Xiaokeping-induced autophagy protects pancreatic ß-cells against apoptosis under high glucose stress.

Xiaokeping (XKP), a prescribed Traditional Chinese Medicine (TCM), has been used to treat patients with type Ⅱ diabetes mellitus for many years; however, the molecular mechanism of its effects is unknown. As the only insulin producer, the pancreatic ß cell plays an important role in diabetes. Whether XKP influences the viability of pancreatic ß cells remains to be substantiated. In the present study, autophagy/apoptosis analyses were used to evaluate the therapeutic effect of XKP on pancreatic ß-cells induced by high glucose levels and to investigate a potential causal molecular mechanism of XKP effect on the cells. The pancreatic ß-cell lines MIN-6 were divided into four groups: control, high glucose (33.3 mmol/L), high glucose with XKP, high glucose with XKP and 3-Methyladenine (3-MA). Immunofluorescence assay was employed to determine autophagosome formation and flow cytometry was used to determine apoptotic rates of the ß cells by the detecting expression of autophagy- and apoptosis-related proteins. High glucose increased the apoptotic rate of ß-cells from 5.37% to 23.24%; however addition of XKP mitigated the rate at 10.92%. Data indicate that autophagy of ß-cells was induced by XKP via the mammalian target of rapamycin (mTOR) pathway. Where the autophagy inhibitor 3-MA was added, the apoptotic rate was 23.94%, similar to the high glucose group rate. The results suggest a potential cytoprotective effect of XKP from high glucose toxicity by its induction of autophagy which may be linked to mTOR-mediated autophagy.

Detection of Huperzine A and Huperzine B in fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate by HPLC / 生物工程学报

In this study, we established a rapid and efficient HPLC method to determine the accumulation of Huperzine A and Huperzine B in the fermentation broth of endophytic fungus Colletotrichum gloesporioides from Huperzia serrate. The chloroform extracts of fermentation broth were dissolved in methanol and filtered before injection for HPLC analysis. The analysis was performed on an Agilent Eclipse plus-C18 column (250 mm×4.6 mm, 5 μm) by isocratic elution. The mobile phase was 0.015 mol/L ammonium acetate-methanol (70:30, V/V), the flow rate was 1 mL/min and the detection wavelength was set at 308 nm. Huperzine A and Huperzine B could be well separated within 25 min. Good linearity of Huperzine A was found in the range of 1.50-48.00 μg/mL (r=0.999 5), and that of huperzine B was in 0.25-7.50 μg/mL (r=0.999 7). The average recoveries of Huperzine A and Huperzine B were 106.83% and 108.06%, respectively (RSD=3.34%, 3.60%). The results demonstrate that this method can detect the content of huperzine A and huperzine B in fermentation broth simply, rapidly, accurately and in good reproducibility. Under the optimized conditions, the accumulated content of huperzine A and huperzine B were measured from the sixth to the fifteenth day. Huperzine A and Huperzine B reached the highest (12.417 0 μg/mL and 4.660 3 μg/mL, respectively) at the fourteenth and eighth days. The analysis methodology could contribute to the future study of huperzine A and huperzine B biosynthesis in C. gloeosporioides, consequently facilitate the development of new drug resources.