effect on the proliferation of hRMECs. Therefore, the cellular proteins from 10 μmol/L 4-HNE-stimulated group and negative control group were acquired and prepared by FASP sample preparation method. Data independent acquisition was used for data acquisition, and the GO analysis and pathway enrichment were performed for analysis of differentially expressed proteins. Results CCK-8 kits detection results showed that theA value of the 10 and 20 μmol/L 4-HNE-stimulated groups were significantly higher than negative control group and 5 μmol/L 4-HNE-stimulated group (F=25.42, P<0.01), while there were no differences between 10 and 20 μmol/L 4-HNE-stimulated groups, and theA value of 50 μmol/L 4-HNE-stimulated groups was lower than negative control. A total of 2710 quantifiable proteins were identified by peoteomics,and 118 proteins were differentially expressed (fold change>1.5,P<0.05). Seventy-two proteins were up-regulated after 4-HNE stimulation, whereas 46 proteins were down-regulated. Particularly, the expressions of Heme oxygenase-1, Sulfoxdoxin-1, Heat shock protein A1B, Thioredoxin reductase-1, Glutathione reductase, ATPase and prothrombin were increased when cells were added in 4-HNE, whereas the expressions of apolipoprotein A1 and programmed cell death protein 4 were decreased. These differentially expressed proteins were mainly involved in the biological processes such as oxidative stress, cell detoxification, and ATPase-coupled membrane transport.Conclusion After stimulated with 4-HNE, the oxidative stress, cell detoxification, and ATPase-coupled membrane transport protein expression may change in hRMECs in order to regulate oxidative stress and growth situation.

Objective: To determine the changes of protein expressions in human retinal microvascular pericytes (HRMPCs) stimulated with high glucose by using quantitative proteomics, which provides new clues for future investigation of diabetic retinopathy (DR). Methods: HRMPCs were divided into two groups.The cells in control group were cultured in DMEM basic medium with 25 mmol/L glucose, while the cells in high glucose group were cultured in DMEM medium with 35 mmol/L glucose.The amount of living cells was measured by cell counting kit-8(CCK-8). The proteins were collected from the two groups and then were digested with trypsin.Peptides of 2 μg were injected into the time of flight-mass spectrometer and the acquisition mode was DDA.The results were further analyzed using bioinformatics software. Results: CCK-8 results showed that the absorbance (A₄₅₀) of HRMPCs in high glucose group was 0.75±0.04, which was significantly lower than 0.91±0.05 in control group (t=5.784, P=0.000 2). In total, 1 972 proteins were identified and 54 of them were significantly different between the two groups (fold change >1.5). Among them, 13 proteins were up-regulated, including CTNNB1 and CTBP2; while 41 proteins were down-regulated, including SQSTM1 and HMGCS1.The differentially expressed proteins were mainly involved in citric acid cycle and aerobic respiration. Conclusions The expressions of many proteins in HRMPCs change under the stimulation of high glucose, which may influence the respiration and the ATP production of cells and eventually induce the loss of pericyte.

Objective To observe the effects of ischemic postconditioning (IPTC) on myocardial infarction sizes (IS) and protein kinase Cα (PKCα) expression in aged rats with post-ischemia reperfusion injury,and to explore the mechanism.Methods A total of 120 male Wistar rats were divided into aged group and adult group.The aged group was randomly divided into control group (n=12,30-min ischemia and 3-h reperfusion),5 s,10 s,30 s and 60 s IPTC groups [n= 12,each;after 30 min occlusion of left coronary artery (LCA),three cycles of 5 s,10 s,30 s,60 s reperfusion respectively followed by the same interval LCA re-occlusion were applied at the beginning of reperfusion].The IS was measured with TTC dye,and PKC expression was investigated by immunohistochemistry.Results Different IPTC intervals had different effects on IS and PKC expression,10 s and 30 s IPTC could reduce IS both in aged rats and adult rats [(55.9±6.0)% and (47.4±5.5)%],IS in 10 s IPTC group in aged rats was (48.1±5.3)%,in adult rats was (39.2±5.7) %;IS in 30 s IPTC group in aged rats was (48.8 ± 6.8) %,in adult rats was (40.2 ± 6.1 ) %.PKCα expression increased in aged and adult rats (all P<0.05).5 s IPTC could increase IS [IS in 5 s IPTC group in aged rats was (63.5±5.4)%,and PKCα expression reduced in aged rats (all P<0.05)].Conclusions IPTC has cardio-protective effect in aged rats suffering from acute myocardial injury during reperfusion,the effect of IPTC is related to reperfusion-reocclusion interval.