The Study On The Mechanism Of Polysaccharides From Atractylodes Macrocephala Koidz Alleviating Fatty Liver Hemorrhage Syndrome In Laying Hens

Fatty liver hemorrhage syndrome（FLHS）is one of the most common noninfectious causes of death in caged laying hens,characterized by significant decrease of egg production in the early stage and acute death with ruptured and hemorrhages liver by necropsy in the end stage.Due to the high incidence,high mortality,and difficult treatment of FLHS,it has severely restricted the development of large-scale livestock and poultry breeding,bringing great loss to our country’s social economy.Although there have been certain studies on mechanisms of FLHS,the research data on the treatment,prevention and control of the disease is still incomplete.With the advent of the“anti-antibiotic”era,Chinese herbal medicine has gradually gained public recognition for its advantages,including remarkable efficacy,no antibiotic residues,and high safety.It has been shown that polysaccharide from Atractylodes Macrocephala Koidz（PAMK）,one of the active components of the traditional Chinese medicine AMK,has many pharmacological effects such as enhancing immunity,improving antioxidant enzyme activity,anti-tumor,and improving gastrointestinal function.Considering the single component and low cost,here we analyzed the structural characteristics of PAMK prepared from water decocting and alcohol precipitation and deeply evaluated the protective effect of PAMK against FLHS in laying hens,providing more evidence for the application of PAMK preventing FLHS.This research is divided into the following three parts:1.Extraction and structural identification of PAMKThe PAMK was extracted by a one-step ethanol precipitation method,and the polysaccharide contend was measured by phenol-sulfuric acid method.And the structure of PAMK was analyzed by high gel permeation chromatography（HPGPC）,gas chromatography-mass spectrometry（GC-MS）,ultraviolet spectrum,infrared spectrum and nuclear magnetic resonance（NMR）.The results showed that the polysaccharide content in PAMK was 91.27%,and the molecular weight of PAMK was 2816 Da,which was composed of glucose and mannose in a molar ratio of 0.582 to 0.418.The UV spectrum of PAMK showed no absorption peak at wavelengths 260 nm and 280nm,indicating that neither nucleic acids nor proteins existed in this polysaccharide.The infrared spectrum showed that the PAMK had intense absorbance band around at 3,353 cm^-1and 2,928 cm^-1.These bands were attributed to the O–H of hydroxyl and C–H stretching vibration,respectively,which represented the characteristic groups of general polysaccharides.The absorption peaks at 931 cm^-1and 887 cm^-1indicated the presence of furanose and pyranose rings in PAMK.NMR spectra showed strong contraction vibration of PAMK at5.32ppm（¹H）and 103.66ppm(¹³C),respectively,indicating the existence ofα-type andβ-type connections in the structure of PAMK.What’s more,in vitro antioxidant test proved that PAMK had a strong scavenging effect on DPPH free radicals,which was positively correlated with the concentration of PAMK.2.The alleviative effect of PAMK on FLHS in laying hensIn this study,the model of FLHS was established by feeding a high energy and low protein（HELP）diet.150 Hy-line brown laying hens were randomly divided into 5 groups（n=30）:CON group（normal diet）,HELP group（HELP diet）,HELP+P-40 group（HELP diet+40mg/kg PAMK）,HELP+P-200 group（HELP diet+200mg/kg PAMK）and HELP+P-400 group（HELP diet+400mg/kg PAMK）.After 12 weeks,the laying hens of the HELP group were depressed,feed intake decreased,and the abdomen showed expanded.The liver was greasy and yellowish brown,with a few bleeding spots on the liver surface.The weekly egg laying rate of the HELP group had a significantly decreased（p<0.01）,while the liver index and the abdominal fat percentage were significantly increased（p<0.01）.Severe vacuolar degeneration of liver cells was observed in HELP group by hematoxylin-eosin（H&E）staining,and a large amount of lipid droplets were observed in HELP group by oil red O staining,suggesting the HELP diet induced hepatic steatosis.Serological tests showed that the aspartate amino transferase（AST）,alanine aminotransferase（ALT）,triglycerides（TG）,total cholesterol（TC）and low-density lipoprotein cholesterol（LDL-C）levels were significantly increased（p<0.01）,and high-density lipoprotein cholesterol（HDL-C）content decreased significantly（p<0.01）.All these results indicated that the model of FLHS was successfully established.Compared with the HELP group,the supplement of PAMK could significantly alleviated hepatocyte steatosis,liver injury and lipid accumulation induced by the HELP diet,indicating that PAMK could alleviate FLHS.3.The mechanism of PAMK alleviating FLHSIn this study,we tested oxidative damage related index.Compared with CON group,the hepatic catalase（CAT）,glutathione catalase（GSH-Px）,total superoxide dismutase（T-SOD）activity was markedly decreased and the contend of malondialdehyde（MDA）were significantly increased in the HELP group（p<0.01）.The q PCR showed that the transcriptional level of CAT,heme oxygenase（HO-1）,GSH-Px and quinone oxidoreductase enzyme（NQO1）in the HELP group were significantly increased（p<0.01）,indicating that long-term feeding of the HELP diet resulted in severe oxidative stress reaction.Compared with the HELP group,the addition of PAMK significantly increased the activities of CAT,GSH-Px and T-SOD in liver tissue,and decreased the level of MDA（p<0.05,p<0.01）.Meanwhile,the transcriptional level of CAT,HO-1,GSH-Px and NQO1 in liver were significantly increased（p<0.05,p<0.01）,suggesting that PAMK alleviated oxidative damage in FLHS laying hens and prevented the occurrence of FLHS.Further,we found that,compared with CON group,the transcriptional level of lipid synthesis related genes in the HELP group liver tissue:acetyl-coa carboxylase（ACC）,fatty acid synthase（FAS）,stearoyl-Co A desaturase-1（SCD-1）significantly raised（p<0.01）,and fatty acidβoxidation related genes:adipose triglyceride lipase（ATGL）,carnitine palmitoyl transferase-1（CPT-1）,peroxisome proliferators-activated receptorsα（PPARα）significantly declined（p<0.01）.However,PAMK significantly down-regulated the transcriptional level of ACC,FAS,and SCD-1,and up-regulated the transcriptional level of ATGL,CPT-1,and PPARα（p<0.05,p<0.01）.It was suggested that PAMK could reduce lipid synthesis and accelerate fatty acidβoxidation to relieve FLHS.