Jun-Hui Du's research while affiliated with Xi'an Jiaotong University and other places

Aim: To investigate the effect of leptin on the angiogenesis of RF/6A cells (monkey retinal choroidal endothelial cells) in vitro and test the cellular signaling in the mechanism. Methods: RF/6A cells were cultured in vitro and randomly divided into four groups: normal control, with leptin at 50, 100, 200 ng/mL for cell counting kit-8 (CCK8). RF/6A cell proliferation and migration were examined by Transwell assays, while RF/6A cell tube formation by Matrigel assay. JAK2, p-JAK2, STAT3, and p-STAT3 protein expression was measured by Western blotting. Cells were then divided into the following treatment groups: control, 100 ng/mL leptin and AG-490 (100 ng/mL leptin+10 µmol/L AG-490) for examinations of RF/6A cellular behaviour again. Analysis of differences was carried out using one-way ANOVA and least significant difference (LSD). Results: RF/6A cell proliferation, migration and cell tube formation were promoted significantly by leptin in a dose-dependent manner (P<0.05). Western blotting showed that leptin up-regulated p-JAK2 and p-STAT3 expression levels. Treatment with the JAK/STAT pathway inhibitor, AG-490, decreased leptin-induced p-JAK2 and p-STAT3 expression, and inhibited cell proliferation, migration and cell tube formation induced by leptin (P<0.05). Conclusion: Leptin can promote RF/6A cell angiogenesis in vitro via activation of the JAK2/STAT3 signaling pathway.

Aim: To observe the role and mechanism of autophagy in retinal pigment epithelial cell (RPE) damaged by high glucose, so as to offer a new idea for the treatment of diabetic retinopathy (DR). Methods: ARPE-19, a human RPE cell line cultured in vitro was divided into the normal control (NC), autophagy inhibitor 3-methyladenine (3-MA), high-glucose (HG), and HG+3-MA groups. Cell viability was detected by CCK-8 assay and the apoptosis rate was measured by flow cytometry. The protein expressions of apoptosis markers, including Bax, Bcl-2, and Caspase-3, as well as autophagy marker including microtubule-related protein 1 light chain 3 (LC3), p62, and mechanistic target of rapamycin (mTOR) were detected by Western blotting. Autophagic flux was detected by transfection with Ad-mCherry-GFP-LC3B. Results: Under high glucose conditions, the viability of ARPE-19 was decreased, and the apoptosis rate increased, the protein expressions of Bax, Caspase-3, and LC3-II/LC3-I were all increased and the expressions of Bcl-2, p62 and p-mTOR decreased, and autophagic flux was increased compared with that of the controls. Treatment with 3-MA reversed all these changes caused by high glucose. Conclusion: The current study demonstrates the mechanisms of cell damage of ARPE-19 through high glucose/mTOR/autophagy/apoptosis pathway, and new strategies for DR may be developed based on autophagy regulation to manage cell death of RPE cells.

Diabetic rats display cognition impairments accompanied by activation of NF-κB signalling and increased Aβ expression. Ghrelin has been suggested to improve cognition in diabetic rats. In this study, we investigated the role of ghrelin on cognition and NF-κB mediated Aβ production in diabetic rats. A diabetic rat model was established with streptozotocin (STZ) injection, and diabetic rats were intracerebroventricularly administered with ghrelin or (D-lys3)-GHRP-6 (DG). Our results showed that diabetic rats had cognition impairment in the Morris water maze test, accompanied by the higher expression of Aβ in the hippocampus. Western blot analysis showed that diabetic rats exhibited significantly decreased levels of GHSR-1a and protein phosphatase 1 (PP1) in the hippocampus and increased activation of the IKK/NF-κB/BACE1 pathway. Chronic ghrelin administration upregulated hippocampal PP1 expression, suppressed IKK/NF-κB/BACE1 mediated Aβ production, and improved cognition in STZ-induced diabetic rats. These effects were reversed by DG. Then, primary rat hippocampal neurons were isolated and treated with high glucose, followed by Ghrelin and DG, PP1 or IKK. Similar to the in vivo results, high glucose suppressed the expression levels of GHSR-1a and PP1, activated the IKK/NF-κB/BACE1 pathway, increased Aβ production. Ghrelin suppressed IKK/NF-κB/BACE1 induced Aβ production. This improvement was reversed by DG and a PP1 antagonist and was enhanced by the IKK antagonist. Our findings indicated that chronic ghrelin administration can suppress IKK/NF-κB/BACE1 mediated Aβ production in primary neurons with high glucose treatment and improve the cognition via PP1 upregulation in diabetic rats.

Aim: To investigate the regulation of vascular endothelial growth factors (VEGF) and pigment epithelium-derived factor (PEDF) expression by autophagy in retinal pigment epithelium (RPE) cells on exposure to hypoxia. Methods: ARPE-19, an RPE cell line, was treated as following: the control group was kept in a normoxic incubator; the hypoxia group was incubated in a hypoxic incubator with 1% O2/5% CO2/94% N2 for 24h; the hypoxia + 3-methyladenine (3-MA) group was pretreated with 10 mmol/L 3-MA for 1h and then in the hypoxic incubator for 24h; and the hypoxia + chloroquine (CQ) group was pretreated with 50 µmol/L CQ for 1h and then in the hypoxic incubator for 24h. The morphology and ultrastructure of the cells was observed by an inverted microscope or a transmission electronic microscope (TEM). Western blot was performed to assay the expression of autophagy-associated markers, including microtubule associated protein 1 light chain 3 B (LC3B), Beclin-1, Atg5 and p62. The concentration of VEGF and PEDF in the culture supernatant was determined by ELISA, and the ratio of VEGF/PEDF was calculated. Results: There were no obvious differences in cell morphology among different groups and autolysosomes could be observed in the cytoplasm in all groups. Compared to the control cells, the LC3B-II/I ratio and levels of Beclin-1 and Atg5 were significantly increased and p62 level was significantly decreased in the hypoxia group. With the increase of VEGF and decrease of PEDF concentration, the VEGF/PEDF ratio was significantly increased in the hypoxia group compared to the control cells. The LC3B-II/I ratio was significantly reduced by 3-MA treatment and increased by CQ treatment. The expressions of Beclin-1 and Atg5 were significantly reduced by 3-MA or CQ treatment, while expression of p62 was increased in the 3-MA or CQ treated cells. The concentration of VEGF was significantly decreased and PEDF increased, thereby the VEGF/PEDF ratio was decreased in the hypoxia + 3-MA group and hypoxia + CQ group compared with that in the hypoxia group. Conclusion: Hypoxia leads to elevated autophagy in RPE cells, and expression of VEGF and PEDF might be regulated by autophagy on exposure to hypoxia to further participate in regulating the formation of retinal neovascularization.

Aim: To explore the state of autophagy and related mechanisms in the murine retinal microvascular endothelial cells (RMECs) under hypoxia stimulation. Methods: The murine RMECs were primarily cultured and randomly divided into three groups: hypoxia group (cultured in 1% O2 environment), hypoxia+autophagy inhibition group [pretreated with 5 mmol/L 3-methyladenine (3-MA) for 4h followed by incubation in 1% O2] and control group (cultured under normoxic condition). The state of autophagy in RMECs was examined by assaying the turnover of light chain 3B (LC3BB) and expression of Beclin-1, Atg3 and Atg5 proteins with Western blotting, by detecting formation of autophagosomes with transmission electron microscopy (TEM) and by counting the number of GFP+ puncta in RMECs. The protein levels of AMPK, P-AMPK, Akt, P-Akt, m-TOR and P-mTOR were also assayed by Western blotting. Results: Primary murine RMECs were successfully cultured. Under hypoxic conditions, the ratio of LC3BB-II/I and the expression of Beclin-1, Atg3 and Atg5 proteins were increased when compared with the control group. In addition, the numbers of autophagosome and the GFP+ puncta were also increased under hypoxia. However, pre-treatment with 3-MA obviously attenuated these changes in autophagy in RMECs under hypoxia. Protein expression of P-Akt and P-AMPK was increased but P-mTOR level was decreased in cells exposed to hypoxia. Conclusion: In murine RMECs autophagy is activated under hypoxia possibly through activation of the AMPK/mTOR signaling pathway.

The formation of neovascularization is a common pathological feature of many ocular vascular diseases, and is an important cause of vision loss in patients. Neovascularization can cause retinal hemorrhage, vitreous hemorrhage, and other serious complications, leading to loss of vision. The treatment of intraocular neovascularization is the focus of ophthalmology research. In recent years, some studies have found that autophagy is closely related to vascular endothelial growth factor and the formation of neovascularization. Autophagy is expected to become a new target for the treatment of intraocular neovascularization. Therefore, this article reviews the research on autophagy and the formation of intraocular neovascularization.

Diabetes mellitus (DM) has been regarded as an important risk factor for Alzheimer’s disease (AD), and diabetic patients and animals have shown cognitive dysfunction. More research has shown that the amyloid-β (Aβ), which is a hallmark of AD, was found deposited in the hippocampus of diabetic rats. This Aβ accumulation is regulated by the receptor for advanced glycation end products (RAGE) and low-density lipoprotein receptor-related protein (LRP-1). However, the expression of RAGE and LRP-1 in diabetic rats is not very clear. In the present study, we used streptozotocin (STZ)-induced diabetic rats to investigate whether the expression of RAGE and LRP-1 is related to Aβ1–42 deposition at the hippocampus, prefrontal lobe, and amygdala in DM. We found that diabetic rats had longer escape latency and less frequency of entrance into the target zone than that of the control group (P < 0.05) in the Morris water maze (MWM) test. The Aβ1–42 expression in the hippocampus and prefrontal lobe significantly increased in the DM group compared to the control group (P < 0.05). RAGE increased (P < 0.05), while LRP-1 decreased (P < 0.05) in the hippocampus tissue and prefrontal lobe tissue of DM rats. The Aβ1–42 deposition was correlated with RAGE positively (P < 0.05), but with LRP-1 negatively (P < 0.05). Further, the expression levels of Aβ1–42, RAGE, and LRP-1 were not changed in the amygdala between the diabetic rats and the control group. These findings indicated that upregulating RAGE and/or downregulating LRP-1 at the hippocampus and the prefrontal lobe contributed to the Aβ1–42 accumulation and then further promoted the cognitive impairment of diabetic rats.

Purpose: To study the effect of autophagy on vitality, migration, and tube formation of RF/6A cells under the condition of D-glucose. Methods: Cultured RF/6A cells were randomly divided into 4 groups (control, low glucose, high glucose, and high glucose with 3-methyladenine [3-MA]). Autophagy-related proteins (Atg7, p62, and LC3) were monitored. Cell vitality, cell migration, tube formation, reactive oxygen species (ROS) production, and apoptosis were assessed. Results: Cell vitality significantly decreased and cell migration and tube formation significantly increased in the high-glucose group (p < 0.05). Pretreatment with 3-MA significantly increased cell viability and inhibited cell migration and tube formation (p < 0.05). ROS production increased in the high-glucose group and decreased in the high-glucose with N-acetylcysteine (NAC) group (p < 0.05). The level of apoptosis increased in the high-glucose group, while it was reduced in the high-glucose with 3-MA group. Conclusion: Autophagy maybe participates in the formation of retinal neovascularization induced by high glucose.

To compare apelin-13, a ligand of G-protein-coupled receptor which has been shown to be involved in retinal angiogenesis, and vascular endothelial growth factor (VEGF) serum levels in type 2 diabetes mellitus (T2DM) with or without retinopathy, and to investigate the relationship between the serum concentration of apelin-13 and diabetes retinopathy. Sixty-nine patients with T2DM were enrolled. Of the 69 patients, 16 had proliferative diabetic retinopathy (PDR group), 23 had non-PDR (NPDR group) and 30 had no retinopathy (T2DM group). Subjects' information, including demographics, medical history, and use of medications were recorded. Their serum samples were collected for measuring the levels of C-reactive protein (CRP), serum lipid and glycosylated hemoglobin. Apelin-13 and VEGF serum levels were measured by enzyme-linked immunosorbent assay. Kruskal-Wallis test and one-way ANOVA were used to compare the differences among these groups. Chi-square test was used to assess categorical variables. Correlations between variables were investigated by Spearman rho correlation test and stepwise regression analysis. All statistical analyses were performed through SPSS 17.0 software. Sex, age, body mass index (BMI), blood pressure, CRP, hemoglobin A1c (HbA1c) have no significantly difference in the three groups. Serum level of apelin-13 was significantly elevated in PDR group as compared with T2DM group (P=0.041). Differences of VEGF serum concentration in the three groups were statistically significant (P=0.007, P=0.007 and P<0.001, respectively). Spearman rho correlation test showed that serum apelin-13 was positively correlated with BMI, serum triglycerides, VEGF, but not with age, duration of diabetes, blood pressure, CRP, HbA1c and total-cholesterol. Stepwise regression analysis showed that BMI also significantly associated with serum apelin-13 (P=0.002), while VEGF and serum triglycerides were irrelevant. This study elucidated a positive association of apelin-13 serum level with PDR, but not with VEGF. Apelin-13 may influence the promotion of PDR but unrelated with VEGF.