| Diarrhea of livestock and poultry is common in breeding industry,and the harm is serious.In the process of breeding,adding antibiotics to feed plays an important role in the prevention and control of livestock and poultry diarrhea.However,long-term unreasonable use of antibiotics leads to the continuous generation of drug-resistant strains and serious threat to food safety.China’s Ministry of Agriculture and Rural Affairs has banned all growth-promoting feed additives,except traditional Chinese medicine,from Jan 1,2020.Schisandra chinensis polysaccharide(SCP)is one of the effective components of Schisandra chinensis,which has the potential effect of alleviating intestinal injury.However,as a macromolecular substance,its application is limited.Nano-selenium,as a new form of selenium,has higher bioavailability,lower toxicity and stronger bioactivity than traditional selenium,and has attracted more and more attention.However,the stability of nano-selenium is poor,and it is easy to aggregate in aqueous solution and convert to elemental selenium without biological activity,which limits the play of biological activity.Nano-selenium modified with polysaccharide as template can improve the stability of selenium and the bioavailability of polysaccharide while combining the advantages of polysaccharide and selenium.In this study,the crude SCP were extracted by water extraction and alcohol precipitation method.The purified SCP was used as the preparation of SCP-Selenium nanoparticles(SCPSe NPs).The protective effect of SCP-Se NPs on intestinal inflammatory injury induced by Lipopolysaccharides(LPS)was explored in vivo,providing a theoretical basis for the clinical application of SCP-Se NPs.1.Extraction,separation and purification of SCP.The crude polysaccharide of Schisandra chinensis was extracted by water extraction and alcohol precipitation.Purification of schisandra polysaccharides by Sevage and DEAE-52 cellulose column chromatography.Determination of sugar content by phenol-sulfuric acid method.The chemical and chemical properties of SCP were analyzed by Molish reaction,Fehring’s reagent reaction and biuret reagent reaction.Determination of SCP monosaccharides by high performance liquid chromatography.The results showed that the extraction rate of crude polysaccharide was 2.9%.The purified sugar content was 89.2%.SCP is a carbohydrate substance without soluble reducing sugar or protein substance.SCP consists of Man,Rib,Rha,Glc A,Gal A,Glc,Xyl,and Ara.2.Preparation and characterization of SCP-Se NPs.SCP-Se NPs were prepared by chemical reduction method,and dual-wavelength colorimetry was used to investigate the influence of SCP concentration on the stability of SCPSe NPs in the preparation process.The surface morphology of SCP-Se NPs was observed by TEM.XRD is used to determine whether SCP-Se NPs is crystal.The element composition of SCP-Se NPs was detected by EDX.The combination mode of SCP and Se NPs was detected by FTIR.The particle size,PDI and Zeta potential of SCP-Se NPs at 0,3,7 and 14 d in different storage environments were detected by laser particle size analyzer.The results showed that the optimal SCP concentration for preparing SCP-Se NPs was 0.1 mg/m L.The SCP-Se NPs had a particle size of 120.8 nm,a PDI of 0.17,and a Zeta potential of-38.3 m V.SCP-Se NPs has a uniform dispersed spherical shape and an amorphous structure.SCP-Se NPs are amorphous and mainly composed of carbon,selenium and oxygen.SCP and Se NPs form new O-H· Se and CO· Se bonds.SCP-Se NPs were stored at room temperature,the particle size and PDI increased on the 7th day,and precipitation was observed on the 14 th day,with no change in potential.The particle size,PDI and potential of SCP-Se NPs stored at 4 ℃ did not change in 14 days.3.The protective effect of SCP-Se NPs on intestinal inflammatory injury induced by LPS in mice was explored.Except blank group,the other groups were intraperitoneally injected LPS,and the mental state,coat condition,fecal characteristics and diarrhea of mice were observed within 6 h.Calculated the Disease activity index(DAI).The changes of intestinal villus height and crypt depth were observed by HE staining.The goblet cells in mouse intestinal tract were observed by AB/PAS staining.Observed expression of Zonula occludens 1(ZO-1)by Immunohistochemical(IHC).The contents of IL-1β,IL-6 and TNF-α in serum were determined by ELISA.The m RNA levels of IL-1β,IL-6,TNF-α,NLRP3,TLR4,My D88 and NF-κB were detected by q RT-PCR.The results showed as follows:(1)The observation of clinical symptoms showed that the model group of mice was depressed,lazy,hypothermic,like to huddle,stand up and have messy and dull hair,diarrhea,contaminated around the anus,smelly feces,and positive occult blood.Compared with model group,mental depression,diarrhea and fecal occult blood of mice in SCP group and SCP-Se NPs group were relieved.(2)The statistical results of DAI score showed that DAI score of the model group was significantly higher than that of the blank group.Compared with model group,DAI score of SCP group was decreased(P > 0.05),and DAI score of SCP-Se NPs group was significantly decreased(P < 0.05).(3)Histopathological examination showed that compared with blank group,the villi of jejunum in model group were seriously damaged,epithelial cells were shed in large numbers,and the villi were broken,shed and atrophy.Villi height was significantly decreased(P < 0.05),crypt depth was significantly increased(P < 0.05),and the ratio of villi height to crypt depth was significantly decreased(P < 0.05).Compared with model group,the villi structure of jejunum in SCP treated mice was repaired,and the ratio of villi height to crypt depth was increased(P< 0.05).The villi in the small intestine treated with SCP-Se NPs had a complete structure,no large area of villi shedding,and the villi were arranged neatly.The villi height was significantly increased,the crypt depth was significantly decreased(P < 0.05),and the ratio of villi height to crypt depth was significantly increased(P < 0.05).(4)Goblet cell number statistics,compared with blank group,goblet cells decreased or even disappeared in model group(P < 0.05),and the thickness of mucous layer decreased.Compared with model group,goblet cell number and mucous area in SCP group and SCP-Se NPs group were increased by different amplitude(P <0.05).(5)The expression of ZO-1 was visible,and the expression level of ZO-1 was decreased in model group compared with blank group(P < 0.05).Compared with model group,the expression level of ZO-1 protein in SCP group was increased(P > 0.05),and that in SCP Se NPs group was increased(P < 0.05).(6)The contents of IL-1β,IL-6 and TNF-α in serum were detected.Compared with blank group,serum contents of IL-1β,IL-6 and TNF-α in model group were significantly increased(P < 0.05).Compared with model group,the contents of IL-1β,IL-6 and TNF-α in serum of SCP and SCP Se NPs groups were significantly decreased(P < 0.05).(7)The m RNA expression levels of IL-1β,IL-6,TNF-α,NLRP3,TLR4,My D88,NF-κB in intestinal tissue.Compared with blank group,the m RNA relative expression levels of IL-1β(P< 0.05),IL-6(P < 0.05),TNF-α(P < 0.05),NLRP3(P < 0.05),My D88(P > 0.05)were increased.Compared with model group,the m RNA relative expression levels of IL-1β(P <0.05),IL-6(P < 0.05),the TNF-α(P < 0.05),NLRP3(P < 0.05),TLR4(P > 0.05),My D88(P >0.05)and NF-κB(P > 0.05)were decreased in the SCP group and SCP-Se NPs group.In summary,the yield of water extraction and alcohou precipitation of SCP was 2.9%,and the sugar content of SCP was 89.2% after separation and purification.The optimal SCP concentration of SCP-Se NPs was 0.1mg/m L,and the obtained SCP-Se NPs particle size was120.8 nm,PDI is 0.17,and Zeta potential is-38.3 m V.The SCP-Se NPs is an amorphous spheroid,which is mainly composed of carbon,selenium and oxygen elements.SCP and Se NPs rely on O-H···Se bond and C-O···Se bond bonding,and are more stable when stored 4 ℃。In the mice model of intestinal inflammatory injury induced by LPS,both SCP and SCP-Se NPs could improve the clinical symptoms of mice,relieve the pathological injury of intestinal tissue,prewent the loss of ZO-1,reduce the contents of IL-1β,IL-6,TNF-a in serum,and reduce the m RNA level of IL-1β,IL-6,TNF-a in tissue.The m RNA level of NLRP3 in tissue was decreased,and the therapeutic effect of SCP-Se NPs was superior to SCP.It is speculated that SCP-Se NPs may ameliorate intestinal inflammatory injury in mice by anti-inflammatory and rapairing intestinal barrier. |
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