High glucose may promote the proliferation and metastasis of hepatocellular carcinoma via E2F1/RRBP1 pathway.

AIMS: Diabetes is considered as one of the important risks in the progression of Hepatocellular carcinoma(HCC). Ribosome binding protein 1 (RRBP1), a rough endoplasmic reticulum protein, plays an essential role in diabetes and various cancer. E2F transcription factor 1 (E2F1), an upstream transcription factor of RRBP1, shows promoting tumor progression effect in multifarious cancers. In this research, we tried to identify whether regulating E2F1/RRBP1 pathway could inhibit the proliferation and metastasis of HepG2 cells induced by high glucose. MAIN METHODS: Proteomic, bioinformatics, molecular biology including RT-qPCR and Western blot, cell biology containing Cell Counting Kit-8 (CCK-8), wound healing assay and transwell assay, and biochemistry analyses incorporating Luciferase assay and CHIP assay were used in this study. KEY FINDINGS: High glucose promoted the proliferation and metastasis of HepG2 cells through up-regulating the expression of RRBP1. Bioinformatics analysis predicted that E2F1 might be the transcription factor of RRBP1. Knocking-down of E2F1 down-regulated mRNA and protein expression levels of RRBP1 in HepG2 cells significantly and suppressed the proliferation, migration and invasion of cells remarkably, Reverse effect was observed in cells that E2F1 was overexpressed. Meanwhile, luciferase and CHIP assay determined that E2F1 could bind to the RRBP1 promoter and promote the transcription of RRBP1. Finally, rescue assay verified the important role of RRBP1/E2F1 axis in the process of HepG2 cells proliferation and metastasis. SIGNIFICANCE: All of the above provided possibility to improve the efficiency of HCC complicated with diabetes treatment by regulating the E2F1/RRBP1 pathway.

C­reactive protein/oxidized low density lipoprotein/ß2­glycoprotein i complexes induce lipid accumulation and inflammatory reaction in macrophages via p38/mitogen­activated protein kinase and nuclear factor­κB signaling pathways.

Oxidized low-density lipoprotein (oxLDL) can bind to ß2-glycoprotein I (ß2GPI) and C-reactive protein (CRP) to form stable complexes, which exert certain effects in diabetic cardiovascular disease. A previous study by our group has confirmed that the resulting complexes promote atherosclerosis in diabetic BALB/c mice. The present study was designed to investigate the effects and potential mechanisms of oxLDL complexes on lipid accumulation and inflammatory reactions in RAW264.7 macrophages cultured in a hyperglycemic environment. Cultured cells were divided into seven groups, which were treated with phosphate­buffered saline (control), CRP, ß2GPI, oxLDL, CRP/oxLDL, oxLDL/ß2GPI or CRP/oxLDL/ß2GPI. The results revealed the formation of foam cells in the oxLDL, CRP/oxLDL, oxLDL/ß2GPI as well as CRP/oxLDL/ß2GPI groups. Compared with oxLDL, the three complexes induced less lipid accumulation (P<0.05) through inhibiting the expression of CD36 mRNA and promoting the expression of and ABCG1 mRNA (P<0.05 vs. oxLDL). Furthermore, the levels of inflammatory factors interleukin (IL)­1ß, IL­6 and tumor necrosis factor­α were elevated in the CRP/oxLDL and CRP/oxLDL/ß2GPI groups (P>0.05 vs. oxLDL), and obvious effects on p38/mitogen­activated protein kinase and nuclear factor (NF)­κB phosphorylation were also observed in these groups (P<0.05 vs. oxLDL). These results suggested that CRP/oxLDL/ßG2P1 complexes may induce lipid accumulation and inflammation in macrophages via the p38/MAPK and NF­κB signaling pathways. However, some differences were observed between the complexes, which may be attributed to the property of each constituent; therefore, further studies are required.

Domain I-IV of ß2-glycoprotein I inhibits advanced glycation end product-induced angiogenesis by down-regulating vascular endothelial growth factor 2 signaling.

Advanced glycation end products (AGEs) are a contributing factor in the angiogenesis that is characteristic of proliferative diabetic retinopathy. However, a previous study made a promising observation that domain I­IV of ß2­glycoprotein I (DI­IV) inhibits angiogenesis in human umbilical vein cells. The present study aimed to confirm the inhibition of AGE­induced angiogenesis in retinal endothelial cells by DI­IV and to investigate the potential underlying mechanisms. The RF/6A rhesus macaque choroid­retinal vascular endothelial cell line was cultured in vitro and treated with AGEs in the presence or absence of different concentrations of DI­IV. The proliferation, migration and tube formation of the RF/6A cells were evaluated using MTS assays, in vitro wound healing assays and in vitro Matrigel angiogenesis assays, respectively. The mRNA expression of vascular endothelial growth factor (VEGF), VEGF receptor (VEGFR) 2, VEGFR 1 and receptor for AGE (RAGE) were quantified by reverse transcription quantitative polymerase chain reaction. The expression of VEGFR­1, VEGFR­2 and the activation of protein kinase B (Akt) and extracellular signal­regulated kinase (ERK) were also assessed by western blot analysis. The results indicated that AGEs promoted the migration, proliferation and tube formation of RF/6A cells in vitro (P<0.05), increased the expression of VEGF, VEGFR­2 and RAGE (P<0.05) and increased the phosphorylation of Akt and ERK (P<0.05). DI­IV inhibited the increase in VEGFR­2 mRNA and protein, but did not inhibit the increase in VEGF or RAGE mRNAs. These results led to the conclusion that DI­IV inhibited AGE­induced angiogenesis in the RF/6A cells, which was accompanied by a downregulation in the expression of VEGFR­2 and its downstream phosphatidylinosol 3­kinase/Akt and mitogen­activated protein kinase/ERK1/2 pathways. These findings provide further support towards the treatment of proliferative diabetic retinopathy by interventions that act via a mechanism similar to that of DI­IV.

Liraglutide ameliorates renal injury in streptozotocin­induced diabetic rats by activating endothelial nitric oxide synthase activity via the downregulation of the nuclear factor­κB pathway.

Accumulating evidence has implicated that liraglutide, one of the human glucagon­like peptide­1 (GLP­1) analogues, elicits protective effects on diabetic nephropathy; however, the mechanism has yet to be fully elucidated. The present study aimed to assess the effect and underlying mechanisms of liraglutide in diabetic nephropathy. Wistar rats with streptozotocin­induced diabetes mellitus were subcutaneously injected with liraglutide or phosphate buffer for 12 weeks at a dose of 0.3 mg/kg/12 h. The biochemical parameters were determined, renal histological examination was performed by hematoxylin and eosin and periodic acid Schiff base staining, and the mRNA levels of nuclear factor κB (NF­κB) and endothelial nitric oxide synthase (eNOS) were assessed by quantitative polymerase chain reaction. Furthermore, the protein expression of NF­κB and eNOS as well as eNOS phosphorylation were examined by western blot analysis and the levels of inflammatory cytokines downstream of NF­κB were evaluated by fluorescence-assisted cell sorting and finally, the eNOS activity and nitric oxide (NO) production were evaluated by ELISA. Liraglutide decreased the levels of total cholesterol, urine, 24-h urinary albumin, blood urea nitrogen, serum creatinine and histological damage. Liraglutide also reduced the expression of NF­κB at mRNA and protein levels; the expression of tumor necrosis factor­α, interferon­Î³, interleukin­6 and monocyte chemoattractant protein­1 were also reduced. By contrast, eNOS phosphorylation, eNOS activity and NO production appeared to have increased. Liraglutide may have a direct beneficial effect on diabetic nephropathy by improving eNOS activity by inhibiting the NF­κB pathway without eliciting a glucose lowering effect.

C-reactive protein/oxidised low-density lipoprotein/ß2-glycoprotein I complex promotes atherosclerosis in diabetic BALB/c mice via p38mitogen-activated protein kinase signal pathway.

BACKGROUND: The aim of this study was to investigate the effect of C-reactive protein/oxidised low-density lipoprotein/ß2-glycoprotein I (CRP/oxLDL/ß2GPI) complex on atherosclerosis (AS) in diabetic BALB/c mice. METHODS: BALB/c mice were fed high-fat and normal diet. Eight weeks later, the mice fed with high-fat diet were injected with streptozotocin (STZ) to induce diabetes. The diabetic mice were respectively injected twice monthly with 20 µg oxLDL, 20 µg ß2GPI, 40 µg oxLDL/ß2GPI complex, 44 µg CRP/oxLDL/ß2GPI complex, and PBS. Aortas were stained with Sudan IV to investigate lipid plaque formation. The infiltration condition of smooth muscle cells (SMCs), macrophages, and T cells in the aortas were determined by immunohistochemistry (IH). The mRNA expressions of receptors associated with lipid metabolism were quantified by real-time PCR. The phosphorylation of p38 mitogen-activated protein kinase (p38MAPK) and MKK3/6 in aorta tissues were assessed by Western blot. The expression of inflammation cytokines was evaluated by protein chip. RESULTS: The lipid plaques were more extensive, the lumen area was obviously narrower, the ratio of intima and media thickness were increased, and the normal internal elastic lamia structure and endothelial cell disappeared (P < 0.05) in the oxLDL and CRP/oxLDL/ß2GPI groups (P < 0.05). CRP/oxLDL/ß2GPI complex dramatically promoted infiltration of SMCs, macrophages, and T cells, improved the mRNA expression of ABCA1 and ABCG1, but reduced the mRNA expression of SR-BI and CD36 and increased the phosphorylation of p38MAPK and MKK3/6 (all P < 0.05). The highest expression levels of IL-1, IL-9, PF-4, bFGF, and IGF-II were detected in the CRP/oxLDL/ß2GPI group (P < 0.05). CONCLUSIONS: CRP/oxLDL/ß2GPI complex aggravated AS in diabetic BALB/c mice by increasing lipid uptake, the mechanism of which may be mediated by the p38MAPK signal pathway.

Preferential enhancement of working memory in mice lacking adenosine A(2A) receptors.

There is evidence that adenosine acting at A(2A) receptors (A(2A)R) can influence striatal plasticity and cognitive functions. We examined spatial working memory in wild-type (WT) and A(2A) receptor knock-out (KO) mice using two assessments: the eight arm radial maze and a repeated trial Morris water maze (MWM) paradigm. Compared to WT littermates, A(2A)R KO mice displayed enhanced working memory as evidenced by a decrease in escape latency in trial 2 compared to trial 1 in the repeated trial MWM, and by a reduction in working memory errors in the radial arm maze. Both MWM and radial maze results indicated that this enhancement of working memory in A(2A)R KO mice was selective for this specific short-term memory. The decrease in escape latency in MWM was detected with an inter-trial interval of 15 s but not with intervals of 10 or 60 min. In the radial maze, spatial reference memory and memory retention after prolonged training (15 days but not 6 days) were not affected by the A(2A)R KO. These results demonstrate preferential improvement in spatial working memory by genetic inactivation of the A(2A)R and support a modulatory role of the A(2A)R in spatial working memory in mice.