The Effects Of Angelica Sinensis Polysaccharide On Metabolic Syndrome Related Diseases And Its Potential Mechanisms

Angelica,which is our traditional Chinese medicine,has been used for thousands of years.Angelica in Chinese medicine is the dry roots of Angelica sinensis(Oliv.Diels),an herb of Minxian County in Gansu Province,China,which is commonly used in blood circulation and tonic pain.Angelica sinensis polysaccharide(ASP),which is isolated from the roots pieces of Angelica,has significant pharmacological activities.Modern medical researches have shown that ASP has antioxidant,immune regulation,anti-anemia,anti-inflammatory,regulating glucose and lipid metabolism disorders and other pharmacological effects.The aim of this study was to clarify the therapeutic effects and mechanisms of ASP on metabolic syndrome-related diseases,including type 2 diabetes mellitus(T2DM),nonalcoholic fatty liver disease(NAFLD)and alcoholic fatty liver disease(AFLD).In this paper,the roots of Angelica sinensis was used as the raw material,ASP was extracted by water and precipitated by alcohol,and the quality of ASP was evaluated by kinds of methods.Animal models of T2DM,NAFLD and AFLD were stablished.Insulin resistance cell model,hepatic steatosis cell model and alcoholic fatty liver cell model were also established in vitro.Mice were orally administered by ASP,and cells were co-cultured with ASP,afterwards the therapeutic effects and molecular mechanisms were evaluated by a series of methods.This study not only clarifies the role and mechanisms of ASP in the treatment of metabolic syndrome-related diseases,but also provides a theoretical basis for its clinical application.It also provides more basis for drug treatment of metabolic syndrome and plays a positive role in improving the quality of people's life.Part One:The extraction,purification and identification of ASPThe preparation of ASP was along with the established and optimized methods:First,crude polysaccharide was obtained by the method of water extraction combined with alcohol precipitation,then the ASP was acquired after repeated freeze-thawing removal,0.8 ?m and 0.45 ?m microporous membrane removal and Mw 3500 dialysis bag removal.At last,the products were placed into the vacuum freeze dryer until a white flocculent ASP.Then the refined ASP was obtained by dextran gel G50 chromatographic separation column separation and purification.The identifications of ASP were determined.The polysaccharide content of ASP was more than 90%,and the weight average molecular weight of ASP was 72900 Da.The UV scan showed that there was almost no nucleic acid and protein.The results showed that ASP was mainly composed of arabinose,glucose and galactose,with the ratio of 1:2.5:7.5.The experiments in this part showed that the polysaccharide extracted from Angelica sinensis had high purity and uniform molecular weight,and it was almost free of impurities.The experiment was easy to repeat,the control was stable and the result was stable.The ASP could be used in the subsequent pharmacological study.Part Two:The therapeutic effects and molecular mechanisms of ASP on T2DMThere are many T2DM patients in China,this disease seriously affects the health of people,but the current drug treatments of diabetes always have many significant side effects due to the single target.However,the side effects of ASP were small and the pharmacological effects of ASP were strong.The aim of this study was to clarify the therapeutic effect of ASP on T2DM mice and provide the theoretical basis for the clinical application of polysaccharide.We used long-term feeding of mice with high-calorie diet,plus streptozotocin(STZ)to destroy islet P cells to establish T2DM mice model,and then the mice were treated with ASP for four weeks.After treatment,the glucose tolerance,the contents of liver mass index,glycosylated hemoglobin(HbA1c),serum TC,serum TG,serum LDL-c and serum HDL-c were measured;Glycokinse(GK)and glucose-6-phosphatase(G6Pase)levels were measured.H&E and oil red O histological staining slices were made to observe the effects ASP.Serum ALT,serum AST and glycogen were detected.The expression of IRS-2,PI3K,Akt,p-Akt and GLUT2 in the liver of mice were detected by Western Blot(WB)method.In addition,to clarify the stimulating effect of ASP on insulin pathway,we also established insulin resistance HepG2 cell model,and the contents of p-IR?,p-IRS1,p-PI3K and p-Akt were detected after treatment of ASP.During the experiment,polydipsia,polyuria,more food and weight loss were observed in T2DM mice.ASP had not effect on the reduced body weight,but could reduce the liver weight index of T2DM mice.FBG and HbA1c in T2DM mice were significantly increased,more than 11.1 mmol/L,glucose tolerance was significantly reduced;ASP treatment significantly reduced the blood glucose,and improved glucose tolerance.In addition,T2DM mice showed significant dyslipidemia and liver injury(increased ALT and AST levels),ASP can significantly control these biomarkers nearly to normal levels.Histological observation showed that the islets of T2DM mice were severely damaged and ASP could repair islets.Liver oil red O slices showed that there was a lot of fat accumulation in T2DM mice.Similarly,ASP could relieve fatty liver.It is noteworthy that ASP could significantly improve the serum insulin levels in mice,suggesting that we ASP might have a unique role in insulin pathway.The results showed that ASP could significantly increase the expression levels of key proteins,suggesting that ASP could stimulate insulin pathway and play hypoglycemic effect.To further clarify this effect,we established the insulin resistance cell model.After ASP intervention,the cell insulin pathway key proteins could be significantly upregulated,and PI3K inhibitor LY294002 could offset the effect of ASP,once again proving that ASP could upregulate insulin pathways.On the other hand,ASP could increase the content of hepatic glycogen in mice and increase the key enzyme GK of glycogen synthase,suggesting that the hypoglycemic effect of ASP was related to its regulation on glycogen synthesis.This part of the experiment proved that ASP could significantly relieve T2DM mice hyperglycemia,hyperlipidemia and liver injury.The hypoglycemic effect of ASP was due to its stimulation of insulin secretion,improvement of the sensitivity of liver insulin pathway,improvement of liver glycogen content.In this part of the experiment,we also found that ASP had a significant effect on liver fat accumulation,which laid the foundation for the following study of NAFLD/AFLD.Part Three:The therapeutic effects and molecular mechanisms of ASP on NAFLDWith the improvement of living standards and the lack of movement,the prevalence of NAFLD in China has risen sharply,which seriously affects public health.However,the clinical field of NAFLD drug treatment is still blank,this part of the experiment aims to clarify the therapeutic effect of ASP on NAFLD,and clear the specific mechanisms.It also provided more basis and new possibilities for NAFLD treatment.In this study,we fed mice with high-fat and high-sugar diet for up to three months to simulate the unhealthy eating habits of NAFLD population and establish animal models.The mice were sacrificed one month after ASP treatement.The FBG and body weight were monitored every week.The oral glucose tolerance test(OGTT)was performed in the final stage of the experiment.Liver weight index,serum TC,serum TG,serum HDL-c and serum LDL-c were measured after the mice were sacrificed Serum ALT and serum AST were also measured.Serum insulin levels were measured and the insulin resistance index was calculated.The insulin-related proteins in the liver of mice were detected.To observe the effects of ASP on NAFLD mice,liver glucokinase(GK),glucose-6-phosphatase(G6Pase),pyruvate kinase(PK)and hexokinase(HK)were measur-ed.The chemical methods were used to detect the contents of hepatic SOD,reactive oxygen species(ROS),malondialdehyde(MDA),reduced glutathione(GSH)and glutathione peroxidase(GSH-PX).And the contents of catalase-related proteins PPAR?,AMPK,p-AMPK and SIRT1 in the liver were measured by WB method.In addition,the levels of autophagy-related protein LC3B in the liver were also explored.To further clarify the regulation of lipid metabolism by ASP,we also used oleic acid to induce the steatosis of HepG2 cell,and cultured with ASP medium.We then detected the contents of LC3B and autophagy upstream regulatory molecule AMPK,clearing the lipid-lowering effect and regulation of cell autophagy by ASP.The formation of NAFLD is recognized as the "two hit" hypothesis,that is,to the"first hit" of simple steatosis and the "second hit" of oxidative stress.Our experimental results showed that serum lipid and fat levels were abnormal in the model mice.Liver oil red O staining and H&E staining were further confirmed the "first hit" of NAFLD mice,there existing many cell vacuoles and much lipid accumulation,but ASP had a significant reversal effects on these abnormal indicators,suggesting that ASP could effectively alleviate the "first hit".The lipid-lowering effect of ASP was due to the increase in lipid metabolic key proteins including PPAR?,SIRT1 and AMPK.By examining oxidative stress markers ROS and MDA,as well as antioxidant enzymes SOD,GSH,GSH-PX and CAT,we found that NAFLD mice had severe oxidative stress in the liver;similarly,ASP significantly increased liver antioxidant capacity of NAFLD mice,removing reactive oxygen species,easing the "second hit." In addition,insulin resistance was also the initiation factor of NAFLD.In the experiment,the resistin resistance index of NAFLD mice was significantly higher than that of normal mice,and ASP could significantly decrease the insulin resistance index and alleviate insulin resistance.According to the research basis of the second part,we speculated that ASP might stimulate insulin pathway in NAFLD mice.The results showed that ASP could increase the contents of IR?,p-IRS1,IRS 1,IRS2,p-PI3K,PI3K,Akt,Akt,membrane-GLUT2 and GSK-3?,and reduce the content of negative regulator p-JNK in insulin pathway.It was proved that ASP could alleviate insulin resistance by stimulating insulin signal pathway of NAFLD mice.Long-term consumption of high-calorie feed led to increased glucose metabolism disorders in mice,with increased FBG and HbA1c,decreased glucose tolerance,decreased glycogen content and abnormal glucose metabolism levels.ASP could improve the abnormal levels of glucose metabolism-related indicators.In addition,the latest study shows that activation of autophagy could effectively improve NAFLD;we detected the liver autophagy key protein LC3-? and LC3-? content,the results showed that autophagy was significantly reduced in NAFLD mice,ASP could significantly improve LC3-? expression,enhance liver autophagy.We had also demonstrated the role of ASP in the activation of hepatocyte autophagy in vitro,and found that this effect is related to its activation of autophagy upstream regulatory factor AMPK.All in all,this study had confirmed that ASP was effective in treating NAFLD by alleviating "two hit".The effects of ASP might be related to the activation of SIRT1-AMPK pathway and hepatic autophagy.In addition,ASP activated insulin pathway,relieved insulin resistance,promoted glucose absorption and accelerated the synthesis of glycogen,thereby effectively improving glucose metabolism disorders.This study clarified the therapeutic effects of ASP on NAFLD and provided new ideas for drug treatment of NAFLD.In addition,in this part of the experiment,we found that ASP could promote autophagy,which laied the foundation for the next part of the mechanism.Part Four:The therapeutic effects and molecular mechanisms of ASP on AFLDThis part of the study to explore the role of ASP on AFLD.At present,the living standards of our people continue to improve,alcohol consumption increased year by year,AFLD has become the second leading factor leading to liver disease.However,there is no AFLD drug treatment;therefore,the development of drugs to treat AFLD is urgent.We established AFLD mouse model by continuous administration of high-dose and high-concentration of liquor,and treated with ASP.Then the blood and liver were collected after the mice were sacrificed.The levels of serum TG,ALT and AST,liver TG and GSH were measured.The liver section was stained with H&E and oil red O to observe the liver steatosis and fat accumulation.Liver PAS staining was also used to observe the distribution of hepatic glycogen.Liver autophagy was observed by transmission electron microscopy.The mRNA levels of LC3B,ATG3 and p62 were detected by RT-PCR.The protein contents of LC3B and p62 were detected by WB method.On the other hand,the fatty liver model of alcoholic HepG2 cells was established in vitro,and the in vitro lipid-lowering effect of ASP was observed by oil red O colorimetric method.Flow cytometry was used to detect intracellular reactive oxygen species(ROS)and the intracellular mitochondrial membrane potential(MMP).Immunofluorescence and WB methods were used to detect the intracellular LC3B contents.In addition,the effect of ASP on intracellular autophagy flux was detected or observed by transfected HepG2 cells with adenovirus expressing mCherry-GFP-LC3B fusion protein,or treated with lysosomal inhibitor chloroquine(CQ).AFLD mice showed significant changes in body weight,yet the liver index did not vary,serum and liver TG levels were significantly increased,oil red O staining showed AFLD mice liver has serious fat accumulation.ASP could not improve the decreased weight of mice.However,ASP significantlt improved fatty liver after treatment.At the same time,serum ALT and AST in AFLD mice were significantly increased,and liver injury was serious.ASP could relieve liver injury significantly.Transmission electron microscopy showed that the liver of the mice treated with ASP had many autophagosomes closed to the lipid droplets,suggesting that the mouse liver might have"lipophagy" effect.We then detected the mRNA or protein levels of autophagy-related proteins LC3B,ATG3 and p62,it was confirmed that ASP could significantly enhance the level of autophagy in AFLD mice.ASP could also significantly alleviate alcohol-induced fat accumulation,ROS accumulation and abnonnal mitochondrial membrane potential in vitro.On the other hand,ASP could significantly promote autophagy,increase the number of autophagosomes and improve autophagy flux.In summary,ASP could effectively treat AFLD,liver injury and lipid metabolism disorders,its significant lipid-lowering effect might be related with the promotion of liver autophagy and upregulation of "lipophagy".However,the pathogenesis of AFLD is complex,ASP treatment might not only with the regulation of autophagy,a more comprehensive mechanism of action remains to be studied.In general,ASP could effectively treat metabolic syndrome-related diseases,lower blood glucose and blood lipids,relieve insulin resistance,promote cell autophagy.The study of this paper provided a sufficient theoretical basis and research basis for the clinical application of ASP.ASP is expected to be developed into the preparation for human body to improve people's health,and carry forward the traditional medicine of our motherland.