Investigation Of Poria Cocos Polysaccharide In Regulating Nrf2/HO-1 Signaling Pathway To Alleviate Non-alcoholic Fatty Liver Disease

Research Background:Non-alcoholic fatty liver disease(NAFLD)is the most common chronic liver disease in the world.At present,western medicine treatment has limitations.Therefore,actively seeking treatment methods to prevent and control NAFLD and prevent it from developing into advanced stages such as cirrhosis.Liver disease is of great significance.Poria cocos is the traditional Chinese medicine,and it is often used in combination with other traditional Chinese medicines for treatment of NAFLD.Poria cocos polysaccharide is the main component of Poria cocos,and studies have shown that Poria cocos polysaccharides has a wide range of therapeutic effects such as anti-oxidant,anti-inflammatory,and anti-tumor.This study mainly focused on the effect and mechanism of Poria cocos and Poria cocos polysaccharides,on NAFLD.Research purposes:To explore the mechanism of Poria cocos and its main component,Poria cocos polysaccharide,to reduce lipid accumulation,inhibit inflammation and oxidative stress,and improve NAFLD.Research methods:1.Zebrafish experiment:The concentration of traditional Chinese medicines was determined according to the 96-hour toxicological experiment.Zebrafish 3 days post fertilization(dpf)were soaked in 0.05%Thioacetamide(TAA)solution for 72 hours to establish zebrafish juvenile NAFLD model.At the same time,the drug intervention group was given Poria cocos solution(25 μg/ml,50 μg/ml,100 μg/ml)or Poria cocos polysaccharide solution(1 mg/ml,2 mg/ml,4 mg/ml).The larval zebrafish were subjected to paraffin section and frozen section,and oil red-O staining,H&E staining,and immunofluorescence staining were used to detect the pathological changes in the liver of zebrafish larvae.RNA was extracted,and qRT-PCR was used to detect the expression of related genes.2.Mice experiment:The 8～10-week-old mice were grouped and fed with Western diet model feed for 6 weeks to establish the NAFLD mice model.The drug intervention groups were given Poria cocos decoction solution(1.67g/kg,3.3g/kg,6.7g/kg)or Poria cocos polysaccharide solution(100mg/kg,200mg/kg,400mg/kg),and atorvastatin tablets(20 mg/kg)as a positive control group.Drug intervention was started at the 7th week of modeling,and it were administered by gavage once every 2 days for 6 weeks.The mice serum and livers were collected after modeling.Serum detection,H&E pathological staining,oil red-O staining,Nile red staining,fluorescent probe detection,immunohistochemical staining,and immunofluorescence staining were used to detect changes in mice livers and protein expression.3.Cell experiment:Selecting mice liver cell line AML-12 cell to culture,first determine the concentration of Poria cocos polysaccharide solution(25 μg/ml,50μg/ml,100 μg/ml)according to the CCK-8 experiment.The NAFLD model of AML-12 cells was induced by Lipid Mixture,and Nrf2 agonist and inhibitor were added to perform cell climbing and cell plating.Oil red-O staining,Nile red staining,ROS probe detection,and western blotting were used to detect cellular lipid droplet accumulation,oxidation response and protein expression.Research results:1.The NAFLD model of zebrafish larvae was successfully induced by TAA.The results of H&E staining showed that compared with the model group,Poria cocos reduced the liver lipid accumulation in zebrafish larvae;Poria cocos polysaccharide significantly reduced the lipid accumulation in the liver of zebrafish larvae,significantly decreased the expression levels of zebrafish inflammatory genes and lipid-related genes,and increased high expression level of Nrf2 gene.2.The results of H&E staining in the liver of the NAFLD mice model showed that compared with the control group,the model group had significant liver lipid accumulation.Poria cocos and Poria cocos polysaccharide treatment groups showed that both of them improved liver steatosis and reduced lipid accumulation in mice,and the improvement in the high concentration group was close to that of the atorvastatin positive drug group.Poria cocos polysaccharide reduce liver inflammation and oxidative stress,increased the expression of Nrf2 and HO-1 proteins,and improved liver antioxidant capacity.3.The results of the AML-12 cell experiment showed that compared with the model group,the Poria cocos polysaccharide treatment group reduced cell lipid droplet deposition,reduced oxidation,and increased the protein expression of Nrf2 and HO-1;the expression of Nrf2 increased after ML385 added to Poria cocos polysaccharide.Conclusion:Poria cocos and Poria polysaccharides cocos decreases NAFLD lipid accumulation.Poria polysaccharides cocos regulates Nrf2/HO-1 signaling pathway and reduces the occurrence of liver inflammation and oxidative stress.