Study On The Mechanism Of Mongolian Medicine WWQZS Against Hyperlipidemia Based On TMM Of “Eliminating Phlegm And Producing Essence”

Wuwei Qingzuo San(WWQZS)(Tonglaga-5 in Mongolian),is dealt with poor appetite,indigestion and stomachache loaded in the Pharmacopoeia of The People's Republic of China-2015 Chinese Edition(CH.P)as well as hyperlipidemia and coronary heart disease loaded in Encyclopedias of Mongolian Studies Medical Science-Medical Volume.As a traditional Mongolian patent medicine and simple preparation,five herbal medicines in WWQZS.In clinical,Mongolian medicine practitioners get accustomed to treating dyslipidemia due to the principles of TMM for helping liver produce essence and eliminate phlegm again.However,the underlying mechanisms of WWQZS for anti-hyperlipidemia remained unclear and has to be further investigated.AIM:A combination of network pharmacology and validation experiments in hyperlipidemia hamster is used to understand the mechanism of WWQZS against hyperlipidemia based on the traditional Mongolian medicine of "Eliminating Phlegm and Producing Essence" for enlarging clinical indications.METHODS:1.Collection of candidate components of WWQZS.Components and their ADME information of all the five herb medicine in WWQZS were obtained from TCMSP and Chemistry Database[DB/OL].We chose the two parameters,namely, oral bioavailability(OB)and drug-like(DL)to obtain the potential active ingredients to evaluate the degree of an oral dose of drugs that distribute to the bloodstream and the structural similarity between the compound and the drugs clinically.Targets related to hyperlipidemia identified in WWQZS2.Collection of putative targets of WWQZS.The putative targets of candidate components from five herbs in WWQZS were obtained from TCMSP developed by professor Wangand protein IDs of the targets were converted to gene IDs in the UniProt(https://www.uniprot.org/).The interaction for these targets was analyzed by interactive venngrapher using TBtools v0.5 too.3.Collection of targets related-to hyperlipidemia.Furthermore,we collected the targets related to hyperlipidemia were collected from following TTD(Therapeutic Target Database;http://bidd.nus.edu.sg/BIDD-Databases/TTD/TTD.asp),OMIM database(Online Mendelian Inheritance in Man;http://www.omim.org/),DrugBank database(https://www.drugbank.ca/)and GeneCards(https://www.genecards.org/).4.PPI information collection.PPI information of putative targets,disease targets was obtained from STRING resource(version 10.5;https://string-db.org/),which collects and integrates the information about known and predicted protein-protein association data.5.Network construction and analysis.Three networks of candidate compounds-targets predicted putative targets and known hyperlipidemia-related targets were constructed and visualized by Cytoscape(version 3.6.1)based on the data previously collected.Then,the intersection of networks between predicted putative targets and known hyperlipidemia-related targets network was combined by Merge,a tool of Cytoscape.Afterward,two topological property of each node,namely degree and betweenness centrality in the interaction network was analyzed by NetwokAnalyzer.6.Gene ontology and KEGG pathway enrichment analysis.Furthermore,GO annotations and KEGG pathways enrichment analysis for two pairs of targets,namely,potential targets related to hyperlipidemia and candidate targets selected by topological properties in PPI networks,were performed by Metascape(http://metascape.org/gp/).7.Experimental design.Six hamsters were fed on NFD,and the remaining eighteen hamsters were supplied with HFD.After the five-weeks induction period,hamsters fed on HFD were randomly divided into three group due to the serum lipid.Hence,all the hamsters were divided into five group again:(1)NFD group:NFD+0.5%CMC-Na;(2)HFDgroup:HFD+0.5%CMC-Na;(3)WWQZSLgro up:HFD+7.8g/kgWWQZS;(4)WWQZSH group:HFD+31.2g/kg WWQZS.8.The livers and bodies weight of hamsters.During the experimental period,body weight was recorded every week.After 8-week supplementation period,the livers were removed and weighed,meanwhile,the largest lobes of the livers were fixed in 10%paraformaldehyde and the others were stored at-80? until use.9.Determination of TC,TG,LDL-C,HDL-C,AST,and ALT in serum.The serum was separated by centrifuging at 3000rpm for 20 min at 4? and stored at-80? until analysis.The concentration of serum TC,TG,LDL-C,HDL-C,ALT,and AST were assessed with an automatic biochemical analyzer.10.Histological analysis.The largest lobe of the livers fixed in 4%formalin and embedded in paraffin.Sections were obtained and later stained with hematoxylin and eosin(H&E)for the histological examination.Cryosections of liver were stained by 0.2%Oil-Red O and counterstained with hematoxylin to visualize the lipid droplets.Tissue sections were then observed with the microscope(Nikon Eclipse Ci,Japan)and software.11.Western blot analysis.Whole cell protein was prepared from isolated liver and extracted by RIPA buffer.Quantified protein lysates were separated by SDS-PAGE,transferred to PVDF membrane(Millipore,United States)with 110V at 4? for 100 min and incubated overnight at 4? with primary antibodies against CYP7Al;SREBP-2;p-AMPKa;HMGCR;Bcl-2;p53 and ?-ACTIN after blocked in TBST containing 5%BSA for 1h at room temperature.Then,membranes were blotted with an appropriate HRP-labeled goat anti-rabbit IgG(H+L)or anti-mouse IgG(H+L)(1:10000,Jackson)secondary antibody for 1h at room temperature.At last,the membranes were washed with TBST three times again and antibodies binding was detected using ECL(WBKLS0500,Millipore).12.Molecular Docking.Identification of compounds from WWQZS and plasma was used by UHPLC-ESI-Q-Orbitrap Plus MS and molecular docking between identified compound and targets was sued by Autodock vina.RESULTS:1.A total of 540 chemicals information were collected from the two websites.Further,screened by two principles(OB?30%,DL?0.1)and added with low OB and DL index but highly active activity,we harvested 212 compounds after duplicated and removed fitted with the two parameters in the WWQZS.At last,the five herbal medicine,Shiliu,Honghua,Bibo,Doukou and Rougui,contributed 34,68,51,52,55 compounds.2.Based on TCMSP,we obtained 219 predicted putative targets for 212 candidate compounds from WWQZS,161 for Shiliu,176 for Honghua,94 for Bibo,136 for Doukou,103 for Rougui.3.Next to 219 predicted putative targets from TCMSP,we gained 349 known targets related to hyperlipidemia based on five existing resources,namely,TTD,OMIM,DrugBank and GeneCards,the network of the compound-putative target,consisting of 429 nodes and 4213 edges,was constructed to understand the complex interaction between compounds and their targets.4.The knowledge of protein-protein interaction(PPI)network helps more system-wide understanding of cellular function for the diseases.According to the STRING resource,we constructed a predicted putative targets network(517 nodes and 14448 edges)and a known hyperlipidemia-related targets network(620 nodes and 14360 edges)Moreover,the two networks were interested in one merge network consisting of 111 nodes and 1847 edges to unravel the mechanism of WWQZS against hyperlipidemia.Then,we selected two topological features,degree and betweenness,as important factors in network construction,in order to identify the candidate targets.Subsequently,based on the median values of degree and betweenness were 31 and 0.0034,45 targets whose network consisted of 45 nodes and 792 edges were identified as candidate targets.5.Enrichment analysis of targets for WWQZS against hyperlipidemia.Due to Metascape,GO enrichment analysis implied two pairs of the genes shared numerous BP that regulation of lipid metabolic processes and lipid localization;cellular response to lipid concerned with metabolism of lipid;regulation of MAP kinase activity and protein kinase activity associated with regulation of kinases;regulation of coagulation responded to wounding;negative regulation of cell proliferation and apoptotic signaling pathway relevant cell cycle and so on.Nevertheless,referring to MF)and CC,there were less shared ways than BP,for example,MF shared lipid and phosphatase binding,receptor ligand,growth factor,receptor ligand,and growth factor activities,etc.And CC showed similar membrane raft,receptor complex,membrane region,and so forth.In addition,KEGG signaling pathways explicated hoperlipidic effect were involved in processes including metabolism associated with AGE-RAGE signaling pathway in diabetic complications,adipocytokine signaling pathway,and insulin signaling pathway;kinase function according to ErbB,MAPK signaling,AMPK signaling and PI3K-Akt signaling pathways;cell cycle related to apoptosis and so on communally in two groups of targets,interestingly,we found the more pathways related to shared lists,potential targets list and fewer ones belonging to candidate targets.6.During the 5-week induction period,the rates of hamsters body mass fed on HFD was faster than the ones fed on NFD.After induction time,the weight of hamsters fed on NFD sustainably increased but the HFD groups begun to fluctuate,interestingly,even the weight had reduced.In general,the weight of WWQZH group descended more the HFD group.Meanwhile,there is no difference in liver/body weight among each group.7.Blood lipid shows that compared with NFD before treatment,the serum TG,TC,LDL-C,HDL-C were all elevated significantly different in HFD hamsters.After administration of the 7.8 or 31.2g/kg concentration of WWQZS for 8 weeks,the serum TC and TG in HFD group significantly rose(P<0.05,n=6)compared with the NFD,but compared to HFD,the TC,TG,LDL-C and HDL-C in the WWQZH group were significantly decreased(P<0.05,n=6),meanwhile,the TG and LDL-C of WWQZSL and WWQZSH groups were reduced but not significantly.8.AST and ALT show that compared with NFD before treatment,the serum ALT increased significantly in HFD hamsters(p<0.05,n=6)but had no influence on AST.After administration of WWQZS for 8 weeks,the serum AST in HFD and WWQZSL groups significantly rose(P<0.05,n=6)compared with the NFD,but compared to HFD,it was significantly decreased in the WWQZH group(P< 0.05,n=6)and decreased in WWQZSL group,meanwhile,it still had no influence on AST.9.Liver sections in NFD group showed the structures of tissue were normal with polygonal edges and clear cell borders,and the nucleus stayed round and clear.In contrast,the HFD group revealed visible histological changes including cell edema,focal degeneration,and necrosis.Likewise,there was degeneration in WWQZSL group but much better the ones in HFD.However,the tissue in WWQZSH nearly returned to normal and cell edema nearly was found with distinct and clear borders.Oil-Red O staining revealed the presence of lipid accumulation in both HFD and WWQZSL groupsbut hepatic lipid accumulation in WWQZSH group was less than one in HFD.10.WWQZS promoted the expression of CYP7A1 and p-AMPKa in two group of WWQZS compared with HFD group,further,WWQZS attenuated the HMGCR protein expression in WWQZSH group compared with HFD group but the upstream SREBP-2 appeared downtrend expressing more than HFD group.11.To assess the effect of WWQZS on cholesterol metabolism in HFD-induced hyperlipidemia hamsters,the protein expression of HMGCR,SREBP-2,CYP7A1 p-AMPKa,p-ACC,p53,Bcl-2 was evaluated in livers of WWQZS-treated hyperlipidemia hamsters by Western blotting.WWQZS promoted the expression of CYP7A1 and p-AMPK?(P<0.05)and p-ACC(P<0.05)in two group of WWQZS compared with HFD group,further,WWQZS attenuated the HMGCR(P<0.05)protein expression in WWQZSH group compared with HFD group but there is no difference on SREBP-2.Meanwhile,due to the protein associated with apoptosis,compared to HFD group,WWQZSL and WWQZSH could downregulate protein expression of p53 and upregulate protein expression of Bcl-2.12.We finally identified 13 compounds under positive and negative ions.Compared with serum samples,piperine appeared at 9 time points in plasma.Taking piperine as an example,the results of molecular docking showed that the free energy between piperine and HMGCR protein was-8.0(kcal/mol).HMGCR protein and piperine played a role through two hydrogen bonds.792 MET residues on HMGCR protein and carbonyl of piperine were built and the ILE at protein 696 binds to the oxygen bond on the five-membered ring of piperine.The free energy between CYP7A1 protein and piperine is-8.1(kcal/mol).HMGCR protein and piperine play a role through a hydrogen bond.The 48-position PHE residue on CYP7A1 protein binds to the carbonyl group of piperine.Conclusion:In conclusion,our results suggest that WWQSZS exerts hoperlipidic and liver-protective effect against hyperlipidemia through multiple targets and pathways,including increasing cholesterol catabolism by upregulating CYP7A1 and inhibiting cholesterol synthesis by upregulating p-AMPKa to suppress the expression of HMGCR in livers and piperine may be one of the basic substances,meanwhile WWQZS may inhabit apoptosis in hepatocyte to protect liver function.Through this research,a new reference is also provided to other researches in the study of ethnopharmacology.