| Rheumatoid arthritis(RA)is a prolonged autoimmune disorder,which is characterized by synovial hysperplasia.It will cause joint pain,swelling,and rigidity of patients,thereby leading to severe physical dysfunction and reduced life quality.Statistics show that approximately 0.5-1.0% of the global population suffers from RA,and approximately one-third of them are likely to be disabled within 20 years.If RA is not treated appropriately,it will gradually exacerbate,finally causing progressive disability and socioeconomic burdens.In addition,the current treatment of RA drugs includes: non-steroidal anti-inflammatory drugs,anti-rheumatic drugs,glucocorticoids,biological agents,etc.Glucocorticoids and non-steroidal anti-inflammatory drugs are effective in easing stiffness and pain but do not retard the course of RA.Diseasemodifying anti-rheumatic drugs can efficiently attenuate joint swelling or deformity,inflammation,and pain,whereas their ability to restrain destruction in joints is very limited.In addition,the adverse effects of these drugs include cardiovascular complications,gastrointestinal lesions,and reproductive toxicity,as well as the high expenses of biological agents,which limit their long-term use.Hence,it is very urgent to develop a new anti-RA drug with more favourable safety profile,more efficient property,and less low side effect.Clematidis Radix et Rhizoma(CRR,Weilingxian in Chinese,Ranunculaceae)is mainly derived from Clematis manshurica Rupr.,Clematis hexapetala Pall.,and Clematis chinensis Osbeck.As an important Traditional Chinese medicine,this herb is used to dispel dampness,dredge meridian,and relieve pain.Previous investigations reported that CRR has been proved to possess great anti-RA synovial hyperplasia effect,but studies on the pharmacodynamic substances of CRR are mostly limited to the total saponins instead of pharmacodynamic monomers.So,the specific pharmacodynamic substances that exert the anti-RA synovial hyperplasia effect are still unclear.Traditionally,CRR in treating rheumatism and dampness should be processed with water,which indicated that the efficient pharmacodynamic substances of CRR should be water-soluble components.In the early stage,our research group has determined the content of more than 20 batches of CRR from different sources,and found that Magnoflorine(MAG)was the main compound of CRR and its content was up to 0.1%,which could be used as an important content determination indicator.Besides,modern pharmacological studies revealed that MAG has good anti-inflammatory,immunomodulatory,neuroprotective,anti-diabetic,and cardiovascular protective effect.However,the anti-RA synovial hyperplasia effect and its molecular mechanisms of MAG has not been systematically elucidated.Therefore,we firstly deduce that MAG could be the effective substance responsible for the anti-RA synovial hyperplasia effect of CRR,and investigate the effect and its mechanism of MAG on anti-RA synovial hyperplasia from the whole-tissue-cell-signaling pathway aspect,in order to sovle the problem of unclear pharmacodynamic substances of CRR.In our study,we establised an in vivo model,freund’s complete adjuvant(CFA)-induced adjuvant-induced arthritis(AIA)rats,and an in vitro model,IL-1β-treated MH7 A cells.In addition,the rat ankle joint morphology,body weight,paw swelling,and arthritis score were assessed,and HE staining as well as ELISA were also applied for the anti-RA efficacy evaluation in vivo.Besides,we also used CCK-8,wound healing,transwell,flow cytometry,immunofluorescent staining,ELISA,q RT-PCR,Western blotting,pathway inhibitors,and si RNA assay to systematically investigate the anti-RA synovial hyperplasia effect of MAG and its mechanisms in vitro and in vivo.The methods and results are listed as follows:1.An in vivo AIA animal study was established by intracutaneously injecting with 100 μl CFA to the right hind metatarsal footpad of Wistar rats.The rats were randomly grouped into five groups(8 rats per group): control group,AIA model group,5 mg/kg MAG group,10 mg/kg MAG group,and 1 mg/kg MTX group.After the rats were intraperitoneally injected with different drugs,the anti-RA synovial hyperplasia effect in vivo was evaluated.The results indicated that compared with AIA model group,MAG(5 mg/kg,10 mg/kg)dose-dependently ameliorated the weight loss,paw swelling in ankle and paw,as well as arthritis score in model rats.The results of ELISA disclosed that MAG and MTX obviously decreased the serum proinflammatory cytokines of AIA rats,including IL-6,IL-17,TNF-α,and IL-1β,and increased the serum anti-inflammatory cytokines of IL-10 of AIA rats.Besides,the results of HE staining of ankle histopathological sections also showed that MAG significantly alleviated the infiltration and invasion of the synovial tissue in the ankle of the AIA model rats.At the same time,the favarable anti-RA effects in MTX group were also observed.Above results indicated that MAG exerts great anti-RA synovial hyperplasia effect in AIA rats.2.An IL-1β-induced fibroblast-like synoviocyte MH7 A cell model was established.After drug treatment for 24 h and 48 h,the cell survival rate of MH7 A cells incubated with MAG(1.25,2.5,5,10,20,and 40 μM)was tested using CCK-8 assay,and the non-toxic MAG concentration was further used to screen the efficacy against MH7A cell proliferation.The results showed that MAG(5,10μM)significantly decreased the MH7A cell proliferation induced by 10 ng/m L IL-1βafter 24 h and 48 h treatment in a concentration-and time-dependent manner.The levels of IL-6,IL-8,COX-2,and i NOS were assessed using q RT-PCR,ELISA,and Western blotting.Besides,DCFH-DA probes were applied to detect the content of reactive oxygen species(ROS)in each group combining fluorescence microscope and flow cytometry,and the curcial proteins in Keap1-Nrf2/HO-1 signalling pathway as well as the Nrf2 nuclear translocation were also determined.The results disclosed that MAG dose-dependently reduced the pro-inflammatory cytokines and ROS production induced by IL-1β,increased the nuclear Nrf2 expression and HO-1 expression,decreased the Keap1 and cytoplasmic Nrf2 expression,and promoted translocation of Nrf2 from cytoplasm to nuclei.Above results suggested that the effect of MAG on reducing pro-inflammatory cytokines and ROS production were associated with activating the Keap1-Nrf2/HO-1 signalling pathway.MH7A cells were transfected with Nrf2 siRNA to knock down the Nrf2 gene level using si RNA assay,and the transfection efficiency and the expression of Nrf2 gene and protein were determined by using q RT-PCR and Western blotting assays.Subsequently,the effect of MAG on ROS production,cell migration,cell invasion,cell apoptosis,and cell cycle were determined using fluorescence microscope,flow cytometry,wound healing,transwell,q RT-PCR,Western blotting after treatment with IL-1β.In addition,The MMPs and apoptosis-related gene and protein levels were also assessed.Results showed that MAG can significantly reduce the ROS production,inhibit the migration and invasion of MH7 A cells,reduce the production of MMPs,promote cell apoptosis,and regulate cell cycle in IL-1β treated MH7 A cells.Nrf2 gene plays an important regulatory role in the anti-RA effect of MAG.NF-κB and PI3K/Akt signaling pathways are widely involved in the growth,migration,invasion,differentiation,inflammation,and other cell processes of synovial fibroblasts.Based on this,Western blotting was utilized to detect the expression of crucial proteins in NF-κB and PI3K/Akt signalling pathways.We also provided evidences that MAG could be a potential inhibitor of NF-κB and PI3K/Akt pathway and regulate the crostalk between PI3K/Akt and NF-κB signalilng pathways as well as the cross talk between Keap1-Nrf2/HO-1 and NF-κB signalilng pathways by using NF-κB and PI3K/Akt inhibitors,BAY11-7082 and LY294002,respectively.These results demonstrated that the antiinflammatory,pro-apoptotic and anti-oxidative effects of MAG are related to the regulation of PI3K/Akt,NF-κB and Keap1-Nrf2/HO-1 signalling pathways as well as the cross-talk among them,and the Nrf2 gene is very crucial for MAG to exert anti-RA synovial hyperplasia effect. |
