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Abstract In order to study the antiinfection effect of phoshporylated Agaricus blazei polysaccharide on mice， mice were drenched phoshporylated A. blazei polysaccharide for 14 d， and an A. blazei polysaccharide control group and a blank control group were also set. The mice in all the three groups were infected with Escherichia coli on the 15th day and the 29th day of the experiment， the body weight of the mice in each group was recorded， as well as the spleen index on the 29th day of infection and the 36th day of the experiment， and the antiE. coli titer in serum was also detected. The results showed that phoshporylated A. blazei polysaccharide could promote and maintain the development of spleen， and improve the immune response of organisms， thereby resisting bacterial infection. Furthermore， phoshporylated A. blazei polysaccharide has better immunoregulation and antiinfection effects than A. blazei polysaccharide， and could play an important role in veterinary clinical treatment and prevention and control of diseases as a immunologic adjuvant or immunopotentiator.
Key words Polysaccharide from Agaricus blazei Murrill； Phosphorylation； antiinfection effect； Mouse； Escherichia coli； body weight； Spleen index； ELISA
Agaricus blazei Murrill （ABM）， belonging to Agaricus in Agaricaceae family of Agaricales in Hymenomycetes of Basidiomycotina， is a kind of fungus used as both food and medicine[1]. A. blazei has good taste， and the polysaccharide extracted from it has miraculous effects in regulating immune function， enhancing immunity， resisting tumor and oxidation， reducing blood fat and preventing diabetes[2-5]. Researches show that polysaccharides have their biological activity changed with the change of spatial structure after molecular modification， and the biological activity could be enhanced， or they could be endowed with more biological activity[6]. Phosphorylated polysaccharides as a type of important polysaccharide derivatives have various kinds of biological activity including immunoregulation， antiviral and antitumor activity. Nagasawa et al.[7]found that after phosphorylation， glucan could promote the mitosis of mouse splenocytes， enhance the expression of CD86 and CD69 on B lymphocyte and dendritic cell surfaces， and promote the secretion of IL10 as well. Liu et al.[8]found that levan has remarkably improved antiherpes virus activity after phosphorylation. Huang et al.[9]performed phosphorylation modification on waterinsoluble polysaccharide （13）αDglucan， and found that its antitumor activity was improved significantly. In this study， an antiinfection experiment was carried out to illustrate the antibacterial infection effect of phoshporylated A. blazei polysaccharide on mice and discuss its action mechanism and protecting effect， and the antiinfection effect of phoshporylated A. blazei polysaccharide was compared with that of A. blazei polysaccharide， so as to expound the immunoregulation effect of A. blazei polysaccharide. This study will provide reference data for clinical application and further study.
Materials
Experimental animals
Sixty Kunming mice （half male half female） were purchased from the Experimental Animal Center of Tianjin Medical University. The mice were 5-8 weeks old， and had a body weight of （20±2） g. They were supplied with sufficient fodder， and unlimited water. The mice were raised in different cages at room temperature under suitable humidity.
Main reagents
Polysaccharide of A. blazei （purity of 48.63）， purchased from Shanghai Kangzhou Fungal Polysaccharide Co.， Ltd.； phosphorylated A. blazei polysaccharide， synthesized by Preventive Veterinary Medicine Laboratory of Tianjin Agricultural College， with phosphate group content of 13.99%， the graft quantity of phosphate of 9.14% and polysaccharide content of 74.28%； HRPGoat antimouse IgG， purchased from Shanghai Beizhuo Biotechnology Co.， Ltd.； skim milk powder， purchased from America BD company； TMB （PR1200）， purchased from Shanghai Yisheng Biotechnology Co.， Ltd.
Phosphate buffer （PBS）： NaCl 8.0 g， KH2PO4 0.2 g， KCl 0.2 g and Na2HPO4·12H2O 3.65 g were added with distilled water to 1 L. The solution was mixing well， and adjusted to pH of 7.4. Finally， the solution was subpackaged， subjected to sutoclaved sterilization and preserved at room temperature.
Coating buffer： Na2CO3 1.59 g and NaHCO3 2.93 g were added with deionized water to 1 L， mixed well and adjusted to pH of 9.6. The solution was subpacged and preserved at 4 ℃.
Cleaning solution （PBST）： The solution was preprared by adding 0.5 ml of Tween20 into 1 L of PBS buffer.
Blocking solution： The blocking solution was prepared by adding 5 g of skim milk powder into 100 ml of PBS buffer.
Antibody diluent： The antibody diluent was prepared by adding 2 g of skim milk powder into 100 ml of PBS buffer.
Stopping solution： The stopping solution was prepared by adding 11 ml of concentrated sulfuric acid into 89 ml of deionized water， followed by mixing well.
Strain Escherichia coli was preserved by the Basic Veterinary Medicine Laboratory， Tianjin Agricultural College.
Main instruments
Microplate Reader （type 680）， purchased from BioRad Laboratories； electroheating standingtemperature cultivator （type DMP9272）， purchased from Shanghai Precision Instruments Co.， Ltd.； centrifuge （type LW250A）， purchased from Shanghai Medical Instruments Factory； aseptic operation table （type SWCJ1D）， purchased from Jiangsu Tongjing Purification Equipment Co.， Ltd.； biochemical incubator （type 2500）， purchased from Changzhou Aohua Instruments Co.， Ltd.； medical vertical refrigerator （type DW40L262）， purchased from Qingdao Haier Co.， Ltd.； ultralow temperature refrigerator （type 902ULTS）， purchased from Thermo Fisher Scientific Inc.； electronic analytical balance （type BS124S）， purchased from Sartorius Scientific Instruments （Beijing） Co.， Ltd.； oscillator （type ZWA）， purchased from Jintan Longsheng Experimental Instruments Factory； pipettor （type 720005）， purchased from Biohit Biotech （Suzhou） Co.， Ltd.； clean bench （type BHC1300IIA2）， purchased from Suzhou Sujie Purifying Equipment Co.， Ltd.
Methods
Grouping and treatments of experimental animals
The mice were fed adaptively for 2 d and randomly divided into three groups， each of which included 18 mice. The second group was drenched A. blazei polysaccharide solution at a dose of 100 mg/kg， and the third group was drenched A. blazei polysaccharide solution at a dose of 100 mg/kg， for 14 d continuously. Meanwhile， the first group was drenched equal amount of normal saline. The body change in each group was recorded. According to the method in reference[10]， on the 15th d， each group was drenched E. coil， according to 0.2 ml/mouse， 1×108 cfu/ml. The resistance and morbidity of mice in each group after the infection with E. coil were observed， the body weight of mice in each group was recorded， and such symptoms as depression， reduction of activity level， reduction of body weight and diarrhea occurring in the first group were selected as detection indices. On the 29th day， 9 mice were selected from each group and subjected to blood collection and anatomy， during which organ diseases were observed， and organs were weighed， followed by calculation of organ index. Mice remaining in each group were drenched E. coli， according to 0.2 ml/mouse， 1×108 cfu/ml. The resistance and morbidity of mice in each group after the infection with E. coli were observed， the body weight change of mice in each group was recorded， and such symptoms as depression， reduction of activity level， reduction of body weight and diarrhea occurring in the first group were selected as detection indices. On the 36th day， 9 mice were taken from each group and subjected to blood collection and anatomy， during which organ diseases were observed， and organs were weighed， followed by calculation of organ index. The collected blood were separated for serum， which was preserved at -20 ℃ for later use. Detection of antiE. coli IgG titer in serum
E. coli holoantigen was diluted with the coating buffer to 1 μg/ml， added into ELISA reaction plate according to 100 μl/hole， and preserved at 4 ℃ overnight. After removal of coating buffer， the holes were washed with the cleaning solution for three times， and during washing， the reaction plate was placed on a 96hole plate oscillator and washed for 5 min at 600 r/min. The blocking solution was added into the ELISA reaction plate according to 300 μl/hole， and the plate was placed into a wet box for incubation at 37 ℃ for 2 h； and the washing step was the same as above. The tobedetected serum was diluted with antibody diluent at a ratio of 1∶100. Diluted serum was then added into the ELISA plate， according to 100 μl/hole， and the plate was placed into a wet box for incubation at 37 ℃ for 1 h. Meanwhile， negative positive was also set. And washing was performed for four times， and the step was the same as above. Into the ELISA plate， developing solution was added according to 100 μl/hole， followed by light reaction for 15 min. The stopping solution was added into the ELISA plate according to 50 μl/hole. The ELISA plate was added into the microplate reader， to read the absorance at 450 and 630 nm. When the OD value of an experimental hole is two times of that of the control hole， it is determinated as positive.
Statistical analysis
The experimental data were treated with SPSS 11.5， and plotting was performed with Graphad Prism software.
It could be seen from Fig. 1 that from the first day of the experiment， the body weight of mice in each group was on the increase， the average body weight of mice in the third group was higher than that of the second group， and the average body weight of mice in the second group was higher than the first group， indicating that the phoshporylated A. blazei polysaccharide had the gaining effect better than A. blazei polysaccharide. After the infection with E. coli， i.e.， on the 16th day and the 30th day of the experiment， the body weight of mice in each group decreased slightly and then increased remarkably， and among the various groups， the body weight restoration effect of the third group was better than those of other two groups， indicating that phoshporylated A. blazei polysaccharide had antiE. coli infection effect than A. blazei polysaccharide in mice.
Organ indices
The results are shown in Fig. 2.
It could be seen from Fig. 2 that on the 14th d after the first infection， i.e.， on the 29th day of the experiment， there was a significant difference in spleen index between the first group and the second group （P<0.05）， and the first group was significantly different from the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide and A. blazei polysaccharide both could promote the growth and development of the spleen. On the 7th day after the second infection， i.e.， on the 36th day of the experiment， there were significant differences between the first group and the third group and between the seond group and the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide could better promote the growth and development of the spleen than A. blazei polysaccharide. Determination of antiE. coli IgG titer in serum
The results are shown in Fig. 3.
It could be seen from Fig. 3 that on the 14th day after the first infection， i.e.， on the 29th day of the experiment， there were significant differences in antiE. coli IgG titer between the first group and the third group and between the second group and the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide could improve the antiE. coli IgG titer in the serum of the mice infected for the first time and enhance the humoral immune response of organism； and on the 7th day after the second infection， i.e.， on the 36th day of the experiment， there was a significant difference in antiE. coli IgG titer between the first group and the second group （P<0.05）， there was a very significant difference between the first group and the third group （P<0.01）， and there was a significant difference between the second group and the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide and A. blazei polysaccharide could improve the antiE. coli IgG titer in the serum of the mice infected for the second time， and phoshporylated A. blazei polysaccharide could better enhance the humoral immune response of organism than A. blazei polysaccharide.
Agricultural Biotechnology2018
Discussion
Spleen is an important peripheral immune organ， the proportion of which in body reflects the immunologic function of organisms to a certain degree and is an important detection index. In this study， after the first time of E. coli infection， phoshporylated A. blazei polysaccharide and A. blazei polysaccharide both could promote growth and development of spleen， thereby improving the antiE. coli infection ability of organisms， which was reflected by the rapider recovery of the body weight of mice in the second group and the third group after the infection. After the second time of E. coli infection， phoshporylated A. blazei polysaccharide had better effect of promoting and maintaining growth and development of spleen than A. blazei polysaccharide， thereby significantly improving the ability of resisting the second time of E. coli infection in organisms， which was reflected by the rapider recovery of body weight of mice in the third group than the first group and the second group.
IgG is a kind of important immunological competence synthesized and secreted by plasmocytes in lymph node and spleen， which is the main component of antibodies and plays a role of protecting organisms in resisting pathogen infection， and the detection of its content is also an important index for the evaluation of humoral immune response. In this study， phoshporylated A. blazei polysaccharide could significantly improve the spleen index of mice and enhance the immune response of organisms， so phoshporylated A. blazei polysaccharide could significantly improve the antiE. coli IgG titer in mouse serum in the first time of infection， and promote the production of humoral immune response， while A. blazei polysaccharide showed no significant effect of improving the antiE. coli IgG titer in mouse serum in the first time of infection. In the second time of infection， phoshporylated A. blazei polysaccharide had a better effect of promoting and maintaining the growth and development of spleen than A. blazei polysaccharide， as well as a better effect of improving the antiE. coli IgG titer in mouse serum in the second time of infection than A. blazei polysaccharide. The research shows that the immunoregulation activity of phosphorylated polysaccharide is related to the absorption of Ca+[11]. Ca+ is the first signal triggering lymphocyte proliferation response， and phosphorylated polysaccharide could inhibited the formation of Ca3（PO4）2 precipitate， thereby facilitating the binding of Ca+ and Ca+ vector A23187， promoting Ca+ influx and stimulating proliferation of B lymphocytes and T lymphocytes[12-13]. Currently， detailed immunoregulation mechanisms of phosphorylated polysaccharide is still not clear and needs further exploration. To sum up， phoshporylated A. blazei polysaccharide could promote and maintain the development of spleen， and improve the immune response of organisms， thereby resisting bacterial infection. Furthermore， phoshporylated A. blazei polysaccharide has better immunoregulation and antiinfection effects than A. blazei polysaccharide， and could play an important role in veterinary clinical treatment and prevention and control of diseases as a immunologic adjuvant or immunopotentiator.
References
[1]GAO PY， MA JF. Chronic toxicity experiment of compound Agaricus blazei Murill Polysaccharide Oral Liquid in Rats[J]. China Animal Husbandry and Veterinary Medicine， 2014， 41（1）： 146-149. （in Chinese）
[2]CUI LR， LI HJ， WANG HY， et al. Study on Agaricus blazei polysaccharide in promoting OVA antigen immunoreaction[J]. Medical Innovation of China， 2014， 11（36）： 18-20. （in Chinese）
[3]GYORFI J， GEOSEL A， KISS M， et al. Gas chromatographymass spectrometry confirmation of the sensory scent features of the most commonly consumed Agaricus bisporus and Agaricus subrufescens exhibiting anticancerous traits[J]. J Med Food， 2013， 16（2）：167-175.
[4]FANTUZZI E， ANASTACIO L R， NICOLI J R， et al. Evaluation of royal sun Agaricus， Agaricus brasiliensis S. Wasser et al.， aqueous extract in mice challenged with Salmonella enterica serovar Typhimurium[J]. Int J Med Mushrooms， 2011， 13（3）：281-288.
[5]LYU J， BAI F， CAO LX. Effects of Agaricus blazei Murrill polysaccharide on serum inflammatory factor and immunologic function of diabetic rats[J]. Chinese Journal of Gerontology， 2016， 36（1）：44-46. （in Chinese）
[6]GAO HJ， YANG YG， AN L， et al. Research progress in molecular modification and pharmacological activity of Chinese materia medica polysaccharides[J]. Chinese Traditional and Herbal Drugs， 2015， 46（7）： 1074-1080. （in Chinese）
[7]NAGASAWA C， NISHIMURA UJ， TOHNO M， et al. Oral administration of phosphorylated dextran regulates immune response in ovalbuminimmunized mice[J]. Asian Austral J Anim， 2010， 23（1）： 106-115. [8]LIU XX， WAN ZJ， SHI L， et al. Preparation and antiherpetic activities of chemically modified polysaccharides from Polygonatum cyrtonema Hua[J]. Carbohydr Polym， 2011， 83（2）： 737-742.
[9]HUANG QL， ZHANG LN. Preparation， chain conformation and antitumor activities of watersoluble phosphated （13）αDglucan from Poria cocos mycelia[J]. Carbohydr Polym， 2011， 83（3）： 1363-1369.
[10]HAN X， XU Y， XIA XZ， et al. Comparative study of different mixture ratio of compound ethyl pyruvate suspension on model of bacterial infection[J]. China Animal Husbandry and Veterinary Medicine， 2014， 41（9）： 216-220. （in Chinese）
[11]OTANI H， SAKAKIBARA I， AOKI T. Immunomodulatory effects of phosphorylated dextrin in mouse spleen cell cultures[J]. J Nutr Sci Vitaminol， 2007， 53（4）： 349-353.
[12]NAKANO T， SALVADOR AS， TAMOCHI J， et al. Phosphorylation of starch and dextrin by dryheating in the presence of phosphate， and their calcium phosphatesolubilizing ability[J]. Nahrung， 2003， 47（4）： 274-278.
[13]TOYOSHIMA S， IWATA M， OSAWA T. Kinetics of lymphocyte stimulation by concanavalin A[J]. Nature， 1976， 264： 447-449.
Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU
Key words Polysaccharide from Agaricus blazei Murrill； Phosphorylation； antiinfection effect； Mouse； Escherichia coli； body weight； Spleen index； ELISA
Agaricus blazei Murrill （ABM）， belonging to Agaricus in Agaricaceae family of Agaricales in Hymenomycetes of Basidiomycotina， is a kind of fungus used as both food and medicine[1]. A. blazei has good taste， and the polysaccharide extracted from it has miraculous effects in regulating immune function， enhancing immunity， resisting tumor and oxidation， reducing blood fat and preventing diabetes[2-5]. Researches show that polysaccharides have their biological activity changed with the change of spatial structure after molecular modification， and the biological activity could be enhanced， or they could be endowed with more biological activity[6]. Phosphorylated polysaccharides as a type of important polysaccharide derivatives have various kinds of biological activity including immunoregulation， antiviral and antitumor activity. Nagasawa et al.[7]found that after phosphorylation， glucan could promote the mitosis of mouse splenocytes， enhance the expression of CD86 and CD69 on B lymphocyte and dendritic cell surfaces， and promote the secretion of IL10 as well. Liu et al.[8]found that levan has remarkably improved antiherpes virus activity after phosphorylation. Huang et al.[9]performed phosphorylation modification on waterinsoluble polysaccharide （13）αDglucan， and found that its antitumor activity was improved significantly. In this study， an antiinfection experiment was carried out to illustrate the antibacterial infection effect of phoshporylated A. blazei polysaccharide on mice and discuss its action mechanism and protecting effect， and the antiinfection effect of phoshporylated A. blazei polysaccharide was compared with that of A. blazei polysaccharide， so as to expound the immunoregulation effect of A. blazei polysaccharide. This study will provide reference data for clinical application and further study.
Materials
Experimental animals
Sixty Kunming mice （half male half female） were purchased from the Experimental Animal Center of Tianjin Medical University. The mice were 5-8 weeks old， and had a body weight of （20±2） g. They were supplied with sufficient fodder， and unlimited water. The mice were raised in different cages at room temperature under suitable humidity.
Main reagents
Polysaccharide of A. blazei （purity of 48.63）， purchased from Shanghai Kangzhou Fungal Polysaccharide Co.， Ltd.； phosphorylated A. blazei polysaccharide， synthesized by Preventive Veterinary Medicine Laboratory of Tianjin Agricultural College， with phosphate group content of 13.99%， the graft quantity of phosphate of 9.14% and polysaccharide content of 74.28%； HRPGoat antimouse IgG， purchased from Shanghai Beizhuo Biotechnology Co.， Ltd.； skim milk powder， purchased from America BD company； TMB （PR1200）， purchased from Shanghai Yisheng Biotechnology Co.， Ltd.
Phosphate buffer （PBS）： NaCl 8.0 g， KH2PO4 0.2 g， KCl 0.2 g and Na2HPO4·12H2O 3.65 g were added with distilled water to 1 L. The solution was mixing well， and adjusted to pH of 7.4. Finally， the solution was subpackaged， subjected to sutoclaved sterilization and preserved at room temperature.
Coating buffer： Na2CO3 1.59 g and NaHCO3 2.93 g were added with deionized water to 1 L， mixed well and adjusted to pH of 9.6. The solution was subpacged and preserved at 4 ℃.
Cleaning solution （PBST）： The solution was preprared by adding 0.5 ml of Tween20 into 1 L of PBS buffer.
Blocking solution： The blocking solution was prepared by adding 5 g of skim milk powder into 100 ml of PBS buffer.
Antibody diluent： The antibody diluent was prepared by adding 2 g of skim milk powder into 100 ml of PBS buffer.
Stopping solution： The stopping solution was prepared by adding 11 ml of concentrated sulfuric acid into 89 ml of deionized water， followed by mixing well.
Strain Escherichia coli was preserved by the Basic Veterinary Medicine Laboratory， Tianjin Agricultural College.
Main instruments
Microplate Reader （type 680）， purchased from BioRad Laboratories； electroheating standingtemperature cultivator （type DMP9272）， purchased from Shanghai Precision Instruments Co.， Ltd.； centrifuge （type LW250A）， purchased from Shanghai Medical Instruments Factory； aseptic operation table （type SWCJ1D）， purchased from Jiangsu Tongjing Purification Equipment Co.， Ltd.； biochemical incubator （type 2500）， purchased from Changzhou Aohua Instruments Co.， Ltd.； medical vertical refrigerator （type DW40L262）， purchased from Qingdao Haier Co.， Ltd.； ultralow temperature refrigerator （type 902ULTS）， purchased from Thermo Fisher Scientific Inc.； electronic analytical balance （type BS124S）， purchased from Sartorius Scientific Instruments （Beijing） Co.， Ltd.； oscillator （type ZWA）， purchased from Jintan Longsheng Experimental Instruments Factory； pipettor （type 720005）， purchased from Biohit Biotech （Suzhou） Co.， Ltd.； clean bench （type BHC1300IIA2）， purchased from Suzhou Sujie Purifying Equipment Co.， Ltd.
Methods
Grouping and treatments of experimental animals
The mice were fed adaptively for 2 d and randomly divided into three groups， each of which included 18 mice. The second group was drenched A. blazei polysaccharide solution at a dose of 100 mg/kg， and the third group was drenched A. blazei polysaccharide solution at a dose of 100 mg/kg， for 14 d continuously. Meanwhile， the first group was drenched equal amount of normal saline. The body change in each group was recorded. According to the method in reference[10]， on the 15th d， each group was drenched E. coil， according to 0.2 ml/mouse， 1×108 cfu/ml. The resistance and morbidity of mice in each group after the infection with E. coil were observed， the body weight of mice in each group was recorded， and such symptoms as depression， reduction of activity level， reduction of body weight and diarrhea occurring in the first group were selected as detection indices. On the 29th day， 9 mice were selected from each group and subjected to blood collection and anatomy， during which organ diseases were observed， and organs were weighed， followed by calculation of organ index. Mice remaining in each group were drenched E. coli， according to 0.2 ml/mouse， 1×108 cfu/ml. The resistance and morbidity of mice in each group after the infection with E. coli were observed， the body weight change of mice in each group was recorded， and such symptoms as depression， reduction of activity level， reduction of body weight and diarrhea occurring in the first group were selected as detection indices. On the 36th day， 9 mice were taken from each group and subjected to blood collection and anatomy， during which organ diseases were observed， and organs were weighed， followed by calculation of organ index. The collected blood were separated for serum， which was preserved at -20 ℃ for later use. Detection of antiE. coli IgG titer in serum
E. coli holoantigen was diluted with the coating buffer to 1 μg/ml， added into ELISA reaction plate according to 100 μl/hole， and preserved at 4 ℃ overnight. After removal of coating buffer， the holes were washed with the cleaning solution for three times， and during washing， the reaction plate was placed on a 96hole plate oscillator and washed for 5 min at 600 r/min. The blocking solution was added into the ELISA reaction plate according to 300 μl/hole， and the plate was placed into a wet box for incubation at 37 ℃ for 2 h； and the washing step was the same as above. The tobedetected serum was diluted with antibody diluent at a ratio of 1∶100. Diluted serum was then added into the ELISA plate， according to 100 μl/hole， and the plate was placed into a wet box for incubation at 37 ℃ for 1 h. Meanwhile， negative positive was also set. And washing was performed for four times， and the step was the same as above. Into the ELISA plate， developing solution was added according to 100 μl/hole， followed by light reaction for 15 min. The stopping solution was added into the ELISA plate according to 50 μl/hole. The ELISA plate was added into the microplate reader， to read the absorance at 450 and 630 nm. When the OD value of an experimental hole is two times of that of the control hole， it is determinated as positive.
Statistical analysis
The experimental data were treated with SPSS 11.5， and plotting was performed with Graphad Prism software.
It could be seen from Fig. 1 that from the first day of the experiment， the body weight of mice in each group was on the increase， the average body weight of mice in the third group was higher than that of the second group， and the average body weight of mice in the second group was higher than the first group， indicating that the phoshporylated A. blazei polysaccharide had the gaining effect better than A. blazei polysaccharide. After the infection with E. coli， i.e.， on the 16th day and the 30th day of the experiment， the body weight of mice in each group decreased slightly and then increased remarkably， and among the various groups， the body weight restoration effect of the third group was better than those of other two groups， indicating that phoshporylated A. blazei polysaccharide had antiE. coli infection effect than A. blazei polysaccharide in mice.
Organ indices
The results are shown in Fig. 2.
It could be seen from Fig. 2 that on the 14th d after the first infection， i.e.， on the 29th day of the experiment， there was a significant difference in spleen index between the first group and the second group （P<0.05）， and the first group was significantly different from the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide and A. blazei polysaccharide both could promote the growth and development of the spleen. On the 7th day after the second infection， i.e.， on the 36th day of the experiment， there were significant differences between the first group and the third group and between the seond group and the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide could better promote the growth and development of the spleen than A. blazei polysaccharide. Determination of antiE. coli IgG titer in serum
The results are shown in Fig. 3.
It could be seen from Fig. 3 that on the 14th day after the first infection， i.e.， on the 29th day of the experiment， there were significant differences in antiE. coli IgG titer between the first group and the third group and between the second group and the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide could improve the antiE. coli IgG titer in the serum of the mice infected for the first time and enhance the humoral immune response of organism； and on the 7th day after the second infection， i.e.， on the 36th day of the experiment， there was a significant difference in antiE. coli IgG titer between the first group and the second group （P<0.05）， there was a very significant difference between the first group and the third group （P<0.01）， and there was a significant difference between the second group and the third group （P<0.05）， indicating that phoshporylated A. blazei polysaccharide and A. blazei polysaccharide could improve the antiE. coli IgG titer in the serum of the mice infected for the second time， and phoshporylated A. blazei polysaccharide could better enhance the humoral immune response of organism than A. blazei polysaccharide.
Agricultural Biotechnology2018
Discussion
Spleen is an important peripheral immune organ， the proportion of which in body reflects the immunologic function of organisms to a certain degree and is an important detection index. In this study， after the first time of E. coli infection， phoshporylated A. blazei polysaccharide and A. blazei polysaccharide both could promote growth and development of spleen， thereby improving the antiE. coli infection ability of organisms， which was reflected by the rapider recovery of the body weight of mice in the second group and the third group after the infection. After the second time of E. coli infection， phoshporylated A. blazei polysaccharide had better effect of promoting and maintaining growth and development of spleen than A. blazei polysaccharide， thereby significantly improving the ability of resisting the second time of E. coli infection in organisms， which was reflected by the rapider recovery of body weight of mice in the third group than the first group and the second group.
IgG is a kind of important immunological competence synthesized and secreted by plasmocytes in lymph node and spleen， which is the main component of antibodies and plays a role of protecting organisms in resisting pathogen infection， and the detection of its content is also an important index for the evaluation of humoral immune response. In this study， phoshporylated A. blazei polysaccharide could significantly improve the spleen index of mice and enhance the immune response of organisms， so phoshporylated A. blazei polysaccharide could significantly improve the antiE. coli IgG titer in mouse serum in the first time of infection， and promote the production of humoral immune response， while A. blazei polysaccharide showed no significant effect of improving the antiE. coli IgG titer in mouse serum in the first time of infection. In the second time of infection， phoshporylated A. blazei polysaccharide had a better effect of promoting and maintaining the growth and development of spleen than A. blazei polysaccharide， as well as a better effect of improving the antiE. coli IgG titer in mouse serum in the second time of infection than A. blazei polysaccharide. The research shows that the immunoregulation activity of phosphorylated polysaccharide is related to the absorption of Ca+[11]. Ca+ is the first signal triggering lymphocyte proliferation response， and phosphorylated polysaccharide could inhibited the formation of Ca3（PO4）2 precipitate， thereby facilitating the binding of Ca+ and Ca+ vector A23187， promoting Ca+ influx and stimulating proliferation of B lymphocytes and T lymphocytes[12-13]. Currently， detailed immunoregulation mechanisms of phosphorylated polysaccharide is still not clear and needs further exploration. To sum up， phoshporylated A. blazei polysaccharide could promote and maintain the development of spleen， and improve the immune response of organisms， thereby resisting bacterial infection. Furthermore， phoshporylated A. blazei polysaccharide has better immunoregulation and antiinfection effects than A. blazei polysaccharide， and could play an important role in veterinary clinical treatment and prevention and control of diseases as a immunologic adjuvant or immunopotentiator.
References
[1]GAO PY， MA JF. Chronic toxicity experiment of compound Agaricus blazei Murill Polysaccharide Oral Liquid in Rats[J]. China Animal Husbandry and Veterinary Medicine， 2014， 41（1）： 146-149. （in Chinese）
[2]CUI LR， LI HJ， WANG HY， et al. Study on Agaricus blazei polysaccharide in promoting OVA antigen immunoreaction[J]. Medical Innovation of China， 2014， 11（36）： 18-20. （in Chinese）
[3]GYORFI J， GEOSEL A， KISS M， et al. Gas chromatographymass spectrometry confirmation of the sensory scent features of the most commonly consumed Agaricus bisporus and Agaricus subrufescens exhibiting anticancerous traits[J]. J Med Food， 2013， 16（2）：167-175.
[4]FANTUZZI E， ANASTACIO L R， NICOLI J R， et al. Evaluation of royal sun Agaricus， Agaricus brasiliensis S. Wasser et al.， aqueous extract in mice challenged with Salmonella enterica serovar Typhimurium[J]. Int J Med Mushrooms， 2011， 13（3）：281-288.
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Editor： Yingzhi GUANG Proofreader： Xinxiu ZHU