Mechanisms Of The Effects Of Enteromorpha Prolifera Polysaccharide-zinc Complex On Intestinal Homeostasis

The intestine is a key site for the digestion and absorption of nutrients and is also the body’s first line against external infections,which can susceptible to oxidative stress or inflammatory damage caused by external stimuli.Thus,a healthy intestinal function is fundamental to the rapid growth and development of the body.The intestinal flora is the key to resist invasive pathogens.One of the ways they provide protection is to crowd out invaders and make other bacteria inaccessible.In the past many years,antibiotics were often used to alleviate the imbalance of intestinal homeostasis,but the increase of antibiotic resistant bacteria further exacerbated the imbalance of intestinal flora,and then damaged the health of the body.Therefore,the research on antibiotic substitutes is urgent.Enteromorpha prolifera polysaccharide is a polysaccharide extracted from the natural green algae marsh moss,which has been shown to have various biological functions such as anti-tumour,anti-virus,antioxidant,immunomodulatory and flora regulation,and has received increasing attention in recent years.Zinc is an essential trace element in animals and plays an important role in their growth and development,immunity and reproduction.Studies have shown that polysaccharide Zn complexes have significant advantages than polysaccharide or other forms of organic Zn alone in modulating immune function and antioxidant capacity.There have been many studies on the use of zinc polysaccharide complexes in animal production,but little research has been done on the use of zinc polysaccharide complexes in intestinal homeostasis.In this experiment,we investigated the effect of Enteromorpha prolifera polysaccharide-zinc(EP-Zn)on weaned piglets as an alternative to antibiotics by adding EP-Zn to the feed,and further investigated the effect of EP-Zn on intestinal homeostasis under physiological and inflammatory conditions.Part I: Effect of EP-Zn replacing antibiotics on intestinal homeostasis in weaned pigletsTo investigate the effect of EP-Zn replacing antibiotics on intestinal homeostasis in weaned piglets.A total of 224 healthy weaned piglets(Duroc × Landrace × Large Yorkshire)of similar genetic background and body weight(5.92±0.19 kg)were randomly divided into two treatments with 7 replicates of 16 piglets per replicate.The antibiotics group was fed 800 mg/kg enduracidin+ 400 mg/kg olaquindox in the basal diet,the EP-Zn group was fed 800 mg/kg EP-Zn in the basal diet.The results showed that the addition of EP-Zn had no significant effect on growth performance,but had the effect on reducing diarrhea rate and improving ileal development.EP-Zn replacement of antibiotics significantly increased(P<0.01)the levels of serum glutathione peroxidase(GSH-Px),total antioxidant capacity(T-AOC)and malondialdehyde(MDA)in weaned piglets,increased(P<0.05)the m RNA and protein expression of HSP70 and Nrf2 in the jejunum and ileum,as well as the m RNA and protein expression of the jejunal and ileal tight junction proteins Occludin,and inhibited(P<0.05)the expression of NF-κB phosphorylation and downstream inflammatory factors such as interleukin-6(IL-6),interleukin-8(IL-8),interleukin-12(IL-12)and tumour necrosis factor-α(TNF-α),thereby protecting the structural integrity of the intestine.In conclusion,EP-Zn can improve the intestinal mucosal barrier damage caused by weaning,and can effectively replace preventive antibiotics to a certain extent to improve the intestinal health of weaned piglets.Part II: Effect of EP-Zn on intestinal homeostasis in C57BL/6J mice under physiological conditionsOn the basis of the previous part of the experiment,this experiment further investigated the effect of EP-Zn on intestinal homeostasis under physiological conditions.Sixty 8-week-old C57BL/6J mice were randomly divided into 5 groups of 12 mice each group according to their body weight for 28 days.350,700,1050 and 1400 mg/kg EP-Zn were added on the diets of C57BL/6J mice to screen the appropriate additive amounts in this experiment and observe the effect of EP-Zn on intestinal barrier.The results showed that 350 mg/kg EP-Zn significantly increased(P<0.05)serum superoxide dismutase(SOD),700 and 1050 mg/kg EP-Zn significantly increased(P<0.05)serum T-AOC,and 700 mg/kg EP-Zn significantly increased(P<0.05)serum MDA in mice.Different concentrations of EP-Zn increased(P<0.05)serum TNF-α,IL-1β,IL-6and decreased(P<0.05)interleukin-10(IL-10)levels in mice to different degrees,and this effect decreased with the increases of EP-Zn.Therefore,350 mg/kg was chosen as the appropriate amount of EP-Zn in this study.Further results showed that EP-Zn significantly increased(P<0.05)the expression of IL-1β,and decreased(P<0.05)the expression of IL-6 and interleukin-17(IL-17)in the ileum.Meanwhile,EP-Zn significantly decreased(P<0.05)the abundance of Coriobacteriaceae＿UCG-002,Brevibacterium,Brachybacterium,Dietzia,unclassified＿f＿Microbacteriaceae and Parasutterella,significantly increased(P<0.05)the abundance of unclassified＿f＿Lachnospiraceae,Atopostipes and unclassified＿f＿Ruminococcaceae.And significantly decreased(P<0.05)the abundance of short-chain fatty acids(acetic acid,propionic acid,isobutyric acid and isovaleric acid)(P<0.05)in the colon.The difference bacteria Coriobacteriaceae＿UCG-002 showed a highly significant(P<0.01)positive correlation with propionic acid,isobutyric acid and isovaleric acid,Brevibacterium showed a highly significant(P<0.01)positive correlation with acetic acid,Dubosiella showed a significant(P<0.05)positive correlation with propionic acid and Akkermansia showed a significant(P<0.05)positive correlation with isovaleric acid.It is evident that under physiological conditions,EP-Zn has a role in regulating the immune activity of the body,possibly by modulating the intestinal microbial barrier and the immune barrier and regulating the secretion of cytokines.Part III: EP-Zn alleviates LPS-induced intestinal inflammation through inhibition of TLR4/NF-B signalling pathwayThis experiment was conducted to further investigate the effect of EP-Zn on the intestinal barrier of C57BL/6J mice in an inflammatory state.Thirty-six 8-week-old C57BL/6J mice were selected and randomly divided into three groups of 12 mice each group according to body weight:control group(NC),LPS-treated group(LPS)and EP-Zn and LPS co-treated group(EP-Zn).Mice were given the basal diet(NC and LPS groups)and EP-Zn(EP-Zn group)diets respectively for 28 days.On day 28 of the experiment,mice in the LPS and EP-Zn groups were injected intraperitoneally with 200 μL LPS(LPS dissolved in sterile Na Cl solution)at a dose of 0.5 mg/kg BW,and mice in the NC group were injected intraperitoneally with the same dose of saline.The results showed that the EP-Zn had no significant effect(P>0.05)on LPS-stimulated body weight loss,liver and spleen swelling in mice,but significantly alleviated(P<0.05)LPS-induced colonic length atrophy,significantly reduced LPS-induced increases in MPO(P<0.05)and DAO(P<0.05)levels,inhibited NF-κB phosphorylation(P< 0.05)and expression of downstream inflammatory factors such as IL-1β,IL-6 and IL-17(P<0.05),while significantly increased(P<0.05)the LPS-induced decrease of Peptostreptococcacea in colon.It can be concluded that EP-Zn may alleviate LPS-induced colonic inflammatory response by inhibiting NF-κB phosphorylation,ameliorating intestinal microbial barrier disruption,and inhibiting the release of inflammatory factors.In summary,EP-Zn replaces antibiotics to increase the level of expression of the intestinal tight junction protein Occludin,protects intestinal integrity and blocks the expression of inflammatory factors,thereby reducing the rate of diarrhea rate.In the physiological state,EP-Zn has a role in modulating the immune activity of the body,regulating the secretion of cytokines by modulating the composition of intestinal microorganisms and short-chain fatty acids.In the inflammatory state,EP-Zn could alleviate LPS-induced colonic inflammation by inhibiting NF-κB phosphorylation and the release of downstream inflammatory factors induced by LPS stimulation.The above findings provide a comprehensive insight into the immunomodulatory and anti-inflammatory activities of EP-Zn,and provide theoretical support for further EP applications in animal feeds.