Administration of Liposomal-Based Pde3b Gene Therapy Protects Mice Against Collagen-Induced Rheumatoid Arthritis via Modulating Macrophage Polarization.

Background: Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation and joint destruction. Despite progress in RA therapy, it remains difficult to achieve long-term remission in RA patients. Phosphodiesterase 3B (Pde3b) is a member of the phosphohydrolyase family that are involved in many signal transduction pathways. However, its role in RA is yet to be fully addressed. Methods: Studies were conducted in arthritic DBA/1 mice, a suitable mouse strain for collagen-induced rheumatoid arthritis (CIA), to dissect the role of Pde3b in RA pathogenesis. Next, RNAi-based therapy with Pde3b siRNA-loaded liposomes was assessed in a CIA model. To study the mechanism involved, we investigated the effect of Pde3b knockdown on macrophage polarization and related signaling pathway. Results: We demonstrated that mice with CIA exhibited upregulated Pde3b expression in macrophages. Notably, intravenous administration of liposomes loaded with Pde3b siRNA promoted the macrophage anti-inflammatory program and alleviated CIA in mice, as indicated by the reduced inflammatory response, synoviocyte infiltration, and bone and cartilage erosion. Mechanistic study revealed that depletion of Pde3b increased cAMP levels, by which it enhanced PKA-CREB-C/EBPß pathway to transcribe the expression of anti-inflammatory program-related genes. Conclusion: Our results support that Pde3b is involved in the pathogenesis of RA, and Pde3b siRNA-loaded liposomes might serve as a promising therapeutic approach against RA.

Gut microbial metabolite targets HDAC3-FOXK1-interferon axis in fibroblast-like synoviocytes to ameliorate rheumatoid arthritis.

Rheumatoid arthritis (RA) is an autoimmune disease. Early studies hold an opinion that gut microbiota is environmentally acquired and associated with RA susceptibility. However, accumulating evidence demonstrates that genetics also shape the gut microbiota. It is known that some strains of inbred laboratory mice are highly susceptible to collagen-induced arthritis (CIA), while the others are resistant to CIA. Here, we show that transplantation of fecal microbiota of CIA-resistant C57BL/6J mice to CIA-susceptible DBA/1J mice confer CIA resistance in DBA/1J mice. C57BL/6J mice and healthy human individuals have enriched B. fragilis than DBA/1J mice and RA patients. Transplantation of B. fragilis prevents CIA in DBA/1J mice. We identify that B. fragilis mainly produces propionate and C57BL/6J mice and healthy human individuals have higher level of propionate. Fibroblast-like synoviocytes (FLSs) in RA are activated to undergo tumor-like transformation. Propionate disrupts HDAC3-FOXK1 interaction to increase acetylation of FOXK1, resulting in reduced FOXK1 stability, blocked interferon signaling and deactivation of RA-FLSs. We treat CIA mice with propionate and show that propionate attenuates CIA. Moreover, a combination of propionate with anti-TNF etanercept synergistically relieves CIA. These results suggest that B. fragilis or propionate could be an alternative or complementary approach to the current therapies.

C-reactive protein lowers the serum level of IL-17, but not TNF-α, and decreases the incidence of collagen-induced arthritis in mice.

The biosynthesis of C-reactive protein (CRP) in the liver is increased in inflammatory diseases including rheumatoid arthritis. Previously published data suggest a protective function of CRP in arthritis; however, the mechanism of action of CRP remains undefined. The aim of this study was to evaluate the effects of human CRP on the development of collagen-induced arthritis (CIA) in mice which is an animal model of autoimmune inflammatory arthritis. Two CRP species were employed: wild-type CRP which binds to aggregated IgG at acidic pH and a CRP mutant which binds to aggregated IgG at physiological pH. Ten CRP injections were given on alternate days during the development of CIA. Both wild-type and mutant CRP reduced the incidence of CIA, that is, reduced the number of mice developing CIA; however, CRP did not affect the severity of the disease in arthritic mice. The serum levels of IL-17, IL-6, TNF-α, IL-10, IL-2 and IL-1ß were measured: both wild-type and mutant CRP decreased the level of IL-17 and IL-6 but not of TNF-α, IL-10, IL-2 and IL-1ß. These data suggest that CRP recognizes and binds to immune complexes, although it was not clear whether CRP functioned in its native pentameric or in its structurally altered pentameric form in the CIA model. Consequently, ligand-complexed CRP, through an as-yet undefined mechanism, directly or indirectly, inhibits the production of IL-17 and eventually protects against the initiation of the development of arthritis. The data also suggest that IL-17, not TNF-α, is critical for the development of autoimmune inflammatory arthritis.

Spin Trapping of Nitric Oxide by Hemoglobin and Ferrous Diethyldithiocarbamate in Model Tumors Differing in Vascularization.

Animal tumors serve as reasonable models for human cancers. Both human and animal tumors often reveal triplet EPR signals of nitrosylhemoglobin (HbNO) as an effect of nitric oxide formation in tumor tissue, where NO is complexed by Hb. In search of factors determining the appearance of nitrosylhemoglobin (HbNO) in solid tumors, we compared the intensities of electron paramagnetic resonance (EPR) signals of various iron-nitrosyl complexes detectable in tumor tissues, in the presence and absence of excess exogenous iron(II) and diethyldithiocarbamate (DETC). Three types of murine tumors, namely, L5178Y lymphoma, amelanotic Cloudman S91 melanoma, and Ehrlich carcinoma (EC) growing in DBA/2 or Swiss mice, were used. The results were analyzed in the context of vascularization determined histochemically using antibodies to CD31. Strong HbNO EPR signals were found in melanoma, i.e., in the tumor with a vast amount of a hemorrhagic necrosis core. Strong Fe(DETC)2NO signals could be induced in poorly vascularized EC. In L5178Y, there was a correlation between both types of signals, and in addition, Fe(RS)2(NO)2 signals of non-heme iron-nitrosyl complexes could be detected. We postulate that HbNO EPR signals appear during active destruction of well-vascularized tumor tissue due to hemorrhagic necrosis. The presence of iron-nitrosyl complexes in tumor tissue is biologically meaningful and defines the evolution of complicated tumor-host interactions.

IGF-1 Genome-Edited Human MSCs Exhibit Robust Anti-Arthritogenicity in Collagen-Induced Arthritis.

Stem cell therapy stands out as a promising avenue for addressing arthritis treatment. However, its therapeutic efficacy requires further enhancement. In this study, we investigated the anti-arthritogenic potential of human amniotic mesenchymal stem cells (AMM) overexpressing insulin-like growth factor 1 (IGF-1) in a collagen-induced mouse model. The IGF-1 gene was introduced into the genome of AMM through transcription activator-like effector nucleases (TALENs). We assessed the in vitro immunomodulatory properties and in vivo anti-arthritogenic effects of IGF-1-overexpressing AMM (AMM/I). Co-culture of AMM/I with interleukin (IL)-1ß-treated synovial fibroblasts significantly suppressed NF-kB levels. Transplantation of AMM/I into mice with collagen-induced arthritis (CIA) led to significant attenuation of CIA progression. Furthermore, AMM/I administration resulted in the expansion of regulatory T-cell populations and suppression of T-helper-17 cell activation in CIA mice. In addition, AMM/I transplantation led to an increase in proteoglycan expression within cartilage and reduced infiltration by inflammatory cells and also levels of pro-inflammatory factors including cyclooxygenase-2 (COX-2), IL-1ß, NF-kB, and tumor necrosis factor (TNF)-α. In conclusion, our findings suggest that IGF-1 gene-edited human AMM represent a novel alternative therapeutic strategy for the treatment of arthritis.

Macrophage-derived mir-100-5p orchestrates synovial proliferation and inflammation in rheumatoid arthritis through mTOR signaling.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by synovial inflammation, causing substantial disability and reducing life quality. While macrophages are widely appreciated as a master regulator in the inflammatory response of RA, the precise mechanisms underlying the regulation of proliferation and inflammation in RA-derived fibroblast-like synoviocytes (RA-FLS) remain elusive. Here, we provide extensive evidence to demonstrate that macrophage contributes to RA microenvironment remodeling by extracellular vesicles (sEVs) and downstream miR-100-5p/ mammalian target of rapamycin (mTOR) axis. RESULTS: We showed that bone marrow derived macrophage (BMDM) derived-sEVs (BMDM-sEVs) from collagen-induced arthritis (CIA) mice (cBMDM-sEVs) exhibited a notable increase in abundance compared with BMDM-sEVs from normal mice (nBMDM-sEVs). cBMDM-sEVs induced significant RA-FLS proliferation and potent inflammatory responses. Mechanistically, decreased levels of miR-100-5p were detected in cBMDM-sEVs compared with nBMDM-sEVs. miR-100-5p overexpression ameliorated RA-FLS proliferation and inflammation by targeting the mTOR pathway. Partial attenuation of the inflammatory effects induced by cBMDM-sEVs on RA-FLS was achieved through the introduction of an overexpression of miR-100-5p. CONCLUSIONS: Our work reveals the critical role of macrophages in exacerbating RA by facilitating the transfer of miR-100-5p-deficient sEVs to RA-FLS, and sheds light on novel disease mechanisms and provides potential therapeutic targets for RA interventions.

High phosphate and calcium induce osteoblastic phenotype switching and calcification of corneal epithelial cells in a Runx2-dependent and synergistic manner; a possible mechanism of chronic kidney disease-associated corneal calcification.

Patients with advanced chronic kidney disease (CKD) have elevated circulating calcium × phosphate product levels and exhibit soft tissue calcification. Besides the cardiovascular system, calcification is commonly observed in the cornea in CKD patients on hemodialysis. Cardiovascular calcification is a cell-mediated, highly regulated process, and we hypothesized that a similar regulatory mechanism is implicated in corneal calcification with the involvement of corneal epithelial cells (CECs). We established a mouse model of CKD-associated corneal calcification by inducing CKD in DBA/2J mice with an adenine and high phosphate diet. CKD was associated with aorta and corneal calcification as detected by OsteoSense staining and corneal Ca measurement (1.67-fold elevation, p < 0.001). In vitro, excess phosphate and Ca induced human CEC calcification in a dose-dependent and synergistic manner, without any influence on cell viability. High phosphate and Ca-containing osteogenic medium (OM; 2.5 mmol/L excess phosphate and 0.6 mmol/L excess Ca over control) increased the protein expression of Runx2 and induced its nuclear translocation. OM increased the expression of the bone-specific Ca-binding protein osteocalcin (130-fold increase, p < 0.001). Silencing of Runx2 attenuated OM-induced CEC calcification. Immunohistology revealed upregulation of Runx2 and overlapping between the Runx2 and the Alizarin red positive areas of calcification in the cornea of CKD mice. This work sheds light on the mechanism of CKD-induced corneal calcification and provides tools to test calcification inhibitors for the prevention of this detrimental process.

Repurposed drug agomelatine is therapeutic against collagen-induced arthritis via iNOS targeting.

BACKGROUND: The most promising biologics tumor necrosis factor α (TNFα) inhibitors are effective in treating rheumatoid arthritis (RA) in only 50-70 % of the cases; thus, new drugs targeting TNFα-mediated inflammation are required. METHODS: Firstly, the drugs that could inhibit FLS proliferation and TNFα induced inflammatory cytokine production were screened. Secondly, treatment effects of the identified drugs were screened in collagen-induced arthritis (CIA) mouse model. Thirdly, the inhibitory effect of the identified drug, agomelatine (AOM), on TNFα induced inflammatory cytokine production and NF-κB activity were confirmed. Fourthly, bioinformatics was applied to predict the binding target of AOM and the binding was confirmed, and the already known inhibitor of target was used to test the treatment effect for CIA mouse model. Finally, the effect of AOM on signaling pathway was tested and on TNFα induced inflammatory cytokine production was observed after inhibiting the target. RESULTS: AOM effectively inhibited TNFα-induced NF-κB activation, NF-κB p65 translocation, and inflammatory cytokines production in vitro and was therapeutic against CIA. The mechanistic study indicated inducible nitric oxide synthase (iNOS) as the binding target of AOM. 1400 W, a known inhibitor of iNOS, could effectively treat CIA by decreasing iNOS activity and the levels of inflammatory cytokines. The inhibitory effect of AOM on TNFα-induced inflammation was further elucidated by 1400 W, or NF-κB p65 inhibitor JSH-23, indicating that AOM is therapeutic against CIA via iNOS/ERK/p65 signaling pathway after binding with iNOS. CONCLUSIONS: AOM is therapeutic against CIA via inhibition of the iNOS/ERK/p65 signaling pathway after binding with iNOS.

Extracorporeal photopheresis reduces inflammation and joint damage in a rheumatoid arthritis murine model.

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.

Systems genetics of influenza A virus-infected mice identifies TRIM21 as a critical regulator of pulmonary innate immune response.

Tripartite motif 21 (TRIM21) is a cytosolic Fc receptor that targets antibody-bound, internalized pathogens for destruction. Apart from this intrinsic defense role, TRIM21 is implicated in autoimmune diseases, inflammation, and autophagy. Whether TRIM21 participates in host interactions with influenza A virus (IAV), however, is unknown. By computational modeling of body weight and lung transcriptome data from the BXD parents (C57BL/6 J (B6) and DBA/2 J (D2)) and 41 BXD mouse strains challenged by IAV, we reveal that a Trim21-associated gene network modulates the early host responses to IAV infection. Trim21 transcripts were significantly upregulated in infected mice of both B6 and D2 backgrounds. Its expression was significantly higher in infected D2 than in infected B6 early after infection and significantly correlated with body weight loss. We identified significant trans-eQTL on chromosome 14 that regulates Trim21 expression. Nr1d2 and Il3ra were among the strongest candidate genes. Pathway analysis found Trim21 to be involved in inflammation and immunity related pathways, such as inflammation signaling pathways (TNF, IL-17, and NF-κB), viral detection signaling pathways (NOD-like and RIG-I-like), influenza, and other respiratory viral infections. Knockdown of TRIM21 in human lung epithelial A549 cells significantly augmented IAV-induced expression of IFNB1, IFNL1, CCL5, CXCL10, and IFN-stimulated genes including DDX58 and IFIH1, among others. Our data suggest that a TRIM21-associated gene network is involved in several aspects of inflammation and viral detection mechanisms during IAV infection. We identify and validate TRIM21 as a critical regulator of innate immune responses to IAV in human lung epithelial cells.

Zishen Yutai Pills Promote Angiogenesis at the Maternal-Fetal Interface in Recurrent Spontaneous Abortion Mice by Regulating miR-187/VEGF Axis.

Ethnopharmacological Relevance: Zishen Yutai pills (ZYP), a traditional Chinese patent medicine, was listed in China in 1981. It is composed of 15 traditional Chinese medicines and has the effects of regulating menstruation, helping pregnancy, and preventing abortion. In clinical practice, it is effective in preventing habitual and threatened miscarriages, and continuing to explore its mechanism of action is very meaningful research. Aim of the Study: To explore the possible mechanism of ZYP promoting angiogenesis at the maternal-fetal interface in recurrent spontaneous abortion (RSA). Materials and Methods: In vitro experiments, placental trophoblast cells (PTCs) were isolated from the placental tissue of RSA mice and divided into six groups: Control group, Model group, ZYP group, miR-187 inhibitor NC group, miR-18 7 inhibitor group, and miR-187 inhibitor+ZYP group. Cell viability and cell cycle were measured using CCK8 and flow cytometry, respectively. The expression levels of miR-187, VEGF, VEGF-R1, and VEGF-R2 were measured using RT-qPCR, WB, and IF staining. Animal experiments first establish an RSA mice model (CBA/J × DBA/2) and then randomly divide the mice into four groups (n=10): normal pregnancy group, RSA model group, ZYP group, and progesterone capsule group. Observed the changes in embryo absorption rate, pathological morphology of decidual tissue, and ultrastructure of vascular endothelial cells in each group of mice. RT-qPCR, WB, and IF staining methods were used to determine the expression of miR-187, VEGF, VEGF-R1, and VEGF-R2. Results: In vitro, ZYP promoted the viability of PTCs and regulated their cell cycle, and ZYP down-regulated miR-187, up-regulated VEGF, VEGF-R1 and VEGF-R2 levels. miR-187 inhibitor showed the same effects, and further ZYP intervention enhanced the effects. In vivo, ZYP remarkably reduced embryo resorption rates, and improved the pathological morphology of decidual tissues and ultrastructure of vascular endothelial cells. Moreover, ZYP down-regulated miR-187, up-regulated VEGF, VEGF-R1 and VEGF-R2. Conclusion: In summary, ZYP can regulate the expression of VEGF via miR-187, then promote the angiogenesis at the maternal-fetal interface, and playing a therapeutic role in RSA.

Kisspeptin prevents pregnancy loss by modulating the immune microenvironment at the maternal-fetal interface in recurrent spontaneous abortion.

PROBLEM: Immune factors are crucial in the development of recurrent spontaneous abortion (RSA). This study aimed to investigate whether kisspeptin regulates immune cells at the maternal-fetal interface and whether G protein-coupled receptor 54 (GPR54) is involved in this process, through which it contributes to the pathogenesis of RSA. METHOD OF STUDY: Normal pregnancy (NP) (CBA/J × BALB/c) and RSA (CBA/J × DBA/2) mouse models were established. NP mice received tail vein injections of PBS and KP234 (blocker of kisspeptin receptor), whereas RSA mice received PBS and KP10 (active fragment of kisspeptin). The changes in immune cells in mouse spleen and uterus were assessed using flow cytometry and immunofluorescence. The expression of critical cytokines was examined by flow cytometry, ELISA, Western blotting, and qPCR. Immunofluorescence was employed to detect the coexpression of FOXP3 and GPR54. RESULTS: The findings revealed that the proportion of Treg cells, MDSCs, and M2 macrophages in RSA mice was lower than that in NP mice, but it increased following the tail vein injection of KP10. Conversely, the proportion of these cells was reduced in NP mice after the injection of KP234. However, the trend of γδT cell proportion change is contrary to these cells. Furthermore, FOXP3 and GPR54 were coexpressed in mouse spleen and uterus Treg cells as well as in the human decidua samples. CONCLUSION: Our results suggest that kisspeptin potentially participates in the pathogenesis of RSA by influencing immune cell subsets at the maternal-fetal interface, including Treg cells, MDSC cells, γδT cells, and M2 macrophages.

Human Adipose Tissue-Derived Stem Cells Inhibit Coronary Artery Vasculitis in a Mouse Model of Kawasaki Disease.

BACKGROUND: Adipose tissue-derived mesenchymal stem cells (ADSCs) are used for the treatment of various diseases because of their rapid proliferation and high anti-inflammatory and tissue repair properties. Kawasaki disease is a systemic vasculitis with coronary arteritis and aneurysms occurring in pediatric patients. In this study, we examined serologically and pathologically whether the administration of human ADSCs (hADSCs) to a mouse model of Kawasaki disease could suppress vasculitis. METHODS: Candida albicans water-soluble fractions were intraperitoneally injected into DBA/2 mice for 5 consecutive days to generate a mouse model of Kawasaki disease. The model mice were intravenously administered hADSCs or phosphate-buffered saline (PBS). Serum samples collected on days 15 and 29 were used to compare cytokine levels. Mouse hearts dissected on day 29 were subjected to hematoxylin and eosin and immunohistological staining using Galectin-1 (Gal-1), a protein involved in cardiovascular homeostasis, and CD44, a cell-surface marker of hADSCs. RESULTS: Comparison of inflammation-related cytokines showed a significant decrease in IL-1α expression at day 15 (P<0.05) and IL-6 expression at day 29 (P<0.01) in the hADSCs-treated group compared to the PBS group. Evaluation by hematoxylin and eosin staining showed decreased inflammatory cell infiltration and a tendency towards increased Gal-1 expression in the hADSCs group. CD44 expression was not observed in both the groups. The survival curve showed that the hADSCs group had a significantly longer survival time (P<0.05). CONCLUSIONS: The present experimental results indicate that hADSCs have an early anti-inflammatory effect, and that Gal-1 may be involved in preventing inflammation and reducing tissue damage.

The effect and mechanism of low-molecular-weight heparin on the decidualization of stromal cells in early pregnancy.

OBJECTIVE: This study aimed to analyze the effect of low-molecular-weight heparin (LMWH) on the decidualization of stromal cells in early pregnancy and explore the effect of LMWH on pregnancy outcomes. METHODS: Recurrent spontaneous abortion (RSA) mouse model (CBA/J × DBA/2) and normal pregnant mouse model (CBA/J × BALB/c) were established. The female mice were checked for a mucus plug twice daily to identify a potential pregnancy. When a mucus plug was found, conception was considered to have occurred 12 h previously. The pregnant mice were divided randomly into a normal pregnancy control group, an RSA model group, and an RSA + LMWH experimental group (n = 10 mice in each group). Halfway through the 12th day of pregnancy, the embryonic loss of the mice was observed; a real-time quantitative polymerase chain reaction was used to detect the messenger ribonucleic acid (mRNA) expressions of prolactin (PRL) and insulin-like growth factor-binding protein 1 (IGFBP1) in the decidua of the mice. Additionally, the decidual tissues of patients with RSA and those of normal women in early pregnancy who required artificial abortion were collected and divided into an RSA group and a control group. Decidual stromal cells were isolated and cultured to compare cell proliferation between the two groups, and cellular migration and invasion were detected by membrane stromal cells. Western blotting was used to detect the protein expressions of proliferating cell nuclear antigen (PCNA), cyclin D1, matrix metalloproteinase- (MMP) 2, and MMP-7 in stromal cells treated with LMWH. RESULTS: Compared with the RSA group, LMWH significantly reduced the pregnancy loss rate in the RSA mice (p < 0.05). Compared with the RSA group, the LMWH + RSA group had significantly higher expression levels of PRL and IGFBP1 mRNA (p < 0.01). LMWH promoted the proliferation, migration, and invasion of human decidual stromal cells; compared with the control group, the expression levels of MMP-2, MMP-7, cyclin D1, and PCNA proteins in the decidual stromal cells of the LMWH group increased (p < 0.05). CONCLUSIONS: The use of LMWH can improve pregnancy outcomes by enhancing the proliferation and migration of stromal cells in early pregnancy and the decidualization of stromal cells.

Jianpi Antai formula prevents miscarriage by repressing M1 polarization of decidual macrophages through ubiquitination of NLRP3 mediated by MARCH7.

ETHNOPHARMACOLOGICAL RELEVANCE: Jianpi Antai Formula (JAF) is an ancient formula from He's gynecology, which has been used clinically for more than 30 years and has significant therapeutic effects on spontaneous abortion (SA). Both macrophage polarization and NLRP3 inflammasome correlate with the occurrence of SA in women with recurrent or threatened miscarriage. Whether JAF prevent SA via mediating activation of decidual macrophage (dMφ) and ubiquitination-associated degradation of NLRP3 remains uncertain. AIM OF THE STUDY: This study aimed to clarify the effects of JAF on pregnancy outcomes and dMφ polarization at the maternal-fetal interface in an SA mouse model, and use in vivo and invitro methods to explore whether JAF can inhibit M1 polarization of dMφ by up-regulating MARCH7-mediated NLRP3 ubiquitination, thereby preventing SA. MATERIALS AND METHODS: The CBA/J × DBA/2 mating method was used to establish an SA model and the dMφs of SA mice were isolated and cultured. Th1-, Th2-, Th17- and Treg-related cytokine levels were evaluated using ELISA. qRT-PCR was used to detect the levels of M1/M2 macrophage-related cytokine mRNA in the decidua, and western blotting was used to detect the expression of NLRP3 inflammasome-related proteins in the decidua and placenta. The expression of M1/M2 markers of dMφ was detected using flow cytometry, ASC speck formation was observed using immunofluorescence, and the ubiquitination level of MARCH7-NLRP3 was detected using co-immunoprecipitation. RESULTS: JAF increased the survival rate of fetuses and the levels of estradiol and progesterone in SA model mice. It also reduced the serum Th1 and Th17-associated cytokine levels and decidual M1 macrophage-associated cytokine levels, while elevating the M2 macrophages in SA mice. NLRP3, caspase-1, ASC, and IL-1ß protein expression in the decidua and placenta were also reduced. si-MARCH7 transfection reversed the effect of JAF on inhibiting the formation of the NLRP3 inflammasome and the activation of macrophages in dMφs of SA mice. CONCLUSION: JAF could effectively prevent and treat SA by repressing M1 polarization of dMφs through NLRP3 ubiquitination and pyroptosis inhibition, which were mediated by MARCH7.

Exosome-mediated delivery of super-repressor IκBα alleviates inflammation and joint damages in rheumatoid arthritis.

BACKGROUND: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA. METHODS: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging. RESULTS: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group. CONCLUSION: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

Intramedullary implant stability affects patterns of fracture healing in mice with morphologically different bone phenotypes.

Almost all prior mouse fracture healing models have used needles or K-wires for fixation, unwittingly providing inadequate mechanical stability during the healing process. Our contention is that the reported outcomes have predominantly reflected this instability, rather than the impact of diverse biological conditions, pharmacologic interventions, exogenous growth factors, or genetic considerations. This important issue becomes obvious upon a critical review of the literature. Therefore, the primary aim of this study was to demonstrate the significance of mouse-specific implants designed to provide both axial and torsional stability (Screw and IM Nail) compared to conventional pins (Needle and K-wires), even when used in mice with differently sized marrow canals and diverse genetic backgrounds. B6 (large medullary canal), DBA, and C3H (smaller medullary canals) mice were employed, all of which have different bone morphologies. Closed femoral fractures were created and stabilized with intramedullary implants that provide different mechanical conditions during the healing process. The most important finding of this study was that appropriately designed mouse-specific implants, providing both axial and torsional stability, had the greatest influence on bone healing outcomes regardless of the different bone morphologies encountered. For instance, unstable implants in the B6 strain (largest medullary canal) resulted in significantly greater callus, with a fracture region mainly comprising trabecular bone along with the presence of cartilage 28 days after surgery. The DBA and C3H strains (with smaller medullary canals) instead formed significantly less callus, and only had a small amount of intracortical trabeculation remaining. Moreover, with more stable fracture fixation a higher BV/TV was observed and cortices were largely restored to their original dimensions and structure, indicating an accelerated healing and remodeling process. These observations reveal that the diaphyseal cortical thickness, influenced by the genetic background of each strain, played a pivotal role in determining the amount of bone formation in response to the fracture. These findings are highly important, indicating the rate and type of tissue formed is a direct result of mechanical instability, and this most likely would mask the true contribution of the tested genes, genetic backgrounds, or various therapeutic agents administered during the bone healing process.

Prevention of Collagen-Induced Arthritis by an Anti-Glycan Monoclonal Antibody Reactive with 6-Sulfo Sialyl Lewis x in DBA/1 Mice.

Rheumatoid arthritis (RA) is an autoimmune disease characterized by synovial tissue inflammation, substantially impacting the quality of life of patients. The interaction between L-selectin and its glycoprotein ligands modified with 6-sulfo sialyl Lewis x (6-sulfo sLex) is known to mediate lymphocyte homing to initiate immune responses. Thus, this process could be a potential therapeutic target for RA. Herein, we explored the preventive effects of an anti-6-sulfo sLex monoclonal antibody (mAb), SF1, on collagen-induced arthritis (CIA) in DBA/1 mice. Mice were administered SF1 from day 21 postfirst immunization with type II collagen (CII), and the effects of SF1 on both clinical and histopathological disease progression evoked by the second immunization were examined. SF1 significantly suppressed clinical features and histological levels associated with arthritis severity. Enzyme-linked immunosorbent assay consistently indicated that SF1 inhibited the production of CII-specific IgG2a. Based on the reverse transcription-quantitative PCR analysis, SF1 suppressed the expression of interferon-γ, a T helper 1 cytokine, as well as that of inflammatory cytokines, including tumor necrosis factor-α and interleukin-1ß, in draining lymph nodes. Collectively, these results indicate that SF1, an anti-sulfated glycan mAb, could be beneficial in preventing CIA in mice and may afford as a novel agent to treat RA.

Exploring the efficacy and mechanism of Glycyrrhizae Radix et Rhizoma in improving collagen-induced arthritis in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Rheumatoid arthritis (RA), a chronic auto-immune disease, will cause serious joint damage and disability. Glycyrrhizae Radix et Rhizoma (GRR) is commonly included in many anti-RA formulas used in the clinical practice in China. AIM OF THE STUDY: To elucidate the alleviation of GRR and its active compounds on RA and the possible engaged mechanism. MATERIALS AND METHODS: The clinical score, paw swelling degree and pain threshold were detected in the collagen-induced arthritis (CIA) in DBA/1 mice. The ankle joints of mice were observed by using X-Ray, hematoxylin-eosin (H&E), masson's trichrome (Masson), and safranin O and fast green (Safranin O) staining. The potential targets of GRR were predicted by network pharmacology and further verified by using enzyme-linked immunosorbent assay (ELISA) and western-blot. Real-time polymerase chain reaction (Real-time PCR) and wound healing assay were conducted in synovial MH7A cells. The interaction between active compounds and potential targets predicted by molecular docking was confirmed by using cellular thermal shift assay (CETSA). RESULTS: GRR (615 mg/kg) obviously alleviated CIA in mice. Network pharmacology implied that GRR might affect angiogenesis and inflammation, among which vascular endothelial growth factor-A (VEGF-A), tumor necrosis factor-α (TNFα), interleukin-1ß (IL-1ß), IL-6 and phosphorylated protein kinase B (AKT) might be the key targets involved in this process. GRR decreased AKT phosphorylation and reduced the elevated levels of TNFα, VEGF-A, IL-1ß and IL-6. Next, in vitro results demonstrated that glycyrrhetinic acid (GA) and isoliquiritigenin (ISL) were two active compounds that inhibited TNFα-induced synovial cell angiogenesis and inflammation. Moreover, GA and ISL actually improved RA in CIA mice. The results of molecular docking and CETSA displayed that ISL and GA might interact with TNF receptor-1 (TNFR1), toll-like receptor-4 (TLR4) and VEGF receptor-2 (VEGFR2), thereby contributing to their inhibition on angiogenesis and inflammation. CONCLUSION: GRR and two active compounds, including ISL and GA, alleviated RA via inhibiting angiogenesis and inflammation.

The slow-release adiponectin analog ALY688-SR modifies early-stage disease development in the D2.mdx mouse model of Duchenne muscular dystrophy.

Fibrosis is associated with respiratory and limb muscle atrophy in Duchenne muscular dystrophy (DMD). Current standard of care partially delays the progression of this myopathy but there remains an unmet need to develop additional therapies. Adiponectin receptor agonism has emerged as a possible therapeutic target to lower inflammation and improve metabolism in mdx mouse models of DMD but the degree to which fibrosis and atrophy are prevented remain unknown. Here, we demonstrate that the recently developed slow-release peptidomimetic adiponectin analog, ALY688-SR, remodels the diaphragm of murine model of DMD on DBA background (D2.mdx) mice treated from days 7-28 of age during early stages of disease. ALY688-SR also lowered interleukin-6 (IL-6) mRNA but increased IL-6 and transforming growth factor-ß1 (TGF-ß1) protein contents in diaphragm, suggesting dynamic inflammatory remodeling. ALY688-SR alleviated mitochondrial redox stress by decreasing complex I-stimulated H2O2 emission. Treatment also attenuated fibrosis, fiber type-specific atrophy, and in vitro diaphragm force production in diaphragm suggesting a complex relationship between adiponectin receptor activity, muscle remodeling, and force-generating properties during the very early stages of disease progression in murine model of DMD on DBA background (D2.mdx) mice. In tibialis anterior, the modest fibrosis at this young age was not altered by treatment, and atrophy was not apparent at this young age. These results demonstrate that short-term treatment of ALY688-SR in young D2.mdx mice partially prevents fibrosis and fiber type-specific atrophy and lowers force production in the more disease-apparent diaphragm in relation to lower mitochondrial redox stress and heterogeneous responses in certain inflammatory markers. These diverse muscle responses to adiponectin receptor agonism in early stages of DMD serve as a foundation for further mechanistic investigations.NEW & NOTEWORTHY There are limited therapies for the treatment of Duchenne muscular dystrophy. As fibrosis involves an accumulation of collagen that replaces muscle fibers, antifibrotics may help preserve muscle function. We report that the novel adiponectin receptor agonist ALY688-SR prevents fibrosis in the diaphragm of D2.mdx mice with short-term treatment early in disease progression. These responses were related to altered inflammation and mitochondrial functions and serve as a foundation for the development of this class of therapy.