The Treatment And Mechanism Of Simiao Pill And Its Main Component Berberine On RA And ILD

Section 1 The Effectof Simiao-Derived Serum on RA-FLSObjectiveIn this study,the effects of Simiao-derived serum on fibroblast-like synovial cells of rheumatoid arthritis(RA-FLS)were studied to explore whether Simiao could achieve anti-inflammatory and anti-proliferative effects by inhibiting MAPK/ ERK pathway.MethodsThe synovium of patients with rheumatoid arthritis were donated by patients undergoing arthroscopy and joint replacement.Primary RA-FLS were isolated and cultured,and the effect of TNF-α and IL-1β on the proliferation of RA-FLS was detected by MTT.Preparation of different concentrations of Simiao-derived serum.The Simiao-derived serum was used to detect RA-FLS,and the proliferation of RA-FLS was detected by ELISA.The m RNA levels of Il-6 and TNF-α were detected by Rt-PCR and the MAPK/ERK signaling pathway was detected by Western blotting.ResultsThe primary RA-FLS cells could be normally passed down within 8 generations in vitro.10ng/ml of IL-1β has obvious proliferation effect on RA-FLS.Compared with normal rat serum,the Simiao-derived serum at different concentrations had no obvious effect on the cell proliferation of RA-FLS and the m RNA levels of IL-6 and TNF-α.ConclusionThe Simiao-derived serum had no obvious effect on the proliferation and secretion of inflammatory factors of RA-FLS.The method of in vitro experiment of Simiao pill needs to be further discussed.Section 2 The Effect and Mechanism of Berberine on RA-FLSObjectiveIn this study,we investigatedwhether berberine could play anti-inflammatory and anti-proliferative effectand regulate Lysophosphatidic acid(LPA)function on rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS)via LPA1.MethodsAll the samples in the experiment were donated from outpatient or patients undergoing arhrpsopy or joint replacemnt.The expression of LPA and lysophosphatidic acid receptor 1(LPA1)in RA,osteoarthritis(OA),healthy control was detected.Primary fibroblast synovial cells(RA-FLS)were isolated from synovial tissues of patients with RA(n=4).The levels of LPA-mediated MAPK signaling pathways,ATX,LPA1,K-ras,c-Raf,P-p38,P-JNK and P-ERK1/2 were detected.To verify whether berberine can regulate LPA function through LPA receptor,Molecular docking was adopted to characterize the binding sites of berberine in the predicted protein targets firstly.Then,RA-FLS were stimulated with berberine,LPA,and the inhibitor(Ki16425)of LPA1 and then the effects on the proliferation,apoptosis,the release of inflammatory mediators of RA-FLS,and MAPK pathway were observed.ResultsBerberinecould significantly inhibit proliferationand the excessive production of IL-6,TNF-α of RA-FLS,promote cell apoptosis at the early stage of RA-FLS,prevent cell divisionmaking cells stop at the G0/G1 phase.Berberine could suppress the expression of K-ras,c-Raf,and p-38/ERK-phosphorylation.Compared with healthy control(n=25),the LPA level of plasma(n=28)and synovia fluid(n=10)were significantly higher in RA patients.Compared with OA patients(n=4),LPA1 were strongly expressed in RA(n=4).There was a positive correlation between plasma LPA level and platelet level in RA patients.Interestingly,compared with control group,there was no significant difference in LPA1 and P-JNK protein levels after berberine treatment.Depending on molecular docking,berberine might be combined with Lys39 A of LPA1 by two hydrogen bonds and Arg124 A by hydrophobic forceaccording to molecular docking.LPA-induced proliferation effect was reversed with the addition of berberine and berberine could inhibit the LPA-induced p-38/ERK-phosphorylation via binding to LPA1.ConclusionOur study found that the level of LPA in plasma was significantly increased in RA patients with RA,and there was no statistical difference between plasma and SFthe LPA level of RA patients.However,we could not get synovial fluid from control group to explore whether RA joints exposed to the microenvironment of high levels of LPA.This suggested that inflammation might alter lipid levels in RA,which might exacerbate inflammation in turn.LPA had a role in promoting proliferation and inflammatory of RA-FLS.Berberine might modulated the function of LPA toinhibit the proliferation and inflammation of RA-FLS via blocking p38/ERK MAPK pathway mediated by LPA1,suggesting its potential lipid-regulating,anti-arthritis and inhibition synovial hyperplasia activity against RA and providing a promising therapeutic target for clinical drug development for those RA patients.Section3 The Mechanism of Simiao Pill on Bleomycin Induced Pulmonary Fibrosis in miceObjectivesInterstitial lung disease(ILD)is a frequent complication of rheumatoid arthritis(RA).This study investigated the therapeutic effect of Simiao pill on bleomycin-induced pulmonary fibrosis in mice and its possible molecular mechanism.MethodsMale C57BL/6 mice were divided into control(normal)group,model group,medium-dose group of Simiao pill(SM-M,4.26 g/kg/d)and high-dose group of Simiao pill(SM-H,8.52g/kg/d)randomly,6 mice in each group.SM groups were administrated with Simiao pill once a day for 3 days before modeling,and the normal group and the model group were administrated with normal saline(NS)of equal volume(-3d).All the mice were modeled with 2.5 mg/kg bleomycin except the normal group(0d).The treatment was continued the next day after modeling until day 22(22d).The body mass,amount of ingestion and the change of behavior after model establishment were observed.On the 23 st day after modeling,all mice were executed.The blood,bronchoalveolarlavagefluid(BALF),lung samples of mice were collected for the analysis of inflammation level and degree of lung fibrosis as well as the expression of Jak2/P-Jak2,Jak3/P-Jak3,P-Stat3,Smad2/3,P-Smad2/3,α-SMA,P-ERK1/2 and so on.ResultsThe weight change after modeling could reflect the formation of pulmonary fibrosis indirectly,and the mice had weight loss one week after modeling,indicating the preliminary establishment of the model.Compared with the control group,the model group had significant weight loss(P<0.001).Compared with the model group,body weight of SM-H group increased significantly(P<0.05).There was no significant difference in body weight between the model group and the SM-M group.SM group significantly improved lung and systemic inflammation in mice.Left lung in mice a transverse HE staining showed that the alveoli of model group were collapse and disorder,alveolar interval were thickening with inflammatory cells infiltration.Compared with model group,lung inflammation score and lung damage of SM group were slight obviously(P< 0.001).TNF-αconcentration in serum and BALF were detected.Compared with the control group,serum TNF-αconcentration of model group increased significantly(P < 0.001);Compared with the model group,the serum TNF-level in the SM group was significantly reduced.Compared with the control group,the concentration of TNF-αin BALF in the model group was significantly increased(P < 0.001).Compared with the model group,TNF-αin BALF in SM group was significantly reduced.Compared with the normal group,the total number of BALF cells in the model group and the SM group increased.Compared with the model group,the total number of BALF cells in the SM-H group was significantly reduced(P<0.01).Compared with the normal group,the proportion of macrophages in the model group decreased,but the number of macrophages was still significantly higher than that in the control group.Compared with the model group,the proportion of macrophages in BALF in SM-H group increased,but the number of macrophages was still significantly lower than that in model group(P< 0.05).SM significantly improved pulmonary fibrosis in mice.Compared with the control group,Masson staining and Sirius red staining showed that collagen deposition was significantly increased in the model group.Compared with the model group,SM group had lower collagen deposition and significantly lower fibrosis score(P< 0.01).The content of hydroxyproline(Hyp)in lung tissue was detected by alkaline hydrolysis,and the content of Hyp in the model group was significantly higher than that in control group(P< 0.001).Compared with the model group,the level of Hyp in SM group decreased significantly(P< 0.05).Immunofluorescence showed that SM inhibited epithelial-mesenchymal transformation.The detection of TGF-β/Smad pathway protein showed that compared with the normal control group,the level of surum TGF-β 1of the model group was significantly increased.Compared with the model group,the level of serum TGF-β 1 of SM group was significantly reduced.Interestingly,TGF-β 1 levels were reversed in BALF.Compared with the control group,TGF-β 1 in BALF in the model group was significantly reduced.Compared with the model group,the level of surum TGF-β 1 of SM group showed an increasing trend.Compared with the control group,the ratios of p-smad2/3 and p-smad2/3 and smad2/3 in the model group increased.Compared with the model group,the ratio of p-smad2/3 and p-smad2/3 to smad2/3 in the SM-H group decreased.Compared with the control group,the expression of P-STAT3 was significantly increased in the model group.Compared with the model group,the expression of P-STAT3 of the SM groups was significantly reduced(P < 0.05).However,there was no statistical difference in the expression of P-JAK2.Immunohistochemical method was used to detect the expression of LPA1.Compared with the control group,the relative expression of LPA1 in the model group was significantly increased.Compared with the model group,the relative expression of LPA1 protein in SM-H group was significantly reduced(P< 0.05).Compared with control group,the relative expression of PLC protein in the model group was significantly decreased.Compared with the model group,the relative expression of PLC protein in the SM-H group was significantly increased(P< 0.05).There was no significant difference in levels of serum LPA and PKC between each group.Compared with control group,the relative expression of P-ERK protein in model group increased significantly.Compared with model group,the relative expression of P-ERK protein in SM-H group was significantly reduced(P< 0.05).ConclusionsOur study found that SM can improve bleomycin-induced pulmonary fibrosis by reducing lung inflammation and collagen deposition in mice,and the effect of SM-H group seemed to be more stable.Its therapeutic effect may be due to SM’s inhibition of epithelial-mesenchymal transformation and TGF-TGF /Smad,JAK2/STAT3,ERK/MAPK pathway,which reflecting the multi-target therapeutic mechanism of TCM compound.There was no statistical difference in level of serum LPA in each group.Due to the lack of LPA1 gene knockout mice,whether SM can act anti-fibrosis effect by regulating LPA function needs to be further explored.