| Objective:To investigate the mechanisms of paeoniflorin liposome(Pae-LS)for the treatment of rheumatoid arthritis(RA)in view of the correlation between different polarized phenotypes of synovial macrophages(SMs)and the RA disease activity.To provide some reference for identifying immunotherapeutic targets and developing liposomal drug products.Methods:1.Data were collected from RA and osteoarthritis(OA)patients with a history of knee arthroplasty or synovectomy who were admitted to the General Hospital of Eastern Theater Command from January 2016 to December 2020.Immunofluorescence double-labeling assay was performed on RA and OA synovial tissues,and the fluorescence of two SMs subsets(CD68+iNOS+SMs and CD68+CD206+SMs)were observed by fluorescence microscopy.Spearman’s rank correlation coefficient was conducted to examine the correlation between the SMs phenotypes and rheumatoid factor(RF),C-reactive protein(CRP),erythrocyte sedimentation rate(ESR),and disease activity score 28(DAS28)-3 scores.The diagnostic performances of macrophage phenotypes for prediction of RA and disease activity were determined by receiver operating characteristic curve analysis.2.Pae-LS conjugated with folic acid and polyethylene glycol was prepared by a thin-film dispersion method.Pae-LS was characterized via a laser particle sizer and a nanoparticle sizer in terms of particle size,polydispersity index,and zeta potential.The Pae content was determined by the high-performance liquid chromatography(HPLC)method,and then the encapsulation efficiency and leakage rate of Pae-LS were calculated.The drug release rate of Pae-LS was measured at pH 5.0,6.0,and 7.0 to assess the pH sensitivity.Free Pae content was analyzed by HPLC at 1,2,4,8,12,24,36,48,and 72 h,and the in vitro drug release rate was subsequently calculated.Particle size and polydispersity index were measured at 4℃,25℃,and 37℃ for 72 h to evaluate the stability of Pae-LS.After Pae-LS treatment of RAW 264.7 macrophages,macrophage viability was determined by the MTT assay to assess the effect of Pae-LS on macrophage activity.Twelve SD rats were randomly divided into two groups:the Pae group(the rats were received Pae injection)and the Pae-LS group(the rats were received Pae-LS injection).Liquid chromatography-mass spectrometry was used to detect drug concentrations in rat plasma at multiple time points for clarifying whether liposomes prolong circulation time.3.In in vitro experiments,the uptake of the fluorescent agent coumarin-6(Cou-6)and Cou-6-LS by RAW 264.7 cells stimulated or not with lipopolysaccharide(LPS)was analyzed by flow cytometry,and their fluorescence intensity values was observed by fluorescence microscopy.The RAW 264.7 cells were grouped and subjected to different stimuli.Group Ⅰ:Control group(untreated RAW 264.7 cells),LPS/γ-interferon(IFN-γ)group(cells treated with LPS+IFN-γ),LPS/IFN-γ+Pae group(cells pretreated with LPS/IFN-γ,followed by adding Pae),LPS/IFN-γ+Pae-LS group(cells pretreated with LPS/IFN-γ,followed by adding Pae-LS).Group Ⅱ:LPS/IFN-γ group(RAW 264.7 cells treated with LPS+IFN-γ),LPS/IFN-γ interleukin(IL)-4 group(cells pretreated with LPS+IFN-γ,followed by adding IL-4),LPS/IFN-γ→IL-4+Pae group(cells pretreated with LPS+IFN-γ,followed by adding IL-4 and Pae),LPS/IFN-γ→IL-4+Pae-LS group(cells pretreated with LPS+IFN-γ,followed by adding IL-4 and Pae-LS).The mRNA and protein expression of tumor necrosis factor-α(TNF-α),IL-1β,IL-6,and IL-10 were detected by RT-qPCR and Western Blot,respectively.RT-qPCR and flow cytometry were used respectively to identify the mRNA and protein expression of surface markers CD68,CD206,and iNOS.The protein expression of nuclear factor-κB(NFκB)p65,IkappaB kinase(IKK)α,phosphorylated signal transducer,and activator of transcription(p-STAT)1 and p-STAT6 were assayed by Western Blot.4.In in vivo experiments,collagen-induced arthritis(CIA)rat models were established.Forty rats were grouped and randomized into 5 groups as Normal group(SD rats injected with saline),CIA group(CIA rats injected with saline),CIA+LS group(CIA rats injected with blank liposomes),CIA+Pae group(CIA rats injected with Pae),and CIA+Pae-LS group(CIA rats injected with Pae-LS).The changes in arthritis index,paw swelling degree,body weight,and histological lesions were determined in each group.In addition,Immune cells were collected from ankle synovial tissue.TNF-α,IL-1β,IL-6,and IL-10 genes and protein expression were measured by RT-qPCR and Western Blot.CD68,CD206,and iNOS protein expression were detected by flow cytometry.The protein expression of NFκB p65,IKKα,p-STAT1,and p-STAT6 was determined by Western Blot.Results:1.The levels of CD68+iNOS+ SMs/CD68+CD206+SMs and CD68+iNOS+SMs in synovial tissues from patients with RA were significantly higher than those in patients with OA,whereas the number of CD68+CD206+SMs was much higher in OA,and the difference was statistically significant(P<0.01).Compared with RA patients achieving clinical remission,expression levels of CD68+iNOS+SMs and CD68+iNOS+SMs/CD68+CD206+SMs were significantly higher in active RA(P<0.01).Furthermore,the number of CD68+iNOS+SMs was positively correlated with RF,CRP,ESR and DAS28-3 scores(P<0.01),and the CD68+iNOS+SMs/CD68+CD206+SMs ratio was also positively correlated with RF(P<0.05),CRP(P<0.01),ESR(P<0.01)and DAS28-3 scores(P<0.01).On the contrary,the number of CD68+CD206+SMs was negatively correlated with CRP,ESR and DAS28-3 scores(P<0.01).In addition,the number of CD68+iNOS+SMs had higher sensitivity and specificity in the assessment of RA disease activity(AUC:0.953,a sensitivity of 0.825,a specificity of 0.875),and the CD68+iNOS+SMs/CD68+CD206+SMs ratio had higher sensitivity and specificity in diagnosising RA(AUC:0.973,a sensitivity of 0,958,a specificity of 0.98).2.The prepared Pae-LS were spherical with a mean particle size of 228.92 nm,a polydispersity index of 0.148,and a zeta potential of-26.82 mV.The drug encapsulation efficiency of Pae-LS was 52.88±3.12%,and the leakage rate was 5.27±0.38%at 4 ℃ and 13.05±0.38%at room temperature.The release rate was 76.00±4.58%at pH 5.0,64.04±4.14%at pH 6.0,and 54.01±3.90%at pH 7.0.The in vivo drug release test showed that the drug release rate gradually increased with time,but maintained a stable state after 36h.The mean particle size and polydispersity index of liposomes did not change significantly within 72 h.There was no statistically significant change in the viability of macrophages with Pae-LS treatment compared to non-Pae-LS-treated macrophages(P>0.05).Plasma drug concentrations in SD rats treated with Pae-LS were approximately twice that of Pae-treated rats after 30 min(P<0.05),which remained at a certain level after 3 h.3.In in vitro experiments,Cou-6-LS treatment for LPS stimulated RAW 264.7 macrophages resulted in markedly higher uptake and fluorescence intensity values than free Cou-6(P<0.01).Compared with the LPS/IFN-γ group,LPS/IFN-γ+Pae treatment resulted in decreased the mRNA and protein expression of TNF-α and IL-6(P<0.01),reduced levels of IL-1β mRNA(P<0.01)and IL-1β protein(P<0.05),increased IL-10 mRNA level(P<0.05)and IL-10 protein level(P<0.01),decreased iNOS mRNA level(P<0.01),elevated CD206 mRNA level(P<0.01),declined NFκB p65,IKKα,and p-STAT1 protein expression(P<0.01),as well as raised p-STAT6 protein expression(P<0.01).In comparison with the LPS/IFN-γ group,LPS/IFN-γ+Pae-LS treatment led to downregulated expression of TNF-α,IL-1β,and IL-6 mRNA and protein(P<0.01),upregulated levels of IL-10 mRNA and protein expression(P<0.01),decreased iNOS mRNA levels(P<0.01),increased CD206 mRNA expression(P<0.01),reduced NFκB p65,IKKα,and p-STAT1 protein expression(P<0.01),elevated p-STAT6 protein expression(P<0.01).Compared to the LPS/IFN-γ+Pae group,the LPS/IFN-γ+Pae-LS group showed a more significant decrease in TNF-α mRNA and protein expression(P<0.01),lower levels of IL-1βmRNA(P<0.01)and IL-1β protein(P<0.05),a more noticeable reduction in IL-6 mRNA expression(P<0.05)and IL-6 protein expression(P<0.01),higher IL-10 mRNA level(P<0.01)and IL-10 protein level(P<0.05),lower iNOS mRNA levels(P<0.01),higher CD206 mRNA expression(P<0.01),a more marked reduction in NFκB p65(P<0.05),p-STAT1(P<0.01)and IKKα(P<0.01)protein expression,and higher p-STAT6 protein level(P<0.01).Moreover,when compared to the LPS/IFN-γ→IL-4 group,LPS/IFN-γ→IL-4+Pae treatment resulted in reduced mRNA and protein levels of TNF-α,IL-1β and IL-6(P<0.01),elevated IL-10 mRNA expression(P<0.05)and IL-10 protein level(P<0.01),decreased iNOS mRNA expression(P<0.01),increased CD206 mRNA level(P<0.01),declined NFκB p65(P<0.01),p-STAT1(P<0.05)and IKKα(P<0.01)protein levels,and elevated p-STAT6 protein expression(P<0.01).Compared with the LPS/IFN-γ→IL-4 group,LPS/IFN-γ→IL-4+Pae-LS treatment led to reduced TNF-α,IL-1β,and IL-6 mRNA and protein levels(P<0.01),increased IL-10 mRNA and protein expression(P<0.01),decreased iNOS mRNA level(P<0.01),upregulated CD206 mRNA expression(P<0.01),reduced NFκB p65,p-STAT1,and IKKa protein levels(P<0.01),increased p-STAT6 protein expression(P<0.01).Compared to the LPS/IFN-γ→IL-4+Pae group,the LPS/IFN-γ→L-4+Pae-LS group displayed lower levels of TNF-α mRNA(P<0.05)and TNF-α protein(P<0.01),more significantly reduced mRNA and protein expression of IL-1β and IL-6(P<0.01),higher IL-10 mRNA level(P<0.05)and IL-10 protein level(P<0.01),lower iNOS mRNA level(P<0.01),higher CD206 mRNA expression(P<0.01),more significant reduction in p-STAT1(P<0.01),NFκB p65(P<0.01)and IKKα(P<0.05)protein expression,and higher p-STAT6 protein expression(P<0.01).4.In in vivo experiments,there were no significant changes in body weight,hind paw swelling,and arthritis index in the CIA+LS group compared with the CIA group(P>0.05).On day 20 after treatment,the rats in the CIA+Pae group and CIA+Pae-LS group showed higher body weight(P<0.01)and lower hind paw swelling and arthritis index(P<0.01)than the CIA group.Compared to the CIA+Pae group,the rats in the Pae-LS group had heavier weight gain(P<0.05),a more marked reduction in hind paw swelling(P<0.01),and lower arthritis index(P<0.05).Additionally,when compared with the CIA group,the treatment with CIA+Pae-LS resulted in a remarkable decrease in histopathological synovitis scores(P<0.01).Noteworthy,Pae-LS treatment is more effective than Pae treatment(P<0.01).In addition,compared with the CIA group,the CIA+Pae group and the CIA+Pae-LS group both showed decreased TNF-α,IL-1β,and IL-6 gene and protein expression,increased IL-10 gene and protein levels(P<0.01),elevated CD68 and CD206 co-expression(P<0.01),reduced CD68 and iNOS co-expression(P<0.01),downregulated NFκB p65,IKKα,p-STAT1 protein expression,and upregulated p-STAT6 protein expression(P<0.01).Also,Pae-LS showed better efficacy than Pae(P<0.01).Conclusion:1.CD68+iNOS+SMs are predominant in the synovial tissue of RA patients.The ratio of CD68+iNOS+SMs/CD68+CD206+SMs and the number of CD68+iNOS+SMs are strongly correlated with RA disease activity,which are expected to be new indicators in diagnosing RA and assessing disease activity.2.Pae-LS inhibits M1 macrophage polarization and promotes M2 phenotype polarization through NFκB and STAT signaling pathways for alleviating RA synovial inflammation,which downregulates inflammatory cytokine expression and induces anti-inflammatory cytokine secretion.Pae-LS may be a candidate drug for the treatment of RA. |
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