Huogu Ⅰ formula prevents steroid-induced osteonecrosis in rats by down-regulating PPARy expression and activating Wnt/LRP5/ i3-catenin signaling

OBJECTIVE： To investigate the effects of Huogu I formula on regulation of lipid metabolism in ste- roid-induced osteonecrosis of the femoral head （SONFH） rats and verify our hypothesis that Huogu I formula regulates lipid metabolism by down-regulating peroxisome proliferator-activated receptor gamma （PPARy） expression and activating Wnt signaling pathways. METHODS： Eighty-five rats were divided into four groups： control, model, Huogu 15 g/kg and Huogu 30 g/kg. Six weeks later, animals were anaesthe- tized, femora were dissected for histopathologicalexamination of the osteonecrotic changes and re- pair processes, micro computed tomography （Mi- cro-CT）-based micro-angiography was performed to assess vascularization. Serum lipid levels were detected by haematological examination. The ex- pressions of PPARy, Wnt3a, low density lipoprotein receptor-related protein 5 （LRP5） and 13-catenin were evaluated by immunohistochemistry, Western blot and quantitative real-time polymerase chain reaction analyses. RESULTS： The incidence of osteonecrosis, ratio of empty lacuna, adipose tissue area and adipocyte perimeter in the bone marrow were dramatically lower in the Huogu ~ formula treatment groups. By micro-CT quantification, Huogu ~ formula treat- ment dose-dependently increased vessel volume, vessel surface, percentage of vessel volume and vessel thickness of the femoral heads of SONFH rats. Levels of serum lipid in Huogu 15 g/kg and Huogu 30 g/kg groups reduced significantly. HuoguⅠformula treatment could suppress the ex- pression of PPARy and increase the expressions of Wnt3a, LRP5 and 13-catenin at both protein and mRNA levels. CONCLUSION： The results of our present study highlight the lipid-lowering potential of Huogu Ⅰ formula, and provide further evidence of the in- volvement of the PPARy inhibition and Wnt/LRPS/ 13-catenin signaling activation in the effects of Huogu Ⅰ formula.     

Ibervillea sonorae (Cucurbitaceae) induces the glucose uptake in human adipocytes by activating a PI3K-independent pathway

Ethnopharmacological relevance

Ibervillea sonorae (S. Watson) Greene (Cucurbitaceae), a plant used for the empirical treatment of type 2 diabetes in México, exerts antidiabetic effects on animal models but its mechanism of action remains unknown. The aim of this study is to investigate the antidiabetic mechanism of an Ibervillea sonorae aqueous extract (ISE).

Materials and methods

Non-toxic ISE concentrations were assayed on the glucose uptake by insulin-sensitive and insulin-resistant murine and human cultured adipocytes, both in the absence or the presence of insulin signaling pathway inhibitors, and on murine and human adipogenesis. Chemical composition of ISE was examined by spectrophotometric and HPLC techniques.

Results

ISE stimulated the 2-NBDGlucose uptake by mature adipocytes in a concentration-dependent manner. ISE 50 µg/ml induced the 2-NBDG uptake in insulin-sensitive 3T3-F442A, 3T3-L1 and human adipocytes by 100%, 63% and 33%, compared to insulin control. Inhibitors for the insulin receptor, PI3K, AKT and GLUT4 blocked the 2-NBDG uptake in murine cells, but human adipocytes were insensitive to the PI3K inhibitor Wortmannin. ISE 50 µg/ml also stimulated the 2-NBDG uptake in insulin-resistant adipocytes by 117% (3T3-F442A), 83% (3T3-L1) and 48% (human). ISE induced 3T3-F442A adipogenesis but lacked proadipogenic effects on 3T3-L1 and human preadipocytes. Chemical analyses showed the presence of phenolics in ISE, mainly an appreciable concentration of gallic acid.

Conclusion

Ibervillea sonorae exerts its antidiabetic properties by means of hydrosoluble compounds stimulating the glucose uptake in human preadipocytes by a PI3K-independant pathway and without proadipogenic effects.     

Isoliquiritigenin impairs insulin signaling and adipocyte differentiation through the inhibition of protein-tyrosine phosphatase 1B oxidation in 3T3-L1 preadipocytes

Isoliquritigenin (ISL) is an abundant dietary flavonoid with a chalcone structure, which is an important constituent in Glycyrrhizae Radix (GR). ISL exhibits anti-oxidant activity, and this activity has been shown to play a beneficial role in various health conditions. However, it is unclear whether the anti-oxidant activity of ISL affects insulin signaling pathway and lipid accumulation of adipocytes. We sought to investigate the effects and molecular mechanisms of ISL on insulin-stimulated adipogenesis in 3T3-L1 cells. We investigated whether ISL attenuates insulin-induced Reactive Oxygen Species (ROS) generation, and whether ISL inhibits the lipid accumulation and the expression of adipogenic-genes during the differentiation of 3T3-L1 cells. ISL blocked the ROS generation, suppressed the lipid accumulation and the expression of adipocyte-specific proteins, which are increased in response to insulin stimulation during adipocyte differentiation of 3T3-L1 cells. We also investigated whether the anti-oxidant capacity of ISL is involved in regulating the molecular events of insulin-signaling cascade in 3T3-L1 adipocytes. ISL restores PTP1B activity by inhibiting PTP1B oxidation and IR/PI3K/AKT phosphorylation during the early stages of insulin-induced adipogenesis. Our findings show that the anti-oxidant capacity of ISL attenuated insulin IR/PI3K/AKT signaling through inhibition of PTP1B oxidation, and ultimately attenuated insulin-induced adipocyte differentiation of 3T3-L1 cells.     

再生障碍性贫血患者骨髓间充质干细胞转化生长因子β1及Smad3表达下调的发病学意义

 目的 研究骨髓间充质干细胞（MSC）中转化生长因子 β1（TGF-β1）与 Smad3 的表达下调与再生障碍性贫血（再障）发生的可能关系。 方法 取 20 例重型再障患者骨髓 MSC，分离制备出 Flk1+CD34–MSC 后进行以下试验：①体外定向诱导 MSC向脂肪细胞分化，倒置显微镜观察成脂分化情况，油红 O染色法鉴定脂肪细胞，用实时荧光定量 PCR 检测分化过程中脂蛋白酯酶（LPL）基因的表达；②用蛋白质印迹法检测MSC 中 TGF-β1 及 Smad3 的表达，用 ELISA 检测 MSC分泌 TGF-β1 的能力；③观察不同剂量（0、5、10、15、20 ng/ml）TGF-β1 对再障患者骨髓 MSC 成脂分化和增殖的影响。以 7 名健康成人的骨髓 MSC 相应检测为对照。 结果 再障患者骨髓 MSC 经 4 d 培养后即分化为脂肪细胞，而健康成人骨髓 MSC 中仅见少量细胞出现脂肪滴。再障患者 MSC 培养 4 和 8 d 时 LPL 基因表达水平分别为 0.091 ± 0.028、0.142 ± 0.033，均高于健康成人骨髓 MSC（分别为 0.021 ± 0.011 及 0.049 ± 0.010，均 P < 0.01）；而 TGF-β1 及 Smad3 表达水平明显低于健康成人骨髓 MSC，TGF-β1 分泌水平（3.4 μg/L ± 0.9 μg/L）也明显低于健康成人骨髓 MSC（11.6 μg/L ± 1.2 μg/L，P < 0.01）。在向脂肪细胞分化过程中，仅加入 5 ng/ml 的 TGF-β1 即可明显抑制再障患者骨髓 MSC 向脂肪细胞定向诱导分化，同时促进其增殖。 结论 再障患者骨髓 MSC 中 TGF-β1 及 Smad3 表达水平的显著下调可能参与了再障的部分发病环节。