Xanthones from Swertia mussotii as multitarget-directed antidiabetic agents.

Oxidative stress has been suggested to play a causative role in the development of obesity-induced insulin resistance and type 2 diabetes. Given the antioxidant potency of previously reported xanthones isolated from Swertia mussotii. These natural products were further evaluated against other targets in diabetes, aldose reductase and α-glucosidase, in order to identify novel multitarget-directed antidiabetic agents. Among the 14 xanthones screened, 1,3,7,8-tetrahydroxyxanthone (6), 1,3,5,8-tetrahydroxyxanthone (7), and 2,3,6,8-tetrahydroxyxanthone-7C-(ß-D-glucoside) (12) were confirmed as good antioxidants and α-glucosidase inhibitors. Xanthone 7 was also confirmed as a potent inhibitor of aldose reductase (ALR2). Xanthone 7 was the most active α-glucosidase and ALR2 inhibitor, with IC50 values of 5.2±0.3 µM and 88.6±1.6 nM, respectively, while compound 12 was shown to be the most active antioxidant. Given the overall profile, xanthone 7 is considered to be the most promising multitarget antidiabetic agent, and may have potential for the treatment of both diabetes and diabetic complications.

Antitumor efficacy of a recombinant adenovirus encoding endostatin combined with an E1B55KD-deficient adenovirus in gastric cancer cells.

BACKGROUND: Gene therapy using a recombinant adenovirus (Ad) encoding secretory human endostatin (Ad-Endo) has been demonstrated to be a promising antiangiogenesis and antitumor strategy of in animal models and clinical trials. The E1B55KD-deficient Ad dl1520 was also found to replicate selectively in and destroy cancer cells. In this study, we aimed to investigate the antitumor effects of antiangiogenic agent Ad-Endo combined with the oncolytic Ad dl1520 on gastric cancer (GC) in vitro and in vivo and determine the mechanisms of these effects. METHODS: The Ad DNA copy number was determined by real-time PCR, and gene expression was assessed by ELISA, Western blotting or immunohistochemistry. The anti-proliferation effect (cytotoxicity) of Ad was assessed using the colorimetry-based MTT cell viability assay. The antitumor effects were evaluated in BALB/c nude mice carrying SGC-7901 GC xenografts. The microvessel density and Ad replication in tumor tissue were evaluated by checking the expression of CD34 and hexon proteins, respectively. RESULTS: dl1520 replicated selectively in GC cells harboring an abnormal p53 pathway, including p53 mutation and the loss of p14(ARF) expression, but did not in normal epithelial cells. In cultured GC cells, dl1520 rescued Ad-Endo replication, and dramatically promoted endostatin expression by Ad-Endo in a dose- and time-dependent manner. In turn, the addition of Ad-Endo enhanced the inhibitory effect of dl1520 on the proliferation of GC cells. The transgenic expression of Ad5 E1A and E1B19K simulated the rescue effect of dl1520 supporting Ad-Endo replication in GC cells. In the nude mouse xenograft model, the combined treatment with dl1520 and Ad-Endo significantly inhibited tumor angiogenesis and the growth of GC xenografts through the increased endostatin expression and oncolytic effects. CONCLUSIONS: Ad-Endo combined with dl1520 has more antitumor efficacy against GC than Ad-Endo or dl1520 alone. These findings indicate that the combination of Ad-mediated antiangiogenic gene therapy and oncolytic Ad therapeutics could be one of promising comprehensive treatment strategies for GC.

[Effects of triterpenoid from Psidium guajava leaves ursolic acid on proliferation, differentiation of 3T3-L1 preadipocyte and insulin resistance].

OBJECTIVE: To investigate the influences of triterpenoid from Psidium guajava Leaves (ursolic acid) on the proliferation, differentiation of 3T3-L1 preadipocyte, and its possible mechanism treat for insulin resistance. METHODS: 3T3-L1 preadipocyte was cultured in vitro. After adding ursolic acid to the culture medium for 48h, the cell viability was tested by MTT assay. Induced for 6 days, the lipid accumulation of adipocyte was measured by Oil Red O staining. The insulin resistant cell model was established with Dexamethasone. Cellular glucose uptake was determined with GOD-POD assays and FFA concentration was determined at the time of 48h. Secreted adiponectin were measured by ELISA. The protein levels of PPARgamma and PTP1B in insulin resistant adipocyte were measured by Western Blotting. RESULTS: Compared with medium control group, 30, 100 micromol/L ursolic acid could increase its proliferation and differentiation significantly (P < 0.05 or P < 0.01). Compared with the model group, ursolic acid at 100 micromol/L could enhance cellular glucose uptake of insulin resistant adipocyte significantly both in basic and insulin stimulation state (P < 0.01), while ursolic acid at 30 micromol/L could already enhance its glucose uptake significantly (P < 0.05), and could already decrease its FFA production significantly (P < 0.05). Ursolic acid at 30 micromol/L could increase the secretion of adiponectin on insulin resistant adipocyte significantly (P < 0.05), up-regulate the expression of PPARgamma protein (P < 0.05), but showed no effect on the PTP1B protein expression (P > 0.05). CONCLUSION: Ursolic acid can improve the proliferation and differentiation of 3T3-L1 preadipocyte, enhance cellular glucose uptake, inhibit the production of FFA, promote the secretion of adiponectin insulin resistant adipocyte, its mechanism may be related to upregulating the expression of PPARgamma protein.

[Nephro-protective effects of total triterpenoids from Psidium guajava leaves on type 2 diabetic rats].

OBJECTIVE: To investigate the nephro-protective effects of total triterpenoids from Psidium guajava leaves (TTPGL) on type 2 diabetic rats. METHODS: Diabetic rats were induced by intraperitoneal injection of streptozotocin (STZ, 35 mg/kg) and a high-fat diet. Diabetic rats were divided into five groups: diabetic model control, low-dose TTPGL-treated (60 mg/kg, L-TTPGL), medium-dose TTPGL-treated (120 mg/kg, M-TTPGL), high-dose TTPGL-treated (240 mg/kg, H-TTPGL) and rosiglitazone-treated (3 mg/kg, RSG). The rats received daily treatment for six weeks. At the end of the period,the levels of fasting blood glucose (FPG), fasting insulin (FINS), creatinine (Cr) and blood urea nitrogen (BUN) in serum were measured. Kidneys for histopathological evaluation were stained with Hematoxylin and Eosin (HE). RESULTS: Compared with normal control group, the level of FPG was increased, the insulin and insulin sensitivity index were decreased in the model group; The levels of BUN and Cr were increased with histopathological changes related to diabetic nephropathy in the kidney, which were the glomerular endothelium and mesangial cell proliferation, capillary narrowed, the base-membrane incrassation, glomerular swelling, cysts narrowed and tubules edema. Compared with the model group, the levels of FPG were decreased, serum insulin and insulin sensitivity index were increased significantly in M-TTPGL and H-TTPGL groups (P<0.01 or P<0.05); The levels of BUN and Cr were decreased significantly (P<0.01 or P<0.05) and the renal structural damages were improved significantly. CONCLUSION: TTPGL could decrease the level of blood glucose of diabetic rat effectively, increase the insulin sensitivity index and protect renal lesions in diabetic rats.

[Effects of extracts of Ginkgo biloba leaves on apelin concentration and expressions in plasma and myocardium of rats with myocardial ischemia injury].

OBJECTIVE: To investigate the changes of apelin in plasma and myocardium of model rats and the protective mechanisms of Extracts of Ginkgo biloba leaves (EGb) on myocardial ischemia injury induced by isoproterenol. METHODS: The model of myocardial ischemia injury was induced by subcutaneous injection of high dose isoproterenol. ELISA (enzyme linked immunosorbent assay) was used to measure the apelin concentration in plasma and myocardium. Semi-Quantitative RT-PCR was used to measure the apelin mRNA level in myocardium. The pathomorphology changes of myocardium was observed with light microscope. EGb was administered for 7 weeks. RESULTS: Compared with the normal control group, the apelin concentration in plasma and myocardium and the apelin mRNA level in myocardium significantly decreased in the model group (P < 0.01). Compared with the model group, the apelin concentration in plasma and myocardium and the apelin mRNA level in myocardium obviously increased in the EGb group (P < 0.01). Meanwhile, the NO content in serum also obviously increased and the pathological damage of myocardium was obviously improved. CONCLUSION: The protective mechanisms of EGb on myocardial ischemia injury may be related to the elevation of apelin contents and apelin mRNA level.

Doxorubicin-induced vasomotion and [Ca(2+)](i) elevation in vascular smooth muscle cells from C57BL/6 mice.

AIM: To explore the action of doxorubicin on vascular smooth muscle cells. METHODS: Isometric tension of denuded or intact thoracic aortic vessels was recorded and [Ca(2+)](i) in isolated aortic smooth muscle cells was measured by using Fluo-3. RESULTS: Doxorubicin induced phasic and tonic contractions in denuded vessels and increased levels of [Ca(2+)](i) in single muscle cells. Treatment with 10 micromol/L ryanodine had no effect on basal tension, but it did abolish doxorubicin-induced phasic contraction. Treatment with 10 mmol/L caffeine induced a transient phasic contraction only, and the effect was not significantly altered by ryanodine, the omission of extracellular Ca(2+) or both. Phenylephrine induced rhythmic contraction (RC) in intact vessels. Treatment with 100 micromol/L doxorubicin enhanced RC amplitude, but 1 mmol/L doxorubicin abolished RC, with an increase in maximal tension. Caffeine at 100 micromol/L increased the frequency of the RC only. In the presence of 100 micromol/L caffeine, however, 100 micromol/L doxorubicin abolished the RC and decreased its maximal tension. Treatment with 10 micromol/L ryanodine abolished the RC, with an increase in the maximal tension. In Ca(2+)-free solution, doxorubicin induced a transient [Ca(2+)](i) increase that could be abolished by ryanodine pretreatment in single muscle cells. The doxorubicin-induced increase in [Ca(2+)](i) was suppressed by nifedipine and potentiated by ryanodine and charybdotoxin. CONCLUSION: Doxorubicin not only releases Ca(2+) from the sarcoplasmic reticulum but also promotes the entry of extracellular Ca(2+) into vascular smooth muscle cells.

[Effects of EGb 761 on the cell apoptosis induced by H2O2 in RIN-m beta cells].

OBJECTIVE: To investigate the effects of ginkgo biloba extract (EGb 761) on apoptosis induced by hydrogen peroxide (H2O2) in RIN-m beta-cells. METHODS: The apoptotic model was made by H2O2 exposed for six hours with a concentration of 500 micromol/L The cytotoxicity was measured by MTT. Hoechst 33258 fluorescent staining were used to detect the protective effect of EGb 761 on the apoptosis of RIN-m beta-cells induced by H2O2. Annexin V-PI double staining of Flow cytometry were used to detect apoptosis quantitively. RESULTS: Compare to control group, after exposed to 500 micromol/L H2O2 for 6 hours, the apoptosis rate incereased and cell survival rate were decreased considerably (P < 0.01). Pretreated for 10 hours with EGb 761, the flow cytometry results showed that the apoptosis rate decreased and cell survival rate were increased considerably (P < 0.01, compared to H2O2 control group). CONCLUSION: EGb 761 can decrease RIN-m beta-cells damage and apoptosis induced by H2O2.

[Detection for alpha-glucosidase-inhibiting polyhydroxyalkaloid ingredients of Commelina communis L. by ESIMS].

For detecting polyhydroxyalkaloids-type alpha-glucosidase-inhibiting ingredients of Commelina communis L grown in China, total alkaloids were obtained from the plant by extraction with water, removal of precipitation after the addition of alcohol, enrichment and purification by ion exchange resin and sephadex LH 20 chromatography. Polyhydroxyalkaloids in the total alkaloids were detected by ion trap electron-spray ionization mass spectra (ESIMS). Several reported and unreported polyhydroxyalkaloids in the plant were detected from the material collected from Jixi county, Anhui province. The crude drug growing in China contains alpha-glucosidase-inhibiting polyhydroxyalkaloids and can be used to therapy in diabetes.

[Study on the effect of Ginkgo biloba extract on the tension of diabetic rat artery].

OBJECTIVE: To investigate the effect and possible mechanism of Ginkgo biloba extract EGB50 on vascular tension of type II diabetes mellitus (DM) induced by hyperglycemia and hyperlipidemia. METHODS: Rats were randomly divided into normal control rats (Control), diabetic rats (DM), diabetic rats oral-treated with higher dose Ginkgo biloba extract EGB50 (H) and diabetic rats treated with lower dose EGB50 (L). The serum levels of Advanced Glycosylation end-products (AGEs), cholesterol (CHOL), triglyceride (TRIG) and superior mesenteric artery tension were quantified and measured. RESULTS: After 5 weeks' oral-treatment, serum CHOL, TRIG and AGEs increased, sustained phase of contractile response in high K+ solution and PD2 of phenylephine decreased in diabetic arteries. The biochemical indexes and the tension of vascular function had less significance in L group, but improved distinctly in EGB50 H group (decreasing rate in high K+ solution: 22.52 +/- 5.48%, vs DM:44. 19 +/- 11.03%, P < 0.05) (PD2 of PE: 6.15 +/- 0.22 vs DM: 6.62 +/- 0.13, P < 0.05). CONCLUSION: EGB50 is capable of reducing serum hyperlipidemia,the concentration of AGEs, thus it can reduce the impairment of the oxidants to endothelium cells of vessels and improve pathological and functional change of artery of diabetes.

Mechanism underlying enhanced endothelium-dependent vasodilatation in thoracic aorta of early stage streptozotocin-induced diabetic mice.

AIM: To investigate the mechanism of the enhanced endothelium-dependent vasodilatation in thoracic aorta of the early stage streptozotocin (STZ)-induced diabetic C57BL/6J mice. METHODS: Radioimmunity was used to detect the metabolite of prostaglandin I2 (PGI2), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), in the blood serum. Vascular muscle tension and phenylephrine (PE)-induced rhythmic activity in the isolated thoracic aorta of mice were also compared. RESULTS: 6-Keto-PGF1 alpha in the serum was significantly higher in STZ-induced diabetic mice than age-matched controls [(1.8+/-1.0) microg./L vs (0.5+/-0.3) microg/L, P<0.01]. PE induced rhythmic activity in both diabetic and control mouse aorta but the amplitude was markedly higher in diabetic mice than in controls [(4.9+/-1.7) % vs (12+/-5) %, P<0.01]. PE, high K+ solution-induced contraction, and acetylcholine (ACh)-induced relaxation [(56+/-10) % vs (81+/-8) %, P<0.01] were notably enhanced in diabetic mice than those in controls. Alone NG-nitro-L-arginine methyl ester (L-NAME) or 6-(phenylamino)-5,8-quinolinedione (LY-83583) abolished the rhythmic activity and ACh-induced relaxation in controls but only partially inhibited them in diabetic mice. Indomethacin did not affect rhythmic activity but depressed ACh-induced relaxation. L-NAME plus indomethacin significantly depressed the rhythmic activity and ACh-induced relaxation than L-NAME alone (P<0.01). Furthermore tetraethylammonium plus L-NAME abolished them in diabetic mice. CONCLUSION: The mechanism that enhanced endothelium-dependent vasodilatation in STZ-induced diabetic mice is due to enhanced production of PGI2 and endothelium-derived hyperpolarizing factor (EDHF). The phenomena maybe only take place in early stage of diabetic mice.

Changes of NF-kB, p53, Bcl-2 and caspase in apoptosis induced by JTE-522 in human gastric adenocarcinoma cell line AGS cells: role of reactive oxygen species.

AIM: To identify whether JTE-522 can induce apoptosis in AGS cells and ROS also involved in the process, and to investigate the changes in NF-kB, p53, bcl-2 and caspase in the apoptosis process. METHODS: Cell culture, MTT, Electromicroscopy, agarose gel electrophoresis, lucigenin, Western blot and electrophoretic mobility shift assay (EMSA) analysis were employed to investigate the effect of JTE-522 on cell proliferation and apoptosis in AGS cells and related molecular mechanisms. RESULTS: JTE-522 inhibited the growth of AGS cells and induced the apoptosis. Lucigenin assay showed the generation of ROS in cells under incubation with JTE-522. The increased ROS generation might contribute to the induction of AGS cells to apoptosis. EMSA and Western blot revealed that NF-kB activity was almost completely inhibited by preventing the degradation of IkBalpha. Additionally, by using Western blot we confirmed that the level of bcl-2 was decreased, whereas p53 showed a great increase following JTE-522 treatment. Their changes were in a dose-dependent manner. CONCLUSION: These findings suggest that reactive oxygen species, NF-kB, p53, bcl-2 and caspase-3 may play an important role in the induction of apoptosis in AGS cells after treatment with JTE-522.

Synergism between heparin and adriamycin on cell proliferation and apoptosis in human nasopharyngeal carcinoma CNE2 cells.

AIM: To measure the effect of addition of heparin to adriamycin (ADM) on cell proliferation and apoptosis in CNE2 cells and investigate the possible molecular mechanisms of heparin and ADM interactions. METHODS: Cell viability and cell cycle were determined by MTT assay and flow cytometry. Apoptosis was evaluated by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and agarose gel electrophoresis. The expression of Bax, Bcl-2, p53, and p21 was examined by Western blot. RESULTS: ADM (5 mg/L) alone inhibited the growth of CNE2 cells, which was magnified when heparin was added. ADM elicited typical apoptotic morphologic changes. Compared with ADM or heparin alone, ADM plus heparin obviously enhanced the number of TUNEL positive cells from 12.6 % 1.1 % to 65.7 % 1.3 %, and the DNA ladder was more clearly observed. After exposure to different concentrations of heparin (with or without ADM) for 24 h, CNE2 cells were accumulated in G0/G1 phase. There was a decrease in the number of cells in S phase by the combined heparin and ADM treatment compared to heparin or ADM alone. The ratio of Bax/Bcl-2 was elevated, and p53 and p21 mRNA were over-expression. CONCLUSION: Heparin and ADM appear to interact in a synergistic manner, which may be related to the over-expression of p53 and p21 mRNA and the elevated ratio of Bax/Bcl-2 mRNA.