Study On The Mechanism Of Gentiana Macrophylla Pall.& Clematis Chinensis Osbeck.couplet Medicines In The Treatment Of Rheumatoid Arthritis Based On Network Pharmacology Analysis

OBJECTIVE To study the curative effect of Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines on the rheumatoid arthritis fibroblast-likesynoviocytes(RA-FLSs).Discover the targets which are acted on active components and Chinese medicine decoction by network pharmacology.The results were verified by pharmacological experiments to discuss the mechanism of action in the treatment of RA METHODS1.Analyze the relationship between Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines and RA by network pharmacology 1)Search the RA targets from Drug Bank and TTD databases and construct the RA database.2)Filtrate the active components of Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines according to the standard(OB ? 30%,DL ? 0.18)by TCMSP database.Add other active components which were common obtained by literatures.3)Retrieve the corresponding targets of each active ingredient by Swiss Target Prediction system.The RA targets were mapped to search the important targets by VENNY.The couplet medicines “active ingredients-important targets” network was constructed to select the important chemical and the key targets.4)The interaction network diagram of the key targets was established through the STRING database.5)David platform was used to carry out KEGG signal pathways and GO biological processes enrichment,and sort out important pathways and important biological processes.2.Verify the results analyzed by network pharmacological by HPLC Use the Agilent 883975-902 ZORBAX SB-C18 column(4.6 mm × 150 mm,5 ? m)to verify.The moving phase A is 0.04% phosphoric acid aqueous solution and the moving phase B is methanol.Use the way of gradient elution: 0~28 min,85% A?77% A;28~35 min,77% A?65% A.0~28 min,15% B?23% B;28~35 min,23% B?35% B;flow rate was 0.8 mL/min;column temperature was 30?;detection wavelength was 245 nm;injection volume was 5? L.To detect whether the Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines decoction contained the important components obtained by network pharmacological analysis.The reliability of the results of network pharmacology analysis was investigated.3.The proliferative activity of the dosed RA-FLSs tested by CCK-8 The study was divided into blank group,positive drug(methotrexate)group,single Clematis chinensis Osbeck.decoction group,single Gentiana macrophylla Pall.decoction group,Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines decoction group,gentiopicrin group,sweroside group and swertiamarin group.Test the proliferative activity of RA-FLSs which was dosed for 24 hours by CCK-8 and microplate reader.Analyze the datas by GraphPad 8.0.4.The apoptosis of the dosed RA-FLSs tested by Annexin V-FITC Test the apoptosis of RA-FLSs which was dosed for 48 hours by Annexin V-FITC and FCM.Analyze the datas by GraphPad 8.0.5.Test the expression level of RF and CRP in the cell medium of RA-FLSs based on Elisa Test the expression level of RF and CRP in the cell medium of RA-FLSs which was dosed for 24 hours by Elisa technique.Analyze the datas by GraphPad 8.0.6.Analyze the docking between the important components and the key target protein of RA-FLSs based on molecular docking.1)Search the structure of the key targets protein by RCSB PDB database.2)Look for the 3D chemical structure of swertiamarin,gentiopicrin and sweroside and positive drug by the Substances module of ZINC database.3)Remove the water molecules and other ligand small molecules in the target protein structure by pyMOL2.2.0 software,and then imported them into Mgltools1.5.6 software for hydrogenation,calculated charge,combined with non-polar hydrogen treatment.4)Integrate the components structure by Mgltools1.5.6 software.5)Run AutoDock Vina 1.1.2 for molecular docking by CMD command.7.The protein expression of RA-FLSs study based on Western Blot The protein expression of Bcl-2,PPARD and c-Fos which came from the core proteins of RA-FLSs were tested by Western-Blot technique.Analyze the bands with ImageJ and the datas by GraphPad.8.The gene expression levels study based on RT-PCR Testing the gene expression of Bcl-2 mRNA,PPARD mRNA and c-Fos mRNA which came from the RA-FLSs core protein by RT-PCR technique,and the datas were analyzed by GraphPad 8.0.RESULTS1.The results of network pharmacological analysis There were 11 kinds of active components which came from Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines such as gentiopicrin,sweroside and swertiamarin,4 key targets such as Bcl-2,PPARD and c-Fos,8 biological processes such as steroid hormone synthesis,arachidonic acid metabolism and drug metabolism,and 13 important pathways such as cancer pathway,metabolic pathway and steroid synthesis pathway were found.2.The results of HPLC analysis The gentiopicrin,sweroside and swertiamarin had a good linear relation in the range of 0.0210 ~ 1.0560 ? g,0.0365 ~ 11.7200 ? g and 0.0162 ~ 1.1420 ? g respectively.The average recovery rates of three ingredients were 99.64%(RSD=2.33%,n=6),100.20%(RSD=0.64%,n=6)and 101.11%(RSD=3.59%,n=6)respectively.The contents of three ingredients in the Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines decoction were 2.13 mg/g,28.36 mg/g and 4.51 mg/g,respectively.3.The results of CCK-8 detection Among the herb decoction groups,each dosed group had an inhibitory effect on cell proliferation significantly compared with the blank group(P<0.01).Compared with the positive medicine group,the single Clematis chinensis Osbeck.group was better than the positive medicine group(P<0.01),but the single Gentiana macrophylla Pall.group was worse than the positive medicine group(P<0.01).Compared within each herb group,the single Clematis chinensis Osbeck.group was the best(P<0.01),the Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines group was the second(P<0.01)and the single Gentiana macrophylla Pall.group was the worst group(P<0.01).Among the important ingredient groups,each dosed group had an inhibitory effect on cell proliferation significantly compared with the blank group(P<0.01).Compared with the positive medicine group,the swertiamarin and gentiopicrin groups were better than the positive medicine group(P<0.01).Compared within each important component group,the gentiopicrin group was the best(P<0.01),the swertiamarin was the second(P<0.01)and the sweroside was the worst group(P<0.01).4.The results of Annexin V-FITC detection Among the herb decoction groups,each dosed group had a significant effect on promoting cell apoptosis compared with the blank group(P<0.01).Compared with the positive medicine group,each groups were worse than the positive medicine group(P<0.01).Compared within each herb group,the single Gentiana macrophylla Pall.group was the worst group(P<0.01,P<0.05).The single Gentiana macrophylla Pall.group was the same as the couplet medicines group.Among the ingredient groups,each dosed group had a significant effect on promoting cell apoptosis compared with the blank group(P<0.01,P<0.05).Compared with the positive medicine group,the gentiopicrin group was better than the positive medicine group(P<0.01)but the sweroside group was worse than the positive medicine group(P < 0.01),the swertiamarin group was same as the positive medicine group.Compared within each important component group,the gentiopicrin group was the best(P<0.01),the sweroside group was the second(P<0.01)and the swertiamarin was the worst group(P<0.01).5.The results of Elisa detection Among the herb decoction groups,the content of RF in the cell medium of each dosed group was reduced significantly compared with the blank group(P<0.01).Compared with the positive medicine group,the single Clematis chinensis Osbeck.group was better than the positive medicine group to reduce the content of RF(P<0.01).Compared within each herb group,the single Clematis chinensis Osbeck.group was the best group to reduce the content of RF(P<0.01).The single Gentiana macrophylla Pall.group was the second(P<0.01).The couplet medicines group was the worst(P<0.05).The content of CRP in the cell medium of each treatment group was reduced significantly compared with the blank group(P<0.01).The content of CRP in each group was similar.Among the important ingredient groups,the content of RF in the cell medium of each dosed group was reduced significantly compared with the blank group(P<0.01).Compared with the positive medicine group,the content of RF in sweroside group was higher than the positive medicine group(P<0.01)and the positive medicine group was better to reduce the content of RF.Compared within each important component group,the content of RF in sweroside group was higher than the gentiopicrin and the swertiamarin group(P<0.01),the gentiopicrin group and the swertiamarin group were better to reduce the content of RF.The gentiopicrin group was the same as the swertiamarin group.The content of CRP in the cell medium of each dosed group was reduced significantly compared with the blank group(P<0.01).Compared with the positive medicine group,the content of CRP in the positive medicine group was lower than the gentiopicrin group and the sweroside group(P<0.01),the positive medicine group was better to reduce the content of CRP.Compared within each important component group,the content of CRP in the swertiamarin group was lowest(P<0.01).The gentiopicrin group was the same as the sweroside group.6.The results of molecular docking The gentiopicrin which docked better than other ingredients with Bcl-2(1GJH)target proteins through GLN-190,THR-7,GLY-5,GLY-8 site protein,whose binding energy is-6.7 kcal/mol.The swertiamarin which docked better than other ingredients with PPARD(3PEQ)target proteins through ASN-227,ASN-343,ALA-342 site protein,whose binding energy is-8.1 kcal/mol.The gentiopicrin which docked better than other ingredients with c-Fos(1K6O)target proteins through ARG-141,ARG-156,ARG-164,GLY-142 site protein,whose binding energy is-6.4 kcal/mol.7.The results of Western Blot detection The expression of Bcl-2 protein.Among the herb decoction groups,the expression of Bcl-2 protein in each dosed group(except the couplet medicines group)was reduced differently compared with the blank group(P<0.01).Compared with the positive medicine group,the expression of Bcl-2 protein in the couplet medicines group was higher than the positive medicine group(P<0.01)and the positive medicine group was better to reduce the expression of Bcl-2.Compared within each herb group,the expression of Bcl-2 protein in the single Gentiana macrophylla Pall.group and the single Clematis chinensis Osbeck.group was lower than the couplet medicines group(P<0.01,P<0.05).The single Gentiana macrophylla Pall.group was same as the single Clematis chinensis Osbeck.group.Among the important ingredient groups,the expression of Bcl-2 protein in each dosed group was reduced differently compared with the blank group(P<0.01,P<0.05).Compared with the positive medicine group,the expression of Bcl-2 protein in the gentiopicrin group was lower than the positive medicine group(P<0.01)and the gentiopicrin group was better to reduce the expression of Bcl-2.Compared within each important component group,the expression of Bcl-2 protein in the gentiopicrin group was lowest(P<0.01)and it was the best to reduce the expression of Bcl-2.And the swertiamarin group was the same as the sweroside group.The expression of PPARD protein.Among the herb decoction groups,the expression of PPARD protein in each dosed group was reduced differently compared with the blank group(P<0.01).Compared with the positive medicine group,the expression of PPARD protein in the single Clematis chinensis Osbeck.group was lower than the positive medicine group(P<0.01)and the single Clematis chinensis Osbeck group was better to reduce the expression of PPARD.Compared within each herb group,the expression of PPARD protein in the single Clematis chinensis Osbeck.group was lowest(P<0.01)and the it was better to reduce the expression of Bcl-2.The single Gentiana macrophylla Pall.group was the same as the couplet medicines group.Among the important ingredient groups,the expression of PPARD protein in each dosed group was reduced differently compared with the blank group(P<0.01).Compared with the positive medicine group,the expression of PPARD protein in the swertiamarin group was lower than the positive medicine group(P<0.05)and it was better to reduce the expression of PPARD.Compared within each important component group,the expression of PPARD protein in the swertiamarin group was lowest(P<0.01)and it was best to reduce the expression of PPARD.And the gentiopicrin group was the same as the sweroside group.The expression of c-Fos protein.Among the herb decoction groups,the expression of cFos protein in the positive medicine group,the single Clematis chinensis Osbeck.Group and the couplet medicines group was reduced differently compared with the blank group(P<0.01,P<0.05).Each group was the same as the positive medicine group.Compared within each herb group,the single Gentiana macrophylla Pall.group was the same as the couplet medicines group.The expression of c-Fos protein in the single Clematis chinensis Osbeck.group was lower than the single Gentiana macrophylla Pall.group(P<0.05)and it was the best to reduce the expression of c-Fos.Among the important ingredient groups,the expression of c-Fos protein in each dosed group(except the swertiamarin group)was reduced differently compared with the blank group(P < 0.01,P < 0.05).Compared with the positive medicine group,the expression of c-Fos protein in the swertiamarin group was higher than the positive medicine group(P<0.05)and it was worse to reduce the expression of c-Fos.Compared within each important component group,the swertiamarin group was the same as the sweroside group.The expression of c-Fos protein in the gentiopicrin group was lowest and it was the best to reduce the expression of c-Fos.8.The results of RT-PCR detection The expression of Bcl-2 mRNA.Among the herb decoction groups,the expression of Bcl-2 mRNA in each dosed group(except the couplet medicines group)was reduced differently compared with the blank group(P<0.01).Compared with the positive medicine group,the expression of Bcl-2 mRNA in the single Clematis chinensis Osbeck.group was lower than the positive medicine group but the couplet medicines group was higher than the positive medicine group(P<0.01,P<0.05),and the single Clematis chinensis Osbeck.group was the best to reduce the expression of Bcl-2 mRNA.Compared within each herb group,the expression of Bcl-2 mRNA in the single Clematis chinensis Osbeck.group was lowest(P<0.01)and it was the best to reduce the expression of Bcl-2 mRNA.The single Gentiana macrophylla Pall.group was second(P<0.01)and the couplet medicines group was the worst(P<0.01).Among the important ingredient groups,the expression of Bcl-2 mRNA in each dosed group was reduced differently compared with the blank group(P<0.01).Each group was the same as the positive medicine group.The expression of PPARD mRNA.Among the herb decoction groups,the expression of PPARD mRNA in single Clematis chinensis Osbeck.group was reduced compared with the blank group(P<0.05).Each group was the same as the positive medicine group and every herb decoction groups were similar.Among the important ingredient groups,the expression of PPARD mRNA in the swertiamarin group was reduced compared with the blank group(P<0.05).The other groups were similar.The expression of c-Fos mRNA.Among the herb decoction groups,the expression of cFos mRNA in each dosed group was reduced differently compared with the blank group(P<0.01).Each group was the same as the positive medicine group and every herb decoction groups were similar.Among the important ingredient groups,the expression of c-Fos mRNA in the positive medicine group and the gentiopicrin group was reduced differently compared with the blank group(P<0.01,P<0.05).Compared with the positive medicine group,the expression of c-Fos mRNA in the sweroside group was higher than the positive medicine group(P<0.05)and it was worse to reduce the expression of c-Fos mRNA.Compared within each important component group,the expression of c-Fos mRNA in the gentiopicrin group was the lowest(P<0.01,P<0.05)and it was the best to reduce the expression.The sweroside group was the same as the swertiamarin group.CONCLUSION The important herb ingredients(swertiamarin,gentiopicrin)from Gentiana macrophylla Pall.and Clematis chinensis Osbeck.couplet medicines were good at inhibiting cell proliferation,promoting cell apoptosis and reducing the secretion of RF and CRP.It also could inhibit the expression of Bcl-2,PPARD and c-Fos protein and the expression of Bcl-2 mRNA,PPARD mRNA and c-Fos mRNA effectively.It's a novel approach for treating RA.Network pharmacology is reliable in analyzing the mechanism of drug therapy.