Comparision On Immune-enhancing And Antioxidant Activities Of Seven Selenizing Polysaccharide And Their Mechanism

Selenium polysaccharide is one of organic selenium compounds.It was easily absorbed and utilized by the organism and possessed duple and higher biological activity of polysaccharide and selenium,such as enhancing immunity,anti-oxidantion,anti-cancer,anti-metal poisoning and so on.However,natural polysaccharides are a far cry from meeting demands.In order to obtain more selenium polysaccharides,selenylation modificaton of natural polysaccharide can be performed.It is reported that there are many selenylation modification methods for polysaccharide such as nitric acid-sodium selenite(NA-SS)method,glacial acetic acid-selenite(GA-SA)method,glacial acetic acid-sodium selenite(GA-SS)method,selenium oxychloride(SOC)method and so on.Through our previous series of tests,seven selenizing polysaccharides(sCAPS2,sDOP2,SCPPS5,SEPS5,sGPS6,sLP6 and sAMP9),which have stronger immune enhancing activity and seven selenizing polysaccharides(sSCP1,sLP2,sCAPS2,sCCPS5,sLBP6,sAMP6 and sEPS7),which have stronger antioxidant activities were screened out,respectively.In this research,the four selenylation modification methods of polysaccharide were comparised firstly.NA-SS method was the best.Then,the immune-enhancing of seven selenizing polysaccharides and antioxidant activities of seven selenizing polysaccharides were further compared through the tests in vitro and vivo.sCCP5,which has the strongest immune enhancing activity and sLBP6,which has the strongest antioxidant activities and their optimum dose were screened,respectively.Effects of sCPPS5 on immune-related cytokine mRNA expression and effects of sCAPS2 on expression of MAPK signal pathway proteins were measured,respectively.The purpose of this study is to research selenylation modification methods of polysaccharide,screen the best selenium polysaccharide with immune-enhancing and the best selenium polysaccharide with antioxidant activities and their mechanism,provide the materials for developing immunopotentiators and antioxidantors.The tests are divided into seven parts as follows:Experiment 1.Comparison of four selenylation modification methods of polysaccharide Garlic polysaccharide(GPS)was extracted by water decoction and ethanol precipitation method and purified by trichloroacetic acid method to eliminate protein,permeate through G-200 Sephadex column and freeze drying,the purified GPS were obtained.Garlic polysaccharide(GPS)was prepared and selenizingly modified respectively by Nitric acid-sodium selenite(NA-SS),Glacial acetic acid-selenous acid(GA-SA),Glacial acetic acid-sodium selenite(GA-SS)and selenium oxychloride(SOC)method,each according to L9(34)orthogonal design of three factors,ratio of selenic reagent to GPS,reaction temperature and time,at three levels to obtain nine selenizing GPSs(sGPSs),SGPS1-SGPS9.Their structures were identified,selenium and polysaccharide content were determined,product,selenium and polysaccharide yield were calculated.The results showed that GPS could be successfully modified by all of four methods,but the selenium yield of NA-SS method was the hightest,its polysaccharide yield was higher and procedure was simpler and more economic.At present,NA-SS method was the best way to selenizingly modify polysaccharide.Experiment 2.Comparison of immune-enhancing activities in vitro of seven selenizing polysaccarides The seven selenium polysaccarides sCAPS2?sDOP2?sCPPS5?SEPS5?sGPS6?sLP6 and sAMP9 were screened in our previous tests and modifier Na2SeO3 were diluted with R/MINI1640 at 11 concentrations were added into the cultured mouse spleen lymphocytes.Their safe concentrations were determined by MTT assay.They were diluted with RPMI-1640 at five concentrations were added into the cultured mouse spleen lymphocytes in single or simultaneous with PHA or LPS,respectively.The changes of the ellular A570 value and the lymphocyte proliferation rate were determined by the same method.The results showed that in single adding,the A570 values of all polysaccadides at 1-5 concentration groups were significantly larger than that of corresponding cell control group,the lymphocyte proliferation rates of sCAPS2?sCPPS5 and sGPS6 groups were significantly higher than other groups.In simultaneous adding with PHA,the A570 values of all polysaccharides at 1-5 concentration groups were significantly larger than that of corresponding PHA control group,the lymphocyte proliferation rates of sCPPS5 group were significantly higher than other groups,the following were sCAPS2 and sGPS6 groups.In simultaneous adding with LPS,the A570 values of all polysaccharides at 1-5 concentration groups were significantly larger than that of corresponding LPS control group,the lymphocyte proliferation rates of sCPPS5 group were significantly higher than other groups,the following is sGPS6 group.These results indicated that selenylation modification could enhance the immune-enhancing activity of different plant-derived polysaccharide,which was correlated with the selenium content and the carbohydrate content and the species of polysaccharide.In general evaluation,the actions of sCAPS2?sCPPS5 and sGPS6 were stronger.Experiment 3.Comparison of immune-enhancing activities in vivo of senven selenizing polysaccarides In Test 1,in order to verify the results about effects of seven kinds of selenium polysaccarides on mouse peripherallymphocyte proliferation in vitro.The adjuvant effects of seven selenium polysaccharide on immune response of mice vaccinated with OVA were comparied.Two hundred 6-week-old ICR female mice were divided randomly into 10 groups.The mice except blank control group were vaccinated with OVA vaccine,repeated vaccination after 2 weeks.At the same time as the first vaccination,the mouse in seven selenium polysaccharide groups were intramuscularly injected respectively 0.4 mL of sCAPS2,sDOP2,sCPPS5,sEPS5,sGPS6,sLP6,sAMP9 at concentration of 0.1 mg·mL-1,in selenium reagents Na2SeO3 control group,with 0.2 mL of Na2SeO3 at concentration of 10 ?g mL-1,in vaccination control and blank control group,with equal volume of physiological saline,once a day for three successive days.On 7,14,21 and 28 days after the first vaccination,the changes of serum IgG content and peripheral lymphocyte proliferation were determined.These results showed that all the seven selenium polysaccharides could enhance the immune-enhancing activity at different levels and sCCPS5?sCAPS2 and sGPS6 possessed the beter efficacy.In Test 2,In order to screen out the selenium polysaccharide with the strongest immune-enhancing activity s and its optimal dose,three selenium polysaccharide sCAPS2,sCPPS5 and sGPS6,which were screened out from test 1,at three concentration were further compared on immune response of mice vaccinated with OVA.Two hundred twenty 6-week-old ICR female mice were divided randomly into 11 groups.The mice except blank control group were vaccinated with OVA vaccine,repeated vaccination after 2 weeks.At the same time as the first vaccination,the mouse in seven selenium polysaccharide groups were intramuscularly injected respectively with 0.4 mL of sCAPS2,sCPPS5 and sGPS6 at concentrations of 0.05 mg·mL-1,0.1 mg·mL-1 and 0.15 mg·mL-1,respectively,in vaccination control and blank control group,with equal volume of physiological saline,once a day for three successive days.On 7,14,21 and 28 days after the first vaccination,the changes of serum IgG,IgM,IL-2,IL-4 and IFN-? were determined.The result showed that theimmunoglobulin and cytokines content of nine selenium polysaccharide groups at the most time points were significantly higher than those of corresponding immunization control group,and the sCCPS5M at the most time points were the highest.These results showed that nine selenium polysaccharide groups all have the stronger immune-enhancing activity and the middle dose of sCPPS5 possessed the strongest efficacy.Experiment 4.Comparison of antioxidant activities in vitro of senven selenizing polysaccarides The seven selenium polysaccarides sSCP1?sLP2?sCAPS2?sCCPS5?sLBP6?sAMP6?sEPS7 were screened in our previous tests and modifier Na2SeO3 were diluted with distilled water at five concentrations to determine their scavenging power of DPPH radical,hydroxyl radical and ABTS radical.The results showed that,at the same concentration,the antioxidant activities of selenium polysaccarides were significantly greater or greater than modifier Na2SeO3 The order of average hydroxyl radical clearance rate in selenium polysaccharide was sCAPS2>sLBP6>sCCPS5>sAMP6>sEPS7>sSCP1>sLP2>Na2SeO3,The order of average DPPH radical clearance rate in selenium polysaccharide was sCAPS2>sLBP6>SCCPS5>sAMP6>sSCP1>sEPS7>Na2SeO3>sLP2,The order of average ABTS radical clearance rate in selenium polysaccharide was sCAPS2>sLBP6>sAMP6>sCCPS5>sEPS7>sSCP1>sLP2>Na2SeO3.The results show that the antioxidant activities of seven selenium polysaccarides were higher than selenium reagent Na2SeO3 in vitro and there was a certain of dose-efficacy relationship.In general evaluation,the antioxidant activities of sCAPS2,sLBP6 and sAMP6 were better.Experiment 5.Comparison of antioxidant activities in vivo of senven selenizing polysaccarides In Testl,in order to verify the results about effects of seven kinds of selenium polysaccarides on antioxidant activity in vitro,the antioxidant activity of the seven selenium polysaccharides were comparied in vivo.Two hundred 6-week-old ICR female mice were divided randomly into 10 groups.The mice except blank control group were vaccinated with OVA vaccine,repeated vaccination after 2 weeks.At the same time as the first vaccination,the mouse in seven selenium polysaccharide groups were intramuscularly injected respectively 0.4 mL of sSCP1?sCAPS2?sLP2?sCCPS5?sAMP6?sLBP6?sEMP7 at concentration of 0.1 mg·mL-1,in selenium reagents Na2SeO3 control group,with 0.2 mL of Na2SeO3 at concentration of 10 ?g·mL-1,in vaccination control and blank control group,with equal volume of physiological saline,once a day for three 7,14,21 and 28 days after the first vaccination,the changes of serum T-AOC activity,GSH-Px activity,SOD activity and MDA content were determined.These results showed that all the seven selenium polysaccarides could enhance the antioxidant activity of mice at different levels and sCAPS2?sLBP6 and sAMP6 possessed the better efficacy.In Test 2,In order to screen out the selenium polysaccharide with the best antioxidant activity of selenium polysaccharides and its optimal dose,three selenium polysaccharides sCAPS2,sAMP6 and sLBP6,which were screened out from test 1,at three concentrations were further comparedon antioxidant activities.Two hundred twenty 6-week-old ICR female mice were divided randomly into 11 groups.The mice except blank control group were vaccinated with OVA vaccine,repeated vaccination after 2 weeks.At the same time as the first vaccination,the mouse in seven selenium polysaccharide groups were intramuscularly injected respectively with with 0.4 mL of CAPS2?sAMP6 and sLBP6 at concentrations of 0.05 mg·mL-1,0.1 mg·mL-1 and 0.15 mg·mL-1,respectively,in vaccination control and blank control group,with equal volume of physiological saline,once a day for three successive days.On 7,14,21 and 28 days after the first vaccination,the changes of serum T-AOC,GSH-Px,SOD and MDA were determined.The result showed that,the activities of T-AOC,GSH-Px and SOD of nine selenium polysaccharide groups at every time points were higher or significantly higher than those of corresponding immunization control group and the value of MDA of nine selenium polysaccharide groups at every time points were lower or significantly lower than that of corresponding immunization control group,the activitives of sLBP6H were the best.These results indicated that,the three selenium polysaccharides at three dose all have the stronger the antioxidant activity,and the high dose of sLBP6 possessed the strongest efficacy.Experiment 6.Effects of sCPPSs on mRNA expression of IL-2,IL-4 and IFN-y in mice splenic lymphocyte In order to study the action mechanism of selenium polysaccharide in immune-enhancing actions,the selenium polysaccharides sCPPS5 on expression of IL-2?IL-4 and IFN-y mRNA of mice spleen lymphocyte was determined,taking non-modiified CPPS as control.The mice spleen lymphocyte were cultivated in adding sCPPS5 and CPPS at three concentrations(3.125?1.563?0.781 ?g·ml-1).After cultivation of 44 h,the cells were collected and total RNA was extracted.The expression of IL-2?IL-4 and IFN-? mRNA was determined by fluorescence quantitative RT-PCR assay.IL-2 mRNA expression of sCPPS5 at 3.125 mg·mL-1 was significantly stronger than that of unmodified CPPS.IFN-y mRNA expression of sCPPS5 at 3.125 ?g·mL-1 and 1.563 ?g·mL-1 were significantly stronger than that of unmodified CPPS.IL-4 mRNA expression of sCPPS5 at 3.125 ?g·ML-1 and 1.563 ?g·mL-1 were significantly stronger than that of unmodified CPPS.The results showed that sCPPS5 at suitabal content could promote the expression of IL-2?IL-4 and IFN-? mRNA that could be one of mechanisms of immune-enhancing.Experiment 7.Anti-hepatic injury and effects of sCAPS2 on expression of MAPK signal pathway proteins In order to study the actionmechanism of selenium polysaccharide in antioxidant activity,the effect against CC14-induced hepatotoxic mice and the effect of protein expression in the MAPK pathway of sCAPS2 were determined.Sixty four 6-week-old ICR female mice were divided randomly into 8 groups.64 mice were randomly divided into 8 groups,sCAPS2 The mice in 6 polysaccharide groups were subcutaneously injected with sCAPS2 and CAPS solutions at 0.05 mg·mL-1,0.1 mg-mL-land 0.15 mg·mL-1 three concentrations..The mice in control group and model group were subcutaneously injected the equal amount normal saline.All the groups were injected once a day for 7 successive.Two hours after the final administration,mice in all groups except model group were injected intraperitoneally with 0.3%(v/v)CCl4 0.25 mL,while the mice in normal group received appropriate vehicle.Twenty-four hours after the CCl4 challenge followed by fasting,the animals were anesthetized for obtaining the blood and sacrificed to collect the livers.The blood and liver were sampled for determination of biochemical indicators TP,ALT,AST and ALP in serum,antioxidative indicatorsT-AOC,MDA,SOD and ROS in liver homogenate,histopathological evaluation in liver and Western blot was used to analysis the protein expression of MAPK pathway in liver.The result showed that sCAPS2 could significantly reduce the contents of ALT,AST and ALP in serum,and also significantly reduce the activities of MDA and ROS and promote the activities of SOD and T-AOC in liver homogenate,the effect of sCAPS2 was stronger than that of CAPS.In the model group,there were obvious pathological changes in the liver,while in sCAPS2 group were near normal.Western blot analysis showed that p-ERK,p-JNK,p-p38 protein expression in the liver of CC14-treated model mouse were significantly elevated when compared with the normal control.sCAPS2 could significantly inhibited the protein expression of p-ERK,p-JNKand p-p38.The result indicated that,sCAPS2 could obviously protect mice against CC14-induced hepatic dysfunction and histopathologic damage,and it was stonger than CAPS.Ameliorates CCl4-induced liver damage following sCAPS2 pretreatment is associated with suppression the protein expression of p-ERK,p-JNK and p-p38 of MAPK pathway,that could be one of mechanisms of antioxidant activity.