Ghrelin inhibits autophagy mediated by AKT/mTOR pathway to ameliorate retinal angiogenesis induced by high glucose stress.

AIM: To observe the effect of ghrelin, a growth hormone-releasing peptide, on retinal angiogenesis in vitro under high glucose (HG) stress and to explore the possible mechanism of autophagy. METHODS: Human retinal microvascular endothelial cells (HRMECs) were treated with high concentration of glucose alone or in combination with ghrelin. The cell migration, tube formation and the expression of the autophagy-related proteins LC3-II/I, Beclin-1, p62, phosphorylated AKT (p-AKT)/AKT and phosphorylated mammalian target of rapamycin (p-mTOR)/mTOR were detected. Then, to clarify the correlation between ghrelin effect and autophagy, AKT inhibitor VIII was adopted to treat HRMECs, and cell migration, tube formation as well as the protein expressions of LC3-II/I, Beclin-1 and p62 were observed. RESULTS: Under HG stress, ghrelin inhibited migration and tube formation of HRMECs. Ghrelin inhibited the increases in the protein levels of LC3-II/I, Beclin-1 and the decreases in the protein levels of p62, p-AKT/AKT and p-mTOR/mTOR induced by HG stress. Moreover, under the action of AKT/mTOR pathway inhibitors, the effects of ghrelin on migration and tube formation were both reduced. In addition, the expression of LC3-II/I and Beclin-1 were significantly up-regulated and the expression of p62 was down-regulated. CONCLUSION: Retinal angiogenesis under in vitro HG stress can be inhibited by ghrelin through activating AKT/mTOR pathway to inhibit autophagy.

Effects of quercetin on diabetic retinopathy and its association with NLRP3 inflammasome and autophagy.

AIM: To investigate the effects of quercetin on diabetic retinopathy (DR) and its association with nucleotide-binding oligomerization domain-like receptors 3 (NLRP3) inflammasome and autophagy using retinal endothelial cell as an experimental model. METHODS: Human retinal microvascular endothelial cells (HRMECs) were cultured in vitro and assigned into the control group, high-glucose (HG) group, and HG+different concentrations of quercetin groups. Cellular viability, migration, and tube formation in these groups was detected by MTT, transwell and matrigel assay, respectively. Expressions of NLRP3, apoptosis-associated speck-like protein (ASC), cysteiny aspartate-specific protease-1 (Caspase-1) as well as microtubule-related protein 1 light chain 3 (LC3) and Beclin-1 were detected by Western blotting. Expressions of IL-1ß and IL-18 were detected by ELISA and cellular autophagy was detected by Cyto-ID® autophagy detection kit. RESULTS: Under an HG condition, the viability, migration, tube formation of HRMECs, and the protein expressions of NLRP3, ASC, Caspase-1, IL-1ß, IL-18, LC3, and Beclin-1 as well as autophagy were all increased. Quercetin inhibited angiogenesis of HRMECs as well as the expressions of NLRP3, ASC, Caspase-1, IL-1ß, IL-18, LC3, Beclin-1, and autophagy of HRMECs under a HG condition. The inhibitory effects of quercetin on angiogenesis, NLRP3 inflammasome and autophagy increased with the increase of its concentration. CONCLUSION: The therapeutic potential of quercetin in retinal neovascularization of DR, and inhibition of NLRP3 inflammasome and autophagy signaling pathway may be involved.

Mitochondrial Gene Heterogeneity and Population Genetics of Haemaphysalis longicornis (Acari: Ixodidae) in China.

INTRODUCTION: Haemaphysalis longicornis is an important ectoparasite of domestic and wild animals that can transmit many pathogens including viruses, fungi, bacteria and protozoa. MATERIALS AND METHODS: In this study, we examined genetic variation and population genetics in three mitochondrial (mt) genes [cox1 (cytochrome c subunit 1), rrnL (large subunit ribosomal RNA) and nad5 (NADH dehydrogenase 5)] among four H. longicornis populations from China. RESULTS: The sizes of the partial sequences of cox1, rrnL and nad5 were 776 bp, 409 bp, 510 bp, respectively. Among the obtained sequences, we identified 22 haplotypes for cox1, 2 haplotypes for rrnL and 17 haplotypes for nad5. Low gene flow and significant genetic differentiation (66.2%) were detected among H. longicornis populations. There was no rapid expansion event in the demographic history of four H. longicornis populations in China. In addition, phylogenetic analyses confirmed that all the Haemaphysalis isolates were H. longicornis which were segregated into two major clades. CONCLUSION: The mt DNA genes provide a potential novel genetic marker for molecular epidemiology of H. longicornis and assist in the control of tick and tick-borne diseases in humans and animals.

Autophagy: a new mechanism for regulating VEGF and PEDF expression in retinal pigment epithelium cells.

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.

Surgical treatment for 42 patients with traumatic annular ciliochoroidal detachment.

AIM: To analyze the clinical features of traumatic annular ciliochoroidal detachment (CCD) with ultrasound biomicroscopy (UBM) images, to investigate the surgical outcomes of ciliary body suturing and the prognostic factors. METHODS: Forty-two patients with traumatic annular CCD who had undergone ciliary body suturing were enrolled for complete ocular examinations, including visual acuity (VA), slitlamp microscopy, tonometer, indirect ophthalscopy and UBM. Comparisons of clinical features were performed among baseline and follow-ups, and the morphologic alterations on UBM images were analyzed between pre- and post-surgery. RESULTS: The mean intraocular pressure (IOP) was 5.54mmHg, and the median VA was 0.1 in traumatic eyes at baseline. The pre-surgical morphological features on UBM images consisted of supraciliochoroidal effusion (33.33%), multilayer splits (40.48%) and CCD with cyclodialysis cleft (26.19%). After surgery, the median VA was 0.4 at the final follow-up. IOPs were significantly increased, which the mean final IOP was to 10.36mmHg (P<0.01). UBM images displayed complete reattachment in 40.48% of patients, partial reattachment in 50.00% of patients and 360-degree detachment in 9.52% of patients. Analyzing the prognostic factors, the significant factors were duration, VA at baseline, ocular laterality (P<0.01), gender, age and the presence of hypotonous maculopathy (P<0.05). CONCLUSION: Ciliary body suturing is the optimized procedure for traumatic annular CCD. UBM is a useful equipment for diagnosis and monitoring post-surgical morphological changes. The periodical detection of IOP and UBM is necessary for the observation of surgical outcomes.