Study On The Effect And Mechanism Of Improvement Of Lipid Metabolism In High-fat Mice By Eucommia Bark/leaf Extract And Fecal Microbiota Transplantation

In traditional medicine,eucommia is mainly used to tonifying liver and kidney,strengthening muscles and bones,and relaxing the fetus.In recent years,studies have found that eucommia also has antihyperlipidemia effect,and is mainly used as eucommia bark.However,eucommia bark grows slowly,and large-scale peeling can easily lead to the death of eucommia trees.Studies have shown that the composition of active ingredients in eucommia leaf is similar to that of eucommia bark,but the content of bioactive compounds is not consistent.As a result,it is uncertain if eucommia leaf may substitute eucommia bark in exerting the same anti-hyperlipidemia effect,as well as the manner and mechanism of anti-hyperlipidemia action.In this investigation,network pharmacology was used to analysis the potential anti-hyperlipidemia targets of eucommia bark and eucommia leaf.To establish a high-fat mouse model induced by high fat diet(HFD),compare the anti-hyperlipidemic effects of Eucommia bark extract(EBE)and Eucommia leaf extract(ELE),and explore the direct mechanism of EBE and ELE in anti-hyperlipidemia.To verify whether the antihyperlipidemia effect of EBE/ELE can play a role from the migration of intestinal flora through fecal microbiota transplantation(FMT)experiment,and explore its indirect anti-hyperlipidemia mechanism mediated by intestinal flora.A lipid accumulation model of HepG2 cells was established by oleic acid(OA)induction,and its mechanism of action was verified under the intervention of inhibitors.The main contents and results are as follows:1.The potential targets of eucommia bark and eucommia leaf were screened by network pharmacology,we will extract eucommia bark extract(EBE)and eucommia leaf extract(ELE)using a 1:10 hot water extraction and conduct HPLC-MS/MS assays,and as well as qualitative analysis of active ingredients in eucommia bark extract and eucommia leaf extract.The composition of the active ingredients in EBE and ELE was compared.Network pharmacology analysis revealed that eucommia bark and leaf had seven identical active chemicals(Kaempferol,Quercetin,Geniposidic acid,Rutin,Protocatechuic acid,Aucubin,and Chlorogenic acid)and sixteen same antihyperlipidemia targets.In addition,KEGG pathway analysis suggests that the signaling route responsible for the anti-hyperlipidemia effect of eucommia bark and leaf may be "PI3K-AKT." The results of HPLC-MS/MS analysis showed that there were eight main compounds common in EBE and ELE,namely Eucommioside,Geniposidic acid,Asperulosidic acid,Chlorogenic acid,Hedyotol C,Quercetin 3-O-β-Dsambubioside,Isoquercetin and Kaempferol-3-O-(6"-acetyl)-β-Dglucopyranoside.Therefore,eucommia leaves contain a variety of active ingredients similar to eucommia bark,and their potential antihyperlipidemia pathway may be "PI3K-AKT".2.By establishing a high-fat mice model by HFD-induced and combined with FMT experiment to explore the effect of EBE,ELE and fecal bacterial suspension containing EBE/ELE on lipid metabolism disorder in hyperlipidemic mice.Long-term HFD induction causes considerable increases in body weight and body length in mice,as well as damage to multiple organ tissues and the occurrence of lipid metabolism disorders.However,EBE/ELE intervention significantly reduced the body weight of high-fat mice and significantly shortened the body length of mice.The results of blood lipid and liver lipid metabolism showed that EBE/ELE intervention could significantly reduce the levels of Leptin,ADPN,TG,TC,LDL-c,ALT,and TBA in the plasma;significantly increase the level of HDL-c in the plasma;significantly reduce levels of TG,TC,ALT,AST,and TBA in liver tissue.In addition,EBE/ELE intervention can significantly reduce blood glucose levels and AUC values in high-fat mice,significantly enhances the antioxidant capacity of liver tissues,and inhibit HFD-induced oxidative stress.The results of histomorphological analysis showed that EBE/ELE intervention can significantly inhibit the production of lipid droplets in the liver by promoting the production of autophagies;significantly reduce the area of fat cells in adipose tissue,thereby inhibiting lipid accumulation in adipose tissue.In the FMT experiment,the fecal bacteria suspension containing EBE/ELE had the effect of improving lipid metabolism disorders.However,by comparing fecal bacterial suspensions containing EBE/ELE healthy mice with fecal bacterial suspensions containing EBE/ELE high-fat mice,we found that the intervention effect of fecal bacterial suspension containing EBE/ELE healthy mice in inhibiting weight gain,lowering blood glucose levels and oxidative stress levels in liver tissues of high-fat mice was significantly better than that of fecal bacterial suspension containing EBE/ELE high-fat mice.In the study of EBE/ELE and the regulation of HFD-induced lipid metabolism disorders in suspensions containing EBE/ELE bacteria,we found no significant difference in the effect of ELE compared with EBE.Therefore,in summary,eucommia leaf can replace eucommia bark and exert antihyper-lipidemia by regulating the level of lipid metabolism.3.Long-term intake of HFD can lead to low-grade inflammation in the body,destroy the integrity of colonic tissue structure,and may lead to disturbances in the structure of the intestinal flora.EBE/ELE intervention may be able to reverse this phenomenon,protect the integrity of colon tissue structure,and improve the structure of intestinal flora in order to play an anti-hyperlipidemia role.As a result,the effects of EBE/ELE intervention on colonic tissue structure,intestinal mucosal barrier function,and intestinal microbiota structure in high-fat mice were investigated using histomorphological analysis,qRT-PCR,and high-throughput sequencing of 16S rRNA.The results showed that EBE/ELE intervention could protect the integrity of colon tissue structure from HFD induction by significantly enhancing the intestinal mucosal barrier function(significantly upregulating the expression of ZO-1 and Occludin genes)and significantly reducing the expression of pro-inflammatory factors(TNF-α,IL-6,and IL1β).In addition,the results of the determination of short-chain fatty acids(SCFAs)showed that EBE/ELE intervention could significantly promote the increase of acetic acid,butyric acid and total acid content,which may be closely related to the change of intestinal flora.The results of 16S rRNA sequencing showed that the intervention in EBE/ELE could significantly reduce the relative abundance of Desulfovibrionaceae and Erysipelotrichaceae,and significantly increase the relative abundance of Ruminococcaceae.Changes in the relative abundance of Desulfovibrionaceae,Erysipelotrichaceae and Ruminococcaceae are closely related to changes in lipid metabolism levels.Therefore,EBE/ELE interventions may exert anti-hyperlipidemia effects by regulating the relative abundance of Desulfovibrionaceae,Erysipelotrichaceae,and Ruminococcaceae.To elucidate whether there is a causal relationship between EBE/ELE and the intestinal flora,we explored through FMT experiments.The results showed that the intervention containing the suspension of fecal bacteria in healthy EBE/ELE mice could significantly promote the content of butyric acid and the production of total acid,and significantly reduce the relative abundance of Erysipelotrichaceae and significantly increase the relative abundance of Ruminococcaceae.Therefore,we believe that EBE/ELE intervention can exert antihyperlipidemia effect by regulating the structure of intestinal flora,and they have a direct causal relationship,but their mechanism of action needs to be further explored.Based on the above results,we also found that both ELE and EBE interventions could improve the disturbance of intestinal flora,and there was no significant difference in the effect of the two.Therefore,by regulating the structure of the intestinal flora to exert antihyperlipidemia pathways,eucommia leaf can replace eucommia bark.4.To explore the direct and indirect mechanisms of EBE/ELE in antihyperlipidemia through qRT-PCR and WB experiments,and explore their internal relationships.The results showed that the EBE/ELE intervention could activate the phosphorylation of AKT by upregulating the expression of P-PI3K protein,thereby significantly downregulating the expression of SREBP-1c,SREBP-1a,and SREBP-2 genes,thereby regulating the level of lipid metabolism in the liver and inhibiting the synthesis of lipids.In addition,EBE/ELE intervention can inhibit the expression of LPL in liver by significantly upregulating the expression of Fiaf in colon tissue,and then inhibit the synthesis of TG in liver tissue.EBE/ELE intervention can inhibit lipid accumulation in adipose tissue by significantly upregulating the expression of GPR41 and GPR43 genes in colonic and epididymal adipose tissue.Changes in Desulfovibrionaceae,Erysipelotrichaceae,and Ruminococcaceae were significantly correlated with the expression of Fiaf,LPL,GPR41,and GPR43 genes.In FMT experiments,we also observed similar results.Therefore,we found that EBE/ELE can exert a direct antihyperlipidemia effect by activating the "PI3K-AKT-SREBP" signaling pathway.It plays an indirect anti hyperlipidemic role by activating the"Fiaf-LPL gut-liver axis" and "SCFAs-GPR41/GPR43 gut-fat axis"circulation mediated by Desulfovibrionacea,Erysipelotaceae,and R um nococcaceae.5.The results of network pharmacology and animal experiments showed that eucommia bark and eucommia leaf can exert direct antihyperlipidemia effects through the "PI3K-AKT" signaling pathway.However,reverse verification is also required through the intervention of inhibitors.A lipid accumulation model of HepG2 cells was established by OA induction,and LY294002(PI3K inhibitor),MK-2206 2HC1(AKT inhibitor)and Fastostain(SREBP inhibitor)inhibitors were added,respectively.The verification results show that EBE/ELE can inhibit the expression of SREBP-1c and SREBP-2 proteins by activating the phosphorylation of PI3K and AKT,thereby inhibiting the accumulation of lipid droplets in HepG2 cells.This study confirmed that the activation of the PI3K-AKT-SREBP signaling pathway is the direct anti-hyperlipidemia mechanism of EBE/ELE,and there was no significant difference between the effect of ELE and EBE.Therefore,based on the pathway of regulating the molecular gene level to improve lipid metabolism disorders,eucommia eucommia leaf can replace eucommia bark to play an anti-hyperlipidemia effect.In summary,EBE/ELE can exert direct and indirect antihyperlipidemia effects by regulating the molecular gene level and improving the structure of intestinal flora,and ELE and EBE have equivalent anti-hyperlipidemia effects.Therefore,in terms of antihyperlipidemia,eucommia leaf can replace eucommia bark for medicinal use,so as to realize the sustainable use of eucommia leaf resources.