Structure Identification And Immunoregulatory Activity Of Polysaccharide From Pyrus Sinkiangensis Yu. And Its Mechanism Of Anti Airway Inflammation

Objective:1.To study the optimum extraction technology of Pyrus sinkiangensis Yu.polysaccharide.2.The polysaccharides of Pyrus sinkiangensis Yu.were separated and the structure of homogeneous polysaccharide of Pyrus sinkiangensis Yu.were analyzed.3.To study the antioxidant activity of the polysaccharides.4.The in vitro immune activity of the isolated polysaccharide was studied to search for the active components.5.To study the immune activity of the acidic polysaccharide from Pyrus sinkiangensis Yu.in vivo.6.To study the therapeutic effect and mechanism of Pyrus sinkiangensis Yu.From acid polysaccharide on asthma airway inflammation in mice.7.To study the antitussive,antiasthmatic and expectorant effects of Pyrus sinkiangensis Yu.polysaccharide.Methods:1.The extraction of Pyrus sinkiangensis Yu.polysaccharide was carried out by water extraction and alcohol precipitation method.The extraction temperature,extraction time and ratio of material to liquid were investigated by single factor.2.The Pyrus sinkiangensis Yu.polysaccharide was separated and purified by a variety of chromatographic columns,and the homogeneous polysaccharide was characterized and analyzed by a variety of spectroscopic and chromatographic methods.3.The antioxidant activity of four homogeneous polysaccharides was evaluated by detecting the reactions of DPPH·free radical,hydroxyl free radical,superoxide free radical,ABTS+and iron reduction capacity respectively.4.RAW264.7 macrophage was selected as the intervention object of polysaccharide,and cell proliferation activity,secretion of cytokines such as NO,TNF-?and IL-6 were detected.5.In vivo immune activity of polysaccharides from Pyrus sinkiangensis Yu.the organ index,NK cell killing ability,lymphocyte proliferation activity,serum IL-2,IFN-?,TNF-?,IL-6,NF-?B,TLR4,AP-1 and NO levels in immunosuppressed mice were studyed.6.The content of IL-4,IL-5,IL-13,classification and number of inflammatory cells in BALF were studyed together with the help of HE,AB-PAS staining to observe the pathological changes in lung tissue in mice,and the immune histochemical method to detect NF-?B p65,I?B?and IKK protein expression in the lung tissue of mice,and WB to detect p-ERK,p-JNK,p-p38 MAPK protein expression in the lung tissue of mice,and PCR to detect Muc5ac m RNA expression in lung tissue.7.The cough relieving and expectorant abilities of Pyrus sinkiangensis Yu.polysaccharide were evaluated by ammonia water,SO₂cough inducing experiment and phenol red experiment to evaluate airway expectorant.Results:1.The influence of sequential extraction conditions on the extraction of Pyrus sinkiangensis Yu.polysaccharide was as follows:temperature>solid-liquid ratio>time,the optimal response surface analysis conditions were as follows:extraction temperature:70?,extraction time:95 min,solid-liquid ratio:1:60,the optimal extraction rate was12.46%.2.Four homogeneous components,namely neutral polysaccharides PSNP-1 and PSNP-2,acid polysaccharides PSAP-1 and PSAP-2,were isolated from the polysaccharide using DEAE-650M ion exchange column,Sepharose 6B and Sephacryl S-300 molecular sieve column.The apparent molecular weights of PSNP-1,PSNP-2,PSAP-1 and PSAP-2 were calculated by HPGPC column analysis as 104.7k Da,24.5k Da,64.6k Da and 38.9k Da,respectively.Results of monosaccharides composition analysis:PSNP-1 was mainly composed of Glc,Xyl and Gal at a ratio of 3:1.6:1,and also contained a small amount of Ara,Fuc and Man.PSNP-2 contained Glc,Xyl,Ara and Gal,with a composition ratio of 2.4:1:2.5:1.1,and a low content of Fuc and Man.These results suggest that PSNP-1 is mainly composed of xyloglucan and PSNP-2 is mainly composed of xyloglucan and arabinogalactan.Methylation analysis results:the main chain of PSNP-1 is mainly composed of 1,4-Glcp and 1,2-Xylp,with a ratio of about 4:1.PSNP-2 mainly consists of 1,5-Araf,1,3,3-Araf,1,3,5-Araf,and t-Araf,with a composition ratio of 10.6:1:7:7.IR spectra,¹H-,¹³C-NMR,HSQC and HMBC showed that the main chain of PSNP-1 was composed of t-Xylp,t-Araf,1,5-Araf,1,2-Galp,1,4,6-Glcp and 1,4-Glcp.There was a long linear 1,5-Araf main chain in the arabinoglycan region,and cytarabine branched at O-3 with a branching degree of 0.64.Except for the decrease of 1,5-Araf content and the increase of 1,4,6-Glcp content,PSNP-2 showed similar characteristics with the branching degree of 0.51.3.PSNP-1,PSNP-2,PSAP-1and PSAP-2 have different scavenging and reducing abilities to DPPH·free radicals,hydroxyl radicals,superoxide radicals,ABTS+and iron ions.4.Results of immune activity in vitro:PSNP-1,PSNP-2,PSAP-1 and PSAP-2 could stimulate cell proliferation to different degrees after the intervention of RAW264.7 macrophages,and increase the release of NO and the secretion of IL-6 and TNF-?.5.In vitro immunological activity results:The levels of organ index,NK cell killing ability,lymphocyte proliferative activity,serum IL-2,IFN-?,TNF-?,IL-6,NF-?B,TLR4,AP-1 and NO in immunosuppressed mice after the intervention of PSAP-1 and PSAP-2 had different degrees of influence.6.BALF test results:the levels of IL-4,IL-5 and IL-13 in airway inflammation mice were significantly reduced after the intervention of Pyrus sinkiangensis Yu.acid polysaccharide(P<0.05).The counts of macrophages,neutrophils,eosinophils and lymphocytes in mice with asthma airway inflammation were decreased to different degrees after the intervention of Pyrus sinkiangensis Yu.acid polysaccharide(P<0.05).Pathological results:The pulmonary histomorphology of mice treated with Pyrus sinkiangensis Yu.with high,medium and low dose of acid polysaccharide was improved to a certain extent compared with the model group,and the mucus secreted by goblet cells in the trachea was also reduced.Immunohistochemical results:The expression of the protein in lung tissue of mice treated with acid polysaccharide of Pyrus sinkiangensis Yu.were significantly decreased.WB results:The expression of P-ERK,P-P38 MAPK and P-JNK proteins in the lung tissue of mice treated with the acid polysaccharide of Pyrus sinkiangensis Yu.were all decreased to different degrees compared with the model group(P<0.05).PCR results:The expression of Muc5ac m RNA in the lung tissue of mice treated with the acid polysaccharide of Pyrus sinkiangensis Yu.was significantly lower than that in the model group(P<0.05).7.Cough induced by concentrated ammonia water and sulfur dioxide:The high,medium and low dose of acidic polysaccharide from Pyrus sinkiangensis Yu.increased the duration of cough and reduced the number of coughs compared with the model group,and the cough relieving effect was obvious.Expectorant test:The high,medium and low dose of the acid polysaccharide from Pyrus sinkiangensis Yu.increased the secretion of airway phenol red compared with the model group(P<0.05).Conclusion:1.The extraction yield of Pyrus sinkiangensis Yu.polysaccharide was high after response surface optimization,which could provide a reference for the extraction process of Pyrus sinkiangensis Yu.polysaccharide in the future.2.Thro?gh the structural identification of PSNP-1,PSNP-2,PSAP-1 and PSAP-2,the homogeneous polysaccharide components in Pyrus sinkiangensis Yu.were systematically analyzed and characterized for the first time,which provided a good basis for future studies on activity.3.PSNP-1,PSNP-2,PSAP-1and PSAP-2 all have certain antioxidant activity in vitro.4.In vitro studies on the intervention of RAW264.7 macrophages showed that all of the four components had certain immunomodulatory activities,which provided the research basis for their activities in animal experiments.5.The results of animal immune experiments of PSAP-1and PSAP-2 showed that they had certain immune activity in vivo,which laid a foundation for the subsequent verification of the therapeutic effect of Pyrus sinkiangensis Yu.polysaccharide on mice with asthma airway inflammation.6.The acidic polysaccharide may have a certain therapeutic effect on airway inflammation in mice by reducing the expression of NF-?B P65,P-P38 MAPK,P-ERK,P-JNK,IL-4,IL-5,and IL-13,and by interfering with the synthesis of NF-?B and MAPK signaling pathway and Muc5ac m RNA.7.The acidic polysaccharide have certain effect of relieving cough and eliminating phlegm,which can provide reference for the future development of drugs or health care products for related diseases.