Study On Anti-inflammatory Activities Of Astragalus Polysaccharides And Their Sulphation Modification Products In Vitro And In Vivo

Antibiotic was first used as growth-promoting additives; it can promote the growth rate ofanimals, increase the feed conversion ratio and prevent diseases. However, antibioticpossessed huge side effect, such as drug resistance and antibiotic residue in meat product. Soa great attention has put on the alternatives of antibiotic. Astragalus polysaccharide is themajor active ingredient of Chinese medical herb Astragalus membranaceus, and possesseswide range of bioactivities. The study is composed of four trails, including in vitro and in vivoexperiments to investigate the anti-inflammatory activity of APS and its sulfated derivatives,aiming to provide theoretical direction to antibiotic alternatives.Experiment1The objective of Experiment1was to determine the effect of APS administration timingand concentration on the expression of inflammatory cytokines and mucosa structuralintegrity in LPS-infected Caco2cells. APS (0,25,50,100and200μg/mL) and1μg/mL LPSwere added into the cells in the following three groups:1) the cells were stimulated with LPSfor24h and then treated with APS for24h (post-addition APS group),2) the cells werepre-treated with APS for24h and then stimulated with LPS for another24h (pre-additionAPS group),3) APS was added simultaneously with LPS for48h (simultaneous addition APSgroup). The relative mRNA expression of inflammatory indicator TNF-α, IL-1β, IL-8andtight junction zonula occludens-1(ZO-1) and occludin was determined using real-timequantitative PCR (RT-qPCR). After48h treatment, snapwell filters were mounted onto anUssing chamber system. Isc was measured through KCl agar bridge connected to anautomatic voltage clamp. The output signals were digitized by a data acquisition board andrecorded with Lab-Trax software. Addition of APS significantly down-regulated theexpression of TNF-α, IL-1β and IL-8(P <0.05) and the Isc levels (P <0.05) of LPS-infectedCaco2cells for all three administration treatments. The minimum anti-inflammatory concentration of APS was50,100, and100μg/mL for pre-, post-and simultaneous additionof APS, respectively. LPS stimulation decreased the mRNA expression of occludin inpre-addition group (P <0.05), and also down-regulated the protein expression of ZO-1andoccludin for all three groups.100μg/mL APS incubation elevated the mRNA and proteinexpression of ZO-1and occludin for post-and pre-addition groups, respectively (P <0.05),and up-regulated ZO-1protein expression in pre-addition group. Results suggested that100μg/mL APS had anti-inflammatory and structure protective properties for LPS-infected Caco2cells, and possessed better anti-inflammatory activity in pre-addition group.Experiment2Experiment2focus on the structural modification of APS. The functions ofpolysaccharides are closely related to their structure. Sulfated modification was conductedusing the chlorosulfonic acid-pyridine method. The sulfur content of SAPS was determinedusing the method by Antonopoulos. DS was calculated according to the equation:DS=(1.62×S%)/(32-1.02×S%). The FT-IR spectrum of SAPS was recorded using theAVATAR330FT-IR instrument and the KBr pellet method in a range of400-4,000cm-1. Thecytotoxicity effect of SAPS was evaluated using the MTT method. The followingconcentrations of SAPS (μg/mL) were added to Caco2cell medium for24h:1,000,500,250,100,50,25and10. The OD570value reflects the relative number of live cells, and the valueincreases with the number of live cells. The results showed that the DS of SAPS was1.4inthis experiment. Compared with the spectra of APS, the SAPS FT-IR spectra showed twocharacteristic absorption bands. The absorption band at1,265cm-1indicated the presence ofan asymmetrical S=O stretching vibration. The other absorption at810cm-1represented asymmetrical C-O-S vibration that was associated with a C-O-SO3group, which confirmedthat APS was successfully sulfated. The addition of SAPS significantly decreased the OD570value at high concentrations (250,500and1,000μg/mL); however, SAPS had no effect atlow concentrations (10,25,50and100μg/mL). Therefore, SAPS supplemental levels of25,50and100μg/mL were used in the following experiment.Experiment3The purpose of this experiment was to determine whether sulfated modification couldenhance the anti-inflammatory activity of SAPS and provide theoretical evidence for thedevelopment of antibiotic alternatives. SAPS (25,50and100μg/mL, abbreviated as SAPS-25,SAPS-50and SAPS-100),100μg/mL APS (APS-100) and1μg/mL LPS were added into thecells in the following three groups:1) the cells were stimulated with LPS for24h and then treated with SAPS or APS for24h (post-addition);2) the cells were pre-treated with SAPS orAPS for24h and then stimulated with LPS for another24h (pre-addition);3) SAPS or APSwas added simultaneously with LPS for48h (simultaneous addition); DMEM culturemedium was used as blank control. The mRNA expression of inflammatory indicators (TNF-α,IL-1β and IL-8), tight junctions (ZO-1and occludin) and toll-like receptor4(TLR4) weremeasured using RT-qPCR. Western blot was used to measure the protein expression of tightjunctions and TLR4. LPS stimulation increased the mRNA expression of inflammatoryindicators (TNF-α, IL-1β and IL-8) and TLR4(P <0.05) for all three treatments, decreasedZO-1and occludin mRNA expression in post-and simultaneous groups (P <0.05), declinedthe protein expression of ZO-1and occludin in pre-addition group. Compared with LPS group,100μg/mL APS and SAPS decreased the mRNA expression of three inflammatory cytokinesfor all three treatments (P <0.05), SAPS-25down-regulated TNF-α, IL-1β and IL-8mRNAexpression in post-and pre-addition groups (P <0.05). APS-100elevated mRNA and proteinexpression of occludin in all three groups, SAPS-100increased the protein expression ofZO-1and occludin in pre-addition group, low concentrations of SAPS (25and50μg/mL)up-regulated ZO-1and occludin mRNA expression (P <0.05). Addition of APS and SAPSdown-regulated the mRNA and protein expression of TLR4for all groups andconcentrations.In conclusion, SAPS exhibited enhanced anti-inflammatory activity byincreasing tight junctions expression, and decreasing inflammatory indicator and TLR4expression. SAPS possessed better anti-inflammatory activity in pre-addition group.Experiment4Experiment4evaluated the anti-inflammatory and immunomodulate activities of APSand SAPS in LPS-infected broiler chickens.180healthy Arbor Acres broiler chicks wererandomly divided to six groups. On16d, the birds were intramuscular injected0.5mL saline,0.5mL saline, APS (4or8mg/kg of BW, abbreviated as APS-4, APS-8) or SAPS (4or8mg/kg of BW, abbreviated as SAPS-4, SAPS-8), respectively, once a day for three successivedays. On days19and20, the birds were intraperitoneally injected0.5mL LPS (1mg/kg ofBW). Saline was used as blank control. The growth performance, immune organ index,plasma index, lymphocyte proliferation, and intestine morphology were measured. Therelative mRNA expression of pro-inflammatory factors (TNF-α, IL-1β and IFN-γ), tightjunctions (ZO-2and occludin) and TLR4was determined by RT-qPCR. The results showedthat addition of LPS significantly down-regulated BWG, FI, villus height and IEL number (P<0.05), and elevated FCR, spleen index, plasma NO concentration, H:L ratio, and NO, TNOS,iNOS concentration in T lymphocyte supernate (P <0.05). LPS-treated birds exhibited higher expression of pro-inflammatory cytokines (IL-1β and IFN-γ), and lower expression of tightjunctions (P <0.05). Administration of SAPS-8increased BWG and villus height (P <0.05).Plasma NO concentration was lower in APS-8group than that in LPS group (P <0.05). BothAPS and SAPS injection elevated IEL number (P <0.05), decreased FCR, H:L ratio, and theproduction of TNOS and iNOS in T, B lymphocyte and B lymphocyte (P <0.05), respectively.SAPS down-regulated the expression of TNF-α and IL-1β in jejunum (P <0.05). In addition,the expression of ZO-2and occludin was higher in high doses of APS and SAPS treatments (P<0.05). The expression of TLR4was lower in low dose of SAPS group (P <0.05). Ourfindings suggested that APS and SAPS possessed growth-promoting, anti-inflammatory andimmunomodulating effect on LPS-stimulus broilers. SAPS possessed higher bioactivities thanAPS for the same dose.In summary, APS protected Caco2cells against LPS infection through down-regulatingproinflammatory cytokines expression and elevating tight junctions expression. Both APS andSAPS possessed in vitro and in vivo anti-inflammatory activitys. APS and SAPS possessedbetter bioactivities in pre-addition group. Moreover, SAPS possessed higher bioactivities thanAPS for the same dose, suggesting that sulfated modification elevated the anti-inflammatoryactivity of APS.