Based On Network Pharmacology-Metabolomics To Explore The Effect Substances And Mechanism Of Huanglian Jiedu Decoction In The Treatment Of Osteoarthritis

Background and Objective:Huanglian Jiedu Decoction（HJD）is a traditional Chinese medicine formula with anti-inflammatory,anti-tumor,microcirculation improvement,and other pharmacological benefits.However,its efficacy and mechanism of action in the treatment of osteoarthritis（OA）,are still unknown.In this study,we analyzed the active ingredients and targets of HJD for the treatment of OA by network pharmacology,and validated the active ingredients in the compound using high performance liquid chromatography（HPLC）.Meanwhile,a hydrogen peroxide-induced human normal chondrocyte cell line（C28/I2）was used to create an in vitro cell model of osteoarthritis,and the protective effects and mechanisms of action of the active components in HJD were explored using a combination of cell biology and metabolomics to establish a foundation for fundamental research and clinical application of HJD in the treatment of OA.Methods:1.A network pharmacological study of HJD in the treatment of OA: screening of the active ingredients of HJD by TCMSP database using oral bioavailability（OB）≥30% and drug-like（DL）≥ 0.18 as screening criteria.Using TCMSP,Swiss Target Prediction and Pharm Mapper databases to screen potential targets of active ingredients,using OMIM,Drugbank and Genecards databases to search for osteoarthritis-related target genes,using String database to construct protein interaction network,and then using Metascape database to perform GO analysis and KEGG pathway enrichment analysis.Lastly,the Cytoscape 3.8.2 software was used to construct a "Huanglian Jiedu Decoction-component-target-osteoarthritis" network to predict its possible targets and molecular mechanisms.2.Study on the chemical substance basis of HJD: The chemical substance basis of HJD was analyzed by using HPLC technique on a Wonda Sil C18 Superb 5 μm column（250 mm × 4.6 mm,5 μm）with a gradient elution of acetonitrile-0.4%phosphoric acid water as the mobile phase at 254 nm and 203 nm.3.In vitro cellular investigations to investigate the active compounds in HJD’s anti-OA properties: An in vitro cell model of OA was constructed using human normal chondrocytes C28/I2 with hydrogen peroxide-induced injury,which were identified by cell morphological observation,toluidine blue staining and alisin blue staining,with a normal control group,a model group,a drug group with different active ingredients of HJD,and a positive control group with heparin（180 U）.CCK-8assay was used to detect the effect of the active ingredient in HJD on the proliferation ability of osteoarthritic chondrocytes.Annexin-V FITC/PI was used to observe cell apoptosis.Scratch assay to detect the effect of active ingredient administration on cell healing ability.RT-q PCR to detect the expression of SOX9,ACAN,COL2A1,COL10A1 and matrix metalloproteinase MMP3 and MMP13 in chondrocytes.Western blot was used to detect the expression of matrix metalloproteinase MMP3,MMP13 and apoptosis-related proteins Bcl-2 and Bax to investigate the protective effects of the active ingredients of HJD on osteoarthritic cells.4.Metabolomic study of the mechanism of action of the active ingredients in HJD on the treatment of OA: Ultra Performance Liquid Chromatography-Time of Flight Mass Spectrometry（UPLC/Q-TOF-MS）technique was used to examine the non-targeted metabolomics of the active ingredients of HJD in a co-culture model with cells.The differential metabolites were screened by multivariate statistical analysis using orthogonal partial least squares-discriminant analysis of OPLS-DA combined with the contribution of each component（VIP>1,P<0.05）.It was identified by database matching to compare the changes of differential metabolites before and after administration,and pathway enrichment analysis was performed to reveal the potential mechanism of action of HJD on the treatment of OA.Results:1.A network pharmacological study of HJD on the treatment of osteoarthritis:We screened 85 active ingredients and 747 potential targets in HJD,and 2037 targets in OA,and obtained 265 common targets and 24 core targets by taking the intersection of both.Subsequently,the "Huanglian Jiedu Decoction-componenttarget-osteoarthritis" network was constructed,and the topological parameters of the main active ingredients of HJD for the treatment of OA were obtained,among which the degree values of Kaempferol,Obacunone and Quercetin were relatively high,suggesting that Kaempferol,Obacunone and Quercetin may be able to play a therapeutic role in osteoarthritis.2.Basic research on the chemical substances of HJD: The HPLC results showed that three effective active ingredients,Kaempferol,Obacunone and Quercetin,were indeed present in the extract of HJD.3.In vitro cellular experiments to study the protective effect of active ingredients in HJD on osteoarthritic chondrocytes: The cultured C28/I2 cell line was identified by cytomorphological observation,toluidine blue staining and alisin blue staining as chondrocytes.The CCK-8 experiment determined that hydrogen peroxide modeling concentration and duration of action were 1.2 m M/L and 24 h.The administration of 5μM Kaempferol,3 μM Obacunone,and 30 μM Quercetin,respectively,for 24 h significantly increased the proliferation of osteoarthritic chondrocytes with hydrogen peroxide-induced injury（P<0.05）.Meanwhile,Annexin-V FITC/PI assay for apoptosis observed that 5 μM Kaempferol,3 μM Obacunone,and 30 μM Quercetin could reduce the total apoptosis rate from 12.53% to 10.96%,10.71%,and 7.89%,respectively,which could attenuate hydrogen peroxide-induced chondrocyte apoptosis to different degrees.In addition,cell scratching experiments showed that the cell healing ability was improved after 5 μM Kaempferol,3 μM Obacunone,and 30 μM Quercetin administration treatments compared to the model group.RT-q PCR results showed that 5 μM Kaempferol,3 μM Obacunone,and 30 μM Quercetin significantly increased SOX9 and COL2A1 m RNA expression（P<0.05）and significantly decreased COL10A1,MMP3,and MMP13 m RNA expression（P<0.05）in hydrogen oxide-induced chondrocytes,and also increased ACAN m RNA expression.The results of Western blot experiment showed that Bax protein expression level was significantly reduced（P<0.05）and Bcl-2 protein expression level was significantly increased（P<0.05）after the administration of 5 μM Kaempferol,3 μM Obacunone and 30 μM Quercetin,and among them,3 μM Obacunone and 30 μM Quercetin could also effectively reduce the expression of MMP3 protein（P<0.05）.In addition,5μM Kaempferol,3 μM Obacunone,and 30 μM Quercetin also reduced MMP13 protein expression,although the changes were not significant.4.Metabolomic study of the mechanism of action of the active ingredients in HJD on the treatment of OA: Non-targeted metabolomics screened a total of 36 differential metabolites between normal chondrocytes,osteoarthritic chondrocytes and three active ingredients,Kaempferol,Obacunone and Quercetin,after administration.KEGG pathway enrichment analysis of differential metabolites between normal chondrocytes and osteoarthritic chondrocytes provided pathways for glycerophospholipid metabolism,arginine and proline metabolism,arginine biosynthesis,ether lipid metabolism,sphingolipid metabolism,glutathione metabolism,cysteine and methionine metabolism,amyl-t RNA biosynthesis,and purine metabolism.Among them,the differential metabolite enrichment pathways of three active ingredients,Kaempferol,Obacunone,and Quercetin,involved glycerophospholipid metabolism,cysteine and methionine metabolism,and purine metabolism.Conclusions:1.The three active ingredients of Quercetin,Obacunone and Kaempferol in HJD have certain therapeutic effects on osteoarthritic diseases.2.Quercetin,Obacunone and Kaempferol could promote the proliferation of osteoarthritic chondrocytes,inhibit apoptosis and enhance the healing ability of cells,while increasing the expression of SOX9,ACAN,COL2A1 and Bcl-2 and decreasing the expression of COL10A1,MMP3,MMP13 and Bax,thus producing a protective effect on chondrocytes.3.The pathogenesis of osteoarthritis involves multiple metabolic pathways,and the three active ingredients,Quercetin,Obacunone,and Kaempferol,mainly function through glycerophospholipid metabolism,cysteine and methionine metabolism,and purine metabolism.