Inhibition Of SOCS3 Research Articles

Longitudinal single cell atlas identifies complex temporal relationship between type I interferon response and COVID-19 severity

Due to the paucity of longitudinal molecular studies of COVID-19, particularly those covering the early stages of infection (Days 1-8 symptom onset), our understanding of host response over the disease course is limited. We perform longitudinal single cell RNA-seq on 286 blood samples from 108 age- and sex-matched COVID-19 patients, including 73 with early samples. We examine discrete cell subtypes and continuous cell states longitudinally, and we identify upregulation of type I IFN-stimulated genes (ISGs) as the predominant early signature of subsequent worsening of symptoms, which we validate in an independent cohort and corroborate by plasma markers. However, ISG expression is dynamic in progressors, spiking early and then rapidly receding to the level of severity-matched non-progressors. In contrast, cross-sectional analysis shows that ISG expression is deficient and IFN suppressors such as SOCS3 are upregulated in severe and critical COVID-19. We validate the latter in four independent cohorts, and SOCS3 inhibition reduces SARS-CoV-2 replication in vitro. In summary, we identify complexity in type I IFN response to COVID-19, as well as a potential avenue for host-directed therapy.

Nifuroxazide repurposing for protection from diabetes-induced retinal injury in rats: Implication of oxidative stress and JAK/STAT3 axis.

The prevalence of diabetes mellitus (DM) is alarmingly increasing worldwide. Diabetic retinopathy (DR) is a prevailing DM microvascular complication, representing the major cause of blindness in working-age population. Inflammation is a crucial player in DR pathogenesis. JAK/STAT3 axis is a pleotropic cascade that modulates diverse inflammatory events. Nifuroxazide (Nifu) is a commonly used oral antibiotic with reported JAK/STAT3 inhibition activity. The present study investigated the potential protective effect of Nifu against diabetes-induced retinal injury. Effect of Nifu on oxidative stress, JAK/STAT3 axis and downstream inflammatory mediators has been also studied. Diabetes was induced in Sprague Dawley rats by single intraperitoneal injection of streptozotocin (50 mg/kg). Animals were assigned into four groups: normal, Nifu control, DM, and DM + Nifu. Nifu was orally administrated at 25 mg/kg/day for 8 weeks. The effects of Nifu on oxidative stress, JAK/STAT3 axis proteins, inflammatory factors, tight junction proteins, histological, and ultrastructural alterations were evaluated using spectrophotometry, gene and protein analyses, and histological studies. Nifu administration to diabetic rats attenuated histopathological and signs of retinal injury. Additionally, Nifu attenuated retinal oxidative stress, inhibited JAK and STAT3 phosphorylation, augmented the expression of STAT3 signaling inhibitor SOCS3, dampened the expression of transcription factor of inflammation NF-κB, and inflammatory cytokine TNF-α. Collectively, the current study indicated that Nifu alleviated DR progression in diabetic rats, suggesting beneficial retino-protective effect. This can be attributed to blocking JAK/STAT3 axis in retinal tissues with subsequent amelioration of oxidative stress and inflammation.

Identification and validation of SOCS1/2/3/4 as potential prognostic biomarkers and correlate with immune infiltration in glioblastoma.

Suppressor of cytokine signalling (SOCS) 1/2/3/4 are involved in the occurrence and progression of multiple malignancies; however, their prognostic and developmental value in patients with glioblastoma (GBM) remains unclear. The present study used TCGA, ONCOMINE, SangerBox3.0, UALCAN, TIMER2.0, GENEMANIA, TISDB, The Human Protein Atlas (HPA) and other databases to analyse the expression profile, clinical value and prognosis of SOCS1/2/3/4 in GBM, and to explore the potential development mechanism of action of SOCS1/2/3/4 in GBM. The majority of analyses showed that SOCS1/2/3/4 transcription and translation levels in GBM tissues were significantly higher than those in normal tissues. qRT-PCR, western blotting (WB) and immunohistochemical staining were used to verify that SOCS3 was expressed at higher mRNA and protein levels in GBM than in normal tissues or cells. High SOCS1/2/3/4 mRNA expression was associated with poor prognosis in patients with GBM, especially SOCS3. SOCS1/2/3/4 were highly contraindicated, which had few mutations, and were not associated with clinical prognosis. Furthermore, SOCS1/2/3/4 were associated with the infiltration of specific immune cell types. In addition, SOCS3 may affect the prognosis of patients with GBM through JAK/STAT signalling pathway. Analysis of the GBM-specific protein interaction (PPI) network showed that SOCS1/2/3/4 were involved in multiple potential carcinogenic mechanisms of GBM. In addition, colony formation, Transwell, wound healing and western blotting assays revealed that inhibition of SOCS3 decreased the proliferation, migration and invasion of GBM cells. In conclusion, the present study elucidated the expression profile and prognostic value of SOCS1/2/3/4 in GBM, which may provide potential prognostic biomarkers and therapeutic targets for GBM, especially SOCS3.

SOCS3 Protein Mediates the Therapeutic Efficacy of Mesenchymal Stem Cells against Acute Lung Injury

Mesenchymal stem cells (MSCs) have been studied as novel therapeutic agents because of their immunomodulatory properties in inflammatory diseases. The suppressor of cytokine signaling (SOCS) proteins are key regulators of the immune response and macrophage modulation. In the present study, we hypothesized that SOCS in MCSs might mediate macrophage modulation and tested this in a bacteria-induced acute lung injury (ALI) mouse model. The macrophage phenotype was observed in RAW264.7 alveolar macrophages exposed to lipopolysaccharide (LPS) in an in vitro model, and in the ALI mouse model induced by tracheal administration of Escherichia coli (1 × 107 CFU in 0.05mL PBS). In LPS-exposed RAW264.7 cells, the levels of markers of M1 macrophages, such as CD86 and pro-inflammatory cytokines (IL-1α, IL-1β, IL-6 and TNF-α), significantly increased, but they significantly reduced after MSC treatment. Meanwhile, the levels of markers of M2 macrophages, such as CD204 and anti-inflammatory cytokines (IL-4 and IL-10), increased after LPS exposure, and further significantly increased after MSC treatment. This regulatory effect of MSCs on M1/M2 macrophage polarization was significantly abolished by SOCS3 inhibition. In the E. coli-induced ALI model, tissue injury and inflammation in the mouse lung were significantly attenuated by the transplantation of MSCs, but not by SOCS3-inhibited MSCs. The regulatory effect of MSCs on M1/M2 macrophage polarization was observed in the lung injury model but was significantly abolished by SOCS3 inhibition. Taken together, our findings suggest that SOCS3 is an important mediator for macrophage modulation in anti-inflammatory properties of MSCs.

LncRNA KIAA0087 suppresses the progression of osteosarcoma by mediating the SOCS1/JAK2/STAT3 signaling pathway

Long noncoding RNAs (lncRNAs), widely expressed in mammalian cells, play pivotal roles in osteosarcoma (OS) progression. Nevertheless, the detailed molecular mechanisms of lncRNA KIAA0087 in OS remain obscure. Here, the roles of KIAA0087 in OS tumorigenesis were investigated. KIAA0087 and miR-411-3p levels were detected by RT-qPCR. Malignant properties were assessed by CCK-8, colony formation, flow cytometry, wound healing, and transwell assays. SOCS1, EMT, and JAK2/STAT3 pathway-related protein levels were measured by western blotting. Direct binding between miR-411-3p and KIAA0087/SOCS1 was validated by a dual-luciferase reporter, RIP, and FISH assays. In vivo growth and lung metastasis were evaluated in nude mice. The expression levels of SOCS1, Ki-67, E-cadherin, and N-cadherin in tumor tissues were measured by immunohistochemical staining. Downregulation of KIAA0087 and SOCS1 and upregulation of miR-411-3p were found in OS tissues and cells. Low expression of KIAA0087 was associated with a poor survival rate. Forced expression of KIAA0087 or miR-411-3p inhibition repressed the growth, migration, invasion, EMT, and activation of the JAK2/STAT3 pathway and triggered apoptosis of OS cells. However, the opposite results were found with KIAA0087 knockdown or miR-411-3p overexpression. Mechanistic experiments indicated that KIAA0087 enhanced SOCS1 expression to inactivate the JAK2/STAT3 pathway by sponging miR-411-3p. Rescue experiments revealed that the antitumor effects of KIAA0087 overexpression or miR-411-3p suppression were counteracted by miR-411-3p mimics or SOCS1 inhibition, respectively. Finally, in vivo tumor growth and lung metastasis were inhibited in KIAA0087-overexpressing or miR-411-3p-inhibited OS cells. In summary, the downregulation of KIAA0087 promotes the growth, metastasis, and EMT of OS by targeting the miR-411-3p-mediated SOCS1/JAK2/STAT3 pathway.

LncRNA TSIX knockdown restores spinal cord injury repair through miR-30a/SOCS3 axis

ABSTRACT Spinal cord injury (SCI) is a serious injury to the central nervous system. Previous studies have discovered that the development of SCI is associated with gene expression. The purpose of this study was to explore the significance of lncRNA TSIX in SCI and its underlying mechanism involved. An in vivo SCI mice model and an in vitro hypoxia-treated HT22 cells model were applied in this study. TSIX and SOCS3 expression in SCI tissues was measured by qRT-PCR, western blot and FISH assay. LV-sh-TSIX was injected into SCI mice intrathecally or subjected to HT22 cells to access the consequent alteration in inflammation response, cell apoptosis and functional recovery through ELISA, immunohistochemistry, TUNEL, flow cytometry assays and BMS scores. Then, the underlying mechanism of TSIX was analyzed by bioinformatics analysis and then confirmed by RIP, RNA pull-down and dual-luciferase reporter assay. It was identified that TSIX was up-regulated in HT22 cells under hypoxia operation and spinal cord tissues of SCI mice. TSIX knockdown improved the lesion size and BMS score and inhibited inflammation and cell apoptosis. MiR-30a was identified as a target for TSIX and SOCS3, and TSIX binds to miR-30a by competing with SOCS3, thereby counteracting miR-30a-mediated SOCS3 inhibition. In addition, LV-sh-TSIX effects were significantly overturned by miR-30a inhibition or SOCS3 over-expression. Knockdown of TSIX improved functional recovery and attenuated the inflammation response and cell apoptosis via miR-30a/SOCS3 axis. These results may provide a potential novel insight for SCI treatment.

The SOCS1 KIR and SH2 domain are both required for suppression of cytokine signaling in vivo

Suppressor Of Cytokine Signaling (SOCS) 1 is a critical negative regulator of cytokine signaling and required to protect against an excessive inflammatory response. Genetic deletion of Socs1 results in unrestrained cytokine signaling and neonatal lethality, characterised by an inflammatory immune infiltrate in multiple organs. Overexpression and structural studies have suggested that the SOCS1 kinase inhibitory region (KIR) and Src homology 2 (SH2) domain are important for interaction with and inhibition of the receptor-associated JAK1, JAK2 and TYK2 tyrosine kinases, which initiate downstream signaling. To investigate the role of the KIR and SH2 domain in SOCS1 function, we independently mutated key conserved residues in each domain and analysed the impact on cytokine signaling, and the in vivo impact on SOCS1 function. Mutation of the SOCS1-KIR or SH2 domain had no impact on the integrity of the SOCS box complex, however, mutation within the phosphotyrosine binding pocket of the SOCS1-SH2 domain specifically disrupted SOCS1 interaction with phosphorylated JAK1. In contrast, mutation of the KIR did not affect the interaction with JAK1, but did prevent SOCS1 inhibition of JAK1 autophosphorylation. In human and mouse cell lines, both mutants impacted the ability of SOCS1 to restrain cytokine signaling, and crucially, Socs1-R105A and Socs1-F59A mice displayed a neonatal lethality and excessive inflammatory phenotype similar to Socs1-null mice. This study defines a critical and non-redundant role for both the KIR and SH2 domain in endogenous SOCS1 function.

LncRNA CASC2 Regulate Cell Proliferation and Invasion by Targeting miR-155/SOCS1 Axis in Hepatocellular Carcinoma.

Long noncoding RNAs (lncRNAs) have been reported to be involved in the development and progression of various human malignancies. However, the role of lncRNA CASC2 in hepatocellular carcinoma (HCC) remains mostly unknown. The aim of this study was to investigate the potential roles and underlying mechanisms of CASC2 in HCC progression. We found that CASC2 expressions were downregulated in HCC tissue samples and cell lines. The clinical assays revealed that lower levels of CASC2 were associated with the TNM stage, lymph node metastasis, and a poorer prognosis specific to HCC patients. Overexpression of CASC2 inhibited the proliferating, migratory, and invasion capacity of HCC cells. Bioinformatics analysis and the luciferase reporter assay revealed that CASC2 worked as a molecular sponge for miR-155. And CASC2 could upregulate SOCS1 expression by inhibiting miR-155 expression in HCC cells. Furthermore, SOCS1 inhibition partially inverses the suppression effect of cell proliferation, migration, and invasion regulated by CASC2 in Huh7 and HepG2 cells. Taken together, our findings identified CASC2 as a tumor suppressor to inhibit HCC development by regulating the miR-155/SOCS1 axis, and CASC2 might be a potential therapeutic target of HCC for future clinical treatment.

Protein interaction, cytotoxic, transcriptomic and proteomic responses to structurally distinct EPAC1 activators in HUVECs

The N-acylsulfonamide derivative, I942, represents the first non-cyclic nucleotide partial agonist of EPAC1. This was soon followed by the identification of the I942 analogues, PW0381, PW0521 and PWO577 and a series of benzofuran oxoacetic acid EPAC1 activators, SY006, SY007 and SY009. Protein interaction, cytotoxicity and EPAC1 activation assays applied here identify PWO577 and SY007 as being effective EPAC1 binders that are well tolerated in HUVECs at concentrations greater than 100 μM and up to 48 h incubation and are effective activators of transfected EPAC1 in U2OS cells. Using RNAseq in HUVECs we show that PWO577 and SY007 regulate approximately 11,000 shared genes, with only few differential gene changes being “off-target”. The genes significantly regulated by both PWO577 and SY007 included a subset of genes normally associated with endothelial activation, including ICAM1, MMP1 and CCL2. Of these, only the expression of MMP1 was markedly increased at the protein level, as determined by LC–MS-based proteomics. Both PWO577 and SY007 suppressed IL-6-induced STAT3 activation and associated downstream gene expression, including inhibition of SOCS3, STAT3, IL6ST and JAK3 genes. Together these results demonstrate the utility of structurally distinct, specific and non-toxic EPAC1 activators. Future modifications will be aimed at eliminating the few noted off-target effects.

Breast cancer cell-derived microRNA-155 suppresses tumor progression via enhancing immune cell recruitment and antitumor function.

Evidence suggests that increased microRNA-155 (miR-155) expression in immune cells enhances antitumor immune responses. However, given the reported association of miR-155 with tumorigenesis in various cancers, a debate is provoked on whether miR-155 is oncogenic or tumor suppressive. We aimed to interrogate the impact of tumor miR-155 expression, particularly that of cancer cell–derived miR-155, on antitumor immunity in breast cancer. We performed bioinformatic analysis of human breast cancer databases, murine experiments, and human specimen examination. We revealed that higher tumor miR-155 levels correlate with a favorable antitumor immune profile and better patient outcomes. Murine experiments demonstrated that miR-155 overexpression in breast cancer cells enhanced T cell influx, delayed tumor growth, and sensitized the tumors to immune checkpoint blockade (ICB) therapy. Mechanistically, miR-155 overexpression in breast cancer cells upregulated their CXCL9/10/11 production, which was mediated by SOCS1 inhibition and increased phosphorylated STAT1 (p-STAT1)/p-STAT3 ratios. We further found that serum miR-155 levels in breast cancer patients correlated with tumor miR-155 levels and tumor immune status. Our findings suggest that high serum and tumor miR-155 levels may be a favorable prognostic marker for breast cancer patients and that therapeutic elevation of miR-155 in breast tumors may improve the efficacy of ICB therapy via remodeling the antitumor immune landscape.

Pneumolysin suppresses the initial macrophage pro-inflammatory response to Streptococcuspneumoniae.

Published data for the Streptococcus pneumoniae virulence factor Pneumolysin (Ply) show contradictory effects on the host inflammatory response to infection. Ply has been shown to activate the inflammasome, but also can bind to MRC-1 resulting in suppression of dendritic cell inflammatory responses. We have used an in vitro infection model of human monocyte-derived macrophages (MDM), and a mouse model of pneumonia to clarify whether pro- or anti-inflammatory effects dominate the effects of Ply on the initial macrophage inflammatory response to S. pneumoniae, and the consequences during early lung infection. We found that infection with S. pneumoniae expressing Ply suppressed tumour necrosis factor (TNF) and interleukin-6 production by MDMs compared to cells infected with ply-deficient S. pneumoniae. This effect was independent of bacterial effects on cell death. Transcriptional analysis demonstrated S. pneumoniae expressing Ply caused a qualitatively similar but quantitatively lower MDM transcriptional response to S. pneumoniae compared to ply-deficient S. pneumoniae, with reduced expression of TNF and type I IFN inducible genes. Reduction of the MDM inflammatory response was prevented by inhibition of SOCS1. In the early lung infection mouse model, the TNF response to ply-deficient S. pneumoniae was enhanced and bacterial clearance increased compared to infection with wild-type S. pneumoniae. Overall, these data show Ply inhibits the initial macrophage inflammatory response to S. pneumoniae, probably mediated through SOCS1, and this was associated with improved immune evasion during early lung infection.

SOCS3 limits TNF and endotoxin-induced endothelial dysfunction by blocking a required autocrine interleukin-6 signal in human endothelial cells.

Increased circulating levels of soluble interleukin (IL)-6 receptor α (sIL-6Rα) are commonly observed during inflammatory responses, allowing for IL-6 signaling in cells that express the ubiquitous receptor subunit gp130 but not IL-6Rα, such as endothelial cells. Activation of Toll-like receptor (TLR)-4 or the tumor necrosis factor (TNF) receptor leads to NF-κB-dependent increases in endothelial IL-6 expression. Thus, we hypothesize that danger signals may induce autocrine IL-6 signaling within the endothelium via sIL-6Rα-mediated trans-signaling. In support of this hypothesis, we recently demonstrated that conditional deletion in the endothelium of the IL-6 signaling inhibitor SOCS3 leads to rapid mortality in mice challenged with the TLR-4 agonist endotoxin through increases in vascular leakage, thrombosis, leukocyte adhesion, and a type I-like interferon response. Here, we sought to directly test a role for sIL-6Rα in LPS-treated human umbilical vein and dermal blood microvascular endothelial cells. We show that cotreatment with sIL-6Rα dramatically increases the loss of barrier function and the expression of COX2 and tissue factor mRNA levels induced by LPS. This cotreatment led to strong activation of STAT1 and STAT3 while not affecting LPS-induced activation of p38 and NF-κB signaling. Similar results were obtained when sIL-6Rα was added to a TNF challenge. JAK inhibition by pretreatment with ruxolitinib or by SOCS3 overexpression blunted LPS and sIL-6R synergistic effects, whereas SOCS3 knockdown further increased the response. Together, these findings demonstrate that IL-6 signaling downstream of NF-κB activation leads to a strong endothelial activation and may explain the acute endotheliopathy observed during critical illness.

Effect of microRNA-455-5p (miR-455-5p) on the Expression of the Cytokine Signaling-3 (SOCS3) Gene During Myocardial Infarction.

To explore the effect of microRNA-455-5p (miR-455-5p) and Cytokine Signaling-3 (SOCS3) expression, a model of the cell damage induced during myocardial infarction was established using H₂O₂. The cell counting Kit-8 (CCK-8) and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) assays were used to detect the cell viability and the expression of miR-455-5p and SOCS3 in cells cultured with different concentrations of H₂O₂. After the selection of the optimum culture concentration, a dual-luciferase reporter gene assay was used to detect the binding between and miR-455-5p and its potential target SOCS3. SOCS3 siRNA was transfected into cardiomyocytes using chitosan nanoparticles as a gene carrier, which led to the knockdown of SOCS3 expression, and the cells were transfected with miR-455-5p mimics and inhibitors. The expression of cardiac protective proteins was detected by western blotting, cell viability was detected by CCK8, and cell apoptosis was detected by flow cytometry. The aim of this study was to investigate the effect of miR-455-5p and SOCS3 expression on the activity and apoptosis of damaged cardiomyocytes, and to identify any protective effect on cardiomyocytes. Finally, after the simultaneous overexpression of SOCS3 and miR-455-5p, and the expression of cardiac protective proteins, cell activity, and apoptosis rate were detected. The results showed that the expression of miR-455-5p decreased in a concentration-dependent manner and that the expression of SOCS3 increased in a concentration-dependent manner when the cells were cultured in different concentrations of H₂O₂. The knockdown of SOCS3 expression promoted an increase in cell activity, an increase in cardiac protective proteins, and a decrease in apoptosis. The upregulation of miR-455-5p significantly inhibited the expression of SOCS3, increased cell activity, inhibited apoptosis, and exerted protective effects in myocardial cells. The overexpression of SOCS3 reversed the inhibition of SOCS3 by miR-455-5p and reduced the protective effect of miR-455-5p on myocardial cells. Therefore, this study showed that the upregulation of miR-455-5p significantly inhibited the expression of SOCS3 and resulted in the increased protection of cells damaged by H₂O₂, which was used as a model of myocardial infarction. These results indicate the potential of miR-455-5p in myocardial protection, suggesting that miRNA may be a resource for myocardial therapy.

MiR-181a is a novel player in the STAT3-mediated survival network of TCR\u03b1\u03b2+ CD8+ T large granular lymphocyte leukemia

T-LGL cells arise as a consequence of chronic antigenic stimulation and inflammation and thrive because of constitutive activation of the STAT3 and ERK pathway. Notably, in 40% of patients, constitutive STAT3 activation is due to STAT3 activating mutations, whereas in 60% this is unknown. As miRNAs are amongst the most potent regulators in health and disease, we hypothesized that aberrant miRNA expression could contribute to dysregulation of these pathways. miRNA sequencing in T-LGL leukemia cases and aged-matched healthy control TEMRA cells revealed overexpression of miR-181a. Furthermore, geneset enrichment analysis (GSEA) of downregulated targets of miR-181a implicated involvement in regulating STAT3 and ERK1/2 pathways. Flow cytometric analyses showed increased SOCS3+ and DUSP6+ T-LGL cells upon miR-181a inhibition. In addition, miR-181a-transfected human CD8+ T cells showed increased basal STAT3 and ERK1/2 phosphorylation. By using TL1, a human T-LGL cell line, we could show that miR-181a is an actor in T-LGL leukemia, driving STAT3 activation by SOCS3 inhibition and ERK1/2 phosphorylation by DUSP6 inhibition and verified this mechanism in an independent cell line. In addition, miR-181a inhibition resulted in a higher sensitivity to FAS-mediated apoptosis. Collectively, our data show that miR-181a could be the missing link to explain why STAT3-unmutated patients show hyperactive STAT3.

SOCS3 is Related to Cell Proliferation in Neuronal Tissue: An Integrated Analysis of Bioinformatics and Experiments.

Glioma is the most common primary malignant tumor that occurs in the central nervous system. Gliomas are subdivided according to a combination of microscopic morphological, molecular, and genetic factors. Glioblastoma (GBM) is the most aggressive malignant tumor; however, efficient therapies or specific target molecules for GBM have not been developed. We accessed RNA-seq and clinical data from The Cancer Genome Atlas, the Chinese Glioma Genome Atlas, and the GSE16011 dataset, and identified differentially expressed genes (DEGs) that were common to both GBM and lower-grade glioma (LGG) in three independent cohorts. The biological functions of common DEGs were examined using NetworkAnalyst. To evaluate the prognostic performance of common DEGs, we performed Kaplan-Meier and Cox regression analyses. We investigated the function of SOCS3 in the central nervous system using three GBM cell lines as well as zebrafish embryos. There were 168 upregulated genes and 50 downregulated genes that were commom to both GBM and LGG. Through survival analyses, we found that SOCS3 was the only prognostic gene in all cohorts. Inhibition of SOCS3 using siRNA decreased the proliferation of GBM cell lines. We also found that the zebrafish ortholog, socs3b, was associated with brain development through the regulation of cell proliferation in neuronal tissue. While additional mechanistic studies are necessary, our results suggest that SOCS3 is an important biomarker for glioma and that SOCS3 is related to the proliferation of neuronal tissue.

Investigation of the role of miR-221 in diabetic peripheral neuropathy and related molecular mechanisms.

Diabetic peripheral neuropathy (DPN) is one of the most common complications of diabetes, but the molecular mechanisms of DPN are still unclear. To investigate the role of miR-221 in DPN and the related molecular mechanisms. Streptozotocin (STZ) was used to establish an in vivo DPN model. An in vitro DPN model was established using high glucose-induced SH-SY5Y cells. The pain condition of rats was measured by evaluating the 50% paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). Serum exosomes were extracted and identified. Expression of miR-221 in serum exosomes and serum SOCS3 expression were determined using reverse-transcription quantitative polymerase chain reaction (RT-qPCR). Western blotting was used to measure the protein levels of SOCS3, bradykinin (BK) and prostaglandin E2 (PEG2). The dual luciferase reporter assay was performed to confirm SOCS3 3'-UTR as a target of miR-221. The serum or cell supernatant levels of PEG2, BK, interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α) were measured using enzyme-linked immunosorbent assay (ELISA). Induction of the lenti-miR-221 inhibitor significantly decreased the expression of miR-221 in DPN rats. Both 50% PWT and PWL values were markedly decreased in DPN rats. When miR-221 was inhibited, the 50% PWT and PWL values were both significantly increased. Knockdown of miR-221 significantly increased the expression of SOCS3 and decreased the expression of NF-κB. Furthermore, knockdown of miR-221 remarkably decreased the expression of PEG2, BK, IL-6, IL-1β, and TNF-α in both STZ-treated DPN rats and high glucose-induced SH-SY5Y cells, which was reversed by inhibition of SOCS3. The dual luciferase reporter assay showed that miR-221 directly targeted and negatively regulated SOCS3. Inhibition of miR-221 can reduce pain and decrease expression of inflammatory factors through targeting SOCS3 in DPN.

Downregulation of miR‑409‑3p suppresses LPS‑induced inflammation in human bronchial epithelial cells through SOCS3/JAK1/STAT3 signaling: The implication for bronchopneumonia

In recent decades, the role of microRNAs (miRs) in the development of pneumonia has been reported by a number of researchers. The present study aimed to investigate the role of miR‑409‑3p in lipopolysaccharide (LPS)‑induced human bronchial epithelial cells and the implication for bronchopneumonia. An invitro inflammation model was established using LPS‑induced BEAS‑2B cells. Cell apoptosis was determined by flow cytometry. Inflammatory factors were detected by ELISA and reverse transcription‑quantitative PCR. Protein levels of Janus kinase 1 (JAK1)/STAT3 and suppressor of cytokine signaling (SOCS)3 were determined by western blotting. Dual‑luciferase reporter assay was performed to confirm the interaction between miR‑409‑3p and SOCS3. LPS treatment significantly increased miR‑409‑3p expression and decreased the expression levels of SOCS3 in BEAS‑2B cells. Dual‑luciferase reporter assay demonstrated that miR‑409‑3p directly targeted and negatively regulated SOCS3. Inhibition of miR‑409‑3p markedly decreased the levels of TNF‑α, IL‑6 and IL‑1β, and suppressed apoptosis induced by LPS, which was reversed by SOCS3‑knockdown. The inhibition of SOCS3 significantly activated JAK1/STAT3 signaling, as well as enhancing the levels of TNF‑α, IL‑6 and IL‑1β, and promoting apoptosis, which was reversed by the JAK1 inhibitor Tofacitinib. Suppression of miR‑409‑3p improved LPS‑induced inflammation through SOCS3 in LPS‑treated BEAS‑2B cells, and this may be caused by regulating JAK1/STAT3 signaling.

POCU1b, the n-Butanol Soluble Fraction of Polygoni Cuspidati Rhizoma et Radix, Attenuates Obesity, Non-Alcoholic Fatty Liver, and Insulin Resistance via Inhibitions of Pancreatic Lipase, cAMP-Dependent PDE Activity, AMPK Activation, and SOCS-3 Suppression.

This study investigated the effects of the n-BuOH soluble fraction of Polygoni Cuspidati 80% ethanol extract (POCU1b) on high-fat diet (HFD)-induced obesity, non-alcoholic fatty liver (NAFL), and insulin resistance (IR) to find a safe and more effective agent. HPLC profiling of POCU1b identified seven marker compounds. POCU1b increased glycerol release, cyclic adenosine monophosphate (cAMP) level, and inhibited phosphodiesterase (PDE) activity. Seven weeks of POCU1b treatment decreased body weight gain, weight and adipocyte size in fat tissues, serum lipids, and triglyceride and lipid droplets in the livers of HFD-fed rats. POCU1b improved blood glucose, insulin sensitivity, and impaired insulin secretion in the pancreas. Further, POCU1b ameliorated adiponectin, leptin, IL-6 and TNF-α levels, increased AMPK and p-ACC expression, activated CPT-1 activity, and suppressed FAS mRNA, SOCS-3 protein expression, and NF-κB DNA-binding activity. When compared with the Xenical®-treated group, a positive group, the action of POCU1b on body weight was more effective than that of Xenical. POCU1b did not show side effects, such as oily spotting and loss of appetite. These results suggest that POCU1b possesses therapeutic or preventive potential for obesity, NAFL and IR via inhibitions of pancreatic lipase and cAMP-dependent PDE activity, AMPK activation, and SOCS-3 suppression, without oily spotting and loss of appetite.

DHA reduces hypothalamic inflammation and improves central leptin signaling in mice

AimsAnti-obesity effects and improved leptin sensitivity from n-3 polyunsaturated fatty acids (n-3 PUFAs) have been reported in diet-induced obese animals. This study sought to determine the beneficial central effects and mechanism of docosahexaenoic acid (DHA, 22:6 n-3) in high-fat (HF) diet fed mice. Main methodsMale C57BL/6J mice were given HF diet with or without intracerebroventricular (icv) injection of docosahexaenoic acid (DHA, 22:6 n-3) for two days. Central leptin sensitivity, hypothalamic inflammation, leptin signaling molecules and tyrosine hydroxylase (TH) were examined by central leptin sensitivity test and Western blot. Furthermore, the expression of hepatic genes involved in lipid metabolism was examined by RT-PCR. Key findingsWe found that icv administration of DHA not only reduced energy intake and body weight gain but also corrected the HF diet-induced hypothalamic inflammation. DHA decreased leptin signaling inhibitor SOCS3 and improved the leptin JAK2-Akt signaling pathways in the hypothalamus. Furthermore, icv administration of DHA improved the effects of leptin in the regulation of mRNA expression of enzymes related to lipogenesis, fatty acid β-oxidation, and cholesterol synthesis in the liver. DHA increased leptin-induced activation of TH in the hypothalamus. SignificanceTherefore, increasing central DHA concentration may prevent the deficit of hypothalamic regulation, which is associated with disorders of energy homeostasis in the liver as a result of a high-fat diet.

A lipid-soluble extract of Pinellia pedatisecta Schott orchestrates intratumoral dendritic cell-driven immune activation through SOCS1 signaling in cervical cancer

Ethnopharmacological relevancePinellia pedatisecta Schott extract (PE) is generated from Pinellia pedatisecta Schott, a traditional Chinese medicinal plant. PE suppresses cervical tumor growth and exhibits effects on dendritic cells (DCs) that lead to modulation of antitumor CD4+ and CD8+ responses. AimsTo explore the underlying mechanisms by which PE modulates tumor-associated dendritic cell (TADC) activation and function. MethodsDCs and TADCs were generated from murine bone marrow and exposed to PE solutions at different doses, as well as to repeated doses separated at different time intervals. Quantitative PCR, Western blot analysis, flow cytometry, and gene silencing were used to analyze the modulatory effects of PE on the SOCS1/JAK2/STAT pathways. Furthermore, we separated human cervical tumor-infiltrated DCs (TIDCs) and conducted an ex-vivo stimulation model to observe the effect of PE. For phenotypic analysis of cultured DCs and ex vivo human specimens, we used flow cytometry to detect the molecular markers associated with cell function. ResultsIn cultured TADCs and human cervical TIDCs, maturation- and functional markers (MHCII, CD80, CD83, CD86, and IL-12) were downregulated, whereas SOCS1 was upregulated. PE enhanced the expression of CD80, CD86, and IL-12 in cervical TIDCs, which induced increased expression of CD107a, GZMB, and perforin in CTLs, and furthermore induced apoptosis in a larger number of tumor cells. In cultured TADCs, PE downregulated SOCS1 expression and activated the phosphorylation of JAK2, STAT1, STAT4, and STAT5 in both dose- and time-dependent manners. The effects of PE upregulating MHCII, CD80, CD86, IL-12 on TADCs were blocked after SOCS1 silencing. ConclusionsIn this study, PE restored the impaired function of cervical TIDCs, thereby eliciting further antitumor CTL responses. The effects of PE on TADCs were mediated through inhibition of SOCS1 and activation of downstream JAK2-STAT1/STAT4/STAT5 pathways. PE may be a potent and effective immunomodulatory drug for antitumor treatment via the blockade of SOCS1 signaling in DCs.