| Background:Rheumatoid arthritis(RA)is an autoimmune disease characterized by synovial inflammation and progressive joint destruction,ultimately to considerable disability.The pathogenesis of RA has not been fully elucidated;there are no effective therapies for RA to date.Disease-modifying antirheumatic drugs(DMARDs)are mainly used in clinical practice,including conventional synthesis DMARDs,biological DMARDs and targeted synthetic DMARDs.Guidelines recommend the conventional synthesis DMARDs especial methotrexate as the first choice for RA treatment,but long-term use of these drugs may cause adverse reactions such as gastrointestinal lesions,liver toxicity,and cardiovascular complications,which lead to intolerance.Biological MDARDs and target synthesis DMARDs are newly developed drugs with promising results,but there are still about 20%of patients do not achive good responses to those drugs.Moreover,those drugs are expensive,and long-term use may also cuase adverse reactions such as respiratory tract infection and blood system toxicity.Therefore,development of new drugs for the treatment of RA has always been one of the key tasks.Traditional Chinese medicine(TCM)is a treasure of the Chinese nation.It has been used for thousands of years and is a valuable resource for finding RA treatment drugs.Terminalia Chebula Retz(TC)has been widely used as ethnic medicine especially in Tibetan and Mongolian medicine,and is known as the "king of all medicines".Some patented drugs,such as "fengshi zhitong pills" and "zachong Shisanwei pills",containing TC as their primary herb medicine are used in clinical practice for the treatment of RA.It has been reported that the more than 80%patients achive treatment goal.However,the active components and the mechanisms of TC for treating RA are remaining unclear,which seriously limited the further application of TC.Therefore,it is urgent to apply modern pharmacological research methods to identify the key active components of TC for treating RA and reveal its molecular mechanisms.Promoting the modern research of Chinese medicine such as Terminalia chebula has great importance.Objective:This study intends to predict the potential active components-targets-signaling pathways of TC for the treatment of RA based on network pharmacology;and then perform qualitative and quantitative analysis of the predicted potential active components by using high-resolution mass spectrometry and high performance liquid mass spectrometry.After that,the LPS induced RAW264.7 cell inflammation model,IL-1β induced MH7A cell inflammations model as well as collagen induced arthritis in rat and mice model were utilized in this study for fully exploring the main active components and molecular mehanisms of TC.Aim to provide new evidence and new strategies for the development of new drugs for TCM for the treatment of RA.Methods and Results:1.Predict the potential active components-targets-signaling pathways of TC for RA treatment based on network pharmacology1.1 Qualify candidate active ingredientsUse the TCM System Pharmacology Technology Platform(TCMSP)and public databases to search for the chemical components in TC that has been reported in the literature.The potential active components should meet the inclusion criteria:1.oral bioavailability OB%>30%and drug-like DL>0.18;2.those who do not meet criteria 1 but has been reported to show clearly anti-inflammation activity.According to the criteria,we screened out 10 potential components,of whom 6 polyphenols and 4 alkaloids.1.2 Qualify the target of active component for RA treatmentThe reverse molecular docking server(DRAR-CPI)is used to predict the potential active components’ targets,and then combined with the TCMSP’s own target,obtaining 186 targets.The RA related targets were collected from the National Center for Biotechnology Information NCBI database and genecard database,obtained 1953 targets.The predicted 186 targets were matched to 1953 RA-related targets,and 47 targets were obtained as the target of TC for RA treatment.According to PPI analysis,TP53,TNF,IL6,CASP3,ESR1,MAPL14,VEGFA,IL10,PTGS2 and BCL2L1 are the top 10 key targets.TP53,CASP3,VEGFA and BCL2L1 are mainly related to cell proliferation and apoptosis;ESR1 is mainly related to hormone regulation;TNF,IL6,MAPK14,IL10 and PTGS are mainly related to inflammation.1.3 Construction the "active components-targets-signaling pathway" networkKEGG pathway analysis was performed based on the 47 predicted targets through the DAVID 6.8 platform.A total of 54 KEGG pathways were involved in the anti-RA process of TC,the leading pathways mainly involved TNF signaling pathway,NOD like receptor(NLR)signaling pathway,and T cell receptor(TCR)Signaling pathway,Toll like receptor(TLR)signaling pathway,nuclear factor-κB(NF-κB)signaling pathway,JAK-STAT signaling pathway,and mitogen-activated protein kinase(MAPK)signalingpathway,etc.The above signaling pathways have crosstalk,and ultimately induce inflammation through NF-κB and MAPK signaling pathways.It suggests that the NF-κB and MAPK signaling pathways play a central role in the treatment of rheumatoid arthritis.2.Qualitative and quantitative analysis as well as in vitro activity evaluation of TC extracts and potential active components based on the predicted results of network pharmacology2.1 Preparation of extractsWe chose TC and processed TC(simmer TC)that produced from Yunnan province,extracting with ultrapure water,50%ethanol aqueous solution and ethanol solution as solvents by using ultrasonic extraction method to obtain water extract,50%ethanol extract and ethanol extract,respectively.2.2 Qualitative analysis of components in different extractsBecause the 50%ethanol extract has both the characteristics of water extract and ethanol extract.Therefore,we use high-resolution mass spectrometry to qualitatively analyze the components in 50%ethanol extract of TC and processed TC based on the predicted results of network pharmacology.As a result,6 polyphenols were all determined while 4 alkaloids were not found.2.3 Quantitative analysis of extract componentsthe Folin-Ciocalteu method and high performance liquid chromatography(HPLC)method were used to quantitatively analyze the total phenol content and each polyphenols in the water extract,50%ethanol extract and ethanol extract of TC and processed TC.Results showed that the total polyphenols content in processed TC is significantly higher than that of unprocessed TC.Among them,the 50%ethanol extract has the highest total polyphenols content,which is significantly higher than the water extract and the ethanol extract,showing that the 50%ethanol water solvent is the most suitable extraction solvent.The result of HPLC analysis is similar to that of total polyphenols.The content of polyphenols obtained from 50%ethanol extract is the highest.The content of each compound listed from high to low in 50%ethanol extract of processed TC is chebulanin>chebulagic acid>gallic acid>corilagin>ellagic acid,among them,chebulanin showed the highest content in TC polyphenols.2.4 Study on the activity of extracts and polyphenols2.4.1 Study on the antioxidant activity of water extract,50%ethanol extract and ethanol extract as well as polyphenols of TC and processed TC by DPPH assayResults showed that the extracts of TC and processed TC have good antioxidant activity.No matter water extract,50%ethanol extract or ethanol extract,the scavenging activity of DPPH of processed TC is higher than that of unprocessed TC.The processed TC 50%ethanol extract has the highest scavenging activity with an IC50 value of 7.305 μg/mL.Under the same concentration,the antioxidant activity of each polyphenols compound is stronger than that of the 50%ethanol extract of processed TC.Among them,gallic acid has strongest antioxidant activity,and then followed by ellagic acid;the rest 4 components are equivalent to each other.2.4.2 Study on the anti-inflammatory activity of water extracts,50%ethanol extracts and ethanol extracts of processed TC and its polyphenols by determination of pro-inflammatory cytokines IL-6 and IL-8 in IL-1β induced MH7 A inflammation modelThree different solvent extracts could significantly inhibit the expression of IL-6 and IL-8 while 50%ethanol extract showed best result,followed by the water extract,and finally the ethanol extract.Among those polyphenolic components,both chebulanin and chebulagic acid have a strong inhibitory effect on IL-6 and IL-8 expression within the concentration range investigated,and the inhibitory effect increases as the concentration increases.2.5 Clarify the main active components of TC by principal components analysisBy using the content,the IC50 of DPPH scavenging activity,the inhibition rate of IL-6 and IL-8 at low,middle and high concentrations as indicators,we used principal components analysis to make a rank of those polyphenols to clarify the main active components.After evaluation,the scores of those 5 components were chebulagic acid(1.26),chebulanin(1.02),corilagin(-0.26),ellagic acid(-0.35)and gallic acid(-1.70),respectively.Chebulagic acid and chebulanin had a significantly high score than that of other three components,and therefore,they are identified as the main anti-arthritic active components of TC.3.The pharmacodynamics study of processed TC 50%ethanol extract and the main active component chebulanin in collagen-induced arthritis model in ratsBecause of the large dosage required for the in vivo pharmacodynamics study,the commercial supply of chebulagic acid and chebulanin is insufficient.We successfully prepared chebulanin by ourselves.Thus,we chose processed TC 50%ethanol extract and chebulanin for pharmacodynamics study in vivo.3.1 Methods3.1.1 Establishment of collagen-induced arthritis(CIA)in ratsWe use bovine type Ⅱ collagen and incomplete Freund’s adjuvant in a ratio of 1:1 to emulsify into emulsion,and then subcutaneously inject 0.2 mL of emulsion into the tail of rat;on day 7,the second immunization with the same reagent in the same way was induced and the rat CIA model was established.3.1.2 Group and drug administration42 SD rats(half male and half male,weight 160±20g)were randomly divided into 7 groups with 6 rats in each group.They are the normal group,the CIA model group,and CIA+low-dosage group of processed TC 50%ethanol extract(crude powder amount 0.625 g/kg,0.5 times of the clinical dosage),CIA+middle-dosage group of processed TC 50%ethanol extract(1.25 g/kg,equal to the clinical dosage),CIA+high-dosage group of processed TC 50%ethanol extract(2.5 g/kg,2 times of the clinical dosage),CIA+chebulanin group(60 mg/kg,equal to the content of chebulanin in high-dosage group)and CIA+positive control group(MTX 2.5mg/kg).50%ethanol extract and chebulanin were administrated by intragastric gavage every day from the day 0 to day 28.MTX was gavaged every 7 days.The normal group and CIA model group were given with the same volume of normal saline solution every day till 28 days.3.2 Results3.2.1 Processed TC 50%ethanol extract and chebulanin can reduce the incidence of CIA in rats and relieve symptomsThe rats in the CIA model group began to develop symptoms around day 10,and the overall incidence was significantly higher than that of other groups.Hind paw swelling is predominant phenomenon after the onset.Although the treatment group also developed disease,the incidence and severity index scores are much lower than that of the CIA model group.As for the right hind paw thickness,the average thickness of the normal group was 0.61±0.06 cm.The thickness of rats in the model group gradually increased with the development of the disease with the average thickness was 0.85±0.17 cm at the end.3.2.2 Processed TC 50%ethanol extract and chebulanin can reduce the expression of pro-inflammatory cytokinesOn the day 21,although some rats had obvious paw swelling,the levels of TNF-α in plasma did not show any difference among theose groups.However,when it comes to day 28,TNF-α in the CIA model group was significantly increased and is much higher than that of normal group.Low,middle and high dosage group of TC and chebulanin also significantly inhibit the expression of TNF-α as compared with the CIA model group.The trends of IL-1βand IL-6 are similar to TNF-α suggesting that 50%ethanol extract of processed TC and chebulanin have good anti-arthritic activity.3.2.3 Processed TC 50%ethanol extract and chebulanin have no toxicity on liver,kidney and immune organsThere was no significant difference in liver and kidney function indexes(creatinine,ALT,AST and ALT/AST),thymus index and spleen index among those groups.4.Preliminary study on the anti-arthritic mechanism of chebulanin4.1 Methods4.1.1 Establishment of CIA model in miceBovine type Ⅱ collagen and complete Freund’s adjuvant were mixed and emulsified at a ratio of 1:1 to emulsion,and 0.1 mL was intradermally injected into the base of the tail of the mouse for primary immune induction;on the day 21,bovine type Ⅱ collagen and incomplete Freund’s adjuvant was mixed and emulsified in a ratio of 1:1 to emulsion,and 0.1 mL was intradermally injected into the base of the tail of the mouse for secondary immune induction;the mouse CIA model was constructed through two inductions.4.1.2 Group and drug administrationThe DBA/1 mice were randomly divided into 5 groups,each with 10 mice,normal control group,CIA model group,CIA+chebulanin prophylactic group,CIA+chebulanin therapeutic group and CIA+positive control group.Among them,the chebulanin prophylactic group was administered intragastrically at a fixed time every day after the second immunization on day 21;the chebulanin therapeutic group and the positive control group were observed for onset of RA and discard mice without disease to guarantee more than 8 mice in each group and given continuous administration for 21 days.The normal control group and CIA model group were given the same volume of drug-free solution by gavage every day for 21 days.4.1.3 LPS-induced RAW264.7 cell inflammation model to study the regulation of chebulanin on MAPK pathway and NF-κB pathwayWe chose different concentrations(25μM,50μM and 100μM)of chebulanin that do not affect cell growth to interfere with LPS stimulated RAW264.7 cells,and observe the effect of chebulanin on the nuclear transcription of MAPK pathway p38 and NF-κB pathway p65 with laser confocal;The nuclear protein extraction kit was used to separate the nuclear protein,and Westen blot was used to detect the expression of p38 and p65 in the nucleoprotein.Finally,p38 pathway inhibitors and NF-κB pathway inhibitors were selected to further verify the influence of chebulanin on the pathway.4.2 Results4.2.1 Chebulanin decreased the incidence of arthritis in CIA miceThe results showed that collagen-induced arthrits developed gradually after booter injection.The incidence of arthritis reached 90%in the CIA model group.In the contract,there was no incidence of arthritis during the prophylactic treatment(p<0.05).At the end of the experiment,the incidence of arthritis was still lower in the prophylactic group compared with that in the CIA model group.The CIA model mice showed obvious joint swelling and redness compared with that in the normal control group.On the other hand,chebulanin and leflunomide treatment could significantly relieve these symptoms of RA.4.2.2 Chebulanin alleviated synovitis and impeded bone destructionWe also used micro-CT and HE staining to further investigate the impact of chebulanin treatment on CIA related pathological changes.3D-reconstruction photographs from the micro-CT images showed that the normal control group had a clear joint structure and smooth joint surface;however,the CIA group exhibited a rough bone surface and obvious erosion in joints and paws.The chebulanin and positive control groups showed significant alleviation of these changes,with little or no erosion.HE staining also confirmed that chebulanin can alleviate joint inflammation and prevent joint destruction.4.2.3 Chebulanin inhibited the secretion of pro-inflammatory cytokinesThe levels of TNF-α and IL-6 in serum were significantly increased in the CIA model group compared with those in the normal control group;whereas,chebulanin prophylactic and therapeutic treatment inhibited the secretion of these pro-inflammatory cytokines,particularly in the therapeutic treatment group.4.2.4 Chebulanin can inhibit phosphorylation of p38,JNK,p65 and IκBαWestern blot analysis of mouse joint tissue proteins showed that the levels of phosphorylate p38(p-p38),p-JNK,p-ERK in the MAPK signaling pathway as well as p-p65 and p-IκBα in NF-κB signaling pathway were significantly higher in the CIA model group as compared with control group.However,chebulanin treatment could significantly inhibit p-p38 and p-JNK but not affect p-ERK.In addition,Chebulanin also significantly inhibited the phosphorylation of p65 and IκBα.4.2.5 Chebulanin inhibits p38 and p65 nuclear transcriptionA significant nuclear translocation of p65 and p38 was observed after LPS stimulation,which was significantly suppressed by chebulanin treatment in a concentration-dependent manner(25-100 μM).Western blot analysis was used to detect nuclear p65 and p38.The results showed that chebulanin suppressed p65 and p38 translocation from the cytosol to the nucleus.4.2.6 Chebulanin has a synergistic effect with pathway inhibitorsBoth chebulanin and BAY 11-8072 or SB203580 could significantly inhibit the release of IL-6 and TNF-α induced by LPS.Concurrent administration of chebulanin and the inhibitors strengthened the effect of chebulanin on LPS-induced inflammatory cytokine release.Conclusions:1.By using network pharmacology,we predicted out that 10 potential active components of TC for RA treatment.They show anti arthritic effect by acting on 47 targets such as such as TP53,TNF-α and IL6 and regulate 54 signaling pathways with NF-κB and MAPK as the main pathways.2.By using ultrapure water,50%ethanol water solution and ethanol solution as extraction solvents,we systematically compared the TC and processed TC in total polyphenols content,antioxidant and anti-inflammatory activities.It has been cleared that processed TC is better than unprocessed TC in terms of total polyphenols content,antioxidant and anti-inflammatory activity.Compared with water extracts and alcohol extracts,50%ethanol extracts have the highest polyphenol content and the strongest antioxidant and anti-inflammatory activities.3.To the best of our knowledge,it is the first time to evaluate the anti-arthritic activity of processed TC 50%ethanol extract.The results showed that 50%ethanol extract of TC can reduce the incidence of arthritis in CIA rats in a dose-dependent manner,relieve joint swelling,and decrease the expression of pro-inflammatory cytokines such as TNF-α,IL-1β and IL-6.4.We systematically compared and comprehensive evaluation 5 active components(gallic acid,corilagin,chebulanin,chebulagic acid and ellagic acid)in terms of content,DPPH free radical scavenging IC50 value and also inhibition rate of IL-6 and IL-8 in low,middle and high concentration.Results showed that chebulagic acid and chebulanin were the main active components of the extracts.5.We reported that chebulanin is the main active components in TC extract for the first time and systematically evaluated its pharmacodynamics and mechanisms for RA treatment.Results showed that chebulanin could reduce incidence and relieve symptoms on CIA-induced arthritis in rats and mice.By inhibiting the phosphorylation and nuclear transcription of MAPK and NF-κB-related proteins,it can further inhibit the expression of pro-inflammatory factors,alleviate joint inflammation,and play a role in RA treatment. |
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