Explore Anti-influenza Virus Pharmacological Mechanism Of Curcumae Rhizoma Based On Systems Pharmacology

Curcumae Rhizoma is a commonly used traditional Chinese medicine in China.It was first published in the theory of medicinal properties,which has the effect of accelerating blood circulation,alleviating pain and digesting food.It is mostly used for anti-tumor and anti-inflammatory clinically.Modern pharmacological research shows that Rhizoma Curcumae has many pharmacological effects,such as anti-tumor,anti-inflammatory,anti-virus and enhancing immunity.However,because of the synergistic effect of multi-component,multi-target and its mutil-component,the pharmacological effects of Curcumae Rhizoma can not be fully explained from a single point of view,and the research on the effective components and target mechanism of Curcumae Rhizoma is not comprehensive yet.This topic explores the pharmacological mechanism of Curcumae Rhizoma and its anti-influenza virus pharmacological mechanism through a series of systematic pharmacological methods,predicts the biding pattern between the active ingredient and each related target protein of influenza with molecular docking method,and then verifies its biological activity in vitro.ObjectiveThrough the network pharmacology technology to explode the pharmacological mechanism of Curcumae Rhizoma and its anti-influenza virus pharmacological mechanism,so as to further clarify the material basis of its efficacy,and construct effective ingredients-drug target proteins–diseases network to preliminarily explain the model of integration of multi-component,multi-target and multiple diseases.Molecular docking method was used to screen its effective components against influenza and explains its multi targeting mechanism,and preliminarily verifies its anti-influenza activity in vitro.Methods1.Study on the network pharmacology of the effective components of Curcumae Rhizoma action on the target of influenza?1?The effective components of Curcumae Rhizoma were obtained by using TCMSP database.?2?The target protein of active components of Curcumae Rhizoma was obtained from PharmMapper Server.?3?The gene name of the target is obtained in the Uniprot database and the corresponding gene disease is obtained in the CTD database.?4?Using network visualization software to construct the active ingredient of Curcumae Rhizoma-target-disease interaction network.?5?The DAVID tool was used to analyze the gene-based?GO?analysis of the active components of Curcumae Rhizoma and the enrichment analysis of the pathway of the target proteins.2.Study on molecular docking of anti-influenza candidate molecules of Curcumae Rhizoma?1?Establish the molecular library of chemical composition of Curcumae Rhizoma.?2?Select and pre-treat the target protein.?3?Calculate and analyze the result of molecular docking.3.Experimental study on an in vitro reagent kit for anti-influenza candidate molecules of Curcumae RhizomaUsing in vitro neuraminidase,p38 kinase and COX-2 inhibitor screening kit to preliminary verify its biological activity and calculate IC50 value.4.Drug toxicity test?MTT?Drug toxicity test?MTT?was used to evaluate the effects of different concentrations of Curcumae Rhizoma active ingredients on MDCK cell proliferation.5.The inhibition effect of Curcumae Rhizoma active ingredients on H1N1influenza virusThe effect of Curcumae Rhizoma active ingredients on the expression of M gene mRNA of H1N1 influenza virus strain was detected by Real Time-PCR on MDCK cells.6.The effect of Curcumae Rhizoma active ingredients on neuraminidase?NA?activityNeuraminidase activity kit was used to determine the effect of active components of Curcumae Rhizoma on neuraminidase activity on MDCK cells infected by H1N1 influenza virus.Results1.Network pharmacology analysis results:Tumor,nervous system diseases and metabolic diseases are most closely with Curcumae Rhizoma and it may exert its biological functions primarily by regulating multiple pathways such as cancer pathway,PPARP signaling pathway,PI3K-Akt signaling pathway and so on;Its effective ingredients may mainly play anti-influenza effert by regulating PI3K-Akt signaling pathway,influenza A signaling pathway and Jak-STAT signaling pathway.2.Molecular docking results:With the protein targets of influenza virus synthesis and inflammation,compared with the threshold value of Total-Score value 6,9 small molecules were predicted have better combination pattern with selected targets.In the match of 83 candidate compounds with the related proteins,Hederagenin,daucosterol,curcumin,dDemethoxycurcumin,demethoxycurcumin and curcumin have the highest matching degree with the active sites of NA,PB2,NP,p38MAPK,COX-2 and TNF-?,respectively.3.The experiment results showed that the selected small molecules showed different degrees of inhibitory activity to the targets.The best inhibitory activity against neuraminidase,p38 kinase and COX-2 are bisdemethoxycurcumin?85.31±2.41?M?,hederagenin?40.38±2.63?M?and hederagenin?153.04±3.63?M?,respectively.4.MTT method was used to determine the half toxic concentration?TC50?of bisdemethoxycurcumin and curcumin on MDCK cells,which were 29.35±2.67?M and 32.15±3.01?M,respectively.And the TC₅₀ of Oseltamivir carboxylic acid was 24.92±2.45mM.5.The results of Realtime-PCR test in vitro showed that the effective components of Curcumae Rhizoma,bisdemethoxycurcumin?0.47±0.11?and curcumin?0.19±0.02?,showed a good inhibitory effect on the replication of H1N1 influenza virus strain?P<0.05?.The inhibitory effect of curcumin was similar to that of the positive drug oseltamivir carboxylic acid?0.09±0.03?.6.The results of neuraminidase activity test indicated that bisdemethoxycurcumin?0.40±0.00 mU/ml?and curcumin?0.48±0.03 mU/ml?significantly decreased the activity level of NA?P<0.05?,and the effect inhibition of NA activity in vitro was comparable to that of the positive drug oseltamivir carboxylic acid?0.44±0.04 mU/ml?.ConclusionThe anti influenza active ingredients of Curcumae Rhizoma may mainly play the role of anti-influenza by regulating PI3K-Akt signaling pathway,influenza A and Jak-STAT signaling pathway.The effective components of Curcumae Rhizoma have certain binding and inhibitory effects on influenza virus protein targets?NA,PB2,NP?and inflammatory related protein targets?p38MAPK,COX-2 and TNF-a?,which indicate the pattern of multi-targeting anti-influenza action.The selected small molecules showed different degrees of inhibitory activity to the targets.In vitro cell experiments,it further confirmed that the active ingredient of Curcumae Rhizoma,bisdemethoxycurcumin and curcumin,inhibit the replication of influenza virus by inhibiting the activity of neuraminidase.The comprehensive use of systematic pharmacology can not only quickly and comprehensively analyze the pharmacological effects of drugs,but also contribute to the discovery of pilot compounds.