Study On The Total Synthesis Of H4 And Its Protective Effect On Non-alcoholic Fatty Liver Disease

In recent decades,the global prevalence of non-alcoholic fatty liver disease has increased significantly,and now it has become a kind of liver disease that does great harm to people’s lives.Non-alcoholic fatty liver disease is usually caused by the accumulation of large amounts of lipids in the liver.In order to treat the disease,drug developers are constantly looking for new compounds in order to obtain drugs with high efficacy and safety.The high safety and efficacy of traditional Chinese medicine in the treatment of non-alcoholic fatty liver has attracted people’s attention.The traditional Chinese medicine Sabia parviflora Wall.ex Roxb.has good pharmacological activity of protecting liver,and alkaloids are usually the active components in the plant.H4 is a new phenanthrene alkaloid glycoside isolated from Sabia parviflora Wall.ex Roxb.However,H4 is difficult to be isolated in large quantities.Therefore,we used the total synthesis method to synthesize H4 from 3-methoxy-4-hydroxyphenylethylamine hydrochloride through 12 steps,and discussed the effect and mechanism of H4 on non-alcoholic fatty liver in vivo and in vitro.1 Total synthesis of H4.In order to fully synthesize H4,using 3-methoxy-4-hydroxyphenethylamine hydrochloride as the raw material,12 steps of reactions were carried out successively:acid amine condensation reaction,cyclization reaction,sodium borohydride reduction reaction,Boc protected amine Group reaction,Heck reaction,deprotection group Boc reaction,Eschweiler-Clark reaction,quaternization reaction,Hoffman elimination reaction,Koenigs-Knorr glycoside reaction,deprotection group methyl Reaction and deacetylation reaction.In the demethylation protection experiment,in order to protect the glycosidic bond from breaking,we have tried demethylation reagents such as boron tribromide,sodium ethyl mercaptan,thiophenol,aluminum trichloride,magnesium iodide and trimethyliodosilane.Finally,magnesium iodide acts as a demethylating reagent to remove methyl successfully,and at the same time,it can also keep the glycosidic bond unbroken.In the next step,we will explore the effect and mechanism of H4 against nonalcoholic fatty liver disease in vivo and in vitro.2 Protective effect of H4 on LO2 cell injury induced by FFA and its preliminary mechanismIn order to study the protective effect of H4 on non-alcoholic fatty liver in vitro,LO2 cells(human normal hepatocytes)were used as experimental objects,and FFA was used to establish the model of non-alcoholic fatty liver.The effect of H4 on the growth of LO2 cells was detected by MTT assay.The results showed that even if the concentration of H4 was more than 40 μg/m L,it had no effect on the activity of LO2 cells.It was inferred that H4 had less cytotoxicity and high safety to LO2.Finally,it was determined that the concentration of H4 was 2.5 μg/m L and 10 μg/min.Twenty four hours after administration,according to the results of apoptosis detection,it was inferred that H4 could significantly reduce the apoptosis induced by free fat acid(FFA).H4 can significantly reduce the activities of AST,ALT and TG content in non-alcoholic fatty liver cells.It is speculated that H4 can reduce lipid accumulation caused by FFA and improve liver dysfunction.In addition,H4 could significantly increase significantly the activities of SOD and GSH-Px and decrease the content of MDA in liver tissue of mice with non-alcoholic fatty liver.It is speculated that H4 has the effect of antioxidant stress.Western blotting analysis showed that after H4 treatment of LO2 cells injured by FFA,the expression of Bax protein,phos-Ik Bα and phos-p65 protein was significantly down-regulated,while the expression of Bcl-2 protein was significantly up-regulated.It is speculated that H4 can reduce the apoptosis and inflammation of nonalcoholic fatty liver cells by regulating Bax/Bcl-2 and NF-κB signaling pathways,thus protecting LO2 cells from nonalcoholic fatty liver injury induced by FFA.3 Study on the protective effect and mechanism of H4 on mice non-alcoholic fatty liver disease model induced by high fat diet.In order to study the protective effect of H4 on non-alcoholic fatty liver in vivo,the model of non-alcoholic fatty liver was established by feeding high-fat diet for 8weeks,and drug intervention was given at the same time.The results showed that after8 weeks,the body weight of mice in H4 groups were significantly lower than that in HFD group,and H4 could improve the blood lipid indexes of non-alcoholic fatty liver disease mice(decreased the level of TG,TC and LDL-C,and increased the level of HDL-C)and the activity of enzymes related to liver injury(decreased the level of ALT,AST,ALP,GGT,LDH).Liver histopathological analysis showed that H4 could significantly reduce the excessive formation and accumulation of lipid droplets in hepatocytes.In addition,H4 could elevate significantly the activities of SOD and GSHPx and decrease significantly the level of MDA in the liver tissue of non-alcoholic fatty liver disease mice.Western blot analysis showed that after H4 treatment,the expression of Bax protein,phos-Ik Bα and phos-p65 protein in liver tissue of non-alcoholic fatty liver disease mice was significantly down regulated,while the expression of Bcl-2protein was significantly up regulated.It is speculated that H4 can protect the liver injury of non-alcoholic fatty liver mice by regulating Bax/Bcl-2 and NF-κB signal pathways in vivo.In this experiment,phenanthrene alkaloid glycoside H4 was synthesized by a simple and feasible method.The protective effect of H4 on non-alcoholic fatty liver in vivo and in vitro was also discussed.The results showed that H4 could improve lipid accumulation,oxidative stress and liver function disorder caused by non-alcoholic fatty liver.In addition,H4 could reduce apoptosis and inflammation by regulating Bax/Bcl-2 and NF-κB signal pathway,thus protecting hepatocyte injury.