The Effects Of Pine Pollen Sulfated Polysaccharides On Lipid Differentiation Of3T3-F442A Preadipocyte

Former research of our laboratory had indicated that60%ethyl alcohol precipitateof the Pinus massoniana pollen after sulfating could be used to improve theintracellular free calcium concentration ([Ca2+]i) of spleen suspension of mice andcould also work on T lymphocytes and B lymphocytes which were both separated frommice lymphocytes. Aqueous extract and ethanol extract of pine pollen and phytosterolsin pine pollen could also have effect on3T3-F442A preadipocyte in promotingdifferentiation. Meanwhile phytosterols in pine pollen could inhibit the expression ofglycometabolism, AMPK and GLUT1. Currently, the research based on the effect ofmasson pollen and sulfated polysaccharides on lipid differentiation in3T3-F442Apreadipocyte and [Ca2+]iis still not clear. The aim of this research is to investigate theeffects of PPM60-B and SPPM60-B which were purified by our laboratory onproliferation and lipid accumulation of3T3-F442A preadipocyte and [Ca2+]iand so onand to study how the SPPM60-B mediate [Ca2+]ito lead to the change of lipiddifferentiation. This experiment was mainly divided into the following several parts:1.We used the extraction technology of hot water poaching method and ethylalcohol deposition to extract masson pollen polysaccharides and used trifluoroaceticacid method to remove impure protein, then we choosed Sephacryl S-400HR toseparate the polysaccharides which precipitated by60%alcohol and got ahomogeneous component named PPM60-B. We used chlorosulfonic acid-pyridinemethod to obtain the sulfated polysaccharide compound named SPPM60-B. Afterusing barium chloride-gelatin turbidimetric method to calculate the substitution degreeof polysaccharide and infrared ray (IR) to detect the characterization of polysaccharide,we validated that polysaccharide esterification was done. Those two kinds ofpolysaccharides could be used for the following experiments.2. MTT assay was used to detect the influences of the above two kinds ofpolysaccharides on the proliferation of3T3-F442A preadipocyte under different concentrations and different hours. The results showed that after preadipocyte wastreated in24-72h, SPPM60-B at10μg/mL,25μg/mL and50μg/mL concentrationshad a greatly significant difference on inhibition of proliferation. After48h treatmentwith SPPM60-B in10μg/mL, the3T3-F442A preadipocyte proliferation inhibitionrate reached to24.92%and the effect of inhibition showed strongly significance.PPM60-B and SPPM60-B in other concentrations at different treatment times showedvarious degree of promoting the proliferation of preadipocyte.3.3T3-F442A preadipocyte was treated with the above two kinds ofpolysaccharides under different concentrations. We used oil red O staining to detectthe morphological change before and after differentiation and used isopropyl alcoholsemi-quantitative and triglyceride test kits to detect the level of adipocytedifferentiation. Experimental results showed that PPM60-B did not have significantinfluence on differentiation of adipocyte. While after treated with SPPM60-B theresults showed that SPPM60-B could inhibit differentiation in low concentration andpromote differentiation in high concentration. SPPM60-B at10μg/mL concentrationhad an obvious inhibitory effect on lipid differentiation than other groups.4. We used double-wavelength fluorecent technique to detect the change of[Ca2+]iof the early stage of differentiation of3T3-F442A preadipocyte which weretreated with the above two kinds of polysaccharides. Experiments confirmed thatPPM60-B did not have significant remarkable influence on the change of [Ca2+]iofpreadipocyte. SPPM60-B had a dose-dependent effect on the change of [Ca2+]i. At200μg/mL, SPPM60-B had the strongest influence on [Ca2+]iof preadipocyte. At thedosage of SPPM60-B at200μg/mL in culture madia, our results identified that thedifferent types of intracellular calcium ion channel inhibitors (LY294002, U73122,LMWH,2-APB and Verapamil) could partly suppress the increase of [Ca2+]iof3T3-F442A preadipocyte. However, low molecular weight heparin and2-APB couldtotally inhibit the increase of [Ca2+]iof3T3-F442A preadipocyte by treating withSPPM60-B.[Ca2+]iwere lower than the control group after treating with those twoinhibitors.In conclusion, the results of this experiment confirmed that the pine pollen sulfated polysaccharides could have a huge influence on proliferation activity and lipiddifferentiation accumulation of3T3-F442A preadipocyte by regulating with the changeof [Ca2+]i. We speculated that the underlying molecular mechanism in3T3-F442Apreadipocyte was through receptor-PI3K-PLC-IP3R–Ca2+signaling pathways toactivate the IP3R channel of ER calcium store so as to lead to the release of Ca2+andthen to activate the membrane surface CRAC pathway to giving rise to the inflow ofextra-cellular calcium ion and lead to further increase of [Ca2+]i. This research has agreat significance on the mechanism of polysaccharides modifying the lipiddifferentiation of adipocyte.