| OBJECTIVE:The anti-arthritis effect of Huangqin Qingre Chubi Capsule(HQC)and its mechanism,especially whether it improves rheumatoid arthritis(RA)through FZD8-Wnt/β-catenin signal axis,were studied using adjuvant arthritis(AA)rats and FLS from RA patients.METHODS:Animal experiment part: Complete Freund’s adjuvant(CFA)was injected into the right hind foot of rats with 0.1 m L intradermal injection to establish AA rat rheumatoid arthritis model.The animals were divided into normal group,model group,HQC low,medium,and high(0.18 、 0.36 、 0.72g/kg)dose groups,and methotrexate(MTX0.75g/kg).The therapeutic effect of HQC on AA rats was evaluated by joint score,hind foot swelling,paw retraction threshold,and body weight.Micro-CT was used to detect the hind paws of AA rats to verify the protective effect of HQC on joint bone erosion.Network pharmacology、Molecular docking and molecular dynamics:Prediction of HQC regulated signal pathway based on network pharmacology,and molecular docking was carried out for the quality markers of FZD8 and HQC,namely baicalin,baicalein,chlorogenic acid,and geniposide.The binding stability of four small molecules with FZD8 was studied through molecular dynamics simulation.Cellular experiment part: The synovium and primary FLS of AA rats,as well as FLS of RA patients,were divided into normal group 、 model group 、 HQC medium dose groupand blank group.Using real-time fluorescence quantitative PCR(RT-qPCR)to detect MMP3、Fibronectin、β-catenin、CCND1 and C-myc m RNA expression.Western blot detection of MMP3、Fibronectin、β-catenin、CCND1and C-myc proteins expression.CCK8 method was used to detect FLS cell proliferation.ELISA method detected IL-1β、IL-6、IL-8 and β-catenin level.The confocal microscope detected β-catenin entry status.Immunofluorescence assay was used to detect the entry of FZD8 into the nucleus.Construct an overexpression vector of FZD8,and detect the success of FZD8 vector construction using RT-qPCR.Then RT-qPCR detected the m RNA expression of catenin,CCND1,and C-myc.Use CCK8 method to detect the proliferation of FLS cells after transfection.RT PCR was used to detect the expression of pathological genes MMP3 and Fibronectin m RNA after transfection.RESULTS:Animal experiment part: Compared with the control group,AA rats developed secondary inflammation on the 14 th or 15 th day after modeling,with redness and swelling in the left hind and forelimbs.Arthritis scores and increased foot swelling were significantly higher in the model group than in the normal group(P<0.05),The above two indexes in the HQC group and MTX group were significantly downregulated(P<0.05).The paw contraction threshold and body weight index of the HQC treatment group and the MTX group were significantly higher than those of the model group(P<0.05).Micro CT scanning of rat ankle joints,Severe bone resorption,joint bone destruction,and joint space enlargement occurred in the model group,and the joint injury of rats in HQC group was significantly reduced.For bone microfracture parameters,the HQC group significantly increased bone mineral density(BMD),bone volume fraction(BV/TV),and bone trabecular thickness(TB.TH)(P<0.05).The bone surface/bone volume(BS/BV)and bone trabecular separation(TB.SP)were significantly reduced(P<0.05).Network pharmacology,molecular docking,and molecular dynamics analysis: The network pharmacology predicted that β-catenin,CCND1 core target genes,and Wnt signaling pathway.FZD8 protein and four compounds in HQC have good spatial binding ability,and the specific binding affinity is: baicalein is-6.8 kcal/mol,baicalin is-7.8kcal/mol,chlorogenic acid is-7.2/kcal/mol and geniposide is-6.0kcal/mol.Molecular dynamics simulations were conducted to investigate the binding stability of four small molecules with FZD8.After 40 ns,the simulation trajectory tended to be stable,and finally Baicalein(red line)was stable around 3.4 ?,Baicalin(purple line)was stable around 3.7 ?,Chlorogenic＿acid(green line)was stable around 3.5 ?and Geniposide(blue line)was stable around 4.0 ?.After the binding conformations of the four complexes reached equilibrium,they were relatively stable without obvious fluctuations.Cellular experiment part: RT-qPCR results showed that HQC significantly inhibited MMP3,Fibronectin 、 β-catenin,CCND1 and C-myc m RNA expression(P<0.05),Western blot results showed that HQC significantly inhibited MMP3、Fibronectin、β-catenin 、 CCND1 and C-myc proteins expression(P<0.05),CCK8 detection also confirmed that HQC can significantly inhibit the proliferation of AA FLS(P<0.05).The expression differences of IL-1β、IL-6 and IL-8 were detected by ELISA in AA FLS of each group(P<0.05),It indicates that HQC can inhibit the expression of the three interleukin factors to varying degrees.HQC significantly inhibited the proliferation of FLS in RA patients and also reduced IL-1β、IL-6 and IL-8(P<0.05).ELISA method was used to detect β-catenin level in the nucleus of RA FLS,The expression ofβ-catenin in the nucleus was observed under confocal microscopy.The results showed that HQC significantly reduced the expression of β-catenin in the nucleus of RA FLS(P<0.05).RT-qPCR confirmed that the expression of FZD8 significantly increased by about 3-fold in RA synovial tissue and FLS(P<0.05),and immunofluorescence further confirmed the above results.After adding HQC containing serum to AA FLS and RA FLS,the level of β-catenin in the cell nucleus significantly decreased(P<0.05).At the same time,in FLS containing HQC and FZD8 overexpression vectors,the level ofβ-catenin protein in the nucleus was restored to some extent(P<0.05).RT-qPCR showed that serum containing HQC inhibited the expression of CCND1 and C-myc in AA FLS(P<0.05),and overexpression of FZD8 may interfere with the effect of HQC.The addition of HQC serum to RA FLS resulted in a decrease in the expression of RA pathological genes Fibronectin and MMP3(P<0.05).The overexpression of FZD8 HQC significantly reversed this effect,which also confirms that HQC inhibits RA pathology and exhibits good anti RA effects through FZD8.RT-qPCR showed that serum containing HQC inhibited the expression of CCND1 and C-myc in AA FLS(P<0.05).This result was also observed in RA FLS.CCK8 method was used to detect the proliferation of FLS cells after transfection(P<0.05).CONCLUSION:In conclusion,this work verified that HQC has obvious antiinflammatory and anti-rheumatic effects in AA rats.The expression of FZD8 was significantly increased in synovial tissue of AA rats and FLS of AA and RA patients,which may be a potential new target for the diagnosis and treatment of RA.We confirmed that HQC improved.RA through the FZD8-Wnt/β-catenin signaling pathway,which provided a clear treatment mechanism for HQC to improve RA,and also provided a basis for clinical promotion of HQC. |
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