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.

[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.

The impact of cardiomotor rehabilitation on endothelial function in elderly patients with chronic heart failure.

BACKGROUND: To explore the effects of cardiac exercise rehabilitation on peripheral blood endothelial progenitor cells (EPC) in elderly patients with chronic heart failure. METHODS: 80 elderly patients with chronic heart failure were selected from March 2017 to March 2019 and randomly divided into two groups (N = 40). The control group was treated routinely and walked freely for 30-60 min every day. The patients in the exercise rehabilitation group developed a cardiac exercise rehabilitation plan. Then, cardiac function and peripheral blood B-natriuretic peptide (BNP) levels in the two groups were compared. The cell viability, proliferation, apoptosis, and invasion ability of EPCs were detected. The levels of the PI3K/AKT pathway and eNOS and VEGF were compared. RESULTS: There were no significant differences in all indexes between the two groups before treatment (P > 0.05), and both improved significantly after treatment (P < 0.05). After treatment, LVEF and LVFS in the exercise rehabilitation group were significantly higher than those in the control group (P < 0.05), and LVEDD and LVESD were significantly lower than those in the control group (P < 0.05). The BNP level in the exercise rehabilitation group was significantly lower than that in the control group (P < 0.05). The cell viability, proliferation, invasion ability of EPC, and the levels of PI3K, AKT, eNOS, and VEGF mRNA and protein in the exercise rehabilitation group were significantly higher than those in the control group. Apoptosis rate was significantly lower than those in the control group (P < 0.05). CONCLUSIONS: Visceral exercise rehabilitation can improve cardiac ejection and myocardial function in elderly patients with chronic heart failure, and can promote the vitality, proliferation, and invasion of peripheral blood EPC, and promote the expression of eNOS and VEGF by upregulating the PI3K/AKT pathway to promote angiogenesis and endothelial function.

Development of solidified self-microemulsifying delivery systems with enhanced stability of sirolimus and extended release.

The application of sirolimus (SRL) as immunosuppressive agent is hampered by its poor water solubility and narrow therapeutic range. The self-microemulsifying drug delivery system (SMEDDS) succeeded in improving the solubility of SRL in our previous work. In this study, the formulation of the SMEDDS was further optimized by investigating the influence of the excipients including the media, antioxidant and organic acid. It was demonstrated that addition of 0.20% of citric acid in SMEDDS most efficiently promoted the stability of SRL under high temperature (40±2°C), high humidity (relative humidity 90±5%) or strong light irradiation (4500±500lx). SMEDDS absorbed by microcrystalline cellulose (MCC) was mixed with hydroxypropyl methylcellulose (HPMC) to prepare tablets. The optimal formulation composed of 15% of HPMC 100 LV with hardness of 120N, which had a sustained release of 12h. Results of X-ray powder diffraction and differential scanning calorimetry demonstrated that SRL in the tablets was in amorphous or molecularly dispersed state. The SMEDDS-tablets presented as promising substrates for water insoluble drugs with enhanced stability and extended release.