Heparin-Derived Oligosaccharides Preparation, Structure Analysis And Their Anti-Asthma Molecular Action Mechanisms

Heparin belongs to the family of glycosaminoglycans (GAGs), the molecular is composed of uronic acid (iduronic acid and glucuronic acid), glucosamine, and their sulfated and acetylizad derives, which connect with l→4 glucosidic bond and form the linear chain polysaccharides. The relative molecular weght (M_r) distribution is between 3kD and 30kD. Heparin-derived oligosaccharides (Oligs) are the products of depolymerised heparin with M_r of lkD~4kD according to different degradation methods. Different degradation procedures produce Oligs with different structures, which have close relationship with bioactivity diversity of them. Besides its antithrombotic and anticoagulant activites, heparin was found to have some other functions, such as inhibiting proliferation of smooth muscle, anti-inflammation activity, and antineoplastic activity.Asthma is a chronic inflammation disease that lots of inflammatory cells, mediators and cytokines involved, with symptoms of airway hyperreactivity (AHR) and airway obstruction. Interleukin-4 (IL-4) and interleukin-5 (IL-5) play an important role in inflammation of asthma. An important characteristic of asthma is that IgE level is higher than that of healthy control. IgE combines several inflammatory cells and cause the cells to release mediators of inflammatory with induction of allergen and enhance the AHR.Signal transduction plays an important role in inflammatory process. Nuclearfactor-KB (NF-kB) can regulate the synthesis of inflammatory protein and participate in inflammatory reaction of asthma.The effects of Oligs with different structures on secretion and activities of asthma inflammatory factors were studied. And Oligs bind to IL-4 and IL-5 and result in the secondary structure of IL-4 and IL-5 variance were also investigated.1 Preparation and purification of OligsDifferent Mx Oligs produced by different degradation methods were separated by Superdex 30 gel filtration chromatography procedure. Fraction Olig NO.8, Olig HO.6 and Olig BH.7 were purified by Superdex 30 chromatography procedure again. The relative unitary Olig fragments showed single band in PAGE are purifed by Q Sepharose? Fast Flow and Spherisorb SAX strong anion exchange chromatography methods.2 Structure analysis of OligsInfrared spectra (IR), NMR spectra ('H-NMR> 13C-NMR) and disaccharide analysis methods were used to determine the main molecular structures of the three Oligs. Hydroxide radical and carbonyl group are showed in the IR spectra of Olig NO.8, Olig HO.6 and Olig BH.7. Amide group was found in Olig NO8 structure. The results of NMR spectra and disaccharide analysis methods show that all of the three Oligs contain four monosaccharides, such as UA/AUA(2S), GlcNS(6S), Ido(2S) and GlcNS/GlcNAc, but the contents of them are different. The major disaccharides produced by heparinase hydrolysis of Olig BH.7 are cc-AUA-2S-[l-4]-GlcNS, a-AUA- [l-4]-GlcNS-6S and a-AUA-2S- [l-4]-GlcNS-6S. The major disaccharides of Olig HO.6 and Olig NO.8 which produced by heparinase hydrolysis are a-AUA-2S-[l-4]-GlcNS-6S, a-AUA-[l-4]-GlcN and a-AUA- [l-4]-GlcN, respectively. 3 Effects of Oligs on cytokines secretion in asthmaAn asthmatic mouse model was induceded by ovalbumin and the inflammation extent of lung histopathological changes was studied. It was found that all of the three Oligs have the effect on relieving inflammatory reaction and treating the pathological changes of inflammation in lung. The effects of the three Oligs onsplenic lymphocytes secreting IL-4 and IL-5 in atopic asthma mouse were investigated. The results showed that IL-4 and IL-5 were markedly increased in the splenic lymphocyte culture supernatants of mouse sensitized with OVA. The contents of mIL-4 and mIL-5 in the cell culture supernatant were 32.93 ±2.13pg/mL and 52.83 ±3.82pg/mL respectively in group OVA and were significant differences versus the normal control group, 0.57±0.07pg/mL and 8.30±0.54pg/mL, respectively, /?<0.05. The mIL-4 contents in group Olig BH(1.77±0.70pg/mL), group Olig NO(2.41± 0.62pg/mL) and group Olig HO(6.42±0.65pg/mL) were significantly lower than that in group OVA ,p<0.05. All of the three Oligs presented significant inhibition effect on splenic lymphocytes to secrete IL-4 in mouse and Olig BH was more effective than the others. The mIL-5 contents in group Olig HO (12.04±0.27pg/mL), group Olig NO(12.53±0.73pg/mL) and group Olig BH(10.60±0.22pg/mL) were significantly lower than that in group OVA, p<0.05. These Oligs presented significant inhibition effect on splenic lymphocytes to secrete IL-5 in mouse and there were no significantly difference compared to each other, p>0.05. 4 Effect of Oligs on inflammatory factor expressionThe effects of Oligs on secretion of IgE, positive expression of CD23 and NF-kB p65 were investigated. Results from the experiments of IgE levels in cell culture supernatants of splenic lymphocytes of mouse showed that the IgE level in group OVA (102.20 + 6.79ng/mL) was elevated compared with control healthy group(7.92± 0.46ng/mL). The levels of IgE in group Olig HO( 15.03 ±3.62ng/mL), Olig NO(21.20 ±2.59 ng/mL) and Olig BH(12.20± 1.87ng/mL) were lower than that of group OVA, p<0.05. Three kinds of Oligs presented inhibitive effect on expression of IgE. The depressant effect of Olig BH was the best one while Olig NO was weaker compared with Olig BH and Olig HO.From the study on lymphocyte CD23 molecular expression, it was known that the CD+23 expression level in group OVA(59.82±3.69)% was significantly higher than that of normal control(4.72±0.45)%. After treatment with three Oligs, the CD+23 expression levels in three therapeutic groups were Olig BH(6.20±0.80)%,Olig NO(18.92±0.79)% and Olig HO(21.22± 1.81)%, which were significantly lower than that in group OVA. The effect of Olig BH was better than Olig HO and Olig NO.The expression of NF-kB p65 in lungs tissues in mouse was determined by immunohistochemical staining method. The experimental results showed that the percentage of positive expression of NF-kB p65 in group OVA(45.84±9.51)% significantly higher than that in normal control group (7.46±2.52)%, p<0.05. The percentage of positive NF-kB p65 in three therapeutic groups were Olig NO(15.52± 1.98)%, Olig HO(27.08±6.62)% and Olig BH(12.32 ±0.96)%. Compared with group OVA, the expression of NF-kB p65 in three Oligs therapeutic groups were significantly decreased and got close to the expression level in normal control group.Summarise the above results, Oligs have inhibitory effects on the expression of inflammation proteins, receptors and transcription factors involved in asthma, and have potential functions for preventing and treating inflammation of asthma. 5 Effect of Oligs on cytokine conformationThe results of ELISA experiments of Oligs reacting with IL-4 and IL-5 showed that Oligs could bind to these cytokines. The secondary structure changes of the Oligs-binding cytokines were determined by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) technique. The results demonstrated that the secondary structures of IL-4 and IL-5 were changed after they binding to Oligs and the ratio of a-helix and (3-sheet conformation was different from original IL-4 and IL-5.In conclusion, the major achievements of this research are as following:(1) It is the first time reported the effects of Oligs on activities and expression of asthma-related inflammatory factors. The results showed that Oligs have inhibitory effects on secretion of IgE, and on expression of CD23 and NF-kB. It demonstrated that Oligs have activities on several links on the whole process of inflammation in asthma.(2) It is the first time determined the secondary structure changes ofOligs-binding IL-4 and IL-5 by ATR-FTIR techniques.