Methylene Blue Reduces Retinal Cell Inflammation, Apoptosis, and Oxidative Stress in a Rat Model of Diabetic Retinopathy via Sirtuin 1 Activation.

Objective: Diabetic retinopathy (DR), characterized by neuronal damage in the retina, is primarily driven by oxidative stress resulting from diabetes (DM). This study investigated the potential effects of methylene blue (MB) on streptozotocin (STZ)-induced DR. Methods: A rat model of DR was established via STZ injection, while a cell model was created using high-glucose (HG) exposure of human retinal microvascular endothelial cells. Evaluation of oxidative stress markers, pro-inflammatory cytokines, and pro-apoptotic proteins was performed based on their expression profiles in human retinal microvascular endothelial cells. Results: MB treatment significantly upregulated the expression of sirtuin 1 (SIRT1), which was found to be downregulated in the retinal tissues of STZ-treated rats and HG-exposed human retinal microvascular endothelial cells, as determined by polymerase chain reaction (PCR). Furthermore, MB therapy effectively suppressed STZ-induced oxidative stress, inflammation, and cell death. Consistent with the in vivo findings, MB activated the expression of SIRT1, thereby protecting HG-treated human retinal microvascular endothelial cells against oxidative stress, inflammation, and apoptosis. Conclusion: These results support the conclusion that MB mitigates DR by activating SIRT1, leading to a reduction of inflammation, apoptosis, and oxidative stress.

Endothelial Progenitor Cells Affect the Growth and Apoptosis of Renal Cells by Secreting Microvesicles Carrying Dysregulated miR-205 and miR-206.

Background: This study investigated the mechanism of microRNA (miRNA, miR) in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) involved in renal function in vivo and in vitro injury repair of rat primary kidney cells (PRKs). Methods: Gene Expression Omnibus analysis of potential target miRNAs in nephrotic rats. Real-time quantitative polymerase chain reaction verified the correlation of these miRNAs and screened the effective target miRNAs and their downstream putative target mRNAs. Western blot analyzes the protein levels of DEAD-box helicase 5 (DDX5) and the activation of the proapoptotic factor caspase-3/9 (cleaved). Dil-Ac-LDL staining, immunofluorescence, and a transmission electron microscope (TEM) were used to identify the successful isolation of EPCs and PRKs and the morphology of MVs. Cell Counting Kit-8 was used to detect the effect of miRNA-mRNA on the proliferation of PRKs. Standard biochemical kits were used to detect biochemical indicators in rat blood and urine. Dual-luciferase analysis of miRNA binding to mRNA was conducted. The effect of miRNA-mRNA interaction on the apoptosis level of PRKs was analyzed by flow cytometry. Results: A total of 13 rat-derived miRNAs were potential therapeutic targets, and miR-205 and miR-206 were screened as the targets of this study. We found that the EPC-MVs alleviated the increase of blood urea nitrogen and urinary albumin excretion and the decrease in creatinine clearance caused by hypertensive nephropathy in vivo. The effect of MVs in improving renal function indicators was promoted by miR-205 and miR-206 and inhibited by knockdown of expressed miR-205 and miR-206. In vitro, angiotensin II (Ang II) promoted growth inhibition and apoptosis of PRKs, and similarly, dysregulated miR-205 and miR-206 affected the induction of Ang II. We then observed that miR-205 and miR-206 cotargeted the downstream target DDX5 and regulated its transcriptional activity and translational levels, while also reducing the activation of proapoptotic factors caspase-3/9. Overexpressed DDX5 reversed the effects of miR-205 and miR-206. Conclusion: By upregulating the expression of miR-205 and miR-206 in MVs secreted by EPC, the transcriptional activity of DDX5 and the activation of caspase-3/9 can be inhibited, thereby promoting the growth of PRKs and protecting the injury caused by hypertensive nephropathy.

[Methylene blue reduces IL-1ß levels by enhancing ERK1/2 and AKT phosphorylation to improve diabetic retinopathy in rats].

Objective To investigate the neuroprotective effect of methylene blue on diabetic retinopathy in rats. Methods Thirty SD rats were randomly divided into blank, control and experimental groups. The control and experimental groups were induced with diabetes by streptozotocin (STZ) intraperitoneal injection. After 6 weeks of successful modeling, the experimental group received intravitreal injection of methylene blue at a dose of [0.2 mg/(kg.d)], while the control group received an equal amount of dimethyl sulfoxide (DMSO) intravitreal injection, both continuously injected for 7 days. ELISA was used to detect the levels of retinal superoxide dismutase (SOD), 8-iso-prostaglandin F2alpha (iPF2α) and interleukin-1ß (IL-1ß) in rats. Western blot analysis was used to detect the expression of retinal extracellular signal-regulated kinase 1/2 phosphorylation (p-ERK1/2) and phosphorylated protein kinase B (p-AKT), and PAS staining was used to detect retinal morphological changes. Results Compared with the blank group rats, the retinal SOD activity in the control and experimental group rats was significantly reduced. iPF2α, IL-1ß and p-ERK1/2 level increased, while p-AKT level decreased. Compared with the control group, the SOD activity of the experimental group rats increased. iPF2α and IL-1ß level went down, while p-ERK1/2 and p-AKT level went up significantly. The overall thickness of the retinal layer and the number of retinal ganglion cells were significantly reduced. Conclusion Methylene blue improves diabetic retinopathy in rats by reducing retinal oxidative stress and enhancing ERK1/2 and AKT phosphorylation.

Protective effects of endothelial progenitor cell microvesicles carrying miR­98­5p on angiotensin II­induced rat kidney cell injury.

With the increasing number of patients with hypertensive nephropathy worldwide, it has posed a major threat to health and studies on its treatment and pathogenesis are imminent. The present study investigated the mechanism through which microRNA (miR)-98-5p in microvesicles (MVs) secreted by endothelial progenitor cells (EPCs) is involved in the repair of angiotensin II (Ang II)-induced injury of rat primary renal kidney cells (PRKs). After isolation of rat renal cortical sections, PRKs were isolated by density gradient centrifugation and identified by immunofluorescence staining. Transmission electron microscopy identifies successful separation of Mvs. An in vitro cell injury model was constructed using Ang II. The Gene Expression Omnibus was used to analyze the differentially expressed genes between diabetic rats and normal rats, and the Kyoto Encyclopedia of Genes and Genomes was used to analyze the signaling pathways involved in these differentially expressed genes. Reverse transcription-quantitative PCR was used to analyze the effect of EPC-MVs on the expression of miRNA induced by Ang II, and the levels of target genes and signaling pathway-related proteins involved were analyzed by western blot. luciferase was used to detect the targeted binding of miR-98-5p to insulin-like growth factor 1 receptor (IGF1R). Enzyme-linked immunosorbent assay was used to analyze the effect of EPC-MVs on Ang II-induced oxidative stress and inflammation levels on PRKs. Cell Counting Kit-8 was used to analyze the effect of EPC-MVs on the cell viability of PRKs induced by Ang II. The results showed that treatment of PRKs with Ang II decreased cell viability, whereas oxidative stress and inflammation were increased. However, EPC-MVs alleviated Ang II-induced damage of the PRKs. During this process, the Ang-II-induced downregulation of miR-98-5p was reversed by EPC-MVs, so miR-98-5p may be a key factor regulating the action of EPC-MVs. Dual-luciferase assay confirmed that miR-98-5p targets IGF1R. It was subsequently demonstrated that EPC-MVs overexpressing miR-98-5p promoted phosphorylation of PI3K/Akt/endothelial nitric oxide synthase (eNOS), and inhibited the oxidative stress and inflammation in PRKs, which were reversed by the overexpression of IGF1R. In conclusion, the results of the present study demonstrated that EPC-MVs with high expression of miR-98-5p can activate the PI3K/Akt/eNOS pathway by regulating IGF1R, as well as protect PRKs from Ang II-induced oxidative stress, inflammation and inhibition of cell viability.

Protective effects of endothelial progenitor cell microvesicles on Ang II­induced rat kidney cell injury.

Chronic hypertension can lead to kidney damage, known as hypertensive nephropathy or hypertensive nephrosclerosis. Further understanding of the molecular mechanisms via which hypertensive nephropathy develops is essential for effective diagnosis and treatment. The present study investigated the mechanisms by which endothelial progenitor cells (EPCs) repair primary rat kidney cells (PRKs). ELISA, Cell Counting Kit­8 and flow cytometry assays were used to analyze the effects of EPCs or EPC­MVs on the oxidative stress, inflammation, cell proliferation, apoptosis and cycle of PRKs induced by AngII. A PRK injury model was established using angiotensin II (Ang II). After Ang II induction, PRK proliferation was decreased, apoptosis was increased and the cell cycle was blocked at the G1 phase before entering the S phase. It was found that the levels of reactive oxygen species and malondialdehyde were increased, while the levels of glutathione peroxidase and superoxide dismutase were decreased. Moreover, the levels of the inflammatory cytokines IL­1ß, IL­6 and TNF­α were significantly increased. Thus, Ang II damaged PRKs by stimulating oxidative stress and promoting the inflammatory response. However, when PRKs were co­cultured with EPCs, the damage induced by Ang II was significantly reduced. The current study collected the microvesicles (MVs) secreted by EPCs and co­cultured them with Ang II­induced PRKs, and identified that EPC­MVs retained their protective effect on PRKs. In conclusion, EPCs protect PRKs from Ang II­induced damage via secreted MVs.

MiR-144 affects proliferation and apoptosis of high glucose-induced AC16 cardiomyocytes by regulating CTRP3/JNK signaling.

BACKGROUND: Diabetic cardiomyopathy (DCM) is a common complication of diabetes and can lead to heart failure, arrhythmia, and sudden death. microRNAs (miRNAs) are reportedly involved in many human disease, including DCM. However, little is known about the biologic functions of miR-144 in DCM progression. METHODS: The expression levels of miR-144 and C1q/TNF-related protein-3 (CTRP3) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was used to determine the protein levels of CTRP3, phosphorylated c-Jun amino-terminal kinase (p-JNK), JNK, Bax, Bcl-2, and cleaved-caspase-3. Cell proliferation and apoptosis were detected by Cell Counting Kit-8 (CCK-8) assay and flow cytometry, respectively. The potential binding sites between miR-144 and CTRP3 were predicted by microRNA.org databases and further determined using a dual-luciferase assay. AC16 cardiomyocytes were cultured in high glucose (HG, 30 mmol/L) to mimic hyperglycemia. RESULTS: MiR-144 expression level was enhanced, while CTRP3 expression was reduced in HG-induced AC16 cardiomyocytes. Knockdown of miR-144 or overexpression of CTRP3 dramatically promoted cell proliferation and reduced apoptosis of AC16 cardiomyocytes treated with HG. Inhibition of miR-144 evidently decreased the protein levels of Bax and p-JNK, but elevated Bcl-2 expression in HG-induced AC16 cardiomyocytes. Moreover, CTRP3 was a direct target of miR-144, and its abrogation reversed the effects of miR-144 knockdown on proliferation and apoptosis in HG-induced AC16 cardiomyocytes. SP600125 (a JNK inhibitor, 10 µmol/L) attenuated the si-CTRP3-mediated inhibition of proliferation and promotion of apoptosis in AC16 cardiomyocytes transfected with anti-miR-144 and stimulated with HG. CONCLUSION: MiR-144 regulates proliferation and apoptosis of HG-induced AC16 cardiomyocytes through targeting the CTRP3/JNK signaling pathway, providing a novel avenue for treatment of DCM.