AIM:To evaluate the influence of scutellarin on the expression of vascular endothelial growth factor ( VEGF) in high glucose-treated human retinal pigment epithelial cell line ARPE-19 and to observe the effects of scutellarin on the protein expression of VEGF, p-ERK and VEGFR2 in the retinas of type II diabetic rats.METHODS: Cultured ARPE-19 cells were divided into normal control group, scutellarin group, high glucose group and high glucose+scutellarin group.The protein levels of VEGF, p-ERK and VEGFR2 were measured by Western blot.The VEGF release in ARPE-19 cells was detected by ELISA.Normal rats were randomly divided into normal control group and scutellarin group.Diabetic rat model was established by feeding with high-fat diet and injecting with streptozocin, and randomly divided into diabetes group and diabetes treated with scutellarin group.After 16 weeks, the eyes were removed.The morphological changes of the retinas were observed under light microscope with HE staining, and histopathological score was recorded.The expres-sion of VEGF in the retinas was observed by the method of immunohistochemistry.RESULTS:Compared with normal con-trol group, the protein levels of VEGF, p-ERK and VEGFR2 in the ARPE-19 cells decreased in scutellarin group, but in-creased in high glucose group.The histopathological score of the retinas showed significant difference among diabetes group, diabetes treated with scutellarin group and normal control group, and no significant difference between normal con-trol group and scutellarin group was observed.The expression of VEGF increased in diabetic group and was significantly higher than that in scutellarin treatment group (P<0.05).CONCLUSION:Scutellarin inhibits the increased protein le-vels of VEGF, p-ERK and VEGFR2 in ARPE-19 cells, and decreases the expression of VEGF in the retinas of diabetic rats.The suppression of the diabetic retinopathy development by scutellarin may be partly involved in the ERK/MAPK pathway.

BACKGROUND:Hyperbranched cationic amylopectin is a kind of nonviral gene vectors with low toxicity and good transfection efficiency. However, searching for more efficient methods to delivery it into the body and making the genes expressed are being explored.
OBJECTIVE:To study the expression of DMAPA-Amp wrapped green fluorescent protein (GFP) transferred by modified carotid injection into cerebral ischemic area.
METHODS:Male Sprague-Dawley rats subjected to middle cerebral artery infarction were randomly divided into two groups after 24 hours:experimental group (injected with GFP entrapped DMAPA-Amp via the internal carotid artery) and control group (injected with GFP entrapped DMAPA-Amp via the tail vein). These rats were put to death and their brain tissue was removed after 7 days. The expression of GFP was detected by quantitative PCR and western blot assay, and immunofluorescence staining was performed to detect the expression of GFP located near cerebrovascular endothelial cel s by frozen section.
RESULTS AND CONCLUSION:Compared with the control group, the expression of GFP was much higher in the experimental group detected by quantitative PCR and western blot (P< 0.05). Additional y, the expression of GFP located near cerebrovascular endothelial cel s by frozen section was also higher than that in the control group. Modified carotid injection could significantly promote the expression of hyperbranched cationic amylopectin derivates and GFP in the brain tissue of Sprague-Dawley rats undergoing middle cerebral artery infarction compared with tail vein injection, which indicates DMAPA-Amp and modified carotid injection may cast new lights on the therapy for angiogenesis of ischemic stroke.

Objective:To explore the protective effect and related mechanism of rebamipide on non-steroid anti-inflammatory drug (NSAID) related small intestinal mucosal injury.Methods:A total of 21 C57BL/6 mice were selected and by random number table method, they were divided into negative control group (0.9% NaCl gavage for four days), indomethacin modeling group (20 mg/kg indomethacin gavage for four days) and rebamipide intervention group (20 mg/kg indomethacin gavage for four hours and then 320 mg·kg -1·d -1 rebamipide gavage for four days), seven mice in each group. After modeling, the injury of mice intestinal mucosa of indomethacin modeling group and rebamipide intervention group was evaluated by gross observation as well as pathological analysis. The serum levels of interleukin (IL)-6, IL-10, trefoil factor 3 (TFF3), prostaglandin E2 (PGE2) and epidermal growth factor (EGF) in mice were detected by enzyme-linked immunosorbent assay (ELISA). The expression of IL-6, IL-10, TFF3, cyclooxygenase 2( COX2) and EGF at mRNA level of mice small intestinal tissues were examined by real-time quantitative polymerase chain reaction (qRT-PCR). And the relative expression of TFF3, COX2 and EGF at protein level of mice small intestinal tissues were determined by Western blotting. Levene test and independent sample t test were used for statistical analysis. Results:The scores of gross observation and histopathology of mice small intestinal mucosa injury of rebamipide intervention group were both lower than those of indomethacin modeling group (2.80±0.45 vs. 4.60±1.14, 1.67±0.52 vs. 3.00±0.71), and the differences were statistically significant ( t=2.667 and 3.618, P=0.029 and 0.006). The mouse serum level of IL-6 and the expression of IL-6 at mRNA level in intestinal tissues of indomethacin modeling group were both higher than those of the negative control group, however the serum level of IL-10 was lower than that of the negative control group ((48.83±5.40) ng/L vs. (40.96±5.92) ng/L, 5.23±2.36 vs. 1.12±0.56, (168.50±10.57) ng/L vs. (186.30±7.77) ng/L), and the differences were statistically significant ( t=2.307, 3.372 and 3.366; P=0.047, 0.007 and 0.012). The expression of IL-6 at mRNA level in mice small intestinal tissues of rebamipide intervention group was lower than that of indomethacin modeling group (1.74±0.82 vs. 5.23±2.36), however, the expression of IL-10 at mRNA level was higher than that of indomethacin modeling group (6.44±3.46 vs. 1.22±0.83), and the differences were statistically significant ( t=3.409 and 3.025, P=0.008 and 0.014). The serum levels of TFF3, PGE2 and EGF, the expression of TFF3 at mRNA level of small intestinal tissues, the relative expression of COX2 and EGF at protein level of small intestinal tissues of indomethacin modeling group were all lower than those of the negative control group ((131.20±16.37) ng/L vs. (150.30±9.66) ng/L, (32.68±6.88) ng/L vs. (41.51±3.20) ng/L, (112.70±17.17) ng/L vs. (138.20±10.10) ng/L, 0.43±0.22 vs. 1.20±0.50, 0.33±0.25 vs. 1.30±0.43, 0.28±0.19 vs. 1.15±0.10), and the differences were statistically significant ( t=2.290, 2.645, 2.867, 3.097, 3.405 and 7.106; P=0.048, 0.021, 0.025, 0.017, 0.027 and 0.002). The mice serum levels of PGE2 and EGF, expression of TFF3, COX2 and EGF at mRNA level of small intestinal tissues, as well as the expression of TFF3 and EGF at protein level of small intestinal tissues of rebamipide intervention group were all higher than those of indomethacin modeling group ((43.55±5.28) ng/L vs. (32.68±6.88) ng/L, (153.30±15.66) ng/L vs. (112.70±17.17) ng/L, 2.48±1.70 vs. 0.43±0.22, 2.95±1.56 vs. 0.88±0.45, 3.97±2.54 vs. 0.98±0.76, 1.47±0.26 vs. 0.72±0.35, 1.08±0.36 vs. 0.28±0.19), and the differences were statistically significant ( t= 2.711, 3.658, 2.656, 2.856, 2.524, 3.013 and 3.435; P=0.024, 0.008, 0.026, 0.019, 0.033, 0.039 and 0.026). Conclusions:Rebamipide alleviates small intestinal mucosal injury induced by indomethacin by inhibiting the expression of inflammatory factors and promoting the expression of intestinal mucosal protective factors suggesting that rebamipide plays a protective role in NSAID related small intestinal injury by maintaining the chemical barrier of the intestinal mucosal.

Objective To investigate the expression of FTO in gastric cancer tissues and the functional significance of FTO in MGC-803 cell line.Methods The FTO mRNA was detected by RT-qPCR in 54 cases of gastric cancer samples and their paired adjacent normal control tissues.The effect of FTO overexpression in MGC-803 on cell proliferation,cell migration and invasion were detected by CCK-8,wound heal and ranswell assays,respectively.Results The expression of FTO mRNA was significantly lower than that of adjacent tissues(P＜O.05).Furthermore,overexpression of FTO in MGC-803 cells inhibited cell proliferation,cell migration and invasion.Conclusions FTO is low expressed in gastric cancer tissues and inhibits gastric cancer cell line MGC-803 proliferation,migration and invasion.FTO is closely associated with the development of gastric cancer.

Objective To study the growth inhibition,apoptosis induction effects of cinobufagin(CB)on human osteosarcoma(OS) cell line U2OS,MG63 and SaO2 in vitro and the underlying mechanism of action of cinobufagin in OS cells.Methods Cell viability was assessed by MTT assay.Cell-cycle status,apoptosis-inducing effects were evaluated by flow cytometry,fluorescent staining and DNA fragmentation assays.Inhibitors of apoptosis proteins (IAPs) and Bcl-2 family proteins including Bax,cleaved-PARP,xIAP,cIAP-1,survivin and p65 were tested by Western blot.Results MTT assay showed that CB could inhibited the growth of U2OS,MG63 and SaO2 cells in a dose-and time-dependent manner.The 48 h IC50 of CB on U2OS,MG63 and SaO2 cells were (104.83± 16.96) nmol/L,(47.07±7.5) nmol/L,and (136.72±10.08) nmol/L respectively.The induction of G2/M cell-cycle arrest was seen in the cells treated with CB.After cells were cultured for 12 h in the presence of 100 nmol/L CB,the percentages of cells in the G0/G1 phase were decreased,while G2/M phase were increased in U2OS,MG63 and SaOS2 cells,respectively.The results showed CB inhibited the proliferation of osteosarcoma cells through blocking the cell cycle in G2/M phase.Induction of apoptosis was confirmed by Hoechst 33258 and Annexin V/PI staining.After treating with 100 nmol/L CB for 48 h,the extents of apoptosis were 33.6％±6.4％,36.4％±7.8％ and 29.3％±5.1％,respectively.These results indicate that the anti-tumor activity of cinobufagin in osteosarcoma cells was due to a G2/M cell cycle arrest and apoptosis inducing effect.Western blot showed that CB could induce the apoptosis related family proteins Bax,cleaved-PARP up-regulation,xIAP,cIAP-1,survivin and p65 downregulation in OS cells.Conclusion CB can inhibit the cell viability and induce G2/M cell cycle arrest and apoptosis in U2OS,MG63 and SaO2 cells.The apoptosis-inducing effect of CB is confirmed by the regulation of apoptosis related proteins IAPs and Bcl-2 in vitro.

Objective To test the expression of HGF and its receptor c-met and to evaluate their relationship to lymphangiogenesis in NSCLC. Methods Immunohistochemical study was used to determine the lymphatic microvessel density (LMVD) and the expression of HGF and its receptor c-met in 113 patients with NSCLC and 20 normal lung tissue samples.Results The levels of expression of HGF (67.3 %) and c-met (74.3 %) in NSCLC tissues were significantly higher than that in normal lung tissues (20.0 %, 25.0 %respectively), (u=4.008, 4.342; both P ＜0.05).The expressions of HGF and c-met in NSCLC tissues had no correlation to age, gender, tumor size and differentiation grade.However, the expression of HGF and c-met was positively correlated to TNM stage and lymph node metastasis.In NSCLC samples, HGF and c-met were significantly associated with LMVD contrast to normal sample (HGF: 16.3051±5.3753 vs 10.9934±4.9668,t =4.58; c-met: 15.6692±5.5386 vs 11.3700±5.3875, t =3.97, both P ＜ 0.05).Conclusion The high expression of HGF and its receptor c-met is closely correlated with LMVD and metastasis of lymph nodes, which indicates that HGF and c-met may play an important role in lymphangiogenesis and lymphatic metastasis in NSCLC.

Objective To investigate treatment and prognosis of patients with osteosarcoma of the extremities.Methods A total of 311 patients with osteosarcoma of the extremities,who had undergone treatment in our institute from 1998 to 2008,were enrolled in this retrospective study.Kaplan-Meier survival curve and Cox regression model were used to analyze the correlation between survival rate and variables including patients' demographics,chemotherapy,surgery,complications,and tumor metastasis.Results Among 311 patients,there were 206 males and 105 females,aged from 5 to 56 years (average,18.6 years).A total of 282 patients underwent aggressive or radical surgery,including 149 cases of limb salvage surgery and 133 cases of amputation surgery.One hundred and five patients underwent standard chemotherapy and 206 patients underwent non-standard chemotherapy.The 5-year survival rate was 57.4％ in patients treated with standard chemotherapy,36.3％ in patients treated with non-standard chemotherapy,16.8％ in patients with lung metastasis,50.7％ in patients without lung metastasis,56.6％ in patients who underwent limb salvage surgery,31.8％ in patients who underwent amputation surgery,44.6％ in patients with Enneking stage Ⅱ B and 33.1％ in patients with Enneking stage Ⅲ.For patients treated by amputation surgery,because non-standard chemotherapy which was performed in most of them and other confounding factors,the 5-year survival rate of them was lower.The Cox regression analysis showed that lung metastasis and non-standard chemotherapy were associated with inferior outcomes.Conclusion Neoadjuvant chemotherapy combined with aggressive or radical surgery could cure about 60％ of patients with osteosarcoma of the extremities.Lung metastasis and non-standard chemotherapy are risk factors that severely affect prognosis.

Objective To study the affect and the related molecular mechanism of glycogen synthase kinase-3β in the proliferation of osteosarcomaand its value in the target therapy of osteosarcoma.Methods The expression level of p-GSK-3β(Ser9)and GSK-3β were detected in human osteoblast cell and osteosarcoma cells by western blot.Observe the effect of GSK-3β inhibitors and siRNA interference on the GSK-3β regulate osteosarcoma cells using apoptosis protein chip.Evaluate the valueof GSK-3β target therapy on osteosarcoma in vivo.Results The expression level of p-GSK-3β (Ser9)was lower in osteosarcoma cells.LiCL,GSK inhibitor Ⅸ,siRNA knockdown could inhibit the cell viability and up-regulated the apoptosis-related protein cleaved-caspase3.The results of the protein array showed that downstream proteins of NF-κB downregulated significantly.The results were validated by western blot,while the downregulation of p-Iκ-Bα and nuclear NF-κB p65 were also observed after LiCL treatment.Inhibition of GSK-3β by either LiCl or specific siRNA resulted in a significant reduction of NF-κB luciferase reporter activity.Furthermore,the NF-κB luciferase reporter activity was significantly increased in CA cell lines,but not in KD cell lines.By contrast,NF-κB-luciferase reporter activity was significantly decreased in stably GSK-3β knockdown cells.GSK3β inhibitor LiCL and shRNA knock down demonstrated a strong cytotoxicity effect on osteosarcoma cells in vivo.Conclusion GSK-3β is in the state of relative active in osteosarcoma in osteosarcoma and important in cell proliferation.GSK-3β regulates cell survival partially through the NF-κB pathway.It is a promising therapeutic target in osteosarcoma.

Objective To study the effects of Interferon(IFN)in chronic hepatitis B patients genotype B and C.Methods 20 cases were genotype B ,23 cases were genotype C,all the patients were treated with 5 million units of IFN-a-lb,im,qod,for 12 months,viral markers,liver function and adverse drug reactions were observed.Results There was no statistically significant difference between B,C genotype in the negative conversion rate of HBV-DNA (60.0% ,39.1%),the negative conversion rate of HBeAg(42.9% ,30.8%),anti-HBe seroconversion rate(35.7% ,23.1 %)and the rate ALT normalization(85.0% ,73.9%)(t = 1.86,0.69,0.68 ,0.79,P ＞ 0.05).Conclusion The results suggested that therapeutic efficacy of IFN-α-lb was not significantly different between B,C genotypes.