ObjectiveTo screen and identify an ideal lead compound with potential inhibitory effects on N-methyl-D-aspartate receptor (NMDAR) from the ZINC15 drug database, promoting drug design and development to improve epilepsy treatment. Methods Potential NMDAR inhibitors were identified through a series of computer-aided structural and chemical virtual screening techniques (Discovery Studio). Structure-based virtual screening was used to predict and further filter candidate compounds based on physicochemical, pharmacological, and toxicological properties. The binding affinity and chemical bond distribution between selected compounds and NMDAR were then analyzed, and the stability of the ligand-NMDAR complex in a natural environment was evaluated. Results The study identified one novel natural compound from the ZINC15 database, with ZINC000096085903 showing low rodent carcinogenicity, no Ames mutagenicity, no developmental toxicity, and ideal physicochemical properties. This compound demonstrated high binding affinity and favorable interaction energy, with the ZINC000096085903-NMDAR complex exhibiting more favorable potential energy than the complex formed by NMDAR and the reference ligand ketamine. Furthermore, molecular dynamics simulation indicated that this complex remains stable in vivo and can inhibit NMDAR similarly to ketamine. Conclusion ZINC000096085903 is an ideal lead compound for NMDAR inhibition. With higher binding affinity and stability when bound to NMDAR, as well as slower metabolism, ZINC000096085903 showed significant potential for long-term epilepsy treatment.
ObjectiveTo investigate the effects of specific farnesiod X receptor(FXR) agonist on growth of colon cancer cells in vitro.
MethodsThe effects of specific FXR agonist(GW4064) on the growth of HCT116 cells of colon cancer were studied in vitro by using MTT and flow cytometry. The mRNA expressions of FXR and vascular endothelial grouth factor(VEGF), were determined by using RT-PCR.
ResultsThe FXR specific agonist GW4064 could increase the FXR mRNA expression of HCT-116 cells of colon cancer, downregulation of VEGF mRNA expression, and had obvious inhibitory effect on growth of HCT-116 cells, and promoted the apoptosis of HCT116 cells in a dose and time dependence.
ConclusionsGW4064 can significantly inhibit colon cancer cells in vitro. FXR may be a potential treatment arget of colon cancer.
Diabetic retinopathy (DR) is one of the most serious complications of diabetes mellitus. Severe diabetic macular edema or proliferative retinopathy may lead to impaired vision or even blindness in diabetic patients. The glucagon-like peptide-1 receptor agonist (GLP-1RA) is now commonly used as novel glucose-lowering agents in the clinical management of type 2 diabetes, but the rapid glycaemic changes associated with the use of the GLP-1RA may aggravate the risk of an increase in the occurrence of short-term potential DR. Potential effects and mechanisms of DR include oxidative stress, vascular endothelial growth factor, inflammation, retinal neurodegeneration, and other cytokines.Whether GLP-1RA leads to the increased risk of DR remains controversial. More basic and clinical studies are needed with the aim of further clarifying the correlation between GLP-1RA and DR risk.
Objective To investigate the inhibitory effects and possible related mechanism of OTX008 [a selective inhibitor of galectin-1 (Galectin-1)] on retinal neovascularization (RNV) in mouse model of oxygen-induced retinopathy (OIR). Methods 7-day-old (P7) C57BL/6J mice were randomly (according to random number table) divided into 4 groups including normal group, OIR group, OIR-OTX008 group and OIR-phosphate buffered saline (PBS) group. To establish the OIR mouse model, mice from all groups except normal group were expose to (75±2)% oxygen for 5 days and then to room air. OIR-OTX008 group received an intravitreal injection of 1 μl (0.25 μg/μl) OTX008 at P12, OIR-PBS group received the equal volume (1 μl) of PBS injection. Mice from 4 groups were euthanized at P17, and retinas were collected for molecular biological analysis and morphological study. RNV was evaluated by counting the number of pre-retinal neovascular nuclei and the whole-mount immunofluorescent staining of mouse retina. Cyrosections of retinas were imaged via confocal microscopy to observe the enrichment of staining of Galectin-1. Protein levels of Galectin-1, Neuropilin-1 and phosphorylation of vascular endothelial growth factor receptor 2 (pVEGFR2) were determined with Western blot. Results At P17, Galectin-1 expressed higher in retinal ganglion cell layer, inner plexiform layer and inner nuclear layer from OIR group and OIR-PBS group than normal group. Galectin-1 expressed less in cryosection retinas from OIR-OTX008 group than OIR group and OIR-PBS group. The numbers of pre-retinal neovascular cell nuclei from OIR group and OIR-PBS group were obviously more than that from normal group (t=9.314,P＜0.05). The number of pre-retinal neovascular cell nuclei from OIR-OTX008 group were obviously lower than those from OIR group and OIR-PBS group (t=8.038, 7.774;P＜0.05). The RNV tufts area (t=13.250, 12.570), non-perfusion area (t=15.590, 12.430) and hypoxic area (t=9.542, 9.928) from OIR-OTX008 group were significantly smaller than those in OIR group and OIR-PBS group (P＜0.05). Protein levels of Galectin-1 (t=24.800, 23.060), Neuropilin-1 (t=4.120, 3.530) and pVEGFR2 (t=25.880, 15.480) in the OIR-OTX008 group were significantly down-regulated than those from OIR group and OIR-PBS group (P＜0.05). Conclusion Intravitreal injection of OTX008 inhibits RNV and ameliorates retinal hypoxia in mice model of OIR possibly through down-regulating Galectin-1, Neurolinpin-1 and pVEGFR2.
ObjectiveTo investigate the effect of pharmacologic delay with pioglitazone, a peroxisome proliferator-activated receptor γ (PPAR-γ) agonist, on extended perforator flap survival in a rat model.
MethodsSeventy male Sprague Dawley rats, weighing 250-300 g, were randomly divided into control group (n=35) and experimental group (n=35). A three-territory flap was made, including two choke zones. Pioglitazone was dissolved in 1.5 mL saline. Oral doses of pioglitazone[10 mg/(kg·d)] was given by gavaged for 5 days in the experimental group, while the same volume of saline was given in the control group at same time point. After 7 days, the flap survival area was measured and angiographic diagnosis was made. The tissue samples were harvested from choke zone Ⅱ for histological study and vascular endothelial growth factor (VEGF) expression detection by immunohistochemical staining. The content of nitric oxide (NO) in choke zones I and Ⅱ was measured at immediate, 1, 3, 5, and 7 days after operation.
ResultsThe flap general change of 2 groups was similar. Varying degrees of necrosis occurred with the extension of time in 2 groups. At 7 days after operation, the flap survival rate was 87.73%±3.25% in the experimental group and 76.07%±2.92% in the control group, showing a significant difference (t=-10.338, P=0.000). The number of true anastomosis in choke zones I and Ⅱ was 5.40±1.14 and 3.00±0.71 in the experimental group, and was 3.20±0.84 and 0.80±0.84 in the control group respectively, showing significant differences between the 2 groups (t=-3.479, P=0.008;t=-4.491, P=0.002). The microvessel density and the expression of VEGF in choke zone Ⅱ of experimental group were (33.16±7.73)/mm2 and 4 368.80±458.23, respectively, which were significantly higher than those of control group[(23.29±5.91)/mm2 and 2 241.24±554.43] (t=5.073, P=0.000;t=-14.789, P=0.000). The content of NO in the experimental group were significantly higher than those in the control group at other time points (P<0.05) except for at immediate after operation.
ConclusionPharmacologic delay with pioglitazone can improve extended perforator flap viability through increasing ischemia-induced angiogenesis and choke vessels vasodilation in rat models.
ObjectiveTo explore the metabolic changes during the differentiation of 3T3-L1 adipocytes caused by the treatment of the transient receptor potential vanilloid 4 (TRPV4)-specific agonist GSK1016790A basing on ultra-performance liquid chromatography-mass spectrometry technology. MethodsMouse 3T3-L1 cells were treated with GSK1016790A at different concentrations (0.1, 1, and 10 μmol/L), and the effect of drugs on cell proliferation was detected by cell counting kit-8 method. A mature adipocyte model was constructed, and GSK1016790A was used to activate TRPV4 channel protein activity and verify the expression levels of TRPV4 and triglycerides. Cell metabolites were collected for metabolomic studies, differential metabolites were screened between groups, and related metabolic pathways were analyzed. Results After GSK1016790A intervened in mature adipocytes, the expression levels of TRPV4 mRNA and triglycerides in cells were significantly upregulated (P<0.05). Metabolomics detection found that GSK1016790A screened a total of 45 differential metabolites such as 2-amino-1,3,4-octadecanetriol, linoleic acid, sphingosine, sphinganine, sn-glycerol-3-phosphate and uridine, mainly involving 13 possible metabolic pathways such as sphingolipid metabolism and biosynthesis of unsaturated fatty acids. Conclusion GSK1016790A may promote adipogenesis in adipocytes by activating TRPV4 channel protein activity, and at the same time participate in regulating metabolic pathways such as the biosynthesis of unsaturated fatty acids pathway and sphingolipid metabolism pathway, affecting lipid metabolism in adipocytes.
Objective To investigate the effects of knocking down Rac1 gene (ras-related C3 botulinum toxin substrate 1) by small hairpin RNA (shRNA) on retinal neovascularization in a mouse model of oxygen-induced retinopathy (OIR). Methods One hundred and eight 7-day-old C57BL/6J mice were divided into three groups randomly.The OIR was induced by Smith protocol in 2 groups. OIR mice received an intravitreal injection of Rac1-shRNA plasmid or the nonsense plasmid in the geneintervention group and control group respectively at the age of postnatal day 11 (P11). Non-OIR mice also received an intravitreal injection of Rac1-shRNA plasmid at P11 as the blankintervention group which lived in the normoxic environment.Retinal neovascularization was investigated on flat-mounts after fluorescence angiography at P15 and P17. Endothelial cell nuclei breaking through the internal limiting membrane were counted on pathological section at P17.The expression of Rac1 and NF-kappa;B p65 subunit was measured by immuohistochemistry, Western blot, real-time polymerase chain reaction (RT-PCR) and in situ hybridization. Results Compared with the blank-control group,the level of Rac1 mRNA in the gene-intervention group decreased obviously（t=4.500，P=0.001）;the retinal non-perfusion areas,fluorescence leakage, neovascularization and the number of endothelial cell nuclei breaking through the internal limiting membrane were reduced significantly（t=6.521,P＜0.001）; the level of NF-kappa;B p65 nuclear translocation decreased（t=16.008,P＜0.001）while the expression of NF-kappa;B p65 mRNA was reduced obviously（t=3.354,P=0.006）, which was positively correlated with the expression of Rac1-mRNA (P=0.012).Conclusion Intravitreal injection of Rac1-shRNA with liposome in mice can effectively inhibit the expression of Rac1,and inhibit the retinal neovascularization under relative hypoxia via blocking the ROS-NF-kappa;B pathway.
ObjectiveTo evaluate the photoreceptor-protective effects of Cdk5 inhibitor Roscovitine on retinal degeneration in Royal College of Surgeons (RCS) rat.
MethodsThe RCS rats were divided into three groups according to postnatal days: the early (17 days), medium (25 days) and late intervention group (35 days). Cdk5 inhibitor Roscovitine were used in the right eyes by intravitreal injection as experimental eyes and Roscovitine solvent dimethylsulfoxide were used in the left as control at postnatal 17, 25, 35 days. Hematoxylin-eosin (HE) staining was used to observe the thickness of outer nuclear layer. The expression of Cdk5 P25 and cleave-caspase 3 in the retina was evaluated by immunohistochemistry. The protein expression of cleave-caspase 3 in the retina was determined by Western blot. The apoptosis of retinal cells was examined by terminal-deoxynucleotidyl transferase mediated nick end labeling.
ResultsHE staining showed that thickness of outer nuclear layer in the early and medium intervention groups were significantly thicker than that in the control group (P < 0.05), particularly in the early intervention group. And there was no significant change in the late intervention group (P > 0.05). The expression level of Cdk5, p25, cleave-caspase 3 in the outer nuclear layer in three intervention groups were lower than that in the control group (P < 0.05), especially in the early intervention group.
ConclusionCdk5 inhibitor Roscovitine can delay the retinitis pigmentosa process in RCS rats by early, medium interventional therapy and may have a certain degree of photoreceptor-protective effects.
ObjectiveTo study the effect of endothelin receptor antagonist on recent outcome of patients undergoing Fontan surgery.
MethodsThirty nine patients who received Fontan procedure from January 2009 to December 2010 in Wuhan Asia Heart Hospital were recruited in the study. There were 25 males and 14 females with mean age of 8.02±4.98 years (ranged from 2.5 to 18.0 years). According to the admission number, the patients were randomly divided into an endothelin receptor antagonists group (bosentan group, n=16) and a control group (n=23). The bosentan group received bosentan treatment by gastric fill or oral according to the recommended dose in three days after surgery for over 7 days. The control group did not receive any pulmonary hypertension targeted therapy. On the 10th day after surgery, indexes including mortality etc in the two groups were compared.
ResultsNo death occurred in the bosentan group. One patient died on the 5th day after operation in the control group. There was no significant difference in the postoperative mortality. The patients in the bosentan group got significantly better results than the control group in cardiac function, incidence of pleural effusion, vasoactive drugs score, and serum B-type natriuretic peptide, albumin, alanine aminotransferase on the 10th day (P < 0.05); while the 6-minute walk distance, transcutaneous oxygen saturation, white blood cell count, C-reactive protein, cardiac troponin I, and creatinine of the two groups showed no statistical difference (P > 0.05).
ConclusionEndothelin receptor antagonists can improve the short-term outcome of patients after Fontan surgery.
Objective To observe the inhibitory effect of kallikrein-binding protein (KBP) on choroidal neovascularization. Methods Forty Brown Norway rats were randomly divided into the KBP groups and the control group, 20 rats in each group, the right eye as the experimental eye. The rats were photocoagulated by 532 nm laser to induce CNV model. One week after laser photocoagulation, the rats were received FFA examination. At the second day after FFA examination, the rats of KBP group were received an intravitreal injection of KBP 5 mu;l (4 mg/ml KBP). The same volume of deionized water was injected into the rats in the control group. The rats of two groups received FFA examination at one, two and three weeks after injection. The expressions of vascular endothelial growth factor and pigment epithelium derived factor were observed using hematoxylin and eosin stain and immunohistochemistry stain. CNV leakage area and the cumulative absorbance of laser spot area were analyzed by Image-Pro plus 6.0 software. Results FFA examination showed that there were CNV and fluorescence leakage at one week after laser photocoagulation; one, two and three weeks after injection, the leakage decreased gradually in KBP group, but increased with time in control group. Compared with control group, the spot area and CNV in KBP group reduced gradually, but CNV was always there in control group. The differences of VEGF (F=1.29) and PEDF (F=6.29) expressions at one week after laser photocoagulation were not statistically significant (P＞0.05). The differences of VEGF and PEDF expressions at one, two and three weeks after injection were statistically significant（VEGF:F=14.16,66.89,24.34; PEDF：F=4.22,62.04,233.05;P＜0.001）.Conclusion Intravitreal injection with KBP can inhibit CNV.
