RESUMO
Objective: To investigate the ameliorative effect of tanshinone â ¡A (Tan) on osteoarticular degeneration in ovariectomized rats (a postmenopausal estrogen deficiency model) and the mechanisms involved. Methods: Eight-week-old female Sprague Dawley (SD) rats were randomly allocated to 5 groups (n=10 each), including a Sham operation group (Sham), an ovariectomy group (OVX), and low, medium, and high-dose Tan groups. Eight weeks after bilateral ovariectomy, the rats in the low, medium, and high-dose Tan groups were treated with Tan at the doses of 5, 10, and 20 mg/kg for a duration of 28 days. Evaluation of the rat articular cartilage was performed using X-ray imaging, anatomical observation, hematoxylin and eosin (H&E) staining, and toluidine blue staining. Immunohistochemistry was performed to assess the expression levels of transforming growth factor ß1 (TGF-ß1), phosphorylated-smad2 (p-Smad2), type â ¡ collagen (Câ ¡), matrix metalloproteinase 9 (MMP-9), and MMP-13 in the cartilage tissue. Results: The knee joints of the OVX rats exhibited narrowed joint spaces, osteophyte formation, cartilage erosion or even localized cartilage cracks, faded methylene blue staining on the cartilage surface, disordered arrangement of chondrocytes, unclear or interrupted tidal line, and increased Kellgren-Lawrence grading, Pelletier grading, Mankin grading, and OARSI scores compared to those of the Sham group (P<0.01), as revealed by X-ray imaging, anatomical observation, and histological examination results. Tan ameliorated the degenerative changes in the knee joint caused by OVX in a dose-dependent manner while improving Kellgren-Lawrence grading, Pelletier grading, Mankin grading, and OARSI scores. Immunohistochemistry findings showed that TGF-ß1, p-Smad2, and Câ ¡ expression levels were significantly increased (P<0.01), while MMP-9 and MMP-13 expression levels were significantly decreased (P<0.01) in the articular cartilage of the Tan group compared to those of the OVX group, with all these effects being dose-dependent. Conclusion: Tan mitigates articular cartilage degeneration in ovariectomized rats, which may be related to the regulation of TGF-ß1/Smad2/MMPs signaling pathway.
Assuntos
Abietanos , Cartilagem Articular , Ovariectomia , Ratos Sprague-Dawley , Transdução de Sinais , Proteína Smad2 , Fator de Crescimento Transformador beta1 , Animais , Feminino , Fator de Crescimento Transformador beta1/metabolismo , Ratos , Abietanos/farmacologia , Abietanos/uso terapêutico , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologia , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Colágeno Tipo II/metabolismoRESUMO
The promoting effects of collagen and its derivatives on bone health have been uncovered. However, the structure and effects of type II collagen peptides from squid cartilage (SCIIP) on osteoarthritis still need to be clarified. In this study, SCIIP was prepared from squid throat cartilage with pretreatment by 0.2 mol/L NaOH at a liquid-solid ratio of 10:1 for 18 h and hydrolyzation using alkaline protease and flavourzyme at 50 °C for 4 h. The structure of SCIIP was characterized as a molecular weight lower than 5 kDa (accounting for 87.7 %), a high glycine level of 35.0 %, typical FTIR and CD features of collagen peptides, and a repetitive sequence of Gly-X-Y. GP(Hyp)GPD and GPAGP(Hyp)GD were separated and identified from SCIIP, and their binding energies with TLR4/MD-2 were - 8.4 and - 8.0 kcal/mol, respectively. SCIIP effectively inhibited NO production in RAW264.7 macrophages and alleviated osteoarthritis in rats through the TLR4/NF-κB pathway. Therefore, SCIIP exhibited the potential for application as an anti-osteoarthritis supplement.
Assuntos
Cartilagem , Colágeno Tipo II , Decapodiformes , Osteoartrite , Animais , Decapodiformes/química , Osteoartrite/tratamento farmacológico , Colágeno Tipo II/metabolismo , Camundongos , Cartilagem/química , Cartilagem/metabolismo , Células RAW 264.7 , Ratos , Masculino , Peptídeos/química , Peptídeos/farmacologia , Ratos Sprague-Dawley , Óxido Nítrico/metabolismo , NF-kappa B/metabolismo , Receptor 4 Toll-Like/metabolismoRESUMO
In order to study the toxic effect and mechanism of triptolide(TP) on the reproductive system of female rats with â ¡ type collagen induced arthritis(CIA), 50 SD rats were randomly divided into normal control group, CIA model group, and three groups receiving TP tablets at clinically equivalent doses of 0. 5, 1, and 2 times, respectively(with TP dosages of 3. 75, 7. 5, and 15 µg·kg~(-1)·d~(-1)), each comprising 10 rats. Intragastric administration was started on the day after the first immunization, once a day, for 42 days.The results were taken on the 21st and 42nd days to calculate the uterine and ovarian organ indexes; pathological and morphological changes in uterus and ovaries were observed under a light microscope; and the levels of estradiol(E_2) and cytochrome P450A1(aromatase,CYP19A1) in ovarian homogenate were detected by ELISA. Furthermore, immunohistochemistry was employed to detect the expression levels of transforming growth factor ß3( TGFß3) pathway-related proteins, mothers against decapentaplegic homolog 3(Smad3) and steroidogenic factor-1(SF-1) in ovarian tissues. In vitro, the mouse Chinese hamster ovary(CHO) cell line was established, and after 24 hours of TP administration(30, 60, 120 nmol·L~(-1)), cell proliferation was detected by the thiazolyl blue tetrazolium bromide(MTT) method, apoptosis by the flow cytometry, and TGFß3, Smad3 and SF-1 protein expression in cells by the Western blot method, and the nuclear entry of SF-1 was detected by immunofluorescence. The results showed that compared with the CIA model group, all TP administration groups showed decreased number of uterine glands, total follicles, mature follicles, and corpus luteum on days 21 and 42 of administration, but there was no statistical difference, and only the administration of 2 times the clinically equivalent dose of TP could significantly increase the number of atretic follicles at 42 days of administration. TP at 3. 75 µg·kg-1·d-1significantly reduced the level of E_2 at 21 days of administration and the expression of TGFß3 and Smad3 factors in ovarian tissues,but had no significant effect on the rate-limiting enzyme in estrogen synthesis CYP19A1. TP at 7. 5 and 15 µg·kg~(-1)·d~(-1) significantly reduced the expression of SF-1 regardless of administration for 21 days or 42 days. TP can significantly promote ovarian cell apoptosis in vitro, with apoptosis mainly concentrated in the late stage of apoptosis after 24 hours of administration. In addition, 60 nmol·L~(-1) TP significantly reduced the protein expression of TGFß3, Smad3 and SF-1 in a dose-dependent manner. In summary, intragastric administration of TP at less than 2 times the clinically equivalent dose for 21 days and 42 days did not cause obvious reproductive damage to the uterus and ovarian tissues of CIA rats, and the number of atretic follicles changed significantly only when the 2 times the clinically equivalent dose was administered for 42 days. TP exerted reproductive toxicity in vivo on reproductive target organs and in vitro on ovarian cells by inhibiting the expression of TGFß3/Smad3/SF-1 pathway.
Assuntos
Diterpenos , Compostos de Epóxi , Ovário , Fenantrenos , Ratos Sprague-Dawley , Útero , Animais , Feminino , Diterpenos/farmacologia , Fenantrenos/toxicidade , Ratos , Compostos de Epóxi/toxicidade , Compostos de Epóxi/administração & dosagem , Ovário/efeitos dos fármacos , Ovário/metabolismo , Útero/efeitos dos fármacos , Útero/metabolismo , Colágeno Tipo II/metabolismo , Proteína Smad3/metabolismo , Proteína Smad3/genética , Humanos , Reprodução/efeitos dos fármacos , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , EstradiolRESUMO
BACKGROUND AND AIM: Autoimmunity refers to the presence of autoantibodies and autoreactive lymphocytes against the structural molecules of an individual's cells or tissues, known as self-antigens or autoantigens. It might exist in the absence of autoimmune disease. However, how autoimmunity develops remains a mystery, despite the discovery of autoantibodies in human cord blood. METHODS: Murine fetuses on day 14 of gestation were subjected to intraperitoneal injection of murine thyroid peroxidase (TPO) peptides or collagen type II (CII) at graded doses via transuterine approach. Postnatally, the recipients were examined for autoantibodies by ELISA and autoreactive lymphocytes by in vitro incorporation of tritium and for the development of autoimmune thyroiditis or arthritis. RESULTS: At one month of age, the recipients did not secrete significant levels of anti-TPO or CII IgG2a in sera until a dose of 0.5 µg TPO or 5.0 µg CII was injected in utero. Serum anti-TPO or CII IgG2a persisted for at least two to four months postnatally. In recipients with elevated autoantibodies, their lymphocytes also showed proliferative responses specifically to TPO or CII. However, the development of autoantibodies and autoreactive lymphocytes was not associated with inflammatory cell infiltration of thyroid glands or paw joints even though anti-TPO or CII IgG2a was enhanced by postnatal TPO or CII challenge. CONCLUSION: Fetal exposure to free autoantigens could be immunogenic, shedding new light on the in utero origin of autoantibodies and autoreactive lymphocytes. The development of autoimmunity requires a threshold intensity of autoantigen exposure in the fetus.
Assuntos
Autoanticorpos , Autoantígenos , Autoimunidade , Feto , Iodeto Peroxidase , Animais , Autoantígenos/imunologia , Autoimunidade/imunologia , Feminino , Camundongos , Autoanticorpos/imunologia , Autoanticorpos/sangue , Iodeto Peroxidase/imunologia , Gravidez , Feto/imunologia , Colágeno Tipo II/imunologia , Imunoglobulina G/imunologia , Imunoglobulina G/sangue , Linfócitos/imunologia , Tireoidite Autoimune/imunologiaRESUMO
BACKGROUND: Mesenchymal stem cells (MSCs) derived from the synovium, known as synovium mesenchymal stem cells (SMSCs), exhibit significant potential for articular cartilage regeneration owing to their capacity for chondrogenic differentiation. However, the microRNAs (miRNAs) governing this process and the associated mechanisms remain unclear. While mechanical stress positively influences chondrogenesis in MSCs, the miRNA-mediated response of SMSCs to mechanical stimuli is not well understood. OBJECTIVE: This study explores the miRNA-driven mechano-transduction in SMSCs chondrogenesis under mechanical stress. METHODS: The surface phenotype of SMSCs was analysed by flow cytometry. Chondrogenesis capacities of SMSCs were examined by Alcian blue staining. High throughput sequencing was used to screen mechano-sensitive miRNAs of SMSCs. The RNA expression level of COL2A1, ACAN, SOX9, BMPR2 and miR-143-3p of SMSCs were tested by quantitative real-time polymerase chain reaction (qRT-PCR). The interaction between miR-143-3p and TLR4 was confirmed by luciferase reporter assays. The protein expression levels of related genes were assessed by western blot. RESULTS: High-throughput sequencing revealed a notable reduction in miR-143-3p levels in mechanically stressed SMSCs. Gain- or loss-of-function strategies introduced by lentivirus demonstrated that miR-143-3p overexpression hindered chondrogenic differentiation, whereas its knockdown promoted this process. Bioinformatics scrutiny and luciferase reporter assays pinpointed a potential binding site for miR-143-3p within the 3'-UTR of bone morphogenetic protein receptor type 2 (BMPR2). MiR-143-3p overexpression decreased BMPR2 expression and phosphorylated Smad1, 5 and 8 levels, while its inhibition activated BMPR2-Smad pathway. CONCLUSION: This study elucidated that miR-143-3p negatively regulates SMSCs chondrogenic differentiation through the BMPR2-Smad pathway under mechanical tensile stress. The direct targeting of BMPR2 by miR-143-3p established a novel dimension to our understanding of mechano-transduction mechanism during SMSC chondrogenesis. This understanding is crucial for advancing strategies in articular cartilage regeneration.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II , Diferenciação Celular , Condrogênese , Células-Tronco Mesenquimais , MicroRNAs , Transdução de Sinais , Estresse Mecânico , Membrana Sinovial , Humanos , Agrecanas/metabolismo , Agrecanas/genética , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Diferenciação Celular/fisiologia , Células Cultivadas , Condrogênese/fisiologia , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genética , Células-Tronco Mesenquimais/metabolismo , MicroRNAs/metabolismo , MicroRNAs/genética , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Membrana Sinovial/citologia , Membrana Sinovial/metabolismoRESUMO
The knee joint has long been considered a closed system. The pathological effects of joint diseases on distant organs have not been investigated. Herein, our clinical data showed that post-traumatic joint damage, combined with joint bleeding (hemarthrosis), exhibits a worse liver function compared with healthy control. With mouse model, hemarthrosis induces both cartilage degeneration and remote liver damage. Next, we found that hemarthrosis induces the upregulation in ratio and differentiation towards Th17 cells of CD4+ T cells in peripheral blood and spleen. Deletion of CD4+ T cells reverses hemarthrosis-induced liver damage. Degeneration of cartilage matrix induced by hemarthrosis upregulates serological type II collagen (COL II), which activates CD4+ T cells. Systemic application of a COL II antibody blocks the activation. Furthermore, bulk RNAseq and single-cell qPCR analysis revealed that the cartilage Akt pathway is inhibited by blood treatment. Intra-articular application of Akt activator blocks the cartilage degeneration and thus protects against the liver impairment in mouse and pig models. Taken together, our study revealed a pathological joint-liver axis mediated by matrikine-activated CD4+ T cells, which refreshes the organ-crosstalk axis and provides a new treatment target for hemarthrosis-related disease. Intra-articular bleeding induces cartilage degradation through down-reulation of cartilage Akt pathway. During this process, the soluble COL II released from the damaged cartilage can activate peripheral CD4+ T cells, differention into Th17 cells and secretion of IL-17, which consequently induces liver impairment. Intra-articular application of sc79 (inhibitor of Akt pathway) can prevent the cartilage damage as well as its peripheral influences.
Assuntos
Linfócitos T CD4-Positivos , Fígado , Animais , Camundongos , Humanos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Fígado/patologia , Fígado/metabolismo , Hemartrose/genética , Hemartrose/patologia , Masculino , Modelos Animais de Doenças , Células Th17/imunologia , Células Th17/patologia , Colágeno Tipo II/genética , Venenos Elapídicos/farmacologia , Feminino , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100 ng/mL) was examined using real-time reverse transcription polymerase chain reaction. Further, to assess the deposition of total glycosaminoglycans during chondrogenic differentiation, ATDC5 cells were stained with Alcian Blue. Moreover, the amount of hyaluronan in the culture supernatant was measured by enzyme-linked immunosorbent assay. The addition of Sema3A to cultured ATDC5 cells increased the expression of Sox9, Runx2, COL2A1, ACAN, HAS2, and COL10A1 during chondrogenic differentiation. Moreover, it enhanced total proteoglycan and hyaluronan synthesis. Further, Sema3A was upregulated in the early stages of chondrogenic differentiation, and its secretion decreased later. Sema3A increases extracellular matrix production and promotes chondrogenic differentiation. To the best of our knowledge, this is the first study to demonstrate the role of Sema3A on chondrogenic differentiation.
Assuntos
Diferenciação Celular , Condrogênese , Semaforina-3A , Animais , Diferenciação Celular/efeitos dos fármacos , Semaforina-3A/metabolismo , Condrogênese/efeitos dos fármacos , Camundongos , Condrócitos/metabolismo , Condrócitos/citologia , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Linhagem Celular , Fatores de Transcrição SOX9/metabolismo , Fatores de Transcrição SOX9/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genética , Agrecanas/metabolismo , Agrecanas/genética , Hialuronan Sintases/metabolismo , Hialuronan Sintases/genética , Glicosaminoglicanos/metabolismo , Colágeno Tipo X/metabolismo , Colágeno Tipo X/genéticaRESUMO
To investigate the mechanism of action of aqueous extract of Strychni Semen(SA) on bone destruction in rats with type â ¡ collagen-induced arthritis(CIA), the SD rats were randomly divided into normal group, model group, low, medium, and high dose(2.85, 5.70, and 11.40 mg·kg~(-1)) groups of SA, and methotrexate group. Except for the normal group, the CIA model was prepared for the other groups. After the second immunization, different doses of SA were given to the low, medium, and high dose groups of SA once a day, and the methotrexate group was given once every three days. 0.3% sodium hydroxymethylcellulose(CMC-Na) was given once a day to the normal and model groups for 28 d. The clinical score of arthritis was evaluated every three days. Micro computed tomography(Micro-CT) method was used to evaluate the degree of bone destruction. Histopathological changes in the joint tissue and the number of osteoclasts in CIA rats were evaluated by hematoxylin-eosin(HE) staining and tartrate-resistant acid phosphatase(TRAP) staining. The expression of interleukin-1ß(IL-1ß) in the joint tissue of rats was detected by immunohistochemistry. Western blot was used to detect key protein expression in mitogen-activated protein kinase(MAPK) and phosphatidylinositol 3-kinase/protein kinase B(PI3K/Akt) signaling pathways in the joint tissue of rats. The results showed that different doses of SA were able to improve the red and swollen inflammatory joint and joint deformity in CIA rats to varying degrees, reduce the clinical score, inhibit synovial inflammation, vascular opacification, cartilage erosion, and bone destruction, and reduce the number of TRAP-positive cells in bone tissue. Micro-CT results showed that the SA was able to increase bone mineral density, bone volume fraction, trabecular reduce, and trabecular number and reduce bone surface/bone volume and trabecular separation/spacing. Different doses of SA could down-regulate the protein expression of IL-1ß, p-JNK, p-ERK, p-p38, PI3K, and p-Akt to varying degrees. In conclusion, SA can improve disease severity, attenuate histopathological and imaging changes in joints, and have osteoprotective effects in CIA rats, and its mechanism of action may be related to the inhibition of the overactivation of MAPK and PI3K/Akt signaling pathways.
Assuntos
Artrite Experimental , Artrite Reumatoide , Ratos , Animais , Colágeno Tipo II , Metotrexato , Proteínas Proto-Oncogênicas c-akt , Sêmen , Microtomografia por Raio-X , Fosfatidilinositol 3-Quinases , Ratos Sprague-Dawley , Artrite Reumatoide/tratamento farmacológico , Artrite Experimental/tratamento farmacológico , Artrite Experimental/induzido quimicamenteRESUMO
Spheroids derived from human mesenchymal stem cells (hMSCs) are of limited use for cartilage regeneration, as the viability of the cells progressively decreases during the period required for chondrogenic differentiation (21 days). In this work, spheroids based on hMSCs and a lactose-modified chitosan (CTL) were formed by seeding cells onto an air-dried coating of CTL. The polymer coating can inhibit cell adhesion and it is simultaneously incorporated into spheroid structure. CTL-spheroids were characterized from a morphological and biological perspective, and their properties were compared with those of spheroids obtained by seeding the cells onto a non-adherent surface (agar gel). Compared to the latter, smaller and more viable spheroids form in the presence of CTL as early as 4 days of culture. At this time point, analysis of stem cells differentiation in spheroids showed a remarkable increase in collagen type-2 (COL2A1) gene expression (~700-fold compared to day 0), whereas only a 2-fold increase was observed in the control spheroids at day 21. These results were confirmed by histological and transmission electron microscopy (TEM) analyses, which showed that in CTL-spheroids an early deposition of collagen with a banding structure already occurred at day 7. Overall, these results support the use of CTL-spheroids as a novel system for cartilage regeneration, characterized by increased cell viability and differentiation capacity within a short time-frame. This will pave the way for approaches aimed at increasing the success rate of procedures and reducing the time required for tissue regeneration.
Assuntos
Diferenciação Celular , Quitosana , Condrogênese , Lactose , Células-Tronco Mesenquimais , Esferoides Celulares , Quitosana/farmacologia , Quitosana/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/citologia , Humanos , Diferenciação Celular/efeitos dos fármacos , Condrogênese/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/citologia , Lactose/farmacologia , Lactose/química , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genéticaAssuntos
Condrócitos , Colágeno Tipo II , Células-Tronco Hematopoéticas , Humanos , Agrecanas/genética , Agrecanas/metabolismo , Cartilagem Articular/citologia , Cartilagem Articular/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Condrócitos/citologia , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Proteínas Hedgehog/metabolismo , Proteínas Hedgehog/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologiaRESUMO
Syndecan-1 (Sdc-1), a transmembrane heparan sulfate protein, is implicated in several pathophysiological processes including rheumatoid arthritis (RA). The exact role of Syndican-1 in this autoimmune disease is still undetermined. This study explores the involvement level of Sdc-1 in the development of RA in a collagen II-induced arthritis mice model. RA was induced in two mice strains (wild-type BALB/c group and Sdc-1 knockout) by collagen II. Mice underwent regular clinical observations and scoring. After sacrifice, leg biopsies were taken from mice for histological examination, using a variety of stains. In addition, proteins were extracted, and molecular assessment of TNF-α was performed using the western blot technique. In the Sdc-1 knockout group, clinical scoring results showed a significantly more severe experimental RA; histology showed a significant increase in bone erosion, cartilage destruction, inflammation, and less granulated mast cells than the wild-type. In addition, molecular assessment of TNF-α showed more increase in expression in the Sdc-1 knockout models compared to the wild-type. Data suggest that lack of Sdc-1 enhances the inflammatory characteristics in RA. However, more molecular studies and investigations are needed to determine its exact role and possible mechanisms involved.
Assuntos
Artrite Experimental , Artrite Reumatoide , Sindecana-1 , Fator de Necrose Tumoral alfa , Animais , Masculino , Camundongos , Artrite Experimental/genética , Artrite Experimental/patologia , Artrite Experimental/imunologia , Artrite Reumatoide/genética , Artrite Reumatoide/patologia , Artrite Reumatoide/metabolismo , Artrite Reumatoide/imunologia , Colágeno Tipo II/genética , Modelos Animais de Doenças , Camundongos Endogâmicos BALB C , Camundongos Knockout , Sindecana-1/genética , Sindecana-1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/genéticaRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disease characterized by inflammatory reactions and tissue damage in the joints. Long-term drug use in clinical practice is often accompanied by adverse reactions. Extracorporeal photopheresis (ECP) is an immunomodulatory therapy with few side effects, offering a potential and safe therapeutic alternative for RA through the induction of immune tolerance. This study aimed to investigate the therapeutic effects of ECP on RA using a collagen-induced arthritis (CIA) murine model, as well as to explore its immunomodulatory effects in vivo. Additionally, particular attention was given to the significant role of monocytes during the ECP process. METHODS: A murine model of rheumatoid arthritis was established by administering two injections of bovine type II collagen to DBA/1J mice. ECP, ECP-MD (mononuclear cells were depleted during the ECP), MTX, and PBS treatment were applied to the CIA mice. During the treatment process, clinical scores and body weight changes of CIA mice were closely monitored. After six treatment sessions, micro-CT images of the hind paws from live mice were captured. Ankle joints and paws of the mice were collected and processed for histological evaluation. Spleen samples were collected to measure the Th17/Treg cells ratio, and serum samples were collected to assess cytokine and anti-type II collagen IgG levels. Monocytes and dendritic cells populations before and after ECP in vitro were detected by flow cytometry. RESULT: ECP therapy significantly attenuated the progression of CIA, alleviated the severity of clinical symptoms in CIA mice and effectively suppressed synovial hyperplasia, inflammation, and cartilage damage. There was an expansion in the percentage of CD3 + CD4 + CD25 + FoxP3 + Tregs and a decrease in CD3 + CD4 + IL17A + Th17 cells in vivo. Furthermore, ECP reduced the serum levels of pro-inflammatory cytokines IL-6 (53.47 ± 7.074 pg/mL vs 5.142 ± 1.779 pg/mL, P < 0.05) and IL-17A (3.077 ± 0.401 pg/mL vs 0.238 ± 0.082 pg/mlL, P < 0.0001) compared with PBS. Interestingly, the depletion of monocytes during the ECP process did not lead to any improvement in clinical symptoms or histological scores in CIA mice. Moreover, the imbalance in the Th17/Treg cells ratio became even more pronounced, accompanied by an augmented secretion of pro-inflammatory cytokines IL-6 and IL-17A. In vitro, compared with cells without ECP treatment, the proportion of CD11b + cells were significantly reduced (P < 0.01), the proportion of CD11c + cells were significantly elevated (P < 0.001) 24 h after ECP treatment. Additionally, the expression of MHC II (P < 0.0001), CD80 (P < 0.01), and CD86 (P < 0.001) was downregulated in CD11c + cells 24 h after ECP treatment. CONCLUSION: Our study demonstrates that ECP exhibits a therapeutic effect comparable to conventional therapy in CIA mice, and the protective mechanisms of ECP against RA involve Th17/Treg cells ratio, which result in decreased IL-6 and IL-17A. Notably, monocytes derived from CIA mice are an indispensable part to the efficacy of ECP treatment, and the proportion of monocytes decreased and the proportion of tolerogenic dendritic cells increased after ECP treatment in vitro.
Assuntos
Artrite Experimental , Artrite Reumatoide , Fotoferese , Camundongos , Animais , Bovinos , Interleucina-17/metabolismo , Modelos Animais de Doenças , Interleucina-6 , Camundongos Endogâmicos DBA , Artrite Reumatoide/tratamento farmacológico , Inflamação , Citocinas/metabolismo , Artrite Experimental/terapia , Colágeno Tipo II , Linfócitos T Reguladores , Células Th17RESUMO
Inositol-requiring enzyme-1 (IRE1) is the master regulator of the unfolded protein response pathway, associated with the endoplasmic reticulum (ER) in sensing and regulating cell stress. The activity of IRE1 is highly explored and well-characterized in cancer and other cells. However, the IRE1 molecular mechanism in chondrocytes is poorly understood. The present study explored the effect of IRE1 on chondrocytes regarding its chondrogenic gene expression and its correlation with different cellular pathways and cell behavior. Chondrocytes transfected with the cDNA of IRE1 reduced the expression of type II collagen, disrupting chondrocyte differentiation as confirmed by western blotting and immunofluorescence. Upon siRNA treatment, the influence of IRE1 on chondrocyte differentiation is restored by reviving the normal expression of type II collagen. Different molecular pathways were explored to investigate the role of IRE1 in causing chondrocyte dedifferentiation. However, we found no significant correlation, as IRE1 induces dedifferentiation through independent pathways. In response to various endoplasmic reticulum (ER) agonists (2-deoxy-D-glucose), and ER stress antagonists (tauroursodeoxycholic acid and salubrinal), IRE1 overexpression did not affect GRP78/94, as implicated in the pathogenesis of ER stress. Moreover, when IRE1 overexpression was correlated with the inflammation pathway, nuclear factor-kappa B (NFκB), IRE1 substantially increased the expression of p50 while decreasing the expression of nuclear factor kappa light polypeptide alpha (IκBα). These results suggest that IRE1 induces dedifferentiation in chondrocytes by modulating inflammatory pathways that cause dedifferentiation by disrupting type II collagen expression.
Assuntos
Desdiferenciação Celular , Condrócitos , Colágeno Tipo II , Estresse do Retículo Endoplasmático , Endorribonucleases , Complexos Multienzimáticos , NF-kappa B , Proteínas Serina-Treonina Quinases , Tioureia/análogos & derivados , Condrócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Animais , Colágeno Tipo II/metabolismo , Colágeno Tipo II/genética , Endorribonucleases/metabolismo , Endorribonucleases/genética , NF-kappa B/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Cinamatos/farmacologia , Tioureia/farmacologia , Células Cultivadas , Transdução de Sinais , Chaperona BiP do Retículo EndoplasmáticoRESUMO
Objective To investigate the impact of imidazole ketone erastin (IKE), a ferroptosis inducer, on pulmonary fibrosis progression in mice with collagen-induced arthritis (CIA), and to understand its potential mechanism. Methods Chick type II collagen emulsified in complete Freund's adjuvant (CFA) was injected into DBA/1 mice, aged 8 to 10 weeks, to induce CIA. Fourteen days later, type II collagen emulsified in incomplete Freund's adjuvant (IFA) was administered to the mice. The mice were randomly divided into a control group, a CIA group and a CIA combined IKE group. The development of arthritis was monitored by evaluating the arthritis scores every two days until day 39 and then the mice were sacrificed for organ collection. The histopathological changes of joints were evaluated by HE staining, Safranin O-fast green staining and toluidine blue staining. The histopathological changes of organs including heart, liver, spleen, lung, and kidney were evaluated by HE staining, and Masson's trichrome staining was used to assess pulmonary fibrosis. The expression levels of smooth muscle actin α (α-SMA), fibroblast activating protein α (FAPα), transforming growth factor ß (TGF-ß), type I collagen (Col1), interleukin 1(IL-1), IL-6, IL-17 and tumor necrosis factor α (TNF-α) were detected by immunohistochemical staining. The expression levels of serum cytokines including IL-17α, IL-17F, TGF-ß1, ITG-ß6, TNF receptor superfamily menber 11B(TNFRSF11B), TNFRSF12A, IL-6, IL-1α, IL-1ß, IL-10, TNF-α, CCL5, CCL2, CXCL9, CXCL1, NADK, EPO, CSF2, TGF-α, CCL20 and CCL3 in serum were detected by Olink mouse exploratory panel. Results Histological staining in the CIA mice administered with IKE model demonstrated that IKE treatment reduced bone absorption and the degree of synovial inflammation when active inflammation was present. CIA mice administered with IKE showed lower expression levels of α-SMA, FAPα, TGF-ß, Col1, IL-1, IL-6, IL-17 and TNF-α, according to the immunohistochemical staining of the lung. In addition, the expression levels of CCL5, CXCL9 and IL-6 were also decreased in serum of CIA mice treated with IKE. Conclusion IKE not only ameliorates joint inflammation and bone damage, but also alleviates the inflammation and the progression of pulmonary fibrosis in CIA mice.
Assuntos
Artrite Experimental , Ferroptose , Imidazóis , Cetonas , Piperazinas , Fibrose Pulmonar , Animais , Camundongos , Colágeno Tipo II , Inflamação , Interleucina-17 , Interleucina-1beta , Interleucina-6/genética , Fibrose Pulmonar/induzido quimicamente , Fator de Crescimento Transformador beta , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Pathogenic single nucleotide variants (SNVs) found in the COL2A1 gene are associated with a broad range of skeletal dysplasias due to their impact on the structure and function of the Col2a1 protein. However, the molecular mechanisms of some nucleotide variants detected during diagnostic testing remain unclear. The interpretation of missense and splicing variants caused by SNVs poses a significant challenge for clinicians. In this work, we analyzed 22 splicing variants in the COL2A1 gene which have been found in patients with COL2A1-associated skeletal dysplasias. Using a minigene system, we investigated the impact of these SNVs on splicing and gained insights into their molecular mechanisms and genotype-phenotype correlations for each patient. The results of our study are very useful for improving the accuracy of diagnosis and the management of patients with skeletal dysplasias caused by SNVs in the COL2A1 gene.
Assuntos
Nucleotídeos , Humanos , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Fenótipo , MutaçãoRESUMO
Objective: To investigate the effects and underlying mechanisms of VX765 on osteoarthritis (OA) and chondrocytes inflammation in rats. Methods: Chondrocytes were isolated from the knee joints of 4-week-old Sprague Dawley (SD) rats. The third-generation cells were subjected to cell counting kit 8 (CCK-8) analysis to assess the impact of various concentrations (0, 1, 5, 10, 20, 50, 100 µmol/L) of VX765 on rat chondrocyte activity. An in vitro lipopolysaccharide (LPS) induced cell inflammation model was employed, dividing cells into control group, LPS group, VX765 concentration 1 group and VX765 concentration 2 group without obvious cytotoxicity. Western blot, real-time fluorescence quantitative PCR, and ELISA were conducted to measure the expression levels of inflammatory factors-transforming growth factor ß 1 (TGF-ß 1), interleukin 6 (IL-6), and tumor necrosis factor α (TNF-α). Additionally, Western blot and immunofluorescence staining were employed to assess the expressions of nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1). Thirty-two SD rats were randomly assigned to sham surgery group (group A), OA group (group B), OA+VX765 (50 mg/kg) group (group C), and OA+VX765 (100 mg/kg) group (group D), with 8 rats in each group. Group A underwent a sham operation with a medial incision, while groups B to D underwent additional transverse incisions to the medial collateral ligament and anterior cruciate ligament, with removal of the medial meniscus. One week post-surgery, groups C and D were orally administered 50 mg/kg and 100 mg/kg VX765, respectively, while groups A and B received an equivalent volume of saline. Histopathological examination using HE and safranin-fast green staining was performed, and Mankin scoring was utilized for evaluation. Immunohistochemical staining technique was employed to analyze the expressions of matrix metalloproteinase 13 (MMP-13) and collagen type â ¡. Results: The CCK-8 assay indicated a significant decrease in cell viability at VX765 concentrations exceeding 10 µmol/L ( P<0.05), so 4 µmol/L and 8 µmol/L VX765 without obvious cytotoxicity were selected for subsequent experiments. Following LPS induction, the expressions of TGF-ß 1, IL-6, and TNF-α in cells significantly increased when compared with the control group ( P<0.05). However, intervention with 4 µmol/L and 8 µmol/L VX765 led to a significant decrease in expression compared to the LPS group ( P<0.05). Western blot and immunofluorescence staining demonstrated a significant upregulation of Nrf2 pathway-related molecules Nrf2 and HO-1 protein expressions by VX765 ( P<0.05), indicating Nrf2 pathway activation. Histopathological examination of rat knee joint tissues and immunohistochemical staining revealed that, compared to group B, treatment with VX765 in groups C and D improved joint structural damage in rat OA, alleviated inflammatory reactions, downregulated MMP-13 expression, and increased collagen type â ¡ expression. Conclusion: VX765 can improve rat OA and reduce chondrocyte inflammation, possibly through the activation of the Nrf2 pathway.
Assuntos
Condrócitos , Dipeptídeos , Osteoartrite , para-Aminobenzoatos , Ratos , Animais , Condrócitos/metabolismo , Metaloproteinase 13 da Matriz/metabolismo , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo , Colágeno Tipo II/metabolismo , Interleucina-6 , Lipopolissacarídeos/farmacologia , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/farmacologia , Inflamação/tratamento farmacológico , Osteoartrite/metabolismo , Fator de Crescimento Transformador beta1/metabolismoRESUMO
The objective of this study was to investigate the effect of low-molecular-weight fish collagen (valine-glycine-proline-hydroxyproline-glycine-proline-alanine-glycine; LMWCP) on H2O2- or LPS-treated primary chondrocytes and monoiodoacetate (MIA)-induced osteoarthritis rat models. Our findings indicated that LMWCP treatment exhibited protective effects by preventing chondrocyte death and reducing matrix degradation in both H2O2-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. This was achieved by increasing the levels of aggrecan, collagen type I, collagen type II, TIMP-1, and TIMP-3, while simultaneously decreasing catabolic factors such as phosphorylation of Smad, MMP-3, and MMP-13. Additionally, LMWCP treatment effectively suppressed the activation of inflammation and apoptosis pathways in both LPS-treated primary chondrocytes and cartilage tissue from MIA-induced osteoarthritis rats. These results suggest that LMWCP supplementation ameliorates the progression of osteoarthritis through its direct impact on inflammation and apoptosis in chondrocytes.
Assuntos
Cartilagem Articular , Osteoartrite , Ratos , Animais , Condrócitos , Hidroxiprolina/efeitos adversos , Hidroxiprolina/metabolismo , Glicina/farmacologia , Peróxido de Hidrogênio/farmacologia , Lipopolissacarídeos/farmacologia , Osteoartrite/induzido quimicamente , Osteoartrite/tratamento farmacológico , Osteoartrite/prevenção & controle , Inflamação/metabolismo , Colágeno Tipo II/farmacologia , Peptídeos/farmacologia , Valina/efeitos adversos , Valina/metabolismo , Células CultivadasRESUMO
SUMMARY: Rheumatoid arthritis (RA) that affects the synovial knee joint causes swelling of the synovial membrane and tissue damage. Interleukin-17A (IL-17A) and the enzyme glycogen synthase kinase-3β (GSK3β) are involved in the pathogenesis of RA. The link between IL-17A, GSK3β, the oxidative stress, and the profibrogenic marker alpha-smooth muscle actin (α-SMA) with and without TDZD-8, GSK3β inhibitor has not been studied before. Consequently, active immunization of rats was performed to induce RA after three weeks using collagen type II (COII) injections. The treated group received daily injection of 1 mg/kg TDZD-8 for 21 days following the immunization protocol (COII+TDZD-8). Blood and synovium tissue samples were harvested at the end of the experiment. RA development was confirmed as corroborated by a substantial increase in blood levels of the highly specific autoantibody for RA, anti-citrullinated protein antibody as well as augmentation of reactive oxidative species (ROS) levels measured as lipid peroxidation. RA induction also increased synovium tissue levels of IL-17A and the profibrogenic marker, α-SMA. All these parameters seemed to be significantly (p<0.0001) ameliorated by TDZD-8. Additionally, a significant correlation between IL-17A, ROS, and α-SMA and biomarkers of RA was observed. Thus, knee joint synovium RA induction augmented IL-17A/GSK3β/ROS/α-SMA axis mediated arthritis in a rat model of RA, which was inhibited by TDZD-8.
La artritis reumatoide (AR) que afecta la articulación sinovial de la rodilla provoca inflamación de la membrana sinovial y daño tisular. La interleucina-17A (IL-17A) y la enzima glucógeno sintasa quinasa-3β (GSK3β) están involucradas en la patogenia de la AR. No se ha estudiadol vínculo entre IL-17A, GSK3β, el estrés oxidativo y el marcador profibrogénico actina de músculo liso alfa (α-SMA) con y sin inhibidor de TDZD-8, GSK3β. En consecuencia, se realizó una inmunización activa de ratas para inducir la AR después de tres semanas usando inyecciones de colágeno tipo II (COII). El grupo tratado recibió una inyección diaria de 1 µg/ kg de TDZD-8 durante 21 días siguiendo el protocolo de inmunización (COII+TDZD-8). Se recogieron muestras de sangre y tejido sinovial al final del experimento. El desarrollo de AR se confirmó como lo corroboró el aumento sustancial en los niveles sanguíneos del autoanticuerpo altamente específico para AR, el anticuerpo antiproteína citrulinada, así como el aumento de los niveles de especies oxidativas reactivas (ROS) medidos como peroxidación lipídica. La inducción de AR también aumentó los niveles de tejido sinovial de IL-17A y el marcador profibrogénico, α-SMA. Todos estos parámetros parecían mejorar significativamente (p<0,0001) con TDZD-8. Además, se observó una correlación significativa entre IL- 17A, ROS y α-SMA y biomarcadores de AR. Por lo tanto, la inducción de AR en la sinovial de la articulación de la rodilla aumentó la artritis mediada por el eje IL-17A/GSK3β/ROS/α-SMA en un modelo de rata de AR, que fue inhibida por TDZD-8.
Assuntos
Animais , Ratos , Artrite Reumatoide , Tiadiazóis/administração & dosagem , Fibrose , Imuno-Histoquímica , Western Blotting , Actinas , Imunização , Espécies Reativas de Oxigênio , Ratos Wistar , Interleucina-17 , Colágeno Tipo II/administração & dosagem , Modelos Animais de Doenças , Glicogênio Sintase Quinase 3 betaRESUMO
Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1ß (IL-1ß)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1ß was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1ß-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.