Triptolide Inhibits Expression of Inflammatory Cytokines and Proliferation of Fibroblast-like Synoviocytes Induced by IL-6/sIL-6R-Mediated JAK2/STAT3 Signaling Pathway

Triptolide,a component of the Chinese herb Tripterygium wilfordii Hook F,has been proved to be effective in the treatment of rheumatoid arthritis(RA).However,its underlying mechanisms on RA have not yet been well established.We observed the inhibitory effect of triptolide on the expression of inflammatory cytokines and proliferation of fibroblast-like synoviocytes(FLS)induced by the complex of interleukin-6(IL-6)and the soluble form of the IL-6 receptor(sIL-6R).Furthermore,to clarify the underlying mechanisms,we treated FLS with the Janus-activated kinase 2(JAK2)inhibitor/signal transducer and activator of transcription 3(STAT3)activation blocker AZD1480.In this study,immunohistochemical staining was used to identify vimentin(+)and CD68(−)in FLS.The FLS proliferation was measured by cell proliferation assay,and the cell cycles were analyzed by flow cytometry.Furthermore,ELISA was used to detect the expression of the inflammatory factors in culture solution.The expression levels of p-JAK2,JAK2,p-STAT3 and STAT3 were investigated through Western blotting analysis.The results showed that IL-6/sIL-6R significantly increased the cell proliferation and expression of inflammatory cytokines,including IL-6,interleukin-1β(IL-1β)and vascular endothelial growth factor(VEGF).Triptolide or AZD1480 inhibited the cell proliferation and inflammatory cytokine expression in IL-6/sIL-6R-stimulated FLS by suppressing JAK2/STAT3.The study suggested that the physiological effects of triptolide on RA were due to its contribution to the inhibition of the inflammatory cytokine expression and FLS proliferation by suppressing the JAK2/STAT3 signaling pathway.It may provide an innovative insight into the effect of triptolide in preventing RA pathogenesis.

Establishment and characterization of a fibroblast-like cell line from Anabarilius grahami(Cypriniformes:Cyprinidae)

Though Yunnan province contains some 562 known species of fish,no cell lines from any of these have been made available to date.To protect germplasm resources and provide an effective tool in solving problems at cellular level of Anabarilius grahami,a fish endemic to Fuxian Lake,Yunnan,China,we established and characterized the major features of a continuous cell line(AGF II)from the caudal fin tissue of A.grahami.This AGF II cell line consists of fibroblast-like cells and has been subcultured more than 60 times over the course of a year.The cell line was maintained in DMEM/F12 supplemented with 10%FBS,with a cellular doubling time of 51.1 h.We continued with more experiments to optimize the culture and storage conditions,and found a variety of interesting results:cells could grow at temperature between 24℃and 28℃,with the optimal temperature of 28℃.Likewise,the growth rate of A.grahami fin cells increased when the FBS proportion increased from 5%to 20%,with the optimal growth at the concentrations of 20%FBS;cells were able to grow in L-15 and DMEM/F12 with optimal growth at L-15;DMSO is a better cryoprotectant than Glycerol,EG and MeOH for AGFII cells with optimal concentration of 5%DMSO.Chromosome analysis also showed that the distribution of chromosome number varies from 38 to 52,with a modal peak at 48 chromosomes,accounting for 39.8%of all cells.Using the same primer pairs specific to mtDNA,the AGF II cell sequences obtained by PCR were identical to those from muscle tissues of A.grahami.Both chromosome analysis and PCR amplification confirmed the AGF II cells were from A.grahami,also indicating that that current long-term artificial propagation of A.grahami has been successful.Finally,we noted that when cells were transfected with pEYFP-N1 and pECFP-N1 plasmid,bright fluorescent signals were observed,suggesting that this cell line may be suitable for use in transfection and future gene expression studies。

Protective effects of Dioscin on TNF-α-induced collagen-induced arthritis rat fibroblast-like synoviocytes involves in regulating the LTB4/BLT pathway

Background and Objective:LTB4 has been shown to be involved in rheumatoid arthritis(RA)pathogenesis.The effect of Dioscin(Dio)on the LTB4 pathway of RA have not been reported yet.This study aimed at further exploring whether Dioscin’s effects on TNF-αinduced collagen-induced arthritis(CIA)rat fibroblast-like synoviocytes(FLS)connected with the LTB4 and its receptor pathway.Materials&Methods:In this experiment,control group,TNF-αgroup,and different concentrations of Dioscin groups were established.Cell viability was evaluated using MTT assay.The levels of LTB4 in the samples of above groups were measured using ELISA.The mRNA expression levels of LTA4H,BLT1,and BLT2 were detected by quantitative real time PCR,while the expression level of LTA4H proteins were detected using western blot.The distribution of LTA4H was assessed by immunofluorescence assay.Results:the LTB4 level of TNF-αgroup in sample supernatant was higher than both control group and Dioscin groups with decreased LTB4 levels(p<0.05).Compared with the control group,the expression of LTA4H was significantly increased in TNF-αgroup(p<0.05),whereas LTA4H expressions were significantly decreased in all Dioscin groups when compared to TNF-αgroup(p<0.05).The mRNA expressions of BLT1 and BLT2 were markedly higher in TNF-αgroup than those in control group while Dioscin treatment significantly inhibited the increased expressions of BLT1 and BLT2 induced by TNF-α(p<0.05).Conclusions:These results firstly demonstrate that the protective effect of Dioscin on TNF-αinduced FLS may involve in its reducing LTB4 production by down-regulating LTA4H expression,and may inhibit its downstream pathway by decreasing LTB4 receptors levels.This findings suggest that dioscin produces a potential therapeutic effects for RA via its influencing LTA4H/LTB4/BLT pathway.

Cell Proliferation Ability of Mouse Fibroblast-Like Cells and Osteoblast-Like Cells on a Ti-6Al-4V Alloy Film Produced by Selective Laser Melting

Successful regeneration of tissues and organs relies on the application of suitable substrates or scaffolds in scaffold-based regenerative medicine. In this study, Ti-6Al-4V alloy films (Ti alloy film) were produced using a three-dimensional printing technique called Selective Laser Melting (SLM), which is one of the metal additive manufacturing techniques. The thickness of produced Ti alloy film was approximately 250 μm. The laser-irradiated surface of Ti alloy film had a relatively smooth yet porous surface. The non-irradiated surface was also porous but also retained a lot of partially melted Ti-6Al-4V powder. Cell proliferation ability of mouse fibroblast-like cells (L929 cells) and mouse osteoblast-like cells (MC3T3-E1 cells) on both the surfaces of Ti alloy film was examined using WST assay. Both L929 and MC3T3-E1 cells underwent cell proliferation during the culture period. These results indicate that selective laser melting is suitable for producing a cell-compatible Ti-6Al-4V alloy film for biomaterials applications.

Characterization of smooth muscle, enteric nerve, interstitial cells of Cajal, and fibroblast-like cells in the gastric musculature of patients with diabetes mellitus

AIM To investigate histologic abnormalities in the gastric smooth muscle of patients with diabetes mellitus(DM).METHODS Full-thickness gastric specimens were obtained from patients undergoing surgery for gastric cancer. H&E stain and Masson's Trichrome stain were performed to assess the degree of fibrosis. Immunohistochemical staining using various antibodies was also performed [antibodies against protein gene product 9.5(PGP9.5), neuronal nitric oxide synthase(n NOS), vasoactive intestinal peptide(VIP), neurokinin-1(NK1) receptor, c-Kit, and platelet-derived growth factor receptor-alpha,(PDGFRα)]. Immunofluorescent staining and evaluation with confocal microscopy were also conducted.RESULTS Twenty-six controls and 35 diabetic patients(21 shortduration patients and 14 long-duration patients) were included. There were no significant differences in basic demographics between the two groups except in mean body mass index(BMI)(higher in the DM group). Proportions of moderate-to-severe intercellular fibrosis in the muscle layer were significantly higher in the DM group than in the control group(P < 0.01). On immunohistochemical staining, c-Kit- and PDGFRα-positive immunoreactivity were significantly decreased in the DM group compared with the control group(P < 0.05). There were no statistically significant differences in PGP9.5, n NOS, VIP, and neurokinin 1 expression. On immunofluorescent staining, cellularity of interstitial cells of Cajal(ICC) was observed to decrease with increasing duration of DM.CONCLUSION Our study suggests that increased intercellular fibrosis, loss of ICC, and loss of fibroblast-like cells are found in the smooth muscle of DM patients. These abnormalities may contribute to changes in gastric motor activity in patients with DM.

GRK2 mediated degradation of SAV1 initiates hyperplasia of fibroblast-like synoviocytes in rheumatoid arthritis

Hyperplasia and migration of fibroblast-like synoviocytes(FLSs)are the key drivers in the pathogenesis of rheumatoid arthritis(RA)and joint destruction.Abundant Yes-associated protein(YAP),which is a powerful transcription co-activator for proliferative genes,was observed in the nucleus of inflammatory FLSs with unknown upstream mechanisms.Using Gene Expression Omnibus database analysis,it was found that Salvador homolog-1(SAV1),the pivotal negative regulator of the Hippo-YAP pathway,was slightly downregulated in RA synovium.However,SAV1 protein expression is extremely reduced.Subsequently,it was revealed that SAV1 is phosphorylated,ubiquitinated,and degraded by interacting with an important serine-threonine kinase,G protein-coupled receptor(GPCR)kinase 2(GRK2),which was predominately upregulated by GPCR activation induced by ligands such as prostaglandin E2(PGE2)in RA.This process further contributes to the decreased phosphorylation,nuclear translocation,and transcriptional potency of YAP,and leads to aberrant FLSs proliferation.Genetic depletion of GRK2 or inhibition of GRK2 by paroxetine rescued SAV1 expression and restored YAP phosphorylation and finally inhibited RA FLSs proliferation and migration.Similarly,paroxetine treatment effectively reduced the abnormal proliferation of FLSs in a rat model of collagen-induced arthritis which was accompanied by a significant improvement in clinical manifestations.Collectively,these results elucidate the significance of GRK2 regulation of Hippo-YAP signaling in FLSs proliferation and migration and the potential application of GRK2 inhibition in the treatment of FLSs-driven joint destruction in RA.

Nuciferine alleviates collagen-induced arthritic in rats by inhibiting the proliferation and invasion of human arthritis-derived fibroblast-like synoviocytes and rectifying Th17/Treg imbalance

Rheumatoid arthritis(RA)is a chronic autoimmune disorder marked by persistent synovial inflammation and joint degradation,posing challenges in the development of effective treatments.Nuciferine,an alkaloid found in lotus leaf,has shown promising anti-inflammatory and anti-tumor effects,yet its efficacy in RA treatment remains unexplored.This study investigated the antiproliferative effects of nuciferine on the MH7A cell line,a human RA-derived fibroblast-like synoviocyte,revealing its ability to inhibit cell proliferation,promote apoptosis,induce apoptosis,and cause G1/S phase arrest.Additionally,nuciferine significantly reduced the migration and invasion capabilities of MH7A cells.The therapeutic potential of nuciferine was further evaluated in a collagen-induced arthritis(CIA)rat model,where it markedly alleviated joint swelling,synovial hyperplasia,cartilage injury,and inflammatory infiltration.Nuciferine also improved collagen-induced bone erosion,decreased pro-inflammatory cytokines and serum immunoglobulins(IgG,IgG1,IgG2a),and restored the balance between T helper(Th)17 and regulatory T cells in the spleen of CIA rats.These results indicate that nuciferine may offer therapeutic advantages for RA by decreasing the proliferation and invasiveness of FLS cells and correcting the Th17/Treg cell imbalance in CIA rats.

Madecassoside impedes invasion of rheumatoid fibroblast-like synoviocyte from adjuvant arthritis rats via inhibition of NF-κB-mediated matrix metalloproteinase-13 expression

Fibroblast-like synoviocytes(FLS) play a pivotal role in Rheumatoid arthritis(RA) pathogenesis through aggressive migration and invasion. Madecassoside(Madec), a triterpenoid saponin present in Centella asiatica herbs, has a potent anti-inflammatory effect. In the present study, Madec exerted an obvious therapeutic effect in reversing the histological lesions in adjuvant-induced arthritis(AIA) rats. To recognize the anti-rheumatoid potentials of Madec, we further investigated whether Madec interfered with FLS invasion and metalloproteinase(MMP) expression. In cultures of primary FLS isolated from the AIA rats, Madec(10 and 30 μmol·L–1) was proven to considerably inhibit migration and invasion of FLS induced by interleukin 1β(IL-1β), but exhibiting no obvious effect on cell proliferation. Madec repressed IL-1β-triggered FLS invasion by prohibiting the expression of MMP-13. Additionally, Madec suppressed MMP-13 transcription via inhibiting the MMP-13 promoter-binding activity of NF-κB. Our results further showed that Madec down-regulated the translocation and phosphorylation of NF-κB as demonstrated by Western blotting and immunofluorescence assays. In conclusion, our results suggest that Madec exerts anti-RA activity via inhibiting the NF-κB/MMP-13 pathway.

Therapeutic effect of neohesperidin on TNF-α-stimulated human rheumatoid arthritis fibroblast-like synoviocytes

During the pathogensis of rheumatoid arthritis(RA),activated RA fibroblast-like synoviocytes(RA-FLSs)combines similar proliferative features as tumor and inflammatory features as osteoarthritis,which eventually leads to joint erosion.Therefore,it is imperative to research and develop new compounds,which can effectively inhibit abnormal activation of RA-FLSs and retard RA progression.Neohesperidin(Neo)is a major active component of flavonoid compounds with anti-inflammation and anti-oxidant properties.In this study,the anti-inflammation,anti-migration,anti-invasion,anti-oxidant and apoptosis-induced effects of Neo on RAFLSs were explored to investigate the underlying mechanism.The results suggested that Neo decreased the levels of interleukin IL-1β,IL-6,IL-8,TNF-α,MMP-3,MMP-9 and MMP-13 in FLSs.Moreover,Neo blocked the activation of the MAPK signaling pathway.Furthermore,treatment with Neo induced the apoptosis of FLSs,and inhibited the migration of FLSs.It was also found that Neo reduced the accumulation of reactive oxygen species(ROS)induced by TNF-α.Taken together,our results highlighted that Neo may act as a potential and promising therapeutic drug for the management of RA.

uPAR promotes tumor-like biologic behaviors of fibroblast-like synoviocytes through PI3K/Akt signaling pathway in patients with rheumatoid arthritis

Urokinase-type plasminogen activator receptor(uPAR),is a multifunctional receptor on cell surface,widely present in endothelial cells,fibroblasts,and a variety of malignant cells.Current studies have suggested that uPAR overexpressed on synovial tissues or in synovial fluid or plasma in patients with rheumatoid arthritis(RA).However,there are limited researches regarding the role of uPAR on fibroblast-like synoviocytes of rheumatoid arthritis(RA-FLSs)and its underlying mechanisms.Here,our studies show that the expression of uPAR protein was significantly higher in fibroblast-like synoviocytes(FLSs)from RA than those from osteoarthritis or traumatic injury patients.uPAR gene silencing significantly inhibited RA-FLSs cell proliferation,restrained cell transformation from the G0/G1 phase to S phase,aggravated cell apoptosis,interfered with RA-FLSs cell migration and invasion,and reduced activation of the PI3K/Akt signaling pathway,which may be associated withβ1-integrin.Cell supernatants from uPAR gene-silenced RA-FLSs markedly inhibited the migration and tubule formation ability of the HUVECs(a human endothelial cell line).Therefore,we demonstrate that uPAR changes the biological characteristics of RA-FLSs,and affects neoangiogenesis of synovial tissues in patients with RA.All of these may be associated with theβ1-integrin/PI3K/Akt signaling pathway.These results imply that targeting uPAR and its downstream signal pathway may provide therapeutic effects in RA.

Targeted inhibition of GRK2 kinase domain by CP-25 to reverse fibroblast-like synoviocytes dysfunction and improve collagen-induced arthritis in rats

Rheumatoid arthritis(RA)is an autoimmune disease and is mainly characterized by abnormal proliferation of fibroblast-like synoviocytes(FLS).The up-regulated cellular membrane expression of G protein coupled receptor kinase 2(GRK2)of FLS plays a critical role in RA progression,the increase of GRK2 translocation activity promotes dysfunctional prostaglandin E4 receptor(EP4)signaling and FLS abnormal proliferation.Recently,although our group found that paeoniflorin-6’-O-benzene sulfonate(CP-25),a novel compound,could reverse FLS dysfunction via GRK2,little is known as to how GRK2 translocation activity is suppressed.Our findings revealed that GRK2 expression up-regulated and EP4 expression down-regulated in synovial tissues of RA patients and collagen-induced arthritis(CIA)rats,and prostaglandin E2(PGE2)level increased in arthritis.CP-25 could down-regulate GRK2 expression,up-regulate EP4 expression,and improve synovitis of CIA rats.CP-25 and GRK2 inhibitors(paroxetine or GSK180736 A)inhibited the abnormal proliferation of FLS in RA patients and CIA rats by down-regulating GRK2 translocation to EP4 receptor.The results of microscale thermophoresis(MST),cellular thermal shift assay,and inhibition of kinase activity assay indicated that CP-25 could directly target GRK2,increase the protein stability of GRK2 in cells,and inhibit GRK2 kinase activity.The docking of CP-25 and GRK2 suggested that the kinase domain of GRK2 might be an important active pocket for CP-25.G201,K220,K230,A321,and D335 in kinase domain of GRK2 might form hydrogen bonds with CP-25.Site-directed mutagenesis and co-immunoprecipitation assay further revealed that CP-25 down-regulated the interaction of GRK2 and EP4 via controlling the key amino acid residue of Ala321 of GRK2.Our data demonstrate that FLS proliferation is regulated by GRK2 translocation to EP4.Targeted inhibition of GRK2 kinase domain by CP-25 improves FLS function and represents an innovative drug for the treatment of RA by targeting GRK2.

Activity of fibroblast-like synoviocytes in rheumatoid arthritis was impaired by dickkopf-1 targeting siRNA

Background:Fibroblast-like synoviocytes(FLSs),resident mesenchymal cells of synovial joints,play an important role in the pathogenesis of rheumatoid arthritis(RA).Dickkopf-1(DKK-1)has been proposed to be a master regulator of bone remodeling in inflammatory arthritis.Here,potential impairation on the activity of FLSs derived from RA to small interfering RNAs(siRNAs)targeting DKK-1 was investigated.Methods:siRNAs targeting DKK-1 were transfected into FLSs of patients with RA.Interleukin(IL)-1β,IL-6,IL-8,matrix metalloproteinase(MMP)2,MMP3,MMP9,transforming growth factor(TGF)-pi,TGF-β2 and monocyte chemoattractant protein(MCP)-1 levels in the cell culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA).Invasion assay and 3H incorporation assay were utilized to investigate the effects of siRNAs targeting DKK-1 on FLSs invasion and cell proliferation,respectively.Western blotting was performed to analyze the expression of nuclear factor(NF)-kB,interleukin-1 receptor-associared kinase(IRAK)1,extracellular regulated protein kinases(ERK)1,Jun N-terminal kinase(JNK)and p-catenin in FLSs.Results:DKK-1 targeting siRNAs inhibited the expression of DKK-1 in FLSs(P<0.01).siRNAs induced a significant reduction of the levels of IL-6,IL-8,MMP2,MMP3 and MMP9 in FLSs compared to the control group(P<0.05).DKK-1 targeting siRNAs inhibited the proliferation and invasion of FLSs(P<0.05).Important molecules of pro-inflammatory signaling in FLSs,including IRAKI and ERK1,were decreased by the inhibition of DKK-1 in FLSs.In contrast,β-catenin,a pivotal downstream molecule of the Wnt signaling pathway was increased.Conclusions:By inhibiting DKK-1,we were able to inhibit the proliferation,invasion and pro-inflammatory cytokine secretion of FLSs derived from RA,which was mediated by the ERK or the IRAK-1 signaling pathway.These data indicate the application of DKK-1 silencing could be a potential therapeutic approach to RA.

Activation of human fibroblast-like synoviocytes by uric acid crystals in rheumatoid arthritis

Hyperuricemia-mediated uric acid crystal formation may cause joint inflammation and provoke the destruction of joints through the activation of inflammasome-mediated innate immune responses.However,the immunopathological effects and underlying intracellular regulatory mechanisms of uric acid crystal-mediated activation of fibroblast-like synoviocytes(FLS)in rheumatoid arthritis(RA)have not been elucidated.Therefore,we investigated the in vitro effects of monosodium urate crystals,alone or in combination with the inflammatory cytokines tumor-necrosis factor(TNF)-a or interleukin(IL)-1b,on the activation of human FLS from RA patients and normal control subjects and the underlying intracellular signaling mechanisms of treatment with these crystals.Monosodium urate crystals were able to significantly increase the release of the inflammatory cytokine IL-6,the chemokine CXCL8 and the matrix metalloproteinase(MMP)-1 from both normal and RA-FLS(all P,0.05).Moreover,the additive or synergistic effect on the release of IL-6,CXCL8 and MMP-1 from both normal and RA-FLS was observed following the combined treatment with monosodium urate crystals and TNF-a or IL-1b.Further experiments showed that the release of the measured inflammatory cytokine,chemokine and MMP-1 stimulated by monosodium urate crystals were differentially regulated by the intracellular activation of extracellular signal-regulated kinase and c-Jun N-terminal kinase pathways but not the p38 mitogen-activated protein kinase pathway.Our results therefore provide a new insight into the uric acid crystal-activated immunopathological mechanisms mediated by distinct intracellular signal transduction pathways leading to joint inflammation in RA.

Tetrandrine inhibits migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes through down-regulating the expressions of Rac1, Cdc42, and Rho A GTPases and activation of the PI3K/Akt and JNK signaling pathways

Tetrandrine(Tet), the main active constituent of Stephania tetrandra root, has been demonstrated to alleviate adjuvant-induced arthritis in rats. The present study was designed to investigate the effects of Tet on the migration and invasion of rheumatoid arthritis fibroblast-like synoviocytes(RA-FLS) and explore the underlying mechanisms. By using cultures of primary FLS isolated from synoviums of RA patients and cell line MH7 A, Tet(0.3, 1 μmol·L-1) was proven to significantly impede migration and invasion of RA-FLS, but not cell proliferation. Tet also greatly reduced the activation and expressions of matrix degrading enzymes MMP-2/9, the expression of F-actin and the activation of FAK, which controlled the morphologic changes in migration process of FLS. To identify the key signaling pathways by which Tet exerts anti-migration effect, the specific inhibitors of multiple signaling pathways LY294002, Triciribine, SP600125, U0126, SB203580, and PDTC(against PI3 K, Akt, JNK, ERK, p38 MAPK and NF-κB-p65, respectively) were used. Among them, LY294002, Triciribine, and SP600125 were shown to obviously inhibit the migration of MH7 A cells. Consistently, Tet was able to down-regulate the activation of Akt and JNK as demonstrated by Western blotting assay. Moreover, Tet could reduce the expressions of migration-related proteins Rho GTPases Rac1, Cdc42, and Rho A in MH7 A cells. In conclusion, Tet can impede the migration and invasion of RA-FLS, which provides a plausible explanation for its protective effect on RA. The underlying mechanisms involve the reduction of the expressions of Rac1, Cdc42, and Rho A, inhibition of the activation of Akt and JNK, and subsequent down-regulation of activation and/or expressions of MMP-2/9, F-actin, and FAK.

针刀疏筋解结术对类风湿关节炎家兔滑膜炎症的影响及作用机制研究

目的观察针刀疏筋解结术对类风湿关节炎(RA)模型家兔膝关节滑膜组织NF-κB/Bcl-2通路活性及肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-6、Bax、caspase-3表达的影响,探讨其抑制RA滑膜炎症的作用机制。方法24只新西兰白兔随机分为正常组、模型组、药物组和针刀组,每组6只,除正常组外,其余组采用卵蛋白+弗氏完全佐剂膝关节腔注射复制RA模型,分别予相应干预,连续18 d。测定家兔膝关节痛阈、膝关节周径,超声观察关节腔积液、滑膜厚度及内部血流信号,HE染色观察膝关节滑膜组织形态,TUNEL染色观察滑膜组织成纤维样滑膜细胞(FLS)凋亡情况,免疫组化检测滑膜组织TNF-α、IL-1β、IL-6表达,RT-PCR检测滑膜组织核因子(NF)-κBp65、Bcl-2mRNA表达,Westernblot检测滑膜组织NF-κBp65、p-NF-κBp65、Bcl-2、Bax、caspase-3蛋白表达。结果与正常组比较,模型组家兔膝关节痛阈降低、膝关节周径增加,超声评分和滑膜组织病理评分增加;滑膜组织FLS凋亡率降低,TNF-α、IL-1β、IL-6表达升高,NF-κBp65、Bcl-2 mRNA和蛋白及p-NF-κBp65蛋白表达升高,Bax、caspase-3蛋白表达降低,差异均有统计学意义(P<0.05)。与模型组比较,药物组和针刀组家兔膝关节痛阈增加、膝关节周径减小,超声评分和滑膜组织病理评分减少;滑膜组织FLS凋亡率升高,TNF-α、IL-1β、IL-6表达降低,NF-κBp65、Bcl-2m RNA和蛋白及p-NF-κBp65蛋白表达降低,Bax、caspase-3蛋白表达升高,差异均有统计学意义(P<0.05)。结论针刀疏筋解结术可能通过降低滑膜组织NF-κB/Bcl-2通路活性,促进FLS凋亡,减少TNF-α、IL-1β、IL-6产生,抑制RA滑膜炎症,减轻膝关节肿胀、提升痛阈。

miR-let-7d-HMGA2通路调控缺氧诱导的类风湿关节炎滑膜成纤维样细胞增殖

目的 研究缺氧在类风湿关节炎(rheumatoid arthritis,RA)滑膜成纤维样细胞(fibroblast-like synoviocytes,FLS)增殖中的作用及机制。方法 采用HE染色法检测RA和对照骨关节炎(osteoarthritis,OA)滑膜组织FLS增生情况。分离原代RA-FLS并采用流式细胞术鉴定;将RA-FLS置于常氧(21%O_(2))或缺氧环境(3%O_(2))中培养,CCK-8法检测细胞增殖水平的变化,RT-PCR检测miR-let-7d表达的变化。通过类似物或抑制物改变miR-let-7d的表达,观察其对RA-FLS增殖、凋亡的影响;探究miR-let-7d靶基因在缺氧诱导的RA-FLS增殖中的作用。结果 FLS在RA滑膜中增生明显,在缺氧环境下RA-FLS增殖加快,miR-let-7d表达水平降低;过表达miR-let-7d抑制RA-FLS增殖,但对细胞凋亡水平无明显影响;进一步的研究证实miR-let-7d靶基因HMGA2参与缺氧诱导的RA-FLS增殖。结论 miR-let-7d-HMGA2通路调控缺氧诱导的RA-FLS增殖。

盐酸小檗碱对强直性脊柱炎患者成纤维样滑膜细胞体外增殖和成骨分化的影响

目的:探讨盐酸小檗碱对强直性脊柱炎(ankylosing spondylitis, AS)患者成纤维样滑膜细胞(fibroblast-like synoviocytes, FLS)体外增殖和成骨分化的影响。方法:从AS患者的髋关节滑膜组织中分离培养FLS,经免疫荧光染色鉴定后进行后续实验。取第3代FLS,以白细胞介素(interleukin, IL)-17、IL-23预处理,再以0μmol·L^(-1)(空白组)、80μmol·L^(-1)(盐酸小檗碱低剂量组)、120μmol·L^(-1)(盐酸小檗碱中剂量组)、200μmol·L^(-1)(盐酸小檗碱高剂量组)的盐酸小檗碱干预后,采用光学显微镜观察细胞增殖情况,以酶联免疫吸附法检测炎症因子水平和骨稳态调节因子水平,通过茜素红染色观察细胞成骨分化情况。结果:(1)炎症因子水平测定结果。盐酸小檗碱低、中、高剂量组IL-17、IL-23、IL-6、肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)水平均低于空白组(IL-17:P=0.046,P=0.005,P=0.004;IL-23:P=0.024,P=0.002,P=0.000;IL-6:P=0.012,P=0.000,P=0.000;TNF-α:P=0.012,P=0.004,P=0.001),盐酸小檗碱中、高剂量组IL-17、IL-23、IL-6、TNF-α水平均低于盐酸小檗碱低剂量组(IL-17:P=0.034,P=0.024;IL-23:P=0.020,P=0.000;IL-6:P=0.000,P=0.000;TNF-α:P=0.012,P=0.002),盐酸小檗碱高剂量组IL-17、IL-23、IL-6、TNF-α水平均低于盐酸小檗碱中剂量组(IL-17:P=0.029;IL-23:P=0.014;IL-6:P=0.005;TNF-α:P=0.026)。(2)FLS增殖情况观察结果。光学显微镜观察结果显示,与空白组相比,盐酸小檗碱低、中、高剂量组细胞间隙增大、凋亡细胞数量增加,变化程度呈现剂量依赖性。(3)FLS成骨分化情况观察结果。与空白组相比,盐酸小檗碱低、中、高剂量组茜素红染色程度均降低,而且降低程度呈现剂量依赖性。(4)骨稳态调节因子水平测定结果。盐酸小檗碱低、中、高剂量组Dickkopf相关蛋白1(dickkopf-related protein 1,DKK-1)和核因子κB受体激活蛋白配体(receptor activator of NF-κB ligand, RANKL)水平均低于空白组(DKK-1:P=0.048,P=0.032,P=0.001;RANKL:P=0.046,P=0.021,P=0.001),盐酸小檗碱中、高剂量组DKK-1和RANKL水平均低于盐酸小檗碱低剂量组(DKK-1:P=0.028,P=0.002;RANKL:P=0.047,P=0.002),盐酸小檗碱高剂量组DKK-1和RANKL水平均低于盐酸小檗碱中剂量组(DKK-1:P=0.018;RANKL:P=0.011)。结论:盐酸小檗碱能够抑制AS患者FLS体外增殖,并通过减少FLS分泌骨稳态调节因子RANKL和DKK-1抑制FLS成骨分化,而且其作用具有剂量依赖性。

汉黄芩素对胶原诱导性关节炎大鼠关节炎症影响的内质网应激途径

背景:类风湿关节炎是一种炎性疾病,已有多项研究表明汉黄芩素具有较好的抗炎作用,但其对类风湿关节炎的确切疗效和具体作用机制尚待阐明。目的:探讨汉黄芩素通过调节内质网应激途径改善胶原诱导性关节炎大鼠关节炎症的作用机制。方法:①动物水平:雌性Wistar大鼠分为健康对照组、关节炎模型组、汉黄芩素治疗组,通过皮下注射牛Ⅱ型胶原与弗式佐剂构建胶原诱导性关节炎大鼠模型,治疗组在成功造模后给予连续28 d汉黄芩素灌胃治疗;在此期间,每隔7 d对各组大鼠进行体质量、关节炎评分和踝关节肿胀度测量;实验结束后,观察关节病理变化,qRT-PCR和Western Blot、免疫组化检测关节中内质网应激通路GRP78、CHOP mRNA和蛋白表达水平。②细胞水平:CCK-8检测汉黄芩素对胶原诱导性关节炎大鼠成纤维样滑膜细胞的细胞毒作用;不同浓度汉黄芩素处理毒胡萝卜素诱导的成纤维样滑膜细胞,ELISA检测细胞上清白细胞介素1β、肿瘤坏死因子α水平,DCFH-DA探针法测定各组细胞内活性氧簇,qRT-PCR和Western Blot检测GRP78、IRE1α、XBP1s、CHOP mRNA和蛋白水平。结果与结论:①与健康对照组相比,关节炎模型组关节炎指数评分、踝关节肿胀度升高(P<0.01),病理切片可见滑膜增生及炎症细胞浸润、软骨破坏和骨质侵蚀,踝关节GRP78、CHOP mRNA和蛋白表达显著增多(P<0.01),主要定位在滑膜组织和关节面;与关节炎模型组相比,汉黄芩素治疗组关节炎指数评分、踝关节肿胀度降低(P<0.05),滑膜增生及炎症细胞减少,软骨破坏和骨质侵蚀减轻,踝关节GRP78、CHOP mRNA和蛋白表达减少(P<0.05),在滑膜组织和关节面显著降低;3组大鼠体质量无显著差别(P>0.05)。②细胞实验中,200μmol/L汉黄芩素组可明显降低成纤维样滑膜细胞存活率(P<0.01);与空白对照组相比,毒胡萝卜素组细胞上清白细胞介素1β、肿瘤坏死因子α水平、活性氧含量、GRP78、IRE1α、XBP1s、CHOP mRNA及蛋白表达显著增多(P<0.05);与毒胡萝卜素组相比,50μmol/L和100μmol/L汉黄芩素组可降低细胞上清白细胞介素1β、肿瘤坏死因子α水平(P<0.05,P<0.01),100μmol/L汉黄芩素组可明显减少活性氧含量(P<0.01)、下调GRP78、IRE1α、XBP1s、CHOP mRNA及蛋白表达水平(P<0.05)。③结果提示汉黄芩素可以有效改善胶原诱导性关节炎大鼠的关节炎症反应,且内质网应激途径可能是其干预的关键靶点。

低强度脉冲超声通过p38/JNK-白细胞介素-6抑制类风湿性关节炎滑膜成纤维细胞增殖的研究

目的:探讨低强度脉冲超声(low-intensity pulsed ultrasound,LIPUS)对类风湿性关节炎滑膜成纤维细胞(fibroblastlike synoviocytes in rheumatoid arthritis,RA-FLS)异常细胞表型的抑制作用及可能机制。方法:酶消化法分离滑膜细胞,显微镜下观察细胞形态,同时用免疫荧光检测Vimentin蛋白表达来鉴定RA-FLS。将细胞进行体外培养并分为4组:对照组、LIPUS组、肿瘤坏死因子-α(tumor necrosis factor,TNF-α)组和TNF-α+LIPUS组或3组:对照组、白细胞介素-6(interleukin-6,IL-6)组和IL-6+LIPUS组。CCK8和EDU实验分别检测LIPUS对RA-FLS细胞活性和增殖的作用,划痕实验和Transwell迁移实验观察LIPUS对RA-FLS迁移能力的影响,RT-q PCR检测RA-FLS中重要的细胞因子、趋化因子和基质金属蛋白酶(matrix metalloproteinases,MMPs)的基因表达,ELISA进一步检测LIPUS对RA-FLS中关键效应分子IL-6蛋白水平的作用,Western Blot检测LIPUS对RAFLS中丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路的影响。结果:分离得到较纯净的RA-FLS。在体外培养的RA-FLS中,首先,LIPUS可以抑制TNF-α诱导的细胞活性(P<0.001)和增殖(P=0.007),但它对其迁移及迁移相关MMPs(MMP2和MMP9)转录水平的作用在组间无显著性差异(P>0.05)。在TNF-α诱导的炎性环境下,LIPUS能够抑制IL-6和白细胞介素-8(interleukin-8,IL-8)在m RNA水平上的高表达(P<0.001),但其对白细胞介素-1β(interleukin-1β,IL-1β)、MMP1和MMP13的抑制作用在组间无显著性差异(P>0.05)。与未处理组相比,LIPUS抑制TNF-α诱导的RA-FLS中IL-6的分泌(P<0.001),同时也抑制IL-6诱导的RA-FLS增殖(P=0.003)。LIPUS能够抑制MAPK信号通路中p38 MAPK(P=0.033)的磷酸化和c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)的磷酸化(P=0.019),但其对细胞外信号调节激酶1/2(extracellular signalregulated kinas 1/2,ERK1/2)蛋白的磷酸化则无显著作用(P>0.05)。结论:LIPUS能够减少炎性状态下RA-FLS的异常增殖而不作用于其迁移,该作用可能与p38/JNK-IL-6信号通路的抑制有关。