Human embryonic stem cell-derived mesenchymal stromal cells suppress inflammation in mouse models of rheumatoid arthritis and lung fibrosis by regulating T-cell function.

BACKGROUND AIMS: Rheumatoid arthritis (RA) is characterized by an overactive immune system, with limited treatment options beyond immunosuppressive drugs or biological response modifiers. Human embryonic stem cell-derived mesenchymal stromal cells (hESC-MSCs) represent a novel alternative, possessing diverse immunomodulatory effects. In this study, we aimed to elucidate the therapeutic effects and underlying mechanisms of hESC-MSCs in treating RA. METHODS: MSC-like cells were differentiated from hESC (hESC-MSCs) and cultured in vitro. Cell proliferation was assessed using Cell Counting Kit-8 assay and Ki-67 staining. Flow cytometry was used to analyze cell surface markers, T-cell proliferation and immune cell infiltration. The collagen-induced arthritis (CIA) mouse model and bleomycin-induced model of lung fibrosis (BLE) were established and treated with hESC-MSCs intravenously for in vivo assessment. Pathological analyses, reverse transcription-quantitative polymerase chain reaction and Western blotting were conducted to evaluate the efficacy of hESC-MSCs treatment. RESULTS: Intravenous transplantation of hESC-MSCs effectively reduced inflammation in CIA mice in this study. Furthermore, hESC-MSC administration enhanced regulatory T cell infiltration and activation. Additional findings suggest that hESC-MSCs may reduce lung fibrosis in BLE mouse models, indicating their potential to mitigate complications associated with RA progression. In vitro experiments revealed a significant inhibition of T-cell activation and proliferation during co-culture with hESC-MSCs. In addition, hESC-MSCs demonstrated enhanced proliferative capacity compared with traditional primary MSCs. CONCLUSIONS: Transplantation of hESC-MSCs represents a promising therapeutic strategy for RA, potentially regulating T-cell proliferation and differentiation.

Proteins extracted from pearl oyster (Pinctada martensii) with efficient accelerated wound healing in vitro through promoting cell proliferation, migration, and collagen formation.

Ethnopharmacological relevance: Pearl oyster (Pinctada martensii) is used in Chinese traditional medicine use in photoprotective, anti-inflammatory, and wound treatment.Aim of the study: This study explored whether the mucus protein of Pearl oyster (protein of Pinctada martensii, PMP) affects human skin fibroblast (HSF) proliferation, migration, collagen-related gene expression related to collagen formation, and in vivo healing effects. Materials and methods: The PMP component was analyzed by LC-MS/MS. The cell viability was evaluated using a CCK-8 kit. The expression genes were measured by reverse transcription polymerase chain reaction. A full-thickness excisional wounding model in Sprague-Dawley (SD) rats was used to test the repairing effect of PMP in vivo, and Hematoxylin-Eosin (H&E) and Masson's Trichrome staining were applied to evaluate skin structure. Results: The components of PMP were identified using LC-MS/MS proteomics, and a total of 3023 proteins were detected. The results of PMP-treated HSF showed that PMP effectively promoted cell proliferation by 1.6-fold and cell migration by 1.5-fold at a concentration of 1 mg/mL. Additionally, PMP treatment up-regulated the expression levels of collagen-related genes COL1A1, COL3A1, and MMP-1 in fibroblasts. Furthermore, PMP was applied in the therapy of full-thickness excisional wounds in rats. The results demonstrated that PMP significantly accelerated wound healing time, resulted in the recovery of dermal and epithelial thickness, and stimulated collagen regeneration. The regenerated skin closely resembled the structure of normal skin. Conclusions: These findings provide solid evidence supporting the potential of PMP as a promising candidate for the treatment of skin wounds.

Silencing of circular RNA circPDE5A suppresses neuroblastoma progression by targeting the miR-362-5p/NOL4L axis.

OBJECTIVE: Neuroblastoma (NB) is the most common extra-cranial solid tumour in early childhood. Circular RNAs (circRNAs) have been implicated in the development of NB. The purpose of the current study was to explore the molecular action of circRNA phosphodiesterase 5 A (circPDE5A) in NB malignant progression. MATERIALS AND METHODS: The expression levels of circPDE5A, miR-362-5p and nucleolar protein 4 like (NOL4L) were assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Cell proliferation was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTS) assay. Cell migration and invasion were evaluated by transwell assay. The levels of glucose consumption and lactate production were measured using the commercial assay kits. Targeted correlations among circPDE5A, miR-362-5p and NOL4L were confirmed by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. In vivo assays were performed to examine the role of circPDE5A in tumour growth in vivo. RESULTS: Our results revealed that circPDE5A was up-regulated in NB tissues and cells. The silencing of circPDE5A suppressed NB cell proliferation, migration, invasion, and glycolysis in vitro and diminished tumour growth in vivo. Moreover, circPDE5A directly targeted miR-362-5p by binding to miR-362-5p. CircPDE5A silencing impeded NB malignant progression in vitro through up-regulating miR-362-5p. Furthermore, NOL4L was a direct target of miR-362-5p, and NOL4L mediated the regulation of miR-362-5p on NB malignant progression in vitro. Additionally, circPDE5A functioned as a regulator of NOL4L expression via targeting miR-362-5p. CONCLUSIONS: Our current findings identified that the knockdown of circPDE5A suppressed NB malignant progression at least in part by the regulation of the miR-362-5p/NOL4L axis, providing a novel rationale for developing circPDE5A as a potential target for NB management.

Methyltransferase-like 3 (METTL3) Epigenetically Modulates Glutathione Peroxidase 4 (GPX4) Expression to Affect Asthma.

Asthma, a prevalent chronic airway inflammatory condition, poses a significant health challenge. In this study, we delved into the regulatory mechanisms governing asthma, focusing on Methyltransferase-like 3 (METTL3). Through an ovalbumin (OVA)-induced mouse model and interleukin-13 (IL-13)-induced cell model, we mimicked the in vivo and in vitro functions of METTL3 in asthma. Our research revealed that METTL3 expression significantly decreased in asthma-induced mice and IL-13-stimulated cells compared to the control group. Moreover, METTL3 overexpression enhanced bronchial epithelial cell viability and proliferation. Mechanistically, we observed elevated levels of total iron, Fe2+, malondialdehyde (MDA), lipid reactive oxygen species (ROS), alongside reduced glutathione (GSH) levels in IL-13-stimulated cells. Remarkably, METTL3 overexpression counteracted these effects, suggesting a pivotal role in mitigating asthma-related oxidative stress. Furthermore, our study highlighted the involvement of N6-methyladenosine methylation (m6A) modification, where METTL3 regulated the m6A modification of glutathione peroxidase 4 (GPX4) RNA, impacting RNA stability. Knockdown of METTL3 suppressed m6A modification on GPX4 RNA, impairing its stability and contributing to IL-13-induced ferroptosis. Interestingly, METTL3 overexpression not only inhibited cell ferroptosis but also alleviated asthma symptoms. Our findings shed light on the epigenetic regulation of asthma through METTL3-mediated m6A modification, offering potential therapeutic avenues for this prevalent inflammatory disease.

Promoting repair of highly purified stromal vascular fraction gel combined with advanced platelet-rich fibrin extract for irradiated skin and soft tissue injury.

Background: To evaluate the effect of highly purified stromal vascular fraction gel (SVFG) combined with advanced platelet-rich fibrin extract (APRFE) in treatment of irradiated skin and soft tissue injury. Methods: The subcutaneous fat and whole blood of 4 rabbits were collected to isolate the SVFG and APRFE, respectively. Forty-eight rabbits were divided into 4 groups to prepare irradiated skin injury models with 25 Gy for 24 hours; corresponding dose were performed subcutaneously injected into wounds. In group A, the rabbits were treated with 0.3 mL APRFE combined with 1 mL SVFG. In group B, the rabbits were treated with 1 mL SVFG. In group C, the rabbits were treated with 0.3 mL APRFE, and group D was treated with 1 mL normal saline. The wound healing was detected on the 2, 5, 9 and 14 d after intervention. The wounds tissue was cut for hematoxylin and eosin (HE) staining to observe the structure and Masson staining to observe the collagen content. The expression of CD31 in each group was detected by immunohistochemistry (IHC), the protein and mRNA levels of K19, hypoxia inducible factor-1 alpha (HIF-1α), vascular endothelial growth factor (VEGF), interleukin 8 (IL-8) and interleukin 10 (IL-10) were detected respectively by Western blot (WB) and reverse transcription-polymerase chain reaction (RT-PCR) on 7, 14 and 28 d after intervention. Results: It is revealed that wound healing rates from 5 to 14 d in group A was significantly higher than that of control. The wounds healing rates in group B and C were significantly higher than that of control after 12 d. Masson staining results showed that the collagen content in group A was significantly higher than that of the other 3 groups on the 7, 14 and 28 d. The results of IHC showed that the expression of CD31 in group A was significantly higher than that of the other 3 groups on 7, 14 and 28 d. WB and RT-PCR results showed that relative expression levels of K19, HIF-1α, VEGF, IL-10 in group A were significantly higher than that of the other 3 groups on 7, 14 and 28 d. However, the relative expression levels of IL-8 in group A was significantly lower than that of the other 3 groups on 7, 14 and 28 d. Conclusions: SVFG combined with APRFE can promote the repair of irradiated skin and soft tissue injury by accelerating angiogenesis, promoting collagen synthesis and reducing inflammation.

Interferon Regulatory Factor 4 Regulates the Development of Polymorphonuclear Myeloid-Derived Suppressor Cells Through the Transcription of c-Myc in Cancer.

The accumulation of myeloid-derived suppressor cells (MDSCs) is one of the major obstacles to achieve an appropriate anti-tumor immune response and successful tumor immunotherapy. MDSCs in tumor-bearing hosts are primarily polymorphonuclear (PMN-MDSCs). However, the mechanisms regulating the development of MDSCs remain poorly understood. In this report, we showed that interferon regulatory factor 4 (IRF4) plays a key role in the development of PMN-MDSCs, but not monocytic MDSCs. IRF4 deficiency caused a significant elevation of PMN-MDSCs and enhanced the suppressive activity of PMN-MDSCs, increasing tumor growth and metastasis in mice. Mechanistic studies showed that c-Myc was up-regulated by the IRF4 protein. Over-expression of c-Myc almost abrogated the effects of IRF4 deletion on PMN-MDSCs development. Importantly, the IRF4 expression level was negatively correlated with the PMN-MDSCs frequency and tumor development but positively correlated with c-Myc expression in clinical cancer patients. In summary, this study demonstrated that IRF4 represents a novel regulator of PMN-MDSCs development in cancer, which may have predictive value for tumor progression.

[Effect of epigallocatechin-3-gallate on liver lipid metabolism in rats with intrauterine growth restriction and related mechanism].

OBJECTIVE: To study the effect of epigallocatechin-3-gallate (EGCG) on liver lipid metabolism in rats with intrauterine growth restriction (IUGR) and related mechanism. METHODS: A rat model of IUGR was established by food restriction during entire pregnancy, and then the rats were randomly divided into an IUGR group and an EGCG group (n=8 each). The rats in the EGCG group were fed with water containing EGCG from after weaning to 10 weeks. Eight pup rats born from the pregnant maternal rats without food restriction were used as the control group. At the age of 13 weeks, body weight was measured. Blood and liver tissue samples were collected to measure fasting total cholesterol (TC), triglyceride (TG), free fatty acid (FFA), fasting plasma glucose (FPG), fasting insulin (FINS), and liver lipids. Homeostasis model assessment of insulin resistance (HOMA-IR) and adipose insulin resistance (adipo-IR) were calculated. Pathological sections of the liver were observed and quantitative real-time PCR was used to measure the mRNA expression of related genes in the liver. RESULTS: At the age of 13 weeks, there was no significant difference in body weight between groups (P=0.067). There were significant differences between groups in FPG, FFA, FINS, HOMA-IR, and adipo-IR (P<0.05). There were no significant differences in the serum levels of TC and TG between groups (P>0.05), while the IUGR group had significantly higher levels of TC and TG in the liver than the EGCG group (P<0.05). Oil red staining showed that the IUGR group had a significant increase in hepatic lipid accumulation, while the EGCG group had certain improvement after EGCG treatment. PCR results suggested that compared with the control group, the IUGR group had significant reductions in the mRNA expression of Ampk and Adipor1 and a significant increase in the mRNA expression of Srebf1 (P<0.05), while EGCG increased the mRNA expression of Ampk and reduced the mRNA expression of Srebf1, with no significant differences in the two indices between the EGCG and control groups (P>0.05). CONCLUSIONS: Early EGCG intervention can down-regulate the de novo synthesis of fatty acids through the Ampk/Srebf1 signaling pathway and reduce hepatic lipid accumulation in IUGR rats by improving insulin resistance of hepatocytes.

Simultaneous induction of Graves' hyperthyroidism and Graves' ophthalmopathy by TSHR genetic immunization in BALB/c mice.

BACKGROUND: Graves' disease is the most common form of autoimmune thyroid disorder, characterized by hyperthyroidism due to circulating autoantibodies. To address the pathological features and establish a therapeutic approach of this disease, an animal model carrying the phenotype of Graves' disease (GD) in concert with Graves' Ophthalmopathy (GO) will be very important. However, there are no ideal animal models that are currently available. The aim of the present study is to establish an animal model of GD and GO disease, and its pathological features were further characterized. METHODS: A recombinant plasmid pcDNA3.1- T289 was constructed by inserting the TSHR A-subunit gene into the expression vector pcDNA3.1, and genetic immunization was successfully performed by intramuscular injection of the plasmid pcDNA3.1-T289 on female 8-week-old BALB/c mice. Each injection was immediately followed by in vivo electroporation using ECM830 square wave electroporator. Morphological changes of the eyes were examined using 7.0T MRI scanner. Levels of serum T4 and TSHR antibodies (TRAb) were assessed by ELISA. The pathological changes of the thyroid and orbital tissues were examined by histological staining such as H&E staining and Alcian blue staining. RESULTS: More than 90% of the immunized mice spontaneously developed goiter, and about 80% of the immunized mice manifested increased serum T4 and TRAb levels, combined with hypertrophy and hyperplasia of thyroid follicles. A significantly increased synthesis of hyaluronic acid was detected in in the immunized mice compared with the control groups. CONCLUSION: We have successfully established an animal model manifesting Graves' hyperthyroidism and ophthalmopathy, which provides a useful tool for future study of the pathological features and the development of novel therapies of the diseases.

Efficacy and safety of mycophenolate mofetil in patients with active moderate-to-severe Graves' orbitopathy.

CONTEXT: Graves' orbitopathy (GO) is a potentially sight-threatening disease for which currently available medical therapy is not reliably successful. Mycophenolate mofetil (MMF) is a selective immunosuppressant used widely in many autoimmune diseases. Preliminary studies have shown that MMF is effective in the treatment of active GO. OBJECTIVE: To evaluate the efficacy and safety of MMF in patients with active moderate-to-severe GO. PATIENTS: One hundred and 74 patients with active moderate-to-severe GO were randomized to receive either MMF or glucocorticoids (GC). MAIN OUTCOME MEASURES: The primary outcome was overall response at the 12th and 24th weeks; the outcome assessments included clinical activity score (CAS), soft tissue involvement, pain, visual acuity, proptosis, diplopia and reduction in eye movements. The secondary outcome was changes in those individual parameters. Adverse effects were recorded at each visit. RESULTS: A greater overall response rate was found in the MMF group compared with the GC group at the 24th week (91·3% vs 67·9%, P = 0·000). MMF therapy showed a better CAS response than GC (92·5% vs 70·5% improved, P < 0·05). Patients treated with MMF showed a significantly improved rate of diplopia and proptosis than patients treated with GC at the 24th week (90·4% and 68·8% improved, respectively). Disease reactivation was not observed in the patients treated with MMF but was observed in five patients after GC therapy. Adverse events occurred in 4 of 80 patients treated with MMF (5%), all of which were mild to moderate. A severe adverse event was only observed in one patient treated with GC but not at all in patients treated with MMF. CONCLUSION: Compared with GC treatment, MMF therapy is more effective and safer for patients with active moderate-to-severe GO.