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OBJECTIVE To investigate the effect of berberine on differentiation of rat bone marrow mesenchymal stem cells (MSCs) to adipocytes and its mechanism. METHODS Rat MSCs were isolated and cultured, adipocytic differentiation was induced with adipogenesis-inducing medium (AIM). Cells were assigned into 6 groups:normal control, AIM group, AIM+berberine 0.1, 0.3, 1 and 3 μmol·L-1 groups, respectively. Morphology characteristics of mesenchymal stem cells were observed under an inverted microscope and adipocyte levels were analyzed by oil O staining. Alkaline phosphatase (ALP) activity was detected using p-nitrophenyl phosphate as a substrate. The cell survival was determined by MTT assay. Expressions of peroxisome proliferator activated receptor γ (PPARγ), fatty acid binding protein (aP2) and CCAAT enhancer-binding protein α (C/EBPα) mRNA were detected by semiquantitative RT-PCR. RESULTS Compared with normal control group, MSCs adipogenic differentiation, PPARγ, aP2 and C/EBPα mRNA expression significantly increased in AIM group (P<0.01), ALP activity in AIM group significantly decreased (P<0.01). Compared with AIM group, berberine inhibited MSCs adipogenic differentiation (P<0.01) and berberine 0.1, 0.3, 1 and 3 μmol·L-1 increased ALP activity by 26%, 54%, 81% and 122%, respectively. Berberine 3 μmol·L-1 significantly downregulated PPARγ expression (0.91±0.10 vs 1.34±0.06) (P<0.01), aP2 (1.05±0.10 vs 1.53±0.09) (P<0.01) and C/EBPα mRNA (1.24±0.06 vs 1.54±0.09) (P<0.01). Berberine had no effect on proliferation of MSCs. CONCLUSION Berberine inhibits differentiation of MSCs into adipocytes, which might be closely related to the downregulation of PPARγ, aP2 and C/EBPα mRNA.
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AIM To investigate the inhibitory effect of paeonol on hydrogen peroxide(H2O2)-induced apoptosis in PC12 cells. METHODS The injury model in PC12 cells was generated by H2O2 treatment. The cell viability was determined using methylthiazolyl tetrazolium reduction assay. Apoptotic cells and reactive oxygen species (ROS) were measured by flow cytometry. Lactate dehydrogenase (LDH) activity and malonyldialdehyde (MDA) content were measured by spectroscope respectively. RESULTS After PC12 cells were treated with H2O2 (100 μmol*L-1) for 10 h,its viability obviously decreased, and apoptotic cells, LDH release into the culture media, ROS and MDA contents in PC12 cells significantly increased. When the cells were pretreated with paeonol (12, 25 and 50 μmol*L-1)for 1 h prior to incubation with H2O2, its viability was greatly increased, and apoptotic cells, LDH release, ROS and MDA contents significantly decreased. CONCLUSION Paeonol protects PC12 cells from H2O2-induced apoptosis and this effect is probably achieved through its antioxidative action.
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BACKGROUND: Sinomenine, an alkaloid monomer extracted from Chinese medicinal herb sinomenium acutum,possesses potent anti-inflammatory, analgesic and immunoinhibitory pharmacological activities. Sinomenine has certain therapeutic effect on rheumatoid arthritis and has been widely applied in the clinic. Dendritic cell (DC) is the known antigen presenting cell with the strongest functions in the body.OBJECTIVE: To observe the effect of different doses of sinomenine on CD80 and CD 86 expression and extracellular interleukin-12 secretion in DCs.DESIGN: A controlled repeated measuring study based on the cells.SETTTNG: Department of Pharmacology, Guangdong Medical College.MATERIALS: The experiment was carried out in the Institute of Immunology, Third Military Medical University of Chinese PLA between September 2004 and May 2005. The peripheral blood of healthy donors was supplied by the blood bank of the Southwest Hospital. RPMI1640 medium was purchased from Hyclone Company. Fluorescein isothiocyanate(FITC)-conjugated mouse monoclonal antibodies against CD80 and CD86 and FITC-conjugated mouse IgG1 were purchased from Bioscience Company. Recombinant human interleukin-4 (rhlL-4), recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and tumor necrosis factor-α (TNF-α) were purchased from PharMingen Company. Sinomenine was supplied by Zhengqing Pharmacy Company in Hunan. ELISA kit specific for IL-12 was purchased from Diaclone Company.METHODS: Peripheral blood was collected from healthy volunteers. 1 ×106 U/L recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and 5×105 U/Lrecombinant human interleukin-4 (rhlL-4)were added to the peripheral blood. After 7 days, DCs were harvested. ①The DCs were counted and cultured at 2×108 L 1 in fresh medium supplemented with TNF-α and different doses of sinomenine(3, 10, 30 mg/L). After 48 hours, 2×105 cells were collected and washed twice with phosphate buffer solution (PBS). Then the expressions of CD80 and CD86 were analyzed using 20 μL FITC-conjugated mouse monoclone antibodies against CD80 or CD86.FITC-conjugated mouse IgG1 was used as control at 4 ℃ away from light for 30 minutes. After staining, cells were washed twice with PBS and fixed in 10 g/L paraformaldehyde. The samples were analyzed on a flow cytometry. ②The DCs were counted and cultured at 2 ×108 L-1 in fresh medium supplemented with TNF-α and different doses of sinomenine (3, 10, 30 mg/L). After 48 hours, the supernatants were collected and the concentration of interleukin-12 (IL-12) was determined in a sandwich ELISA using kit specific for IL-12 according to the manufacture's instruction.MAIN OUTCOME MEASURES: ①Effect of different doses of sinomenine on the expressions of CD80 and CD86 on DCs. ②Effect of different doses of sinomenine on the level of IL-12 secreted by DCs.RESULTS : ① Expressions of CD80 and CD86 on DCs: There were no significant differences in the percentage of cells positive and fluorescence intensity for CD80 and CD86 on DC between different doses of sinomenine groups and control groups (P ＞ 0.05). ② IL-12 level secreted by DCs: From ELISA measurement, we found that IL-12 level of the sinomenine 3 mg/L, 10 mg/L and 30 mg/L groups was (863.1±33.7), (668.8±31.9), (512.6±29.6) ng/L, respectively,which was higher than that of control group [ (922.2±36.6),P ＜ 0.05-0.01]. The level of IL-12 in the supernatants of DCs co-cultured with sinomenine was significantly lower compared with control, which was dose-dependent.CONCLUSTON: Sinomenine can inhibit the level of IL-12 secreted by DCs in dose-dependent manner, but do not influence the expressions of CD80 and CD86 in DCs; The therapeutic effect of sinomenine on rheumatoid arthritis may be related to its inhibition to secretion of IL-12 in DCs.