| ObjectiveTo identify the major bioactive compounds with analgesic effects in Wu-tou decoction(WTD)and to investigate the underlying pharmacological mechanism against neuropathic pain(NP).Methods1.Observation of the analgesic effect of WTD on NPAnimals and treatmentIn this study,male ICR mice at the age of 8 weeks old were used.After the construction of SNL or Sham surgeries,mice accepted five kinds of treatment.In the WTD-treatment groups,mice were orally treated with WTD at the dose of 3.15,6.30 and 12.60 g/kg respectively which was equally to 0.5,1 and 2 times of clinical daily dosage.The treatment was performed for 21 days.Mice in the Sham or SNL group,accepted the same volume of distilled water.Measurement of mechanical and heat hyperalgesiaThe 50%paw withdrawal threshold was analyzed at 1(1 h),3(0,1,2,3,4,and 24h),7,10,14,17 and 21 d after WTD administration.Measurement of heat hyperalgesiaThe paw withdrawal latency was measured at 2(1 h),4(0,1,2,3,4,and 24 h),8,12,16,18,and 20 d after WTD administration.At every time point,the paw withdrawal latency was detected for three times with the interval of 5 min.ELISAAccording to the manufacturer's protocols,we performed ELISA to detect the expression levels of cytokines and chemokines in the isolated L5 dorsal horn from SNL mice.Whole-genome microarray analysisThe whole-genome microarray analysis was performed to detect the gene information of spinal cord dorsal horn in mice of the Sham group,the SNL group and the SNL-WTD(12.60 g/kg)group.The total RNA was extracted and purified for whole-genome microarray detection with Agilent Whole Mouse Genome Microarray including 41174 coding gene probes.DEG screeningSignificant DEGs of different groups were identified using the criteria of a |log2-fold change(FC)|>0.5 and P value<0.05.DEGs were screened based on the hierarchical clustering analysis with the usage of the heat map package in R.Cluster analysis of the DEGs was performed by Cluster 3.0 based on the Euclidean distance.Collection of known NP-related genesThe collection of known NP-related genes were performed from two databases:? DrugBank database and?The Online Mendelian Inheritance in Man database.The drug-target interactions were selected with FDA approved drugs for NP and human gene or protein targets.Network construction and analysisGene-gene interaction networks were built up based on the STRING database.When the combined score of gene-gene interaction was higher than the median value it was selected for further study.Navigator software(version 2.2.1)was used to visualize the interaction networks.Function and pathway enrichment analysesKyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses were operated on the base of the Database for Annotation,Visualization and Integrated Discovery(DAVID),and the pathway data was collected from the FTP service of KEGG.Only KEGG pathways with P-values<0.05(corrected using the Bonferroni method)were selected.2.The identification of the bioactive compounds of WTD on NP and the investigation on its network mechanismsCollection of putative targets of WTDBased on the structure information collected from the TCM Database and the chemical compounds of the five herbs contained in the water extract of WTD we detected,the putative targets of WTD were predicted according to the structure similarity principle.Collection of known NP-related genesKnown NP-related genes were collected from DrugBank database,Human Phenotype Ontology,Therapeutic Target Database and DisGeNET-a database of gene-disease associations.Construction of the "NP-related gene--WTD-putative target" interaction networkThe "NP-related gene--WTD-putative target" interaction network was constructed using the links between the known NP-related genes and the putative targets of WTD,which were obtained from the public database STRING.Navigator software was used for the network visualization.The values of three topological features,including node's degree,betweeness and closeness,were calculated to evaluate the topological importance of the nodes in the network and chose the major network targets.Pathway enrichment analysisKEGG pathway enrichment analysis was performed on DAVID,KEGG pathways with P-values<0.05 were selected for further study.Molecular DockingMolecular docking simulation was performed to calculate the binding efficiency of BAC and the corresponding target proteins by Ledock and Pymol.The protein structure information of the candidate targets of WTD was collected from RCSB protein data bank and their resolutions have been carefully checked.The structures of PAE and LIQ were prepared by ChemDraw.The docking score(kcal/mol)indicates the binding energy between the ligand and the receptor.The higher absolute value of docking score represents the stronger binding efficiency of BAC and the corresponding target proteins.Surface Plasmon Resonance(SPR)experimentsBiacore T200 was used to measure the direct binding affinities between compounds and CCL5(GE healthcare).CCL5 was immobilized on a CM5 sensor chip,and it was fixed at 30 ng/?L and the immobilization level of GLU was about 8000 RU.Different concentrations of compounds containing 5%DMSO were serially injected into the channel to evaluate the binding affinity.A reference channel was only activated and blocked to eliminate compound unspecific binding to the surface of the chip.Regeneration buffer of 10 mM NaOH was added after each cycle of PAE.Extra wash with 30%DMSO was also added to remove the last remaining sample in the pipeline.The association constants(ka),the dissociation constants(kd)and the equilibrium dissociation constants(K0)of the compounds were obtained by fitting the data sets to 1:1 Langmuir binding model using Biacore T200 Evaluation Software.3.Evaluation of the analgesic effect and mechanisms validation of WTD and its bioactive compounds against NPAnimals and treatmentTwo parts of in vivo experiments were operated with male SD rats(180-200 g).In the first part,a total of 100 rats were randomly divided into seven groups(n=10,per group)including the Sham,SNL,SNL-WTD-treatment,SNL-the two-BAC combination-treatment,SNL-MVC-treatment,SNL-MVC+WTD-treatment and SNL-MVC+BAC-treatment,SNL-PAE,SNL-LIQ and SNL-PGB groups.The drug treatment was performed for three days via oral administration starting on the fourth day after SNL or Sham operation.In the second part,a total of 40 rats were randomly divided into four groups(n=10,per group)including the Sham,MIP-treatment,MIP+WTD-treatment and MIP+BAC-treatment groups.The treatment was performed for three consecutive days post the implantation of the intrathecal catheter.Induction of SNL modelIn brief,rats were anesthetized via isoflurane inhalation anesthesia.To expose the L5 spine nerve clearly,the transverse process of the sixth left lumbar was cut off.After that,the L5 nerve was carefully separated and tightly ligated with a surgical suture,followed by washing the skin with normal saline and sutured.The Sham rats were kept the naked L5 spine nerved un-ligatured.Measurement of mechanical hypersensitivityRats were acclimatized in individual clear boxes on the wire-mesh platform,to allow access to the ventral surface of the hind paws.The mechanical hypersensitivity was estimated by the sensitivity to the stimulation of von Frey hairs.The von Frey filaments(1.0-100.0 g)were presented perpendicularly to the plantar surface of the injected paw and held in this position for 5-8 s with enough force to cause a slight bend in the filament.Positive responses included an abrupt withdrawal of the hind paw or flinching behavior immediately following removal of the stimulus.A median paw withdrawal threshold was decided according to an adaptation of Dixon's up-down method.The 50%paw withdrawal threshold was analyzed at 0,0.5,1,1.5,2,3 h and 2,3 days after drugs administration,respectivelyMeasurement of cold hypersensitivityBriefly,20?L acetone was sprayed gently to the middle of the plantar surface of the hind paw by a 1 mL syringe from a short distance.The amount of times that rats were licking and/or shaking the hind paw during 5 min after acetone application was recorded and used as an index of responsiveness to cold hypersensitivity.The licking and/or shaking times was measured at 0,0.5,1,1.5,2,3 h and 2,3 days after drugs administration,respectively.The implantation of the intrathecal catheterFirstly,the rat was deeply anesthetized and then accepted the laminectomy to remove the thoracic spine.Then,the polyethylene drain was inserted into the spinal cord and located at the L4 to L5 level.The next,to judge whether the location of the PE drain was appropriate,5?L lidocaine(200 mg in 10 mL)was injected into the intrathecal catheter followed by saline,and the hind legs of rats were completely paralyzed post the administration of lidocaine was regarded as the positive signs.Rats without evidence of neurologic deficit or paralysis after the surgery and lidocaine injection were selected for further experiments in this study.ImmunohistochemistryImmunohistochemistry was performed to detect the expression patterns and cellular localization of GFAP,NeuN and CCL5 proteins in the dorsal horn of the L5 region of the rat spinal cord.In this study,anti-GFAP antibody(1:100,abcam,ab10062),anti-NeuN antibody(1:100,abcam,ab104224)and CCL5 polyclonal antibody(1:100,Elabscience,E-AB-17864)were used as the primary antibodies,a horseradish peroxidase conjugated secondary antibody were used for half an hour at room temperature and diamino benzidine was used as a substrate.RT-PCR analysisRT-PCR analysis was performed to detect the mRNA expression levels of CCL5,CCR5,GNAI1,SRC,PIK3CA and AKT,GAPDH was used as the internal control for normalization and quantification.Relative quantification of gene expression was assessed by the comparative cycle threshold(CT)method,after all experiments were performed for three times.Western blot analysisThe protein expression levels of CCL5,CCR5,GNAI1,p-SRC,p-PIK3CA and p-AKT in the dorsal horn of L5 spinal cord tissue were detected by western blot analysis and GAPDH was used as the internal control for normalization and quantification.All experiments were performed in triplicate.ELISAThe serum levels of tumor necrosis factor-alpha(TNF-?),interleukin(IL)-1? and IL-6 in different groups were detected by ELISA.ELISA kits were purchased from Elabscience Biotechnology Co,Ltd(Wuhan,China)and all experiments were carried out according to the manufacturer's protocols and the results were measured at 450 nm.Statistical analysisStatistical analyses were conducted by SPSS statistical software.For all quantification analyses,one-way ANOVA analyses were used.P-values less than 0.05 were considered significant.Data were represented as the meanąs.Results1.WTD produced analgesic action in a mouse SNL modelCompared with the Sham group,SNL surgery led to both rapid(1-2d)and persistent(21 d)mechanical hyperalgesia and heat hyperalgesia(allP<0.05).In addition,the expression levels of IL-1? and TNF-? secreted by microglia and the protein levels of CCL2 and CXCLlsecreted by astrocytes in the spinal cord dorsal horn at maintenance phase(10 d)after SNL also were significantly higher than those of Sham group(all P<0.05),which indicating that SNL induced glial activation in the spinal cord.WTD(3.15-12.60 g/kg)dose-dependently attenuated mechanical allodynia and heat hyperalgesia,which lasted for at least 2 h,with an optimal inhibitory effect 1 h post-WTD administration.The analgesic effect was sustained for at least one week when WTD was daily administered.Importantly,the time-course effect profile of WTD was similar from the first day to the seventh day,indicating that there was absence of drug tolerance.Furthermore,WTD treatment clearly decreased the expression levels of IL-1?,CCL2 and CXCL1 in the spinal cord dorsal horn in SNL mice dose-dependently(all P<0.05).In total 567 DEGs were identified between SNL and Sham-operated mice.Among these,18 genes(ADCY1,ADRA2A,B4GALT3,BRAF,BTG2,CHRNA4,DYNC1H1,EGFR,GL01,HTR1D,IL1R1,PDGFRA,PDPK1,PGR,PNPLA6,SCN1B,TEK,WARS)were identified as known NP-related genes.More importantly,unsupervised hierarchical clustering and scatter plot analyses of DEGs displayed good differentiation of the SNL samples and the Sham samples.The SNL-induced NP imbalance network consists of 756 nodes and 4774 edges.Based on the degree value of nodes,248 hub genes were regarded as SNL-induced NP-related genes,including many known genes involved in the pathways related to glial cell activation,neuro-immune responses and neuroinflammation according to the pathway enrichment analysis(all P<0.05).Altogether 442 DEGs were distinguished from the comparison between WTD samples and SNL samples.The unsupervised hierarchical clustering and scatter plot analyses of DEGs showed good differentiation of the above two groups.The WTD-SNL-network consists of375 nodes and 3077 edges.According to the 4 topological features of nodes including degree,closeness,betweenness and k-coreness,94 major nodes were selected.Pathway enrichment analysis showed that the major nodes in the WTD-SNL-network were significantly associated with the pathways that were involved in glial cell activation and neuroinflammation(all P<0.05).2.The identification of the bioactive compounds of WTD on NP and the investigation on its network mechanismsIn our previous study,a total of 162 chemical compounds were identified in the water extract of WTD,among them 8 chemical compounds were identified as the candidate bioactive compounds of WTD due to the good bioavailability and druggability.In total,1744 putative targets of the formula were predicted.Especially,WTD shared 107 targets with known FDA approved analgesic agents.Functionally,the putative targets of WTD were significantly enriched into various NP-related pathways,such as Chemokine signaling and NF-kappa B signaling pathways(all P<0.05).Based on the analysis of the "NP-related genes-WTD putative targets",a total of 453 hubs were screened.After the calculation of the three topological features(degree,betweenness and closeness)of each node in the direct interaction network of the above hubs,130 major hubs were identified as the candidate targets of WTD against NP,which were significantly associated with various neuro inflammation related pathways and the neurotransmitter transportation regulatory pathways.Notably,six candidate targets of WTD against NP(CCL5,CCR5,GNAI1,SRC,PIK3CA and AKT)were involved into the Chemokine signaling pathway which was the top enriched one.According to the docking result,the docking scores of PAE or LIQ to CCL5,CCR5,GNAI1,SRC,PIK3CA and AKT were all lower than-5.45 kcal/mol,indicating that the two BACs had strong binding efficiency to their candidate targets.The results of Surface plasmon resonance(SPR)assay further verified the binding affinities of PAE and LIQ to CCL5 protein.In details,both PAE(KD value was 6.667 ?M)and LIQ(KD value was 10.85?M)may bind CCL5 protein with micromolar affinity.The pharmacokinetics of PAE and LIQ after the single treatment of the two-BAC combination or WTD in the plasma of the normal rats at four time points were detected.The time to reach the maximum plasma concentration for both PAE and LIQ after received the two-BAC combination was 5 min,less than that of WTD(120 min and 30 min respectively),indicating that the rapid absorption of the two-BAC combination.The maximum plasma concentrations of PAE(668 ng/mL)and LIQ(17.1 ng/mL)in rats receiving the two-BAC combination were about 4.64-fold and 1.29-fold higher than that in rats receiving WTD(144 ng/mL and 13.3 ng/mL)respectively.Thus,Paeoniflorin and Liquiritin were identified as the two major bioactive compounds of WTD against NP.3.Evaluation of the analgesic effect and mechanisms validation of WTD and its bioactive compounds against NPThe SNL rat model and MIP-induced NP model were led to the mechanical allodynia and cold hypersensitivity successfully(all P<0.05).In contrast to the SNL group,all treatments showed significant inhibition effect of mechanical allodynia and cold hypersensitivity since 0.5 to 2.5 hours post drug administration(all P<0.05)and demonstrated similar effect in the next two days.According to the immunohistochemistry result,compared to the Sham group,the immureactive score of CCL5 protein in the SNL group was significantly increased,but was effectively reversed by the treatment of the two-BAC-combination and WTD.In addition,the real-time PCR and western blot results also proved that,SNL significantly upregulated the gene and protein levels of CCL5,CCR5,GNAI1,SRC,PIK3CA and AKT in the dorsal horn of L5 spinal cord tissues(all P<0.05),which were markedly reduced by the administration of the two-BAC combination or WTD(all P<0.05).ELISA result indicating that,SNL led to the increase of TNF-?,IL-1?,and IL-6 at protein level(all P<0.01),which were effectively reduced by the treatment groups(all P<0.05).ConclusionWe clarified the gene expression patterns of SNL mice in spinal cord tissue and demonstrated that WTD produced a marked analgesic effect on NP by inhibiting glial cell activation and neuroinflammation.Moreover.Paeoniflorin and Liquiritin were identified as the two major bioactive compounds of WTD against NP.Further experiments verified that the combination of Paeoniflorin and Liquiritin effectively alleviated NP by suppressing CCL5,CCR5,GNAI1,SRC,PIK3CA and AKT expression at both gene and protein level,which were similar to WTD.Importantly,the administration of Paeoniflorin and Liquiritin alone didn't exert as prominently therapeutic effects as that of the two-BAC-combination did Collectively,Paeoniflorin and Liquiritin may be the main BACs of WTD in alleviating NP,and the combination of them may be a promising agent for pain control. |
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