Study On The Alleviation Of Fengshi Gutong Capsule On Collagen-Induced Rheumatoid Arthritis In Mice

Backgrounds:Fengshi Gutong capsule(FSGT)indexed in Chinese Pharmacopoeia has functions including warming meridians and dispersing cold,and relieving pain by dredging collaterals.Rheumatoid arthritis(RA)is a chronic autoimmune disease with erosive arthritis as its main clinical manifestation,it highly caused disability.There is currently no effective drug for RA treatment.FSGT is generally used for the treatment of RA in clinic.However,the material basis and engaged mechanism of FSGT in improving RA are still unclear.Objective:This study aims to observe the alleviation of RA provided by FSGT,to explore the engaged mechanism,and further to find the active compounds.This study will provide experimental basis for the application of FSGT in RA treatment in clinic.Methods:1.The alleviation of FSGT on collagen-induced arthritis(CIA)in mice was conducted.DBA/1 mice were immunized at the base of the tail by intradermal injection with 100μg chick type II collagen(CII)(2 mg/m L)that was emulsified in an equal volume of complete Freund adjuvant(CFA)(4 mg/m L).Second immunization with 100 μg of CII in incomplete Freund adjuvant(IFA)was administered on day 21 after the first immunization.Different doses of FSGT(100,300,900 mg/kg)were given to mice by intragastric administration;and methotrexate(MTX)(2 mg/kg)was given to mice by intraperitoneal injection.The intensity of arthritis was assessed by using a clinical scoring system.The swelling degree of hind paw in mice was measured by using electronic Vernier caliper.The change in joint space and cartilage surface calcification were detected by using X-ray.The pain of mice was assessed by measuring mechanical hyperalgesia via 50% mechanical paw withdrawal threshold(PWT).Synovitis,pannus and cartilage damage were evaluated via hematoxylin-eosin staining(H&E),Masson’s trichrome(Masson)and Safranin O and fast green staining(Safranin O),respectively.Serum content of high-sensitivity cardiac troponin I(hs-c Tn I)was measured by using enzyme-linked immunosorbent assay(ELISA)and serum activity of creatine kinase(CK)was detected by using kits.The electrocardiogram was measured by Power Lab within 24 h and at 4 weeks after drug administration.Cardiomyocyte hypertrophy and collagen deposition in the heart was measured by H&E and Masson staining.2.The maximum tolerance test of FSGT was conducted in mice.A single administration of FSGT with maximum allowable concentration and maximum allowable volume was given to mice,and the time and the number of death of mice were observed for 14 days.3.Network pharmacology analysis: The active ingredients from FSGT,including compounds recorded in Pharmacopoeia and literature report,were obtained via traditional Chinese medicine systems pharmacology database and analysis platform(TCMSP)with oral bioavailability(OB)≥30% and drug-likeness(DL)≥0.18 as filter conditions.The protein targets of FSGT were obtained via TCMSP and Swiss target prediction.And then the active ingredients and the protein targets of FSGT were imported into Cytoscape 3.6.1 software to establish an active ingredients-targets network.The protein targets of RA and arthralgia were obtained via Gene Cards databases after median screening.The protein targets of FSGT,RA and arthralgia were uploaded into the Venny 2.1.0 platform to get common targets.The common targets were analyzed by using the String database to obtain the protein-protein interaction(PPI)network,and then they were imported into Metascape database for gene ontology(GO)and kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analyses to obtain the preliminary mechanism involved in the alleviation provided by FSGT on RA and arthralgia.4.Serum contents of rheumatoid factor(RF),vascular endothelial growth factor(VEGF),tumor necrosis factor α(TNFα)and interleukin 6(IL-6)were measured by ELISA.And the expression of phosphorylated protein kinase B(Akt),interleukin-1β(IL-1β)and intercellular cell adhesion molecule-1(ICAM-1)in synovial and cartilage tissues were measured by using western-blot(WB)assay.5.The m RNA expression of IL-1β and inducible nitric oxide synthase(i NOS)were measured in RAW264.7 cells after cells were incubated with lipopolysaccharide(LPS)for 24 h by using real-time polymerase chain reaction(Real-time PCR)to find the active anti-inflammatory compounds.6.The inhibition of compounds on human umbilical vein endothelial cells(HUVEC)migration was detected in cells when cells were incubated with VEGF for 18 h or 24 h to find the active anti-angiogenic compounds.7.The alleviation of collagen-induced arthritis in mice provided by Aconiti Radix Cocta and Aconiti Kusnezoffii Radix Cocta was conducted.DBA/1 mice were immunized at the base of the tail by intradermal injection with 100 μg CII(2 mg/m L)emulsified in an equal volume of CFA(4 mg/m L).Second immunization with 100 μg of CII in IFA was administered on day 21 after the first immunization.FSGT(900 mg/kg),ARC&AKR(TOX)(46,410 mg/kg)and non-ARC&AKR(n TOX)(54,490 mg/kg)were given to mice by intragastric administration(i.g.),and MTX(2 mg/kg)was given to mice by intraperitoneal injection(i.p.).The intensity of arthritis was assessed by using a clinical scoring system.Electronic Vernier caliper was used to measure the swelling degree of hind paw in mice.X-ray was used to detect changes in joint space and cartilage surface calcification.PWT was used to measure mechanical hyperalgesia to assess the pain of mice.Synovitis,pannus and cartilage damage were evaluated via H&E,Masson and Safranin O staining,respectively.Serum content of hs-c Tn I was measured by ELISA.The electrocardiogram was measured by Power Lab within 24 h and at 4 weeks after drug administration.Cardiomyocyte hypertrophy and collagen deposition in the heart was measured by H&E and Masson staining.Results:1.FSGT(300,900 mg/kg)obviously reduced the increased clinic score,alleviated the swelling and pain in the hind paws,the narrowed joint space and the increased infiltration of immune cells,and decreased the increased synovial cell proliferation,cartilage surface erosion and pannus formation in ankle joint of CIA mice.However,FSGT didn’t increase serum hs-c Tn I content and CK activity,and it also didn’t cause abnormal electrocardiogram,cardiomyocyte hypertrophy and collagen deposition in the heart.2.The maximum tolerated dose of FSGT in mice was 11.5 g/kg,which is about 32 times that of the oral dose in adults.3.Totally 130 active compounds and 279 compounds-related targets were obtained.38 common targets were analyzed by String and Metascape database: PPI networks showed that the top 5 genes were IL-6,TNFa,C-C motif chemokine 2(CCL2),VEGFA,ICAM1.GO items showed that inflammation took a large proportion,and KEGG pathways indicated that phosphatidylinositide 3-kinases(PI3K)-AKT signaling pathway and nuclear factor-k-gene binding(NFκB)signaling pathway were the critical pathways in the FSGT-provided alleviation on RA.4.FSGT obviously reduced the increased serum contents of RF,VEGF,IL-6 and TNFα in CIA mice,decreased the increased IL-1β and ICAM-1 protein expression and phosphorylation of Akt in synovium and cartilage from CIA mice.5.The results of RAW264.7 cells in vitro showed that hypaconitine(HA)(5,10 μM),benzoylhypaconine(BHA)(5,10 μM),pseudoephedrine hydrochloride(PSE)(5,10 μM),myricetin(MYR)(0.5,1 μM),uvaol(UVA)(5,10 μM),glycyrrhetnic acid(GA)(10 μM),isoliquiritigenin(ISL)(1,5 μM),quercetin(QUER)(5,10 μM)and kaempferol(KAE)(5,10 μM)all obviously reduced the increased IL-1β and i NOS m RNA expression in LPS-stimulated RAW264.7 cells.6.The results of HUVEC migration in vitro showed that BHA(5,10 μM),kaempferide(KF)(0.5,1 μM),PSE(5,10 μM),GA(5,10 μM),ISL(5,10 μM),QUER(0.6,3 μM)and KAE(5,10 μM)obviously reduced the increased cell migration when HUVECs were incubated with VEGF for 18 h or 24 h.7.ARC&AKR(410 mg/kg)and FSGT(900 mg/kg)obviously reduced the increased clinic score,swelling and pain in the hind paws,narrowed joint space,the increased infiltration of immune cells,the enhanced synovial cell proliferation,cartilage surface erosion and pannus formation in ankle joint of CIA mice.However,it cannot improve RA after removing ARC&AKR in FSGT.Besides,ARC&AKR(410 mg/kg)also cannot increase serum hs-c Tn I level,and cause abnormal electrocardiogram,cardiomyocyte hypertrophy or collagen deposition in the heart.Conclusion:1.FSGT can alleviate experimental RA in CIA mice by relieving the formation of synovitis and pannus.Meanwhile,it had a good analgesic effect with no obvious side effects especially cardiotoxicity.The engaged mechanism may be associated with inhibiting inflammation and angiogenesis via decreasing the expression of inflammatory cytokines and VEGF thourgh reducing the phosphorylation of Akt.2.The results in vitro indicated that BHA,PSE,GA,ISL,QUER and KAE have both anti-inflammatory and anti-angiogenic activities,which may greatly contribute to the FSGT-provided alleviation on RA.3.ARC&AKR can alleviate experimental RA in CIA mice by relieving the formation of synovitis and pannus.Meanwhile,it had a good analgesic effect with no obvious side effects especially cardiotoxicity.FSGT obviously lost the pharmacological activity of improving RA after the elimination of ARC and AKR.These results suggest that ARC and AKR may contribute greatly to the FSGT-provided alleviation on RA.