Kaempferol Alleviates Injury in Human Retinal Pigment Epithelial Cells via STAT1 Ubiquitination-Mediated Degradation of IRF7.

BACKGROUND: Retinal pigment epithelial (RPE) cells have a pivotal function in preserving the equilibrium of the retina and moderating the immunological interaction between the choroid and the retina. This study primarily focuses on delineating the protective effect offered by Kaempferol (Kae) against RPE cell damage. METHODS: Bioinformatics analysis was performed on the GSE30719 dataset to identify hub genes associated with RPE. Subsequently, we analyzed the impact of Kae on RPE apoptosis, cell viability, and inflammatory response through cell experiments, and explored the interaction between hub genes and Kae. RESULTS: Based on the GSE30719 dataset, nine hub genes (ISG15, IFIT1, IFIT3, STAT1, OASL, RSAD2, IRF7, MX2, and MX1) were identified, all of which were highly expressed in the GSE30719 case group. Kae could boost the proliferative activity of RPE cells caused by lipopolysaccharide (LPS), as well as reduce apoptosis and the generation of inflammatory factors (tumor necrosis factor receptor (TNFR), interleukin-1beta (IL-1ß)) and cytokines (IL-1, IL-6, IL-12). STAT1 was shown to inhibit cell proliferation, promote apoptosis, and secrete IL-1/IL-6/IL-12 in LPS-induced RPE cells. Moreover, IRF7 was found to interact with STAT1 in LPS-induced RPE cells, and STAT1 could maintain IRF7 levels through deubiquitination. In addition, we also found that the protective effect of Kae on LPS-induced RPE cell injury was mediated through STAT1/IRF7 axis. CONCLUSION: This study provided evidence that Kae protects RPE cells via regulating the STAT1/IRF7 signaling pathways, indicating its potential therapeutic relevance in the diagnosis and management of retinal disorders linked with RPE cell damage.

Mesenchymal Stem Cell Exosomal miR-146a Mediates the Regulation of the TLR4/MyD88/NF-κB Signaling Pathway in Inflammation due to Diabetic Retinopathy.

Diabetic retinopathy (DR) is the main cause of vision loss in diabetic patients, which cannot be completely resolved by typical blood sugar control. Inflammation influences the development of DR, so reducing the inflammatory response in DR patients is crucial to the prevention of DR. Therefore, we explored the regulatory effect of bone marrow mesenchymal stem cell (BMSC) exosomes on inflammation in DR mice. In order to analyze the mechanism of action, we used BMSC exosomal miR-146a to treat microglias in DR mice to observe cellular changes and expression of inflammatory factors. It was found that BMSC exosomal miR-146a reduced the levels of proliferating cell antigen and B-cell lymphoma-2 in microglias of DR mice and increased Bcl-2-related X with cysteine aspartic protease-3. By analyzing the expression of inflammatory factors, we found that BMSC exosomal miR-146a reduced the levels of TNF-α, IL-1ß, and IL-6, which suggested that miR-146a can alleviate inflammation in DR mice. Further exploration found that miR-146a reduced the activity of TLR4 and increased the activity of MyD88 and NF-κB. Furthermore, the overexpression of TLR4 reversed the effects of miR-146a on the proliferation, apoptosis, and inflammation of microglias. Our study demonstrated that BMSC exosomal miR-146a can regulate the inflammatory response of DR by mediating the TLR4/MyD88/NF-κB pathway, providing an experimental basis for the prevention and treatment of DR.

Case Report: A Case of Cystoid Macular Edema in Retinitis Pigmentosa With Central Retinal Vein Occlusion.

Significance: Cystoid macular edema (CME) is a common complication of retinitis pigmentosa (RP). However, CME in RP with central retinal vein occlusion (CRVO) is rare. Prompt administration of anti-vascular endothelial growth factor (anti-VEGF) medication can achieve a satisfactory prognosis. Purpose: This report describes a case of using anti-VEGF medication to treat CME secondary to RP with impending or mild CRVO. Case Report: A 26-year-old female presented for blurred vision in both eyes. Best-corrected visual acuity (BCVA) was 20/50 in the right eye and finger-counting in the left eye. According to ophthalmic examinations, CME secondary to RP in the right eye and CME secondary to RP with impending or mild CRVO in her left eye can be diagnosed. Central macular thickness (CMT) was 554 µ m in the right eye and 831 µm in the left eye. Only the left eye was treated with a single intravitreal injection of anti-VEGF medication. One month later, BCVA increased to 20/200 and CMT decreased to 162 µm in the left eye. Interestingly, BCVA in the right eye also had an improvement (20/40) and intraretinal fluid decreased significantly. However, 3 months after injection, these improvements of both eyes were not maintained. Conclusion: This is the second case of RP with CRVO. Intravitreal injection of anti-VEGF medication for addressing CME secondary to RP with CRVO is an effective treatment, but it needs to be reinjected.

MiR-192 attenuates high glucose-induced pyroptosis in retinal pigment epithelial cells via inflammasome modulation.

Diabetic retinopathy is one of the most characteristic complications of diabetes mellitus, and pyroptosis plays acrucial role in the onset and development of diabetic retinopathy. Although microRNA-192 (miR-192) has been demonstrated to be involved in diabetic retinopathy progression, to the best of our knowledge, its potential and mechanism in cell pyroptosis in diabetic retinopathy have not been studied. The present study demonstrated that high glucose (HG) contributes to the pyroptosis of retinal pigment epithelial (RPE) cells in a dose-dependent manner. The results revealed that miR-192 was weakly expressed in HG-induced RPE cells. Furthermore, overexpression of miR-192 abrogated the role of HG in RPE cell pyroptosis. Based on the bioinformatics analysis, a dual-luciferase reporter assay, and an RNA pull-down assay, FTO α-ketoglutarate-dependent dioxygenase (FTO) was demonstrated to be a direct target of miR-192. Additionally, upregulation of FTO abolished the effects of miR-192 on RPE cells treated with HG. Nucleotide-binding domain leucine-rich repeat family protein 3 (NLRP3) inflammasome activation is vital for cell pyroptosis, and FTO functions as a pivotal modulator in the N6-methyladenosine modifications of various genes. Mechanistically, FTO enhanced NLRP3 expression by facilitating demethylation of NLRP3. In conclusion, the present results demonstrate that miR-192 represses RPE cell pyroptosis triggered by HG via regulation of the FTO/NLRP3 signaling pathway.

Nanofluorescence Probes to Detect miR-192/Integrin Alpha 1 and Their Correlations with Retinoblastoma.

We developed a novel nanostructure DNA probe for the in situ detection of ITGA1 and miR-192 in retinoblastoma (RB) and to study the correlation between ITGA1 and miR-192 expression and RB development. ITGA1 and miR-192 nanostructure DNA probes were carried by silica particles and coated by dioleoyl-trimethy-lammonium-propane, which enhances their organizational compatibility and infiltration capacity. This probe has stable physicochemical properties and high specificity and sensitivity to detect ITGA1 and miR-192 in situ both in RB cell lines and RB tissues. Using ITGA1 and miR-192 nanostructure DNA probes in RB tissue and cell lines, we found that the expression of ITAG1 drastically increased, but to the contrary, miR-192 was not expressed. After transfection, ITGA1 and miR-192 were overexpressed or silenced in RB116 cells, and we found that ITGA1 could effectively increase the activity and invasion of this RB cell line and reduce its apoptosis level, while miR-192 antagonized this tumor-promoting effect. Therefore, miR-192 can be used as an early biomarker of RB, and ITGA1 may be a new prognostic marker and therapeutic target for the treatment of RB.

Adipose mesenchymal stem cells-secreted extracellular vesicles containing microRNA-192 delays diabetic retinopathy by targeting ITGA1.

Diabetic retinopathy (DR) has characteristics of early loss of capillary pericytes, contributing to aberrant endothelial proliferation and angiogenesis. The function of extracellular vesicles (Evs) derived from mesenchymal stem cells (MSCs) in angiogenesis and endothelial proliferation were investigated in the present study. In particular, the role of microRNA-192 (miR-192) was described. Firstly, the GSE60436 data set was applied to screen out that integrin subunit α1 (ITGA1) was overexpressed in DR. Subsequently, streptozotocin (STZ) was used to induce diabetes in rats, which was later subjected to intravitreal injection of targeted shRNAs. ITGA1 knockdown alleviated inflammation and angiogenesis in STZ-induced diabetic retina. Evs were extracted from MSCs and injected into rat vitreous. Meanwhile, human retinal microvascular endothelial cells, Müller cells, and retinal pigment epithelium cells were exposed to high glucose. MSC-derived Evs relieved inflammatory response and angiogenesis by shuttling miR-192. miR-192 targeted and negatively regulated ITGA1, thereby ameliorating diabetic retinal damage. Our study established that miR-192 released by Evs from MSCs could delay the events of the inflammatory response and angiogenesis in DR and may represent a possible therapeutic approach for the treatment of DR.

MiRNA-144-3p inhibits high glucose induced cell proliferation through suppressing FGF16.

As a major cause of blindness, diabetic retinopathy (DR) is often found in the developed countries. Our previous study identified a down-regulated miRNA: miR-144-3p in response to hyperglycemia. The present study aims to investigate the role of miR-144-3p in proliferation of microvascular epithelial cells. Endothelial cells were treated with different concentrations of glucose, after which miR-144-3p were detected with real-time PCR assay. MiR-144-3p mimics or inhibitors were used to increase or knockdown the level of this miRNA. Western blotting assay and ELISA assay were used to measure the expression and concentration of VEGF protein. 5-Bromo-2-deoxyUridine (BrdU) labeled cell cycle assay was used to detect cells in S phase. MiRNA targets were predicted by using a TargetScan tool, and were further verified by luciferase reporter assay. In the present study, we focussed on a significantly down-regulated miRNA, miR-144-3p, and investigated its role in high glucose (HG) induced cell proliferation. Our data showed that miR-144-3p mimics significantly inhibited HG induced cell proliferation and reduced the percentage of cells in S phase. HG induced up-regulation of VEGF was also prohibited by miR-144-3p mimics. Through wound-healing assay, we found that miR-144-3p suppressed cell migration after HG treatments. Moreover, we predicted and proved that fibroblast growth factor (FGF)16 is a direct target of miR-144-3p. Finally, miR-144-3p attenuated HG induced MAPK activation. In conclusion, we demonstrated that miR-144-3p inhibited high glucose-induced cell proliferation through suppressing FGF16 and MAPK signaling pathway, suggesting a possible role of miR-144-FGF16 in the development of DR.

Construction and immunocompetence of recombinant DNA E.coli ghosts expressing Treponema pallidum adhesin Tp0751 / 中国免疫学杂志

Objective:To construct the recombinant DNA E.coli ghosts (EBGs) expressing Treponema pallidum adhesin Tp0751 (pcD/Tp0751-BG) and determine its immunocompetence in immunized mice,and provide a potential novel method for syphilis vaccine developing.Methods:The recombinant eukaryotic expression plasmid pcDNA3.1(+)/Tp0751 was constructed and loaded into empty EBGs to create pcD/Tp0751-BG.The loading rate was determined accordingly.Macrophages cell line RAW264.7 was transfected with pcD/Tp0751-BG,and the expression of recombinant Tp0751(rTp0751) protein was detected by Western blot(WB).For immuno-competence in mice,the female BALB/c mice were randomly divided into 6 groups,including three control groups,A (PBS),B (EBG),C (empty pcDNA),and experimental group D(naked pcD/Tp0751),E (pcD/Tp0751-BG) and F (pcD/Tp0751-BG+rTp0751).All the mice were immunized as indicated for three times by intramuscular injection at two weeks intervals.The levels of specific IgG in sera and SIgA in genital tract lavage fluid were measured by ELISA.Levels of lymphocyte proliferation and IFN-γ secretion in spleen cells were measured by CCK-8 Cell Counting Kit and ELISA as well,respectively.Results:The loading rate of pcD/Tp0751 to EBGs was 76.1%.WB showed that the target recombinant protein pcD/Tp0751 expressed in RAW264.7 cells was active with Tp-infected rabbit sera.The titers of specific IgG and SIgA in group D,E,F gradually increased to significantly higher level as compared to the control groups (P<0. 01),which reached its peak at wk 8 after last immunization(the titers of IgG and SIgA were 1 :102 400 and 1 :12 800 in group F,respectively). Higher levels of specific IgG and SIgA were observed in groups E and F as compared to group D after first boost (P<0. 01),with groups F higher than group E after last boost(P<0. 01). At wk 8 after the last boost,the stimulation index (SI) and levels of IFN-γ in group D,E,F were all significant higher than the control groups (P<0. 01), with group E and F higher than group D (P<0. 01),and group E higher than group F (P<0. 05). Conclusion: The recombinant DNA EBGs of T. pallidum adhesin Tp0751 (pcD/ Tp0751-BG) possesses the immunocompetence to induce not only strong mucosal and systemic humoral immune response but also systemic cellular immune response in BALB/ c mice. The heterologous boost can be more efficient than homologous boost during immunization process.

SiRNA directed against annexin II receptor inhibits angiogenesis via suppressing MMP2 and MMP9 expression.

BACKGROUND/AIMS: Annexin II receptor (AXIIR) is able to mediate Annexin II signal and induce apoptosis, but its role in angiogenesis remains unclear. This study tries to investigate the role of AXIIR in angiogenesis and the plausible molecular mechanism. METHODS/RESULTS: RNA interference technology was used to silence AXIIR, and the subsequent effects in vitro and in vivo were evaluated thereafter. Our data indicated that human umbilical vein endothelial cells (HUVECs) expressed AXIIR and knockdown of AXIIR significantly inhibited HUVECs proliferation, adhesion, migration, and tube formation in vitro and suppressed angiogenesis in vivo. Furthermore, AXIIR siRNA induced cell arrest in the S/G2 phase while had no effect on cell apoptosis. We found that these subsequent effects might be via suppressing the expression of matrix metalloproteinase 2and matrix metalloproteinase 9. CONCLUSION: AXIIR participates in angiogenesis, and may be a potential therapeutic target for angiogenesis related diseases.