Effects Of Pine (Pinus Massoniana) Pollen Polysaccharide And Its Ester On Mouse Pancreaticβ-cell Insulin Secretion

Pinus massoniana pollen polysaccharides modified by sulfation, Sulfate groupswith steric and electrostatic repulsion can change the the polysaccharides’originalspatial structure, which becomes easier for them to combine with the specific domainof the protein.It affects their function and biological activity changes. Former researchof our laboratory: the Pinus massoniana pollen polysaccharides precipitated by 60%alcohol （PPM60） and its sulfated derivative （SPPM60） can increase intracellular freecalcium concentration in rat myocardial cells, splenocytes of mice and human chronicmyelogenous leukemia cells; they can also reduce intracellular free calciumconcentration in primary rat vascular smooth muscle cells. Intracellular free Ca2+concentration can be one of important factors affecting insulin secretion.But it is still unclear whether PPM60 and SPPM60 role in pancreaticβ-cells,causing changes in intracellular calcium concentration, leading to the secretion ofinsulin. This research aims to discuss the mechanism of PPM60 and SPPM60 oninsulin secretion in mouse pancreaticβ-cell in vitro. Provide ideas for the use of Pinusmassoniana pollen polysaccharides and develop drugs that can treat diabetesadjuvant.The experiment is divided into the following sections.1. Selected PPM60 precipitated by 60% alcohol. Three components PPM60-A,PPM60-B, PPM60-C were obtained separately by Sephacryl S-400HR gel filtrationcolumns, whose molecular weights were uniform. PPM60 and its purified componentswere chemically modified by chlorosulfonic acid-pyridine method, named SPPM60,SPPM60-A, SPPM60-B and SPPM60-C. The the degree of their sulfate were 1.494,1.58, 1.31, 1.19 respectively.The UV, IR spectrum of SPPM60 showed thepolysaccharides characteristic absorption peak and the characteristic absorptions ofsulfated ester bond（C-O-S and S=O）, indicating that this polysaccharide has beensulfated. 2. enzyme-linked immunosorbent assay method was used to detect the insulinsecretion in the MIN6 cells supernatant which was stimulated by PPM60 or SPPM60.SPPM60 of 200μg/mL stimulating for one hour showed a significant role inpromoting insulin secretion of MIN6 cells. But PPM60 almost made no effect oninsulin secretion. Both L-type calcium channel specific inhibitor Verapamil and theintracellular calcium store of IP3 receptor inhibitor Low Molecular Weight Heparin,made a certain degree inhibition of insulin secretion caused by the SPPM60, but notsignificant.3. MIN6 cells were treated with the calcium fluorescence probe Fura 2-AM,using a fluorescence spectrophotometer to detect [Ca²⁺]_ichanges effected by SPPM60.Within 5 min, SPPM60 increased [Ca²⁺]_isignificantly compared with the controlgroup. L-type calcium channel blocker Verapamil partially inhibited the rise of [Ca²⁺]_icaused by SPPM60. Also, IP3 receptor inhibitor Low Molecular Weight Heparin（LMWH） inhibited the rise of [Ca²⁺]_icaused by SPPM60, but not down to the blanklevel.These results suggest that: （1） SPPM60 had significant role in promoting insulinsecretion of MIN6 cells. However PPM60 have little impact on insulin secretion.（2）SPPM60 might promote insulin secretion in MIN6 cells by increasing intracellularfree calcium concentration. （3） The rise of intracellular free calcium concentrationmight have relationship to the open of voltage-dependent calcium channels in cellmembrane and the IP3-sensitive calcium store.