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The purpose of this study was to analyze the mechanism by which irisin affects β‐cell pyroptosis in type 2 diabetes mellitus (T2DM). The in vivo T2DM model was established by raised with high‐fat diet and intraperitoneally injection of streptozocin. Min6 cells were divided into four groups: negative control (NC), high glucose (HG), HG + irisin, and HG + irisin+3‐MA. The cell viability was determined by CCK‐8 assay. Dual‐luciferase gene reporter assay was conducted to confirm the binding between miR‐19b‐3p and SOCS3. The expression level of FNDC5 and GSDMD was visualized using the immunofluorescence assay. The protein level of FNDC5, Beclin1, LC3II/I, NLRP3, cleaved‐caspase‐1, GSDMD‐N, STAT3, p‐STAT3, and SOCS3 was determined by Western blotting. The secretion of irisin, lactate dehydrogenase (LDH), and insulin was checked by ELISA. In vivo results showed that pathological changes in islet tissues with declined number of β cells, elevated FBG value, decreased FIN and HOMA‐β value, elevated autophagy‐associated proteins expressions, and activated NLRP3 signaling in T2DM mice, which were dramatically reversed by FNDC5 overexpression. Furthermore, the declined level of miR‐19b‐3p and p‐STAT3, as well as the upregulation of SOCS3, was greatly rescued by FNDC5 overexpression. The in vitro data confirmed the binding site between SOCS3 and miR‐19b‐3p. SOCS3 was downregulated and p‐STAT3 was upregulated in miR‐19b‐3p mimic‐treated Min6 cells. In HG‐stimulated Min6 cells, the elevated cell viability, increased production of insulin, decreased release of LDH, and inactivated NLRP3 signaling induced by irisin were abolished by miR‐19b‐3p inhibitor and STAT3 inhibitor. The increased level of autophagy‐related proteins and activated SOCS3/STAT3 axis induced by irisin in HG‐stimulated Min6 cells were abolished by miR‐19b‐3p inhibitor. The inhibitory effect of irisin against NLRP3 signaling in HG‐stimulated Min6 cells was abrogated by 3‐MA. In conclusion, irisin alleviated the pyroptosis of β cells in T2DM by inhibiting NLRP3 signaling through miR‐19b‐3p/SOCS3/STAT3 axis mediated autophagy. [ABSTRACT FROM AUTHOR]

Background: Pathological angiogenesis causes significant vision loss in neovascular age-related macular degeneration and other retinopathies with neovascularization (NV). Neuronal/glial-vascular interactions influence the release of angiogenic and neurotrophic factors. We hypothesized that botulinum neurotoxin serotype A (BoNT/A) modulates pathological endothelial cell proliferation through glial cell activation and growth factor release. Methods: A laser-induced choroidal NV (CNV) was employed to investigate the anti-angiogenic effects of BoNT/A. Fundus fluorescence angiography, immunohistochemistry, and real-time PCR were used to assess BoNT/A efficacy in inhibiting CNV and the molecular mechanisms underlying this inhibition. Neuronal and glial suppressor of cytokine signaling 3 (SOCS3) deficient mice were used to investigate the molecular mechanisms of BoNT/A in inhibiting CNV via SOCS3. Findings: In laser-induced CNV mice with intravitreal BoNT/A treatment, CNV lesions decreased > 30%; vascular leakage and retinal glial activation were suppressed; and Socs3 mRNA expression was induced while vascular endothelial growth factor A (Vegfa) mRNA expression was suppressed. The protective effects of BoNT/A on CNV development were diminished in mice lacking neuronal/glial SOCS3. Conclusion: BoNT/A suppressed laser-induced CNV and glial cell activation, in part through SOCS3 induction in neuronal/glial cells. BoNT/A treatment led to a decrease of pro-angiogenic factors, including VEGFA, highlighting the potential of BoNT/A as a therapeutic intervention for pathological angiogenesis in retinopathies. [ABSTRACT FROM AUTHOR]

Introduction: Long noncoding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) has been shown to be involved in Parkinson’s disease (PD) progression, but its mechanism needs to be further explored. Methods: Mice were injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to induce PD mice models, and BV2 cells were treated with lipopolysaccharides (LPS) to mimic PD cell models. MALAT1 expression and suppressor of cytokine signaling 3 (SOCS3) protein level were examined using quantitative real-time PCR and Western blot, respectively. Cell functions were tested by cell counting kit 8 assay and flow cytometry. The interaction between MALAT1 and SOCS3 was confirmed using RNA pull-down and RIP assays. Results: MALAT1 was upregulated in MPTP-induced PD mice and LPS-induced BV2 cells. Silencing of MALAT1 increased viability, while inhibiting apoptosis and inflammation in LPS-induced BV2 cells. Besides, MALAT1 enhanced the SOCS3 promoter methylation to decrease its expression by recruiting DNMT1, DNMT3A, and DNMT3B. Furthermore, SOCS3 knockdown eliminated sh-MALAT1-mediated the inhibition effect on LPS-induced BV2 cell injury. In vivo, MALAT1 silencing ameliorated neurological impairment and neuroinflammation in MPTP-induced PD mice. Conclusion: Our data revealed that MALAT1 worsened PD processes via inhibiting SOCS3 expression by increasing its promoter methylation. [ABSTRACT FROM AUTHOR]

Abstract Long non-coding RNAs (lncRNAs) have emerged as pivotal regulators in numerous biological processes, including macrophage-mediated inflammatory responses, which play a critical role in the progress of diverse diseases. This study focuses on the regulatory function of lncRNA brain and reproductive organ-expressed protein (BRE) antisense RNA 1 (BRE-AS1) in modulating the inflammatory activation of monocytes/macrophages. Employing the THP-1 cell line as a model, we demonstrate that lipopolysaccharide (LPS) treatment significantly upregulates BRE-AS1 expression. Notably, specific knockdown of BRE-AS1 via siRNA transfection enhances LPS-induced expression of interleukin (IL)-6 and IL-1β, while not affecting tumor necrosis factor (TNF)-α levels. This selective augmentation of pro-inflammatory cytokine production coincides with increased phosphorylation of Janus kinase (JAK)2 and signal transducer and activator of transcription (STAT)3. Furthermore, BRE-AS1 suppression results in the downregulation of suppressor of cytokine signaling (SOCS)3, an established inhibitor of the JAK2/STAT3 pathway. Bioinformatics analysis identified binding sites for miR-30b-5p on both BRE-AS1 and SOCS3 mRNA. Intervention with a miR-30b-5p inhibitor and a synthetic RNA fragment that represents the miR-30b-5p binding site on BRE-AS1 attenuates the pro-inflammatory effects of BRE-AS1 knockdown. Conversely, a miR-30b-5p mimic replicated the BRE-AS1 attenuation outcomes. Our findings elucidate the role of lncRNA BRE-AS1 in modulating inflammatory activation in THP-1 cells via the miR-30b-5p/SOCS3/JAK2/STAT3 signaling pathway, proposing that manipulation of macrophage BRE-AS1 activity may offer a novel therapeutic avenue in diseases characterized by macrophage-driven pathogenesis.

Spinal microglial polarization plays a crucial role in the pathological processes of neuropathic pain following peripheral nerve injury. Accumulating evidence suggests that milk fat globule epidermal growth factor-8 (MFG-E8) exhibits anti-inflammatory effect and regulates microglial polarization through the integrin β3 receptor. However, the impact of MFG-E8 on microglial polarization in the context of neuropathic pain has not yet been investigated. In this study, we evaluated the effect of MFG-E8 on pain hypersensitivity and spinal microglial polarization following spared nerve injury (SNI) of the sciatic nerve in mice. We determined the molecular mechanisms underlying the effects of MFG-E8 on pain hypersensitivity and spinal microglial polarization using pain behavior assessment, western blot (WB) analysis, immunofluorescence (IF) staining, quantitative polymerase chain reaction (qPCR), enzyme-linked immunosorbent assay (ELISA), and small interfering RNA (siRNA) transfection. Our findings indicate that SNI significantly increased the levels of MFG-E8 and integrin β3 expressed in microglia within the spinal cord of mice. Additionally, we observed that intrathecal injection of recombinant human MFG-E8 (rhMFG-E8) alleviated SNI induced-mechanical allodynia and thermal hyperalgesia. Furthermore, the results suggested that rhMFG-E8 facilitated M2 microglial polarization and ameliorated neuroinflammation via integrin β3/SOCS3/STAT3 pathway in the spinal cord of mice with SNI. Importantly, these effects were negated by integrin β3 siRNA, or SOCS3 siRNA. These results demonstrate that MFG-E8 ameliorates peripheral nerve injury induced-mechanical allodynia and thermal hyperalgesia by driving M2 microglial polarization and mitigating neuroinflammation mediated by integrin β3/SOCS3/STAT3 pathway in the spinal cord of mice. MFG-E8 may serve as a promising target for the treatment of neuropathic pain. [ABSTRACT FROM AUTHOR]

Accumulating evidence has demonstrated that M1 microglial polarization and neuroinflammation worsen the development of neuropathic pain. However, the mechanisms underlying microglial activation during neuropathic pain remain incompletely understood. Myeloid-epithelial-reproductive tyrosine kinase (Mer), which is a member of the Tyro-Axl-Mer (TAM) family of receptor tyrosine kinases, plays a crucial role in the regulation of microglial polarization. However, the effect of Mer on microglial polarization during neuropathic pain has not been determined. In this study, western blotting, immunofluorescence analysis, quantitative polymerase chain reaction (qPCR), and enzyme-linked immunosorbent assay (ELISA) were used to examine the role of Mer in pain hypersensitivity and microglial polarization in rats with chronic constriction injury (CCI) of the sciatic nerve. The results indicated that Mer expression in microglia was prominently increased in the spinal cords of rats subjected to CCI. Furthermore, treatment with recombinant protein S (PS, an activator of Mer) alleviated mechanical allodynia and thermal hyperalgesia, promoted the switch in microglia from the M1 phenotype to the M2 phenotype, and ameliorated neuroinflammation in rats subjected to CCI. However, the use of suppressor of cytokine signalling 3 (SOCS3) siRNA abolished these changes. These results indicated that Mer regulated M1/M2 microglial polarization and neuroinflammation and may be a potential target for treating neuropathic pain. [ABSTRACT FROM AUTHOR]

Decidualisation of the endometrium is a key event in early pregnancy, which enables embryo implantation. Importantly, the molecular processes impairing decidualisation in obese mothers are yet to be characterised. We hypothesise that impaired decidualisation in obese mice is mediated by the upregulation of leptin modulators, the suppressor of cytokine signalling 3 (SOCS3) and the protein tyrosine phosphatase non-receptor type 2 (PTPN2), together with the disruption of progesterone (P4)-signal transducer and activator of transcription (STAT3) signalling. After feeding mice with chow diet (CD) or high-fat diet (HFD) for 16 weeks, we confirmed the downregulation of P4 and oestradiol (E2) steroid receptors in decidua from embryonic day (E) 6.5 and decreased proliferation of stromal cells from HFD. In vitro decidualised mouse endometrial stromal cells (MESCs) and E6.5 deciduas from the HFD showed decreased expression of decidualisation markers, followed by the upregulation of SOCS3 and PTPN2 and decreased phosphorylation of STAT3. In vivo and in vitro leptin treatment of mice and MESCs mimicked the results observed in the obese model. The downregulation of Socs3 and Ptpn2 after siRNA transfection of MESCs from HFD mice restored the expression level of decidualisation markers. Finally, DIO mice placentas from E18.5 showed decreased labyrinth development and vascularisation and fetal growth restricted embryos. The present study revealed major defects in decidualisation in obese mice, characterised by altered uterine response to E2 and P4 steroid signalling. Importantly, altered hormonal response was associated with increased expression of leptin signalling modulators SOCS3 and PTPN2. Elevated levels of SOCS3 and PTPN2 were shown to molecularly affect decidualisation in obese mice, potentially disrupting the STAT3-PR regulatory molecular hub. [ABSTRACT FROM AUTHOR]

Suppressors of cytokine signaling (SOCS) proteins are important regulators of the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway. Within the SOCS family, SOCS3 is one of the most potent inhibitors of cytokine signaling. However, there is limited knowledge regarding the function of SOCS3 on regulating type I interferon (IFN) signaling in fish. In this study, the complete open reading frame (ORF) of SOCS3 from the large yellow croaker (Larimichthys crocea, LcSOCS3) was cloned and characterized. The ORF of LcSOCS3 was 618 nucleotides in length and encoded a protein containing 205 amino acids. LcSOCS3 had the typical domain architecture of the SOCS family, including an SRC homology 2 (SH2) domain, a SOCS box, an additional kinase inhibition region (KIR), and an extended SH2 subdomain (ESS). Phylogenetic analysis revealed that LcSOCS3 was clustered with other fish SOCS3s and most closely related to the SOCS3 of Collichthy lucidus. LcSOCS3 mRNA was detected in all organs or tissues examined, and its expression was significantly increased in both head kidney and spleen tissues, and primary head kidney leukocytes after poly(I:C) stimulation. Overexpression of LcSOCS3 significantly promoted Spring viremia of carp virus (SVCV) replication, resulting in a more severe cytopathic effect, increased viral titer, enhanced copy number of the SVCV-G gene, and decreased expression levels of IFN1, IRF7, ISG15, Viperin, PKR, and Mx in epithelioma papulosum cyprinid (EPC) cells. Silencing of LcSOCS3 correspondingly up-regulated the expression of IFNi, IFNh, PKR, Viperin, and Mx in large yellow croaker head kidney (LYCK) cells. Additionally, LcSOCS3 was shown to interact with Signal Transducer and Activator of Transcription 1 (STAT1) which may inhibit STAT1 translocating into the nucleus. This speculation was supported by the increased phosphorylation level of STAT1 in head kidney leukocytes after LcSOCS3 silencing. These results indicated that LcSOCS3 functioned as a potential negative regulator of type I IFN signaling in large yellow croaker through its interaction with STAT1. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) have been progressively recognized as critical regulators in the pathology and pathophysiology of central nervous system disease. However, the potential role of circRNAs in intracerebral hemorrhage (ICH) is still largely unclear. Here, we demonstrate that circTrim37 expression was significantly upregulated at 3 days after ICH by circular RNA microarray and qPCR assays. Overexpression of circTrim37 could significantly ameliorate brain injury volume, brain edema, neurologic deficits, and inflammation in vivo after ICH. CircTrim37 promotes M2 polarization while restrains M1 polarization in vitro. Furthermore, circTrim37 acts as an endogenous sponge for miR-30c-5p, thereby inhibiting miR-30c-5p activity, leading to the upregulation of SOCS3 and making the balance of microglial response towards an M2 phenotype. Taken together, our results indicate the participation of circTrim37 and its coupling mechanism in ICH and provide a novel therapeutic target for ICH. [ABSTRACT FROM AUTHOR]

BackgroundNeuropathic pain (NPP) is a multifaceted pain syndrome that occurs as a consequence of physical injury or underlying diseases, with an incidence rate of 7%-10%, NPP poses a significant clinical challenge as current treatment options are ineffective. The accumulation of apoptotic cells and neuroinflammation play crucial roles in the pathological mechanisms of NPP. Here, we aim to investigate strategies for effectively clearing apoptotic cells and provide therapeutic interventions for NPP.MethodsCCI mice were treated with different concentrations of ozone (15μg, 30μg, 45μg) to investigate the effects on the accumulation of apoptotic cells and neuroinflammation. In vitro, the phagocytic function of BMDM towards apoptotic neutrophils after ozone treatment was examined.ResultsWe found ozone at a concentration of 30μg significantly alleviated mechanical hypersensitivity in CCI mice and ozone significantly upregulates the phagocytic activity of BMDM. Furthermore, we investigated the mechanisms and found ozone could activate AMPK, upregulate Gas6 (but not Protein S), activate MerTK (a key receptor involved in apoptosis), and enhance the phagocytic function of BMDM towards apoptotic neutrophils. It caused the promotion of SOCS3 expression and the suppression of inflammatory factors IL-1β, IL-6, and TNF-a. Interestingly, the effect of ozone in alleviating CCI-induced pain was abolished by the AMPK inhibitor CC and the MerTK receptor inhibitor UNC2541.ConclusionOzone facilitated macrophage clearance of apoptotic cells, decreased neuroinflammation by activation of p-AMPK/Gas6/MerTK/SOCS3 signaling pathway, which may become an effective therapeutic approach for neuropathic pain after further clinical validation.

Ferulic acid (FA) is a natural phenolic acid with a strong protective effect against disorder of hepatic lipid metabolism. To elucidate the effect of FA on metabolic associated fatty liver disease (MAFLD), the physiological parameters of mice fed high-fat diet (HFD) with or without FA were examined, and the liver transcriptome and metabolome were evaluated. FA significantly inhibited increases in liver weight and decreased the serum triglyceride levels and prevented HFD-induced hepatic steatosis. Omic data showed that FA might reduce the deposition of carnitine C16:1 to alleviate HFD-induced liver damage by reducing the expression of SOCS3 in vivo. Oleic acid induced steatosis in Hepa1-6 cells was used to verify the hepatoprotective effect of FA in vitro. Treatment with FA suppressed cellular lipid droplet deposition and decreased the expression of SOCS3 and adipogenesis markers. This work indicates the potential of FA as a novel drug for the alleviation of hepatic steatosis.

Based on the analgesic and anti-inflammatory effects of clonidine in previous studies, we hypothesized that clonidine could accelerate wound healing in rats by regulating the expression of related cytokines. In this study, the wound healing effect of clonidine was evaluated using an excision wound model in diabetic rats and a HaCaT cell model. The wounds were treated daily with topical clonidine. The results analyzed by ImageJ2 software show that the wounds of the rats that were treated with 15 ng/mL clonidine recovered faster, and the wound size was also significantly reduced compared to the control group. Western blot assays determined that clonidine induced an increase in the expression of vascular growth factors, namely, Ang-1, Ang-2, and VEGF. Moreover, clonidine demonstrated a rescuing effect on JAK2 within the JAK/STAT pathway by inhibiting SOCS3 expression, leading to decreased SOCS3 levels and increased expression of JAK2 and phospho-STAT3. Histopathological analysis revealed that clonidine promoted complete epithelial repair and minimized inflammation in skin tissue. Additionally, clonidine stimulated HaCaT cell proliferation in vitro and enhanced cellular energy levels in the presence of AGEs. In conclusion, clonidine promoted vascular growth and wound healing by stimulating the expression of cytokines that are beneficial for wound healing.

Abstract Background Diabetic nephropathy (DN) is a life-threatening renal disease and needs urgent therapies. Wogonin is renoprotective in DN. This study aimed to explore the mechanism of how wogonin regulated high glucose (HG)-induced renal cell injury. Methods Diabetic mice (db/db), control db/m mice, and normal glucose (NG)- or HG-treated human tubule epithelial cells (HK-2) were used to evaluate the levels of suppressor of cytokine signaling 3 (SOCS3), Toll-like receptor 4 (TLR4), inflammation and fibrosis. Lentivirus was used to regulate SOCS3 and TLR4 expressions. After oral gavage of wogonin (10 mg/kg) or vehicle in db/db mice, histological morphologies, blood glucose, urinary protein, serum creatinine values (Scr), blood urea nitrogen (BUN), superoxide dismutase (SOD), glutathione (GSH), and reactive oxygen species (ROS) were assessed. RT-qPCR and Western blot evaluated inflammation and fibrosis-related molecules. Results HG exposure induced high blood glucose, severe renal injuries, high serumal Src and BUN, low SOD and GSH, and increased ROS. HG downregulated SOCS3 but upregulated TLR4 and JAK/STAT, fibrosis, and inflammasome-related proteins. Wogonin alleviated HG-induced renal injuries by decreasing cytokines, ROS, Src, and MDA and increasing SOD and GSH. Meanwhile, wogonin upregulated SOCS3 and downregulated TLR4 under HG conditions. Wogonin-induced SOCS3 overexpression directly decreased TLR4 levels and attenuated JAK/STAT signaling pathway-related inflammation and fibrosis, but SOCS3 knockdown significantly antagonized the protective effects of wogonin. However, TLR4 knockdown diminished SOCS3 knockdown-induced renal injuries. Conclusion Wogonin attenuates renal inflammation and fibrosis by upregulating SOCS3 to inhibit TLR4 and JAK/STAT pathway.

Background: Diabetic nephropathy (DN) is a life-threatening renal disease and needs urgent therapies. Wogonin is renoprotective in DN. This study aimed to explore the mechanism of how wogonin regulated high glucose (HG)-induced renal cell injury. Methods: Diabetic mice (db/db), control db/m mice, and normal glucose (NG)- or HG-treated human tubule epithelial cells (HK-2) were used to evaluate the levels of suppressor of cytokine signaling 3 (SOCS3), Toll-like receptor 4 (TLR4), inflammation and fibrosis. Lentivirus was used to regulate SOCS3 and TLR4 expressions. After oral gavage of wogonin (10 mg/kg) or vehicle in db/db mice, histological morphologies, blood glucose, urinary protein, serum creatinine values (Scr), blood urea nitrogen (BUN), superoxide dismutase (SOD), glutathione (GSH), and reactive oxygen species (ROS) were assessed. RT-qPCR and Western blot evaluated inflammation and fibrosis-related molecules. Results: HG exposure induced high blood glucose, severe renal injuries, high serumal Src and BUN, low SOD and GSH, and increased ROS. HG downregulated SOCS3 but upregulated TLR4 and JAK/STAT, fibrosis, and inflammasome-related proteins. Wogonin alleviated HG-induced renal injuries by decreasing cytokines, ROS, Src, and MDA and increasing SOD and GSH. Meanwhile, wogonin upregulated SOCS3 and downregulated TLR4 under HG conditions. Wogonin-induced SOCS3 overexpression directly decreased TLR4 levels and attenuated JAK/STAT signaling pathway-related inflammation and fibrosis, but SOCS3 knockdown significantly antagonized the protective effects of wogonin. However, TLR4 knockdown diminished SOCS3 knockdown-induced renal injuries. Conclusion: Wogonin attenuates renal inflammation and fibrosis by upregulating SOCS3 to inhibit TLR4 and JAK/STAT pathway. [ABSTRACT FROM AUTHOR]

Purpose: Despite lung cancer is one of the leading causes of cancer-related deaths, it remains hard to discover effective diagnostic and therapeutic approaches. Moreover, the five-year survival rate is relatively lower than other tumors. So urgent needs for finding a new theranostic target to treat lung cancer effectively. This study aims to present SOCS3 and NOD2 proteins as novel targets for diagnosis and therapy. Methods: We first confirmed SOCS3 expression level in patients' tissues. Then, we applied knockdown and overexpression of SOCS3 on lung cancer cell lines and performed proliferation, migration, and invasion assay. After that, we found NOD2 is a target of SOCS3 and introduced overexpression of NOD2 to A549 for verifying reduced tumorigenicity of lung cancer cells. Results: We identified protein expression level of SOCS3 was frequently higher in tumor tissues than adjacent normal tissues. Truly, overexpression of SOCS3 promoted proliferation, migration, and invasion capacity of lung cancer cells. We found that SOCS3 interacts with NOD2 and SOCS3 ubiquitinates NOD2 directly. Furthermore, lung cancer tissues with higher SOCS3 expression showed lower NOD2 expression. We confirmed overexpression of NOD2 leads to suppressed tumorigenicity of lung cancer cells, and these effects occurred through MAPK pathway. Conclusion: Collectively, our work reveals novel roles of SOCS3 in lung tumorigenesis and proposes SOCS3 as a promising biomarker candidate for therapeutic and diagnostic target for lung cancer. [ABSTRACT FROM AUTHOR]

The suppressor of cytokine signaling 3 (SOCS3) is a key signaling molecule that regulates milk synthesis in dairy livestock. However, the molecular mechanism by which SOCS3 regulates lipid synthesis in goat milk remains unclear. This study aimed to screen for key downstream genes associated with lipid synthesis regulated by SOCS3 in goat mammary epithelial cells (GMECs) using RNA sequencing (RNA-seq). Goat SOCS3 overexpression vector (PC-SOCS3) and negative control (PCDNA3.1) were transfected into GMECs. Total RNA from cells after SOCS3 overexpression was used for RNA-seq, followed by differentially expressed gene (DEG) analysis, functional enrichment analysis, and network prediction. SOCS3 overexpression significantly inhibited the synthesis of triacylglycerol, total cholesterol, non-esterified fatty acids, and accumulated lipid droplets. In total, 430 DEGs were identified, including 226 downregulated and 204 upregulated genes, following SOCS3 overexpression. Functional annotation revealed that the DEGs were mainly associated with lipid metabolism, cell proliferation, and apoptosis. We found that the lipid synthesis-related genes, STAT2 and FOXO6, were downregulated. In addition, the proliferation-related genes BCL2, MMP11, and MMP13 were upregulated, and the apoptosis-related gene CD40 was downregulated. In conclusion, six DEGs were identified as key regulators of milk lipid synthesis following SOCS3 overexpression in GMECs. Our results provide new candidate genes and insights into the molecular mechanisms involved in milk lipid synthesis regulated by SOCS3 in goats. [ABSTRACT FROM AUTHOR]

Differential activation of macrophages is associated with poor progression of breast cancer (BC). Many reports have elucidated the important involvement of exosomes produced by cancer cells in remodeling the macrophage activation phenotype to promote tumor expansion and invasion. However, the underlying mechanisms by which exosomes secreted by BC cells facilitate macrophage M2 polarization remain enigmatic and worth exploring. In this study, quantitative real-time PCR (RT-qPCR) was used to investigate miR-191-5p expression in BC tumor tissues and cells. Cell counting kit 8 (CCK-8), transwell, and flow cytometry were applied to assess the functional role of miR-191-5p in BC. Isolated nano-vesicles were identified using transmission electron microscopy and western blotting. We also observed that miR-191-5p was significantly elevated in BC clinical samples and that inhibition of miR-191-5p hindered the growth and metastasis of BC cells. Importantly, BC cells successfully accelerated macrophage M2-like polarization by directly transferring exosomes to macrophages, resulting in increased miR-191-5p levels in macrophages. Mechanistically, exosomal miR-191-5p directly inhibited the suppressors of cytokine signaling 3 (SOCS3) expression in macrophages and aggravated macrophage M2 polarization. Similarly, si-SOCS3 transfected macrophages boosted BC cell migration and invasion in a positive feedback manner. Overall, our results manifested a pro-growth and pro-metastatic role between the two cells by elucidating the crucial role of exosomal miR-191-5p in stimulating M2 macrophage polarization and mediating communication between BC cells and macrophages. These findings opened up new horizons for the development of BC therapeutic strategies. [ABSTRACT FROM AUTHOR]

Intervertebral disc degeneration (IDD) poses a significant health burden, necessitating a deeper understanding of its molecular underpinnings. Transcriptomic analysis reveals 485 differentially expressed genes (DEGs) associated with IDD, underscoring the importance of immune regulation. Weighted gene co‐expression network analysis (WGCNA) identifies a yellow module strongly correlated with IDD, intersecting with 197 DEGs. Protein–protein interaction (PPI) analysis identifies ITGAX, MMP9 and FCGR2A as hub genes, predominantly expressed in macrophages. Functional validation through in vitro and in vivo experiments demonstrates the pivotal role of FCGR2A in macrophage polarization and IDD progression. Mechanistically, FCGR2A knockdown suppresses M1 macrophage polarization and NF‐κB phosphorylation while enhancing M2 polarization and STAT3 activation, leading to ameliorated IDD in animal models. This study sheds light on the regulatory function of FCGR2A in macrophage polarization, offering novel insights for IDD intervention strategies. Key points: This study unveils the role of FCGR2A in intervertebral disc (IVD) degeneration (IDD).FCGR2A knockdown mitigates IDD in cellular and animal models.Single‐cell RNA‐sequencing uncovers diverse macrophage subpopulations in degenerated IVDs.This study reveals the molecular mechanism of FCGR2A in regulating macrophage polarization.This study confirms the role of the NF‐κB/STAT3 pathway in regulating macrophage polarization in IDD. [ABSTRACT FROM AUTHOR]

Fluid overload in hemodialysis patients (HD) has been proven to be associated with inflammation. Elevated levels of the pro-inflammatory cytokine interleukin-6 (IL-6) appear to be inadequately counterbalanced by the anti-inflammatory cytokine interleukin-10 (IL-10). We initiated a cross-sectional study enrolling 40 HD patients who were categorized by a bioimpedance measurement in normovolemic (N; 23) and hypervolemic (H; 17) groups to test whether IL-10- and IL-6-related signal transduction pathways (signal transducer of transcript 3: STAT3) and/or a post-transcriptional regulating mechanism (miR-142) are impaired by hypervolemia. IL-10/IL-6 transcript and protein production by PBMCs (peripheral blood mononuclear cells) were determined. Phospho-flow cytometry was used to detect the phosphorylated forms of STAT3 (pY705 and pS727). miR-142-3p/5p levels were detected by qPCR. Hypervolemic patients were older, more frequently had diabetes, and showed higher CRP levels. IL-10 transcripts were elevated in H patients but not IL-10 protein levels. In spite of the elevated mRNA expression of the suppressor of cytokine expression 3 (SOCS3), IL-6 mRNA and protein expression were increased in immune cells of H patients. The percentage of cells staining positive for STAT3 (pY705) were comparable in both groups; in STAT3 (pS727), however, the signal needed for full transactivation was decreased in H patients. miR-142-3p, a proven target of IL-10 and IL-6, was significantly elevated in H patients. Insufficient phosphorylation of STAT3 may impair inflammatory and anti-inflammatory cytokine signaling. How far degradative mechanisms induced by elevated miR-142-3p levels contribute to an inefficient anti-inflammatory IL-10 signaling remains elusive. [ABSTRACT FROM AUTHOR]

Due to the urgent need to create appropriate treatment techniques, which are currently unavailable, LPS-induced sepsis has become a serious concern on a global scale. The primary active component in the pathophysiology of inflammatory diseases such as sepsis is the Gram-negative bacterial lipopolysaccharide (LPS). LPS interacts with cell surface TLR4 in macrophages, causing the formation of reactive oxygen species (ROS), TNF-α, IL-1β and oxidative stress. It also significantly activates the MAPKs and NF-κB pathway. Excessive production of pro-inflammatory cytokines is one of the primary characteristic features in the onset and progression of inflammation. Cytokines mainly signal through the JAK/STAT pathway. We hypothesize that blocking of TLR4 along with TNFR1 might be beneficial in suppressing the effects of STAT1/STAT3 due to the stimulation of SOCS3 proteins. Prior to the LPS challenge, the macrophages were treated with antibodies against TLR4 and TNFR1 either individually or in combination. On analysis of the macrophage populations by flowcytometry, it was seen that receptor blockade facilitated the phenotypic shift of the M1 macrophages towards M2 resulting in lowered oxidative stress. Blocking of TLR4/TNFR1 upregulated the SOCS3 and mTOR expressions that enabled the transition of inflammatory M1 macrophages towards the anti-inflammatory M2 phenotype, which might be crucial in curbing the inflammatory responses. Also the reduction in the production of inflammatory cytokines such as IL-6, IL-1β due to the reduction in the activation of the STAT1 and STAT3 molecules was observed in our combination treatment group. All these results indicated that neutralization of both TLR4 and TNFR1 might provide new insights in establishing an alternative therapeutic strategy for LPS-sepsis.

Abstract Background Polycystic ovary syndrome (PCOS) is a frequent and complicated endocrine disease that remains a major reason for infertility. Bushenhuoluo Decotion (BSHLD) has been validated to exhibit curative effects on PCOS. This study was aimed to explore the potential mechanism underlying the therapeutic action of BSHLD. Methods PCOS rat model was induced by dehydroepiandrosterone (DHEA). Serum hormone and cytokines levels and ovarian pathological alterations were measured to assess ovarian function. Exosomes (Exos) were identified by Transmission electron microscopy and Nanoparticle Tracking Analysis. RT-qPCR, Western blotting, immunohistochemical staining, and immunofluorescence staining were performed to detect molecule expressions. Proliferation and pyroptosis of granulosa cells (GCs) were evaluated by CCK-8 and flow cytometry, respectively. The binding relationship between miR-30a-5p and suppressor of cytokine signaling 3 (SOCS3) was verified by dual luciferase reporter and RIP assays. Results BSHLD treatment improved serum hormone abnormality, insulin sensitivity, and ovarian morphologic changes of PCOS rats. Moreover, BSHLD treatment restrained the excessive autophagy and pyroptosis in ovarian tissues of PCOS rats. Moreover, BSHLD reduced the expression of miR-30a-5p in serum, serum-derived Exos, and ovarian tissues, thus inhibiting autophagy and NLRP3-mediated pyroptosis in GCs. Mechanistically, SOCS3 was proved as a target of miR-30a-5p and could activate mTOR/P70S6K pathway to repress autophagy. The inhibitory effect of miR-30a-5p deficiency on autophagy and pyroptosis of GCs was attenuated by rapamycin. Conclusion Collectively, BSHLD suppressed autophagy and pyroptosis to improve POCS by regulating exosomal miR-30a-5p/SOCS3/mTOR signaling.

Background: The family of Suppressor of Cytokine Signaling (SOCS) acts as a controller of the duration and intensity of cytokine function by negatively regulating the JAK-STAT signaling pathway. SOCS’ role in inflammatory diseases in animal models is well demonstrated. However, its role in the development of human disease is still under investigation. SOCS3 plays an important role in tumor development where its downregulation has been implicated in the pathogenesis of various solid tumors such as triple-negative breast cancer. Aim: The aim of this work was to study (1) the expression of SOCS3 in smokers’ lungs and its relation to the degree of inflammation and (2) SOCS3 regulation by microRNA (miRNA) in alveolar-macrophage (AM)-derived extracellular vesicles (EVs) in bronchoalveolar lavage (BAL). Methods: Group A: 35 smokers’ [19 with COPD (SC) and 16 without COPD (S)] and 9 nonsmokers (NS); SOCS3, TNFα in AM, and CD8+ T cells were quantified by immunohistochemistry, in lung tissue. Group B: additional 9 SC, 11 S, and 5 NS; AM-EVs expressing SOCS3 (CD14+SOCS3+) and SOCS3 suppressors miRNA-19a-3p and 221-3p in EVs were quantified by flow cytometry and PCR, in BAL. Results: The percentage of SOCS3+ AM was higher in SC [68 (6.6–99)%] and S [48 (8–100)%] than in NS [9.6 (1.9–61)%; p = 0.002; p = 0.03] and correlated with % of TNFα+AM (r = 0.48; p = 0.0009) and CD8+ T cells (r = 0.44; p = 0.0029). In BAL, the CD14+SOCS3+ EVs/μL were increased in SC [33 (21–74)] compared to S [16 (8–37); p = 0.03] and NS [9 (7–21); p = 0.003]. Conversely, miRNA-19a-3p and miRNA-221-3p expression were increased in S when compared to SC [19 (2–53) vs. 3 (0.6–8); p = 0.03 and 3 (0.005–9.6) vs. 0.2 (0.08–0.7); p = 0.05]. Conclusions: The suppressor function of SOCS3 in COPD seems to be overridden by other factors and does not follow the animal-model paradigm. Expression of SOCS3 in BAL macrophage-derived EVs might be useful to assess the degree of inflammation and possible progression of COPD. Downregulation of SOCS3, by miRNA, in smokers without COPD might contribute to the risk of developing cancer in these patients. [ABSTRACT FROM AUTHOR]

This study aimed to identify and evaluate drug candidates targeting the kinase inhibitory region of suppressor of cytokine signaling (SOCS) 3 for the treatment of allergic rhinitis (AR). Utilizing an artificial intelligence (AI)-based new drug development platform, virtual screening was conducted to identify compounds inhibiting the SH2 domain binding of SOCS3. Luminescence assays assessed the ability of these compounds to restore JAK-2 activity diminished by SOCS3. Jurkat T and BEAS-2B cells were utilized to investigate changes in SOCS3 and STAT3 expression, along with STAT3 phosphorylation in response to the identified compounds. In an OVA-induced allergic rhinitis mouse model, we measured serum levels of total IgE and OVA-specific IgE, performed real-time PCR on nasal mucosa samples to quantify Th2 cytokines and IFN-γ expression, and conducted immunohistochemistry to analyze eosinophil levels. Screening identified 20 hit compounds with robust binding affinities. As the concentration of SOCS3 increased, a corresponding decrease in JAK2 activity was observed. Compounds 5 and 8 exhibited significant efficacy in restoring JAK2 activity without toxicity. Treatment with these compounds resulted in reduced SOCS3 expression and the reinstatement of STAT3 phosphorylation in Jurkat T and BEAS-2B cells. In the OVA-induced allergic rhinitis mouse model, compounds 5 and 8 effectively alleviated nasal symptoms and demonstrated lower levels of immune markers compared to the allergy group. This study underscores the promising nonclinical efficacy of compounds identified through the AI-based drug development platform. These findings introduce innovative strategies for the treatment of AR and highlight the potential therapeutic value of targeting SOCS3 in managing AR. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 3 (SOCS3) plays a significant role in the process of myocardial adaptation to chronic hypoxia. SOCS3 finely regulates cell signaling cross-talk that occurs between NF-κB and STAT3 during the compensatory protective response. However, the role and mechanism of SOCS3 in hypoxic cardiomyocytes are not fully understood. In the study, we investigated the effect of SOCS3 on the p65 and STAT3 signaling pathways and further examined the potential molecular mechanism involved in regulating apoptosis. Our data showed that SOCS3 silencing could upregulate Ac-p65, p-p65, and p-STAT3 expression in nuclear extracts of H9c2 cells that received hypoxic treatment for 24, 48, and 72 h. SOCS3 silencing also remarkably increased the DNA-binding activity of the p65 motif in hypoxic cultivated H9c2 cells. We also found that SOCS3 knockdown increased cleaved-caspase-3, Bax, and PUMA expression and decreased cleaved PARP and Bcl-2 in expression in hypoxic H9c2 cells. Silencing of SOCS3 caused an increase in LDH leakage from injured cardiomyocytes and reduced cell viability under conditions of hypoxic stress. Furthermore, SOCS3 silencing enhanced the apoptosis of H9c2 cells at 72 h of hypoxia. These findings suggest that knockdown of SOCS3 leads to excessive activation of the NF-κB pathway, which, in turn, might promote apoptosis under conditions of chronic hypoxia. Diagram showing the mechanism of SOCS3 in hypoxic cardiomyocytes. Silencing SOCS3 could induce the apoptosis of H9c2 cells by regulating the apoptosis-associated gene expression, which may be accomplished by activation of the p-STAT3/NF-κB signaling pathway and rely on hypoxia condition. [ABSTRACT FROM AUTHOR]

Background: Asthma associated with obesity is a chronic disease characterized by earlier airway remodeling, severe wheezing, and increased insensitivity to hormone therapy. Reticuline, a bioactive compound of Magnoliae Flos, exerts anti‐inflammatory activity and can inhibit neutrophil recruitment. Thus, this study investigated the role of reticuline in obesity‐related asthma. Methods: The BALB/c mice fed a low‐fat diet (LFD) and high‐fat diet (HFD) were intranasally challenged with house dust mites (HDMs) or ovalbumin (OVA). Reticuline (0.25 mg/kg) was administrated into mice by intragastrical gavage. Airway hyper‐responsiveness was examined after the final challenge. Body weight was measured, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. The number of inflammatory cells in BALF was estimated. Histological changes were assessed by performing hematoxylin–eosin staining, and production of proinflammatory cytokines and IgE was examined by ELISA kits. Related pathways were studied with western blotting. Results: Reticuline suppressed airway resistance and inflammatory infiltration in lung tissue and reduced inflammatory cell recruitment in BALF in obesity mice with asthma. Additionally, the levels of IL‐17A, IL‐1β, IL‐5, macrophage inflammatory protein 2, and regulated on activation, normal T cell expressed and secreted in the lung were reduced by reticuline. Mechanistically, reticuline inactivated the JAK2/STAT3/SOCS3 and p38 MAPK/NF‐κB signaling pathways in obesity‐related asthma. Conclusion: Reticuline alleviates airway inflammation in obesity‐related asthma by inactivating the JAK2/STAT3/SOCS3 and p38 MAPK/NF‐κB signaling pathways. [ABSTRACT FROM AUTHOR]

Background: Gout is the most common inflammatory arthritis in adults. Gout is an arthritic disease caused by the deposition of monosodium urate crystal (MSU) in the joints, which can lead to acute inflammation and damage adjacent tissue. Hyperuricemia is the main risk factor for MSU crystal deposition and gout. With the increasing burden of gout disease, the identification of potential biomarkers and novel targets for diagnosis is urgently needed. Methods: For the analysis of this subject paper, we downloaded the human gout data set GSE160170 and the gout mouse model data set GSE190138 from the GEO database. To obtain the differentially expressed genes (DEGs), we intersected the two data sets. Using the cytohubba algorithm, we identified the key genes and enriched them through GO and KEGG. The gene expression trends of three subgroups (normal control group, intermittent gout group and acute gout attack group) were analyzed by Series Test of Cluster (STC) analysis, and the key genes were screened out, and the diagnostic effect was verified by ROC curve. The expression of key genes in dorsal root nerve and spinal cord of gout mice was analyzed. Finally, the clinical samples of normal control group, hyperuricemia group, intermittent gout group and acute gout attack group were collected, and the expression of key genes at protein level was verified by ELISA. Result: We obtained 59 co-upregulated and 28 co-downregulated genes by comparing the DEGs between gout mouse model data set and human gout data set. 7 hub DEGs(IL1B, IL10, NLRP3, SOCS3, PTGS2) were screened out via Cytohubba algorithm. The results of both GO and KEGG enrichment analyses indicate that 7 hub genes play a significant role in regulating the inflammatory response, cytokine production in immune response, and the TNF signaling pathway. The most representative hub genes SOCS3 and PTGS2 were screened out by Series Test of Cluster, and ROC analysis results showed the AUC values were both up to 1.000. In addition, we found that PTGS2 expression was significantly elevated in the dorsal root ganglia and spinal cord in monosodium urate(MSU)-induced gout mouse model. The ELISA results revealed that the expression of SOCS3 and PTGS2 was notably higher in the acute gout attack and intermittent gout groups compared to the normal control group. This difference was statistically significant, indicating a clear distinction between the groups. Conclusion: Through cross-species comprehensive analysis and experimental verification, SOCS3 and PTGS2 were proved to be new biomarkers for diagnosing gout and predicting disease progression.

Abstract Background Gout pain seriously affects the quality of patients' life. There is still no effective treatment. The inflammatory response is the main mechanism of gout. Here, we found that ozone can reduce the inflammatory reaction in the joints of gouty mice and relieve gout pain, and we further explore its protective mechanism. Methods MSU was used to establish the gouty mice model. Nociception was assessed by Von Frey hairs. Cell signaling assays were performed by western blotting and immunohistochemistry. The mouse leukemia cells of monocyte macrophage line RAW264.7 were cultured to investigate the effects of ozone administration on macrophage. Results Ozone reduced inflammation, relieved gout pain and improved the paw mean intensity and duty cycle of the gouty mice. Ozone increased the phosphorylation of AMP-activated protein kinase (AMPK), induced suppressor of cytokine signaling 3 (SOCS3) expression and inhibited metallopeptidase 9 (MMP9) expression. In vivo, ozone activated AMPK to induce Gas6 release, and upregulated MerTK/SOCS3 signaling pathway to reduce inflammation in mouse macrophage line RAW264.7. Inhibitors of AMPK and MerTK, respectively abolished the analgesic and anti-inflammatory effects of ozone in vivo and in vitro. Gas6 knockout cancelled the protectively effects of ozone on gout pain and the paw mean intensity and duty cycle of gouty mice. Additionally, the level of Gas6 and protein S in plasma of patients with hyperuricemia was significantly higher than that of healthy contrast group. Conclusion Ozone reduces inflammation and alleviates gout pain by activating AMPK to up-regulate Gas6/MerTK/SOCS3 signaling pathway.

Abstract Background Acute myocardial infarction (AMI) has two clinical characteristics: high missed diagnosis and dysfunction of leukocytes. Transcriptional RNA on leukocytes is closely related to the course evolution of AMI patients. We hypothesized that transcriptional RNA in leukocytes might provide potential diagnostic value for AMI. Integration machine learning (IML) was first used to explore AMI discrimination genes. The following clinical study was performed to validate the results. Methods A total of four AMI microarrays (derived from the Gene Expression Omnibus) were included in bioanalysis (220 sample size). Then, the clinical validation was finished with 20 AMI and 20 stable coronary artery disease patients (SCAD). At a ratio of 5:2, GSE59867 was included in the training set, while GSE60993, GSE62646, and GSE48060 were included in the testing set. IML was explicitly proposed in this research, which is composed of six machine learning algorithms, including support vector machine (SVM), neural network (NN), random forest (RF), gradient boosting machine (GBM), decision trees (DT), and least absolute shrinkage and selection operator (LASSO). IML had two functions in this research: filtered optimized variables and predicted the categorized value. Finally, The RNA of the recruited patients was analyzed to verify the results of IML. Results Thirty-nine differentially expressed genes (DEGs) were identified between controls and AMI individuals from the training sets. Among the thirty-nine DEGs, IML was used to process the predicted classification model and identify potential candidate genes with overall normalized weights > 1. Finally, two genes (AQP9 and SOCS3) show their diagnosis value with the area under the curve (AUC) > 0.9 in both the training and testing sets. The clinical study verified the significance of AQP9 and SOCS3. Notably, more stenotic coronary arteries or severe Killip classification indicated higher levels of these two genes, especially SOCS3. These two genes correlated with two immune cell types, monocytes and neutrophils. Conclusion AQP9 and SOCS3 in leukocytes may be conducive to identifying AMI patients with SCAD patients. AQP9 and SOCS3 are closely associated with monocytes and neutrophils, which might contribute to advancing AMI diagnosis and shed light on novel genetic markers. Multiple clinical characteristics, multicenter, and large-sample relevant trials are still needed to confirm its clinical value.

Abstract Background Islet transplantation is used therapeutically in a minority of patients with type 1 diabetes (T1D). However, successful outcomes are hampered by early islet β-cell loss caused by immune rejection and autoimmunity. Recent studies have demonstrated that mesenchymal stromal cells can enhance islet function both in vitro and in vivo by secreting ligands that activate islet G-protein coupled receptors (GPCRs). Stromal cell-derived factor 1 (SDF-1) is an MSC-secreted GPCR ligand, whereas the suppressor of cytokine signaling 3 (SOCS3) is a negative regulator of STAT3-activating cytokines. Here, we determined whether improvement in islet function mediated by exogenous SDF-1 is impaired by SOCS3 in experimental models of T1D. Methods Isolated islets were cultured for 48 h with SDF-1. Cytokine-induced apoptosis was measured immediately. Islets from Socs3 −/− mice were pre-cultured with exogenous SDF-1 and transplanted underneath the kidney capsule of C57BL/6 mice with streptozotocin-induced diabetes. Blood glucose levels were monitored for 28 days. AMD3100, an antagonist of the SDF-1 ligand CXCR4, was administered subcutaneously to islet transplanted mice to inhibit CXCR4 before and after transplantation. Results SDF-1 protected islet cells from cytokine-induced apoptosis in vitro. SOCS3-knockout (KO) islets pretreated with SDF-1 were effective in reducing blood glucose in non-obese diabetic mice in vivo. We found that SDF-1 elicits localized immunosuppression in transplanted SOCS3-KO islets. Immunomodulation was observed when SOCS-KO islets were preconditioned with SDF-1. Gene expression and flow cytometric analyses revealed significantly decreased immune cell infiltration, inflammatory cytokines, and concomitant increases in FOXP3+ regulatory T cells, alternatively activated M2 macrophages, and dendritic cell phenotypes. Administration of AMD3100 impaired the SDF-1-mediated improvement in SOCS3-KO islet function and local immune suppression. Conclusion SDF-1 improves the function of islet grafts in autoimmune diabetes through regulation by CXCR4; however, the presence of SOCS3 reverses the protective effect of SDF-1 on islet grafts. These data reveal a molecular pathway that can elicit localized immunosuppression and delay graft destruction in transplanted islets.

Autism is a widespread neurodevelopmental disorder. Although the research on autism spectrum disorders has been increasing in the past decade, there is still no specific answer to its mechanism of action and treatment. As a pro-inflammatory microRNA, miR-301a is abnormally expressed in various psychiatric diseases including autism. Here, we show that miR-301a deletion and inhibition exhibited two distinct abnormal behavioral phenotypes in mice. We observed that miR-301a deletion in mice impaired learning/memory, and enhanced anxiety. On the contrary, miR-301a inhibition effectively reduced the maternal immune activation (MIA)-induced autism-like behaviors in mice. We further demonstrated that miR-301a bound to the 3′UTR region of the SOCS3, and that inhibition of miR-301a led to the upregulation of SOCS3 in hippocampus. The last result in the reduction of the inflammatory response by inhibiting phosphorylation of AKT and STAT3, and the expression level of IL-17A in poly(I:C)-induced autism-like features in mice. The obtained data revealed the miR-301a as a critical participant in partial behavior phenotypes, which may exhibit a divergent role between gene knockout and knockdown. Our findings ascertain that miR-301a negatively regulates SOCS3 in MIA-induced autism in mice and could present a new therapeutic target for ameliorating the behavioral abnormalities of autism.

BackgroundThe family of Suppressor of Cytokine Signaling (SOCS) acts as a controller of the duration and intensity of cytokine function by negatively regulating the JAK-STAT signaling pathway. SOCS’ role in inflammatory diseases in animal models is well demonstrated. However, its role in the development of human disease is still under investigation. SOCS3 plays an important role in tumor development where its downregulation has been implicated in the pathogenesis of various solid tumors such as triple-negative breast cancer.AimThe aim of this work was to study (1) the expression of SOCS3 in smokers’ lungs and its relation to the degree of inflammation and (2) SOCS3 regulation by microRNA (miRNA) in alveolar-macrophage (AM)-derived extracellular vesicles (EVs) in bronchoalveolar lavage (BAL).MethodsGroup A: 35 smokers’ [19 with COPD (SC) and 16 without COPD (S)] and 9 nonsmokers (NS); SOCS3, TNFα in AM, and CD8+ T cells were quantified by immunohistochemistry, in lung tissue. Group B: additional 9 SC, 11 S, and 5 NS; AM-EVs expressing SOCS3 (CD14+SOCS3+) and SOCS3 suppressors miRNA-19a-3p and 221-3p in EVs were quantified by flow cytometry and PCR, in BAL.ResultsThe percentage of SOCS3+ AM was higher in SC [68 (6.6–99)%] and S [48 (8–100)%] than in NS [9.6 (1.9–61)%; p = 0.002; p = 0.03] and correlated with % of TNFα+AM (r = 0.48; p = 0.0009) and CD8+ T cells (r = 0.44; p = 0.0029). In BAL, the CD14+SOCS3+ EVs/μL were increased in SC [33 (21–74)] compared to S [16 (8–37); p = 0.03] and NS [9 (7–21); p = 0.003]. Conversely, miRNA-19a-3p and miRNA-221-3p expression were increased in S when compared to SC [19 (2–53) vs. 3 (0.6–8); p = 0.03 and 3 (0.005–9.6) vs. 0.2 (0.08–0.7); p = 0.05].ConclusionsThe suppressor function of SOCS3 in COPD seems to be overridden by other factors and does not follow the animal-model paradigm. Expression of SOCS3 in BAL macrophage-derived EVs might be useful to assess the degree of inflammation and possible progression of COPD. Downregulation of SOCS3, by miRNA, in smokers without COPD might contribute to the risk of developing cancer in these patients.

Abstract Background Asthma associated with obesity is a chronic disease characterized by earlier airway remodeling, severe wheezing, and increased insensitivity to hormone therapy. Reticuline, a bioactive compound of Magnoliae Flos, exerts anti‐inflammatory activity and can inhibit neutrophil recruitment. Thus, this study investigated the role of reticuline in obesity‐related asthma. Methods The BALB/c mice fed a low‐fat diet (LFD) and high‐fat diet (HFD) were intranasally challenged with house dust mites (HDMs) or ovalbumin (OVA). Reticuline (0.25 mg/kg) was administrated into mice by intragastrical gavage. Airway hyper‐responsiveness was examined after the final challenge. Body weight was measured, and bronchoalveolar lavage fluid (BALF) and lung tissues were collected. The number of inflammatory cells in BALF was estimated. Histological changes were assessed by performing hematoxylin–eosin staining, and production of proinflammatory cytokines and IgE was examined by ELISA kits. Related pathways were studied with western blotting. Results Reticuline suppressed airway resistance and inflammatory infiltration in lung tissue and reduced inflammatory cell recruitment in BALF in obesity mice with asthma. Additionally, the levels of IL‐17A, IL‐1β, IL‐5, macrophage inflammatory protein 2, and regulated on activation, normal T cell expressed and secreted in the lung were reduced by reticuline. Mechanistically, reticuline inactivated the JAK2/STAT3/SOCS3 and p38 MAPK/NF‐κB signaling pathways in obesity‐related asthma. Conclusion Reticuline alleviates airway inflammation in obesity‐related asthma by inactivating the JAK2/STAT3/SOCS3 and p38 MAPK/NF‐κB signaling pathways.

Background: Gout pain seriously affects the quality of patients' life. There is still no effective treatment. The inflammatory response is the main mechanism of gout. Here, we found that ozone can reduce the inflammatory reaction in the joints of gouty mice and relieve gout pain, and we further explore its protective mechanism. Methods: MSU was used to establish the gouty mice model. Nociception was assessed by Von Frey hairs. Cell signaling assays were performed by western blotting and immunohistochemistry. The mouse leukemia cells of monocyte macrophage line RAW264.7 were cultured to investigate the effects of ozone administration on macrophage. Results: Ozone reduced inflammation, relieved gout pain and improved the paw mean intensity and duty cycle of the gouty mice. Ozone increased the phosphorylation of AMP-activated protein kinase (AMPK), induced suppressor of cytokine signaling 3 (SOCS3) expression and inhibited metallopeptidase 9 (MMP9) expression. In vivo, ozone activated AMPK to induce Gas6 release, and upregulated MerTK/SOCS3 signaling pathway to reduce inflammation in mouse macrophage line RAW264.7. Inhibitors of AMPK and MerTK, respectively abolished the analgesic and anti-inflammatory effects of ozone in vivo and in vitro. Gas6 knockout cancelled the protectively effects of ozone on gout pain and the paw mean intensity and duty cycle of gouty mice. Additionally, the level of Gas6 and protein S in plasma of patients with hyperuricemia was significantly higher than that of healthy contrast group. Conclusion: Ozone reduces inflammation and alleviates gout pain by activating AMPK to up-regulate Gas6/MerTK/SOCS3 signaling pathway. [ABSTRACT FROM AUTHOR]

Retinoblastoma (RB) is an intraocular tumor in children. Accumulated evidence confirms that microRNAs (miRNAs) exert critical functions in RB. This research aimed to investigate the miR‐452‐5p function in RB. MiR‐452‐5p expressions in RB were tested with quantitative real‐time polymerase chain reaction (PCR). MiR‐452‐5p functions in RB were evaluated via Cell Counting Kit‐8, 5‐Ethynyl‐2′‐deoxyuridine assay, flow cytometry, Western blot, and Transwell. MiR‐452‐5p mechanism in RB was assessed using bioinformatics software Starbase and dual‐luciferase reporter gene assay. Meanwhile, miR‐452‐5p function in RB in vivo was examined by constructing tumor xenografts in nude mice, immunohistochemistry, and Western blot assays. MiR‐452‐5p was overexpressed in RB tissues and cells, and miR‐452‐5p expression was positively correlated with RB clinicopathology including the Largest tumor base (mm) and Differentiation. Functionally, miR‐452‐5p knockdown restrained RB cell proliferation, invasion, epithelial–mesenchymal transition (EMT), and facilitated cell apoptosis. Mechanistically, suppressors of cytokine signaling (SOCS3) knockdown restored the inhibitory effects of miR‐452‐5p knockdown on RB cells. Meanwhile, in vivo studies further corroborated that miR‐452‐5p knockdown reduced RB tumor growth, EMT, and accelerated apoptosis in vivo. Also, miR‐452‐5p knockdown increased SOCS3 protein levels, and decreased phosphorylated Janus kinase 2/Janus kinase 2 (JAK2), phosphorylated signal transducer and activator of transcription 3/signal transducer and activator of transcription 3 (STAT3) in vivo. MiR‐452‐5p accelerated RB cell growth and invasion by SOCS3/JAK2/STAT3. [ABSTRACT FROM AUTHOR]

Obesity is a major risk factor for the development of nonalcoholic fatty liver disease (NAFLD), and the subcutaneous white adipose tissue (scWAT) is the primary lipid storage depot and regulates lipid fluxes to other organs. Our previous work identified genes upregulated in scWAT of patients with NAFLD: SOCS3, DUSP1, and SIK1. Herein, we knocked down (KD) their expression in human adipose-derived mesenchymal stem cells (hADMSCs) using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology and characterized their phenotype. We found that SOCS3, DUSP1, and SIK1 expression in hADMSC-derived adipocytes was not critical for adipogenesis. However, the metabolic characterization of the cells suggested that the genes played important roles in lipid metabolism. Reduction of SIK1 expression significantly increased both de novo lipogenesis (DNL) and palmitate-induced lipogenesis (PIL). Editing out SOCS3 reduced DNL while increasing isoproterenolinduced lipolysis and insulin-induced palmitate accumulation. Conversely, DUSP1 reduced PIL and DNL. Moreover, RNAsequencing analysis of edited cells showed that these genes not only altered lipid metabolism but also other biological pathways related to inflammatory processes, in the case of DUSP1, extracellular matrix remodeling for SOCS3, or cellular transport for SIK1. Finally, to evaluate a possible adipocyte-hepatocyte axis, human hepatoma HepG2 cells were cocultured with edited hADMSCs-derived adipocytes in the presence of [3H]-palmitate. All HepG2 cells cultured with DUSP1-, SIK1-, or SOCS3-KD adipocytes decreased [3H]-palmitate accumulation compared with control adipocytes. These results support our hypotheses that SOCS3, DUSP1, and SIK1 regulate multiple aspects of adipocyte function, which may play a role in the progression of obesityassociated comorbidities, such as NAFLD. [ABSTRACT FROM AUTHOR]

Background: Acute myocardial infarction (AMI) has two clinical characteristics: high missed diagnosis and dysfunction of leukocytes. Transcriptional RNA on leukocytes is closely related to the course evolution of AMI patients. We hypothesized that transcriptional RNA in leukocytes might provide potential diagnostic value for AMI. Integration machine learning (IML) was first used to explore AMI discrimination genes. The following clinical study was performed to validate the results. Methods: A total of four AMI microarrays (derived from the Gene Expression Omnibus) were included in bioanalysis (220 sample size). Then, the clinical validation was finished with 20 AMI and 20 stable coronary artery disease patients (SCAD). At a ratio of 5:2, GSE59867 was included in the training set, while GSE60993, GSE62646, and GSE48060 were included in the testing set. IML was explicitly proposed in this research, which is composed of six machine learning algorithms, including support vector machine (SVM), neural network (NN), random forest (RF), gradient boosting machine (GBM), decision trees (DT), and least absolute shrinkage and selection operator (LASSO). IML had two functions in this research: filtered optimized variables and predicted the categorized value. Finally, The RNA of the recruited patients was analyzed to verify the results of IML. Results: Thirty-nine differentially expressed genes (DEGs) were identified between controls and AMI individuals from the training sets. Among the thirty-nine DEGs, IML was used to process the predicted classification model and identify potential candidate genes with overall normalized weights > 1. Finally, two genes (AQP9 and SOCS3) show their diagnosis value with the area under the curve (AUC) > 0.9 in both the training and testing sets. The clinical study verified the significance of AQP9 and SOCS3. Notably, more stenotic coronary arteries or severe Killip classification indicated higher levels of these two genes, especially SOCS3. These two genes correlated with two immune cell types, monocytes and neutrophils. Conclusion: AQP9 and SOCS3 in leukocytes may be conducive to identifying AMI patients with SCAD patients. AQP9 and SOCS3 are closely associated with monocytes and neutrophils, which might contribute to advancing AMI diagnosis and shed light on novel genetic markers. Multiple clinical characteristics, multicenter, and large-sample relevant trials are still needed to confirm its clinical value. [ABSTRACT FROM AUTHOR]

Polycystic ovary syndrome (PCOS) is one of the most common causes of infertility in women. Approximately half of the diagnosed individuals also experience the metabolic syndrome. Central and peripheral resistance to the hormones insulin and leptin have been reported to contribute to both metabolic and reproductive dysregulation. In PCOS and preclinical PCOS animal models, circulating insulin and leptin levels are often increased in parallel with the development of hormone resistance; however, it remains uncertain whether these changes contribute to the PCOS state. In this study, we tested whether central actions of protein tyrosine phosphatase 1B (PTP1B) and suppressor of cytokine signaling 3 (SOCS3), negative regulators of insulin and leptin signaling pathways, respectively, play a role in the development of PCOS-like phenotype. A peripubertal dihydrotestosterone (DHT) excess PCOS-like mouse model was used, which exhibits both metabolic and reproductive dysfunction. Mice with knockout of the genes encoding PTP1B and SOCS3 from forebrain neurons were generated, and metabolic and reproductive functions were compared between knockout and control groups. DHT treatment induced mild insulin resistance but not leptin resistance, so the role of SOCS3 could not be tested. As expected, DHT excess abolished estrous cycles and corpora lutea presence and caused increased visceral adiposity and fasting glucose levels. Knockout mice did not show any rescue of reproductive dysfunction but did have reduced adiposity compared to the control DHT mice. These data suggest that negative regulation of central insulin signaling by PTP1B is not responsible for peripubertal DHT excess-induced reproductive impairments but may mediate its increased adiposity effects. [ABSTRACT FROM AUTHOR]

Abstract Background The transmembrane receptor Kremen2 has been reported to participate in the tumorigenesis and metastasis of gastric cancer. However, the role of Kremen2 in non-small cell lung cancer (NSCLC) and the underlying mechanism remain unclear. This study aimed to explore the biological function and regulatory mechanism of Kremen2 in NSCLC. Methods The correlation between Kremen2 expression and NSCLC was assessed by analyzing the public database and clinical tissue samples. Colony formation and EdU assays were performed to examine cell proliferation. Transwell and wound healing assays were used to observe cell migration ability. Tumor-bearing nude mice and metastatic tumor models were used to detect the in vivo tumorigenic and metastatic abilities of the NSCLC cells. An immunohistochemical assay was used to detect the expression of proliferation-related proteins in tissues. Western blot, immunoprecipitation and immunofluorescence were conducted to elucidate the Kremen2 regulatory mechanisms in NSCLC. Results Kremen2 was highly expressed in tumor tissues from NSCLC patients and was positively correlated with a poor patient prognosis. Knockout or knockdown of Kremen2 inhibited cell proliferation and migration ability of NSCLC cells. In vivo knockdown of Kremen2 inhibited the tumorigenicity and number of metastatic nodules of NSCLC cells in nude mice. Mechanistically, Kremen2 interacted with suppressor of cytokine signaling 3 (SOCS3) to maintain the epidermal growth factor receptor (EGFR) protein levels by preventing SOCS3-mediated ubiquitination and degradation of EGFR, which, in turn, promoted activation of the PI3K-AKT and JAK2-STAT3 signaling pathways. Conclusions Our study identified Kremen2 as a candidate oncogene in NSCLC and may provide a potential target for NSCLC treatment.

Background: Cancer stem cells (CSCs) are different from regular cancer cells because of their self-renewal feature and differentiation potential, which establishes the backbone of the vital role of CSCs in the progress and drug resistance of hepatocellular carcinoma (HCC). The objective of this study was to evaluate the effects of blood exosome-derived miRNA-30d-5p on the stemness and gemcitabine resistance of HCC cells and the underlying mechanisms. Methods: The expression data of HCC-related miRNAs and mRNAs were downloaded from TCGA database and analyzed for differences. Employing the databases of starBase, TargetScan, miRDB, and mirDIP, we conducted target gene prediction upstream of mRNA. The expression of miRNA-30d-5p and SOCS3 mRNA was assayed by qRT-PCR, and the binding between them was validated by dual luciferase assay. CCK-8 was employed to evaluate cell viability and the IC50 value of gemcitabine. Cells were subjected to a sphere-forming assay to assess their ability to form spheres. Western blot was applied to evaluate the levels of cell surface marker proteins (Nanog, CD133, and Oct4) and exosome markers (CD9, CD81, and FLOT1). Results: Bioinformatics analysis found that SOCS3 expression was down-regulated in HCC. qRT-PCR showed that SOCS3 expression was notably lower in HCC cell lines than in normal liver cell WRL68. At the cellular functional level, SOCS3 overexpression inhibited the viability, sphere-forming ability, stemness, and gemcitabine resistance of HCC cells. Bioinformatics analysis demonstrated that miRNA-30d-5p was the upstream regulator of SOCS3 and highly expressed in HCC tissues and cells. Dual luciferase assay demonstrated that miRNA-30d-5p could bind SOCS3. Rescue experiments showed that upregulating SOCS3 could reverse the effects of miRNA-30d-5p overexpression on the viability, sphere-forming ability, and gemcitabine sensitivity of HCC cells. Conclusions: Blood exosome-derived miRNA-30d-5p promoted the stemness and gemcitabine resistance of HCC cells by repressing SOCS3 expression. Hence, the miRNA-30d-5p/SOCS3 axis might be a therapeutic target for chemotherapy resistance and a feasible marker for the prognosis of HCC patients.

Tumor microenvironment is characterized by the occurrence of significant changes due to disrupted signaling pathways that affect a broad spectrum of cellular activities such as proliferation, differentiation, signaling, invasiveness, migration, and apoptosis. Similarly, a downregulated suppressor of cytokine signaling 3 (SOCS3) promotes increased JAK/STAT function due to aberrant cytokine signaling, which results in increased cell proliferation, differentiation, and migration. Multiple carcinomas including breast cancer, prostate cancer, hepatocellular carcinoma, pancreatic cancer, and colorectal cancer involve the disruption of SOCS3 expression due to microRNA overexpression. MicroRNAs are small, conserved, and non-coding RNA molecules that regulate gene expression through post-transcriptional inhibition and mRNA destabilization. The aim of this study was to identify putative microRNAs that interact with SOCS3 and downregulate its expression. In this study, miRWalk, TargetScan, and miRDB were used to identify microRNAs that interact with SOCS3, whereas RNA22 was utilized to identify the binding sites of 238 significant microRNAs. The tertiary structures of shortlisted microRNAs and SOCS3 regions were predicted through MC Sym and RNAComposer, respectively. For molecular docking, HDOCK was used, which predicted 80 microRNA-messengerRNA complexes and the interactions of the top 5 shortlisted complexes were assessed. The complexes were shortlisted on the basis of least binding affinity score and maximum confidence score. This study identifies the interactions of known (miR-203a-5p) and novel (miR-6756-5p, miR-6732-5p, miR-1203, miR-6887-5p) microRNAs with SOCS3 regions due to their maximum interactions. Identifying the interactions of these microRNAs with SOCS3 will significantly advance the understanding of oncomiRs (miRNAs that are associated with cancer development) in tumor development due to their influence on SOCS3 expression. These insights will assist in future studies to understand the significance of miRNA-SOCS3-associated tumor development and progression. [ABSTRACT FROM AUTHOR]

Background: Chemotherapy-induced peripheral neuropathy (CIPN) is a severe adverse reaction to chemotherapeutics, which seriously affects the outcome of chemotherapy and patients' quality of life. Although it is commonly seen, it lacks effective treatment. Our previous study found that ozone could alleviate neuropathic pain. Damage-associated molecular patterns (DAMPs) or Toll-like receptor 4 (TLR4) or tissue factor (TF)-mediated neuroinflammation and microcirculation disturbance is the main reason for CIPN. Suppressors of cytokine signaling (SOCS) 3 is an endogenous negative feedback regulator of inflammation via TLR4 inhibition. Materials and Methods: Oxaliplatin (L-OHP) was used to establish mice's CIPN model. Nociceptive responses were assessed by observing the ICR mice's incidence of foot regression in mechanical indentation response experiments. Cell signaling assays were performed by Western blotting and immunohistochemistry. The mouse leukemia cells of monocyte--macrophage line RAW 264.7 were cultured to investigate the effects of ozone administration on macrophage. Results: Ozone decreased the expression of TF in the blood and sciatic nerve. It upregulated the adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK)-SOCS3 axis to relieve CIPN and inhibit TF expression in vivo. SOCS3 expression was induced by ozone to inhibit the p38/TF signaling in RAW 246.7 cells. Ozone also prevented L-OHP-induced sciatic nerve demyelination. Microglia activation was inhibited, and c-Fos and calcitonin gene-related peptide (CGRP) expression was decreased in the spinal dorsal horn via ozone. Conclusions: In this study, we demonstrated that ozone could alleviate CIPN by upregulating the AMPK-SOCS3 axis to inhibit TF expression, which is a potential treatment for CIPN. [ABSTRACT FROM AUTHOR]

Objective: To investigate the expression inhibition and correlation of indoleamine 2, 3-dioxygenase （IDO） and cytokine signal transduction inhibitor protein-3 （SOCS3） in placenta of patients with hypertensive disorder complicating pregnancy （HDP）. Methods: The expressions of IDO and SOCS3 in placenta of HDP were detected by Western blot method, and the expressions and correlation of them in placenta were analyzed. Results: IDO and SOCS3 were expressed in placenta of patients with HDP and normal pregnancy, and their expression was negatively correlated with the progression of HDP. There was a positive correlation between the expressions of IDO and SOCS3 in placenta of the three groups, and the correlation in preeclampsia group was higher than that in HDP group and normal pregnancy group, and the correlation in HDP group was significantly higher than that in normal pregnancy group. Conclusion: IDO and SOCS3 play an important role in immune tolerance in the third trimester of pregnancy, and may be involved in the pathogenesis of HDP. IDO and SOCS3 expressions and the degree of correlation can pre-reveal the progression of HPD. [ABSTRACT FROM AUTHOR]

Suppressor of cytokine signaling 3 (SOCS3) is a negative regulatory protein that has been identified as a key inhibitory regulator of JAK/STAT signaling pathway. However, the mutual regulatory relationship between SOCS3 and JAK2/STAT3 signaling pathway after vocal fold injury remains unclear. In this study, we used small interfering RNA (siRNA) to investigate the mechanism of SOCS3 regulating of fibroblasts through JAK2/STAT3 signaling pathway after vocal fold injury. Our data shows that SOCS3 silencing promotes the transformation of normal vocal fold fibroblasts (VFFs) into an fibrotic phenotype and activates the JAK2/STAT3 signaling pathway. JAK2 silencing significantly inhibits the increase in type I collagen and α-smooth muscle actin (α-SMA) secretion in VFFs induced by TGF-β but has no significant effect on normal VFFs. The silencing of SOCS3 and JAK2 reverses the fibrotic phenotype of VFFs induced by SOCS3 silencing. Therefore, we suggest that SOCS3 can affect the activation of vocal fold fibroblasts by regulating the JAK2/STAT3 signaling pathway after vocal fold injury. It provides a new insight for promoting the repair of vocal fold injury and preventing the formation of fibrosis. [ABSTRACT FROM AUTHOR]

Nonalcoholic fatty liver disease is caused by an imbalance in lipid metabolism and immune response to pose a risk factor for liver fibrosis. Recent evidence indicates that M2 macrophages secrete transforming growth factor-β1, which contributes to liver fibrosis. Galectin-12 has been demonstrated to regulate lipid metabolism and macrophage polarization. The purpose of this study is to investigate the role of galectin-12 in the development of nonalcoholic fatty liver disease and fibrosis. Liver tissue from wild-type C57BL/6 mice fed with a high-fat diet containing cholesterol and cholic acid for 4–12 weeks was used to examine galectin-12 expression and its correlation with nonalcoholic fatty liver disease. Furthermore, the effects of galectin-12 on M2 macrophages during the progression of nonalcoholic fatty liver disease were investigated by studying Kupffer cells from galectin-12 knockout mice and doxycycline-inducible Gal12−/–THP-1 cells. Ablation of galectin-12 promoted M2 polarization of Kupffer cells, as indicated by higher levels of M2 markers, such as arginase I and chitinase 3-like protein 3. Furthermore, the activation of signal transducer and activator of transcription 6 was significantly higher in Gal12−/− macrophages activated by interleukin-4, which was correlated with higher levels of transforming growth factor-β1. Moreover, Gal12−/− macrophage-conditioned medium promoted hepatic stellate cells myofibroblast differentiation, which was indicated by higher α-smooth muscle actin expression levels compared with those treated with LacZ control medium. Finally, we demonstrated that galectin-12 knockdown negatively regulated the suppressor of cytokine signaling 3 levels. These findings suggested that galectin-12 balances M1/M2 polarization of Kupffer cells to prevent nonalcoholic fatty liver disease progression. [ABSTRACT FROM AUTHOR]