SOCS3 Expression Research Articles

Hepatic stellate cell-intrinsic role of SOCS1 in controlling hepatic fibrogenic response and the pro-inflammatory macrophage compartment during liver fibrosis.

Hepatic stellate cells (HSC) become activated, differentiate to myofibroblasts and produce extracellular fibrillar matrix during liver fibrosis. The hepatic fibrogenic response is orchestrated by reciprocal interactions between HSCs and macrophages and their secreted products. SOCS1 can regulate several cytokines and growth factors implicated in liver fibrosis. Here we investigated the role of SOCS1 in regulating HSC activation. Mice lacking SOCS1 in HSCs (Socs1ΔHSC) were generated by crossing Socs1fl/fl and LratCre mice. Liver fibrosis was induced by carbon tetrachloride and evaluated by Sirius red staining, hydroxyproline content and immunostaining of myofibroblasts. Gene expression of pro-fibrogenic factors, cytokines, growth factors and chemokines were quantified by RT-qPCR. The phenotype and the numbers of intrahepatic leukocyte subsets were studied by flow cytometry. The impact of fibrosis on the development of diethyl nitrosamine-induced hepatocellular carcinoma was evaluated. Socs1ΔHSC mice developed more severe liver fibrosis than control Socs1fl/fl mice that was characterized by increased collagen deposition and myofibroblast differentiation. Socs1ΔHSC mice showed a significant increase in the expression of smooth muscle actin, collagens, matrix metalloproteases, cytokines, growth factors and chemokines in the liver following fibrosis induction. The fibrotic livers of Socs1ΔHSC mice displayed heightened inflammatory cell infiltration with increased proportion and numbers of Ly6ChiCCR2+ pro-inflammatory macrophages. This macrophage population contained elevated numbers of CCR2+CX3CR1+ cells, suggesting impaired transition towards restorative macrophages. Fibrosis induction following exposure to diethyl nitrosamine resulted in more numerous and larger liver tumor nodules in Socs1ΔHSC mice than in Socs1fl/fl mice. Our findings indicate that (i) SOCS1 expression in HSCs is a critical to control liver fibrosis and development of hepatocaellular carcinoma, and (ii) attenuation of HSC activation by SOCS1 regulates pro-inflammatory macrophage recruitment and differentiation during liver fibrosis.

High and Low Levels of ABCB1 Expression Are Associated with Two Distinct Gene Signatures in Lung Tissue of Pulmonary TB Patients with High Inflammation Activity.

P-glycoprotein (encoded by the ABCB1 gene) has a dual role in regulating inflammation and reducing chemotherapy efficacy in various diseases, but there are few studies focused on pulmonary TB patients. In this study, our objective was to identify a list of genes that correlate with high and low levels of ABCB1 gene expression in the lungs of pulmonary TB patients with different activity of chronic granulomatous inflammation. We compared gene expression in two groups of samples (with moderate and high activity of tuberculomas) to identify their characteristic gene signatures. Gene expression levels were determined using quantitative PCR in samples of perifocal area of granulomas, which were obtained from 65 patients after surgical intervention. Subsequently, two distinct gene signatures associated with high inflammation activity were identified. The first signature demonstrated increased expression of HIF1a, TGM2, IL6, SOCS3, and STAT3, which correlated with high ABCB1 expression. The second signature was characterized by high expression of TNFa and CD163 and low expression of ABCB1. These results provide insight into various inflammatory mechanisms and association with P-gp gene expression in lung tissue of pulmonary TB patients and will be useful in the development of a host-directed therapy approach to improving the effectiveness of anti-TB treatment.

The Role and Mechanism of Ferroptosis Mediated by METTL3-m6A Modification in Regulating Radioresistance of Esophageal Cancer

Radioresistance remains the major cause of recurrence within two years after radical radiotherapy in approximately 50% of patients with esophageal cancer (ESCC). Recently, it has been reported that enhancing ferroptosis can reverse tumor radioresistance. However, the underlying mechanisms remain elusive. The aim of this study was to elucidate the role and mechanism of ferroptosis mediated by METTL3 N6-methyladenosine (m6A) modification in regulating radioresistance of Esophageal Cancer. We hypothesize that inhibition of ferroptosis confers radioresistance in Esophageal Cancer by METTL3-induced m6A modification which increase the expression of m6A in the 3'UTR region of SOCS6 and inhibits its expression. To explore the relationship between radioresistance and ferroptosis. Firstly, ferroptosis was analyzed by 4-HNE staining in ESCC specimens; Furthermore, cell death was detected by propidium iodide (PI) or SYTOX Green staining combined with microscopy or flow cytometry in ESCC radioresistance cells and parental cells, and Iron Assay Kit,C11-BODIPY were used to examine whether ferroptosis were inhibited in radioresistance cells. Subsequently, MeRIP-seq and MeRIP-PCR were performed for discussing the relationship between ferroptosis and m6A. Finally, Mettl3 depleted cells were generated with CRISPR/Cas9-mediated knockout system. Then, m6A methylation level, sensitivity to radiation and ferroptosis of Mettl3 depleted cells by EpiQuik m6A RNA methylation quantification kit, colony formation, C11-BODIPY and Western Blot. Firstly, we found that the level of lipid peroxide 4-HNE in ESCC samples that recurred after radical radiotherapy was significantly lower than that in radiotherapy (40Gy), and the ferroptosis of radioresistance cells was inhibited. MeRIP-seq found that the level of m6A in radioresistance cells increased significantly, and then the KEGG pathway analysis of MeRIP-seq results showed that hypermethylation of m6A was closely related to ferroptosis signal pathway. and we also found that the level of m6A was significantly decreased by adding agonist (Erastin), whereas the modification level was significantly increased by adding inhibitor Fer-1. Meanwhile, we found that the level of m6A was significantly decreased in radioresistance cells silencing METTL3, while the ferroptosis was activated, which caused the radiosensitivity of ESCC. Lastly, MeRIP-PCR data showed that METTLT3 induced a significant increase in m6A level in 3' UTR region of SOCS6 mRNA and inhibited its expression, which eventually led to radioresistance of ESCC. m6A-regulated ferroptosis inhibition confers radioresistance of ESCC. METTL3 up-regulated m6A in SOCS6 3' UTR and inhibited the expression of SOCS6, which caused the inhibition of ferroptosis.

Curcumin alleviated dextran sulfate sodium-induced colitis by recovering memory Th/Tfh subset balance.

Restoration of immune homeostasis by targeting the balance between memory T helper (mTh) cells and memory follicular T helper (mTfh) cells is a potential therapeutic strategy against ulcerative colitis (UC). Because of its anti-inflammatory and immunomodulatory properties, curcumin (Cur) is a promising drug for UC treatment. However, fewer studies have demonstrated whether Cur can modulate the mTh/mTfh subset balance in mice with colitis. To explore the potential mechanism underlying Cur-mediated alleviation of colitis induced by dextran sulfate sodium (DSS) in mice by regulating the mTh and mTfh immune homeostasis. Balb/c mice were administered 3% and 2% DSS to establish the UC model and treated with Cur (200 mg/kg/d) by gavage on days 11-17. On the 18th d, all mice were anesthetized and euthanized, and the colonic length, colonic weight, and colonic weight index were evaluated. Histomorphological changes in the mouse colon were observed through hematoxylin-eosin staining. Levels of Th/mTh and Tfh/mTfh cell subsets in the spleen were detected through flow cytometry. Western blotting was performed to detect SOCS-1, SOCS-3, STAT3, p-STAT3, JAK1, p-JAK1, and NF-κB p65 protein expression levels in colon tissues. Cur effectively mitigates DSS-induced colitis, facilitates the restoration of mouse weight and colonic length, and diminishes the colonic weight and colonic weight index. Simultaneously, it hinders ulcer development and inflammatory cell infiltration in the colonic mucous membrane. While the percentage of Th1, mTh1, Th7, mTh7, Th17, mTh17, Tfh1, mTfh1, Tfh7, mTfh7, Tfh17, and mTfh17 cells decreased after Cur treatment of the mice for 7 d, and the frequency of mTh10, Th10, mTfh10, and Tfh10 cells in the mouse spleen increased. Further studies revealed that Cur administration prominently decreased the SOCS-1, SOCS-3, STAT3, p-STAT3, JAK1, p-JAK1, and NF-κB p65 protein expression levels in the colon tissue. Cur regulated the mTh/mTfh cell homeostasis to reduce DSS-induced colonic pathological damage, potentially by suppressing the JAK1/STAT3/SOCS signaling pathway.

Milk thistle nano-micelle formulation promotes cell cycle arrest and apoptosis in hepatocellular carcinoma cells through modulating miR-155-3p /SOCS2 /PHLDA1 signaling axis

BackgroundHepatocellular Carcinoma (HCC) is a prevalent form of liver cancer that causes significant mortality in numerous individuals worldwide. This study compared the effects of milk thistle (MT) and nano-milk thistle (N-MT) on the expression of the genes that participate in apoptosis and cell cycle pathways in Huh-7 and HepG2 cells.MethodsIC50 values of MT and N-MT were determined using the MTT assay. Huh-7 and HepG2 cell lines (containing mutant and wild-type TP53 gene, respectively) were incubated with MT and N-MT for 24h and 48h and the impact of MT and N-MT on the proliferation of these cell lines was evaluated through a comparative analysis. Cell cycle and apoptosis were assessed by flow cytometry after 24h and 48h treatment in the cell lines mentioned. Real-time PCR was used to analyze miR-155-3p, PHLDA1, SOCS2, TP53, P21, BAX, and BCL-2 expression in the cell lines that were being treated.ResultsN-MT reduces cancer cell growth in a time and concentration-dependent manner, which is more toxic compared to MT. Huh-7 was observed to have IC50 values of 2.35 and 1.7 μg/ml at 24h and 48h, and HepG2 was observed to have IC50 values of 3.4 and 2.6 μg/ml at 24 and 48h, respectively. N-MT arrested Huh-7 and HepG2 cells in the Sub-G1 phase and induced apoptosis. N-MT led to a marked reduction in the expression of miR-155-3p and BCL-2 after 24h and 48h treatments. Conversely, PHLDA1, SOCS2, BAX, and P21 were upregulated in the treated cells compared to untreated cells, which suggests that milk thistle has the potential to regulate these genes. N-MT reduced the expression of TP53 in Huh-7 cells after mentioned time points, while there was a significant increase in the expression of the TP53 gene in HepG2 cells. No gene expression changes were observed in MT-treated cells after 24h and 48h.ConclusionN-MT can regulate cancer cell death by arresting cell cycle and inducing apoptosis. This occurs through the alteration of apoptotic genes expression. A reduction in the expression of miR-155-3p and increase in the expression of SOCS2 and PHLDA1 after N-MT treatment showed the correlation between miR-155-3p and PHLDA1/SOCS2 found in bioinformatics analysis. While N-MT increased TP53 expression in HepG2, reduced it in Huh-7. The findings indicate that N-MT can function intelligently in cancer cells and can be a helpful complement to cancer treatment.

Combinational application of the natural products 1-deoxynojirimycin and morin ameliorates insulin resistance and lipid accumulation in prediabetic mice

BackgroundPrediabetes, a stage characterized by chronic inflammation, obesity and insulin resistance. Morin and 1-deoxynojirimycin (DNJ) are natural flavonoids and alkaloids extracted from Morus nigra L., exhibiting anti-hyperglycemic efficacy. However, the benefits of DNJ are shadowed by the adverse events, and the mechanism of morin in anti-diabetes remains under investigation. PurposeIn this study, the combinational efficacy and mechanisms of DNJ and morin in ameliorating insulin resistance and pre-diabetes were investigated. MethodsThe mice model with prediabetes and Alpha mouse liver-12 (AML-12) cell model with insulin resistance were established. The anti-prediabetic efficacy of the drug combination was determined via analyzing the blood glucose, lipid profiles and inflammatory factors. The application of network pharmacology provided guidance for the research mechanism. ResultsIn our study, the intervention of morin ameliorated the insulin resistance via activating the Peroxisome proliferator-activated receptor γ (PPARγ). However, PPARγ activation leaded to the lipid accumulation in prediabetic mice. The combination of 5 mg/kg dose of DNJ and 25 mg/kg morin effectively hindered the progression of T2DM by 87.56%, which was achieved via inhibition of Suppressors of cytokine signaling 3 (SOCS3) and promotion of PPARγ as well as SOCS2 expression. Furthermore, this treatment exhibited notable capabilities in combating dyslipidemia and adipogenesis, achieved by suppressing the Cluster of differentiation 36/ Sterol-regulatory element binding proteins-1/ Fatty acid synthetase (CD36/Serbp1/Fas) signaling. ConclusionThis research confirmed that the drug combination of DNJ and morin in ameliorating insulin resistance and lipid accumulation, and revealed the potential mechanisms. In summary, the combination of DNJ and morin is an underlying alternative pharmaceutical composition in T2DM prevention.

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.

Regulation of High-fat Diet-induced Liver Fibrosis by SOCS1 Expression in Hepatic Stellate Cells

Regulation of High-fat Diet-induced Liver Fibrosis by SOCS1 Expression in Hepatic Stellate Cells

Evaluation of SOCS5 mRNA and its association with serum IL-12 levels and rs41379147 SNP in various subsets of allergic disorders: A case control study

BackgroundThe SOCS proteins act as suppressors of cytokine signaling by impeding certain signaling pathways. SOCS5, a constituent of the SOCS family, has been associated with the management of allergic reactions, primarily by impeding the signaling of interleukin-4 (IL-4), which is known to have a cardinal function in accelerating the development of an allergic reaction. The key goal of our research was to explore the probable ramifications of the SOCS5 single nucleotide polymorphism (SNP) namely rs41379147 on the expression of SOCS5 mRNA and serum IL-12 levels, as well as to analyze the interaction between SOCS5 genotypes and various clinicopathological parameters in atopic diseases. MethodsThe study involved the enrollment of 314 subjects comprising 154 atopic individuals and 160 healthy controls. PCR-RFLP was employed to conduct SNP analysis. Real-Time PCR was employed to quantify SOCS5 mRNA. The enzyme-linked immunosorbent assay (ELISA) technique was used for the quantification of interleukin-12 and total IgE levels in the serum while as chemiluminescence was used to determine Vitamin D levels. ResultsThe PCR-RFLP analysis indicated a lack of statistically significant variation in genotypic and allelic frequencies between the cases and controls (p > 0.05) for − 9147 C/T SNP either in total atopy (OR-0.70, 95% CI=0.43-1.12, p =0.15), and on subgroup stratifications of chronic urticaria (OR-0.81, 95 % CI = 0.42–1.59, p = 0.61), allergic rhinitis (OR-0.63, 95 % CI = 0.33–1.19, p = 0.16) and bronchial asthma (OR-0.66,95% CI = 0.29–1.4, p=0.32). There was reduced mRNA expression of SOCS5 in total atopic cases, allergic rhinitis, bronchial asthma and chronic urticaria in comparison to controls which advocates the fact that SOCS5 has a protective role in allergic disease development. Despite the reduced amounts of IL-12 in total atopic cases and different allergic disorders in comparison to controls, IL-12 showed significant positive correlation with SOCS5 mRNA expression (p < 0.05). ConclusionSOCS5 SNP rs41379147(C/T) does not pose any significant risk towards the development of any allergic disorder and has no impact on the expression of SOCS5 and IL-12. Our study has shown the reduced mRNA expression of SOCS5 among individuals diagnosed with chronic urticaria, allergic rhinitis and bronchial asthma and the expression of SOCS5 showed complete dependence on the cytokine milieu of IL12. The modulation of SOCS5 and IL-12 may represent potential curative targets for treating the menace of allergic diseases and present promising avenues for future investigation.

Tartary buckwheat root polysaccharides ameliorate non-alcoholic fatty liver disease via the IL6-SOCS3-SREBP1c pathway.

Our previous study demonstrated that Tartary buckwheat root polysaccharides (TBRP) could reduce insulin resistance in diabetes mellitus by inhibiting SOCS3-stimulated IRS1 protein degradation. However, whether TBRP has the efficiency to treat non-alcoholic fatty liver disease (NAFLD) is still undetermined. This investigation aimed to examine the effects of TBRP on a high-fat diet (HFD)-triggered NAFLD, and elucidate the underlying molecular mechanisms. Briefly, TBRP toxicity in hepatoma (BEL7404) and pancreatic cancer (BxPC3) cells and zebrafish embryos developmental models, were evaluated in-vitro and in-vivo, respectively. TBRP inhibited cellular lipid accumulation by suppressing fat synthesis, furthermore, it improved body weight gain, liver weight, liver-to-body weight ratio, serum lipids triglyceride, total cholesterol, ALT, LDL-C, HDL-C, and AST levels in the NAFLD mice model. Additionally, TBRP treatment also lowered the nitric oxide content. The qPCR assay revealed that mRNA expression of TNF, IL1β, and IL6 was also markedly reduced in TBRP-treated NAFLD mice. The expression of SOCS3, SREBP1c, and STAT3 was elucidated by western blot analysis, which indicated that TBRP markedly decreased the gene expression for de novo fat synthesis by the SOCS3-SREBP1c pathway. These findings reveal that TBRP ameliorates NAFLD via the IL6-SOCS3-SREBP1c signaling pathway and therefore, may represent a promising approach for NAFLD treatment.

Gold nanoparticles attenuate the interferon-γ induced SOCS1 expression and activation of NF-κB p65/50 activity via modulation of microRNA-155-5p in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) is a very aggressive form of cancer that grows and spreads very fast and generally relapses. Therapeutic options of TNBC are limited and still need to be explored completely. Gold nanoparticles conjugated with citrate (citrate-AuNPs) are reported to have anticancer potential; however, their role in regulating microRNAs (miRNAs) in TNBC has never been investigated. This study investigated the potential of citrate-AuNPs against tumorigenic inflammation via modulation of miRNAs in TNBC cells. Gold nanoparticles were chemically synthesized using the trisodium-citrate method and were characterized by UV-Vis spectrophotometry and dynamic light scattering studies. Targetscan bioinformatics was used to analyze miRNA target genes. Levels of miRNA and mRNA were quantified using TaqMan assays. The pairing of miRNA in 3'untranslated region (3'UTR) of mRNA was validated by luciferase reporter clone, containing the entire 3'UTR of mRNA, and findings were further re-validated via transfection with miRNA inhibitors. Newly synthesized citrate-AuNPs were highly stable, with a mean size was 28.3 nm. The data determined that hsa-miR155-5p is a direct regulator of SOCS1 (suppressor-of-cytokine-signaling) expression and citrate-AuNPs inhibits SOCS1 mRNA/protein expression via modulating hsa-miR155-5p expression. Transfection of TNBC MDA-MB-231 cells with anti-miR155-5p markedly increased SOCS1 expression (p<0.001), while citrate-AuNPs treatment significantly inhibited anti-miR155-5p transfection-induced SOCS1 expression (p<0.05). These findings were validated by IFN-γ-stimulated MDA-MB-231 cells. Moreover, the data also determined that citrate-AuNPs also inhibit IFN-γ-induced NF-κB p65/p50 activation in MDA-MB-231 cells transfected with anti-hsa-miR155-5p. Newly generated citrate-AuNPs were stable and non-toxic to TNBC cells. Citrate-AuNPs inhibit IFN-γ-induced SOCS1 mRNA/protein expression and deactivate NF-κB p65/50 activity via negative regulation of hsa-miR155-5p. These novel pharmacological actions of citrate-AuNPs on IFN-γ-stimulated TNBC cells provide insights that AuNPs inhibit IFN-γ induced inflammation in TNBC cells by modulating the expression of microRNAs.

The role of mediator suppressor of cytokine signaling (SOCS), toll-like receptor 3 (TLR-3) and nuclear factor kappa B (NFκB) on cytokine production during dengue virus infection.

Inability to understand the pathogenesis of severe dengue, in particular the control mechanism of immune responses, has led to high mortality rate for patients with dengue shock syndrome (DSS). The aim of this study was to determine the control mechanism of cytokine production by mediator suppressor of cytokine signaling (SOCS), toll-like receptor 3 (TLR-3) and nuclear factor kappa B (NFκB) during DENV infection. Peripheral blood mononuclear blood cells (PBMC), isolated from healthy individuals, were infected with dengue virus (DENV)-2 strain SJN-006 Cosmopolitan genotype (isolated from Bali, Indonesia). The relative gene expression of SOCS-3, TLR-3, NFκB, and the cytokine genes (interleukin (IL)-6, IL-8, interferon inducible protein 10 (IP-10), and macrophage inflammatory protein-1 beta (MIP-1β)) were measured using qRT-PCR at 6, 12 and 24 hours post infection (hpi). Student t-test and Mann-Whitney test were used to compare the gene expressions while causal correlations were analyzed using regression test and path analyses. DENV-2 infection increased the gene expression of SOCS-3, TLR-3, and NFκB after 12 and 24 hpi. The expression of IL-6, IL-8, IP-10, and MIP-1β genes was increased and peaked at different times post-infection. NFκB and SOCS-3 genes likely have role in the upregulation of IL-8 and IL-6 gene expression, respectively. MIP-1β gene expression was significantly induced by both NFκB and SOCS-3. In conclusion, our study suggested that SOCS-3, TLR-3, and NFκB are important in regulating the production of IL-6, IL-8, IP-10, MIP-1β during early phase of DENV-2 infection. This enriches our understanding on pathogenesis pathway of DENV-associated cytokine storm.

Resistin stimulates PC-3 prostate cancer cell growth through stimulation of SOCS3 and SOCS5 genes.

Resistin and suppressors of cytokine signaling (SOCSs) have been reported to regulate prostate cancer (PCa) cell proliferation and survival, respectively. Whether any of the SOCS molecules mediate the mitogenic effect of resistin on PCa cells is unknown. Using PC-3 human PCa cells, we found that resistin upregulates the expression of SOCS3 and SOCS5 mRNA, but not SOCS7 mRNA, in a dose- and time-dependent manner. The resistin-induced increases in SOCS3 and SOCS5 expression and cell proliferation were prevented by pretreatment with specific inhibitors of the TLR4, ERK, p38 MAPK, JNK, PI3K, and JAK2 proteins. However, pretreatment with a TLR2 inhibitor had no effect on resistin-mediated SOCS3 and SOCS5 expression. In addition, the effects of resistin on SOCS3, SOCS5, and SOCS7 mRNA levels were cell type-specific. Overexpression of either SOCS3 or SOCS5 enhanced further resistin-stimulated growth of PC-3 cells, whereas silencing SOCS3 or SOCS5 antagonized resistin-increased cell growth. Further PCa tissue analysis demonstrated higher levels of RETN, TLR4, SOCS3, and SOCS5 mRNAs in cancer tissues than benign prostate hyperplasia and indicated positive correlations among RETN, TLR4, and SOCS5. These data suggest that SOCS5, TLR4, and, to a lesser extent, SOCS3 can mediate the mitogenic effect of resistin on PC-3 PCa cells.

Store-operated calcium entry mediates hyperalgesic responses during neuropathy.

Neuropathic pain (NP), resulting from nerve injury, alters neural plasticity in spinal cord and brain via the release of inflammatory mediators. The remodeling of store-operated calcium entry (SOCE) involves the refilling of calcium in the endoplasmic reticulum via STIM1 and Orai1 proteins and is crucial for maintaining neural plasticity and neurotransmitter release. The mechanism underlying SOCE-mediated NP remains largely unknown. In this study, we found SOCE-mediated calcium refilling was significantly higher during neuropathic pain, and the major component Orai1 was specifically co-localized with neuronal markers. Intrathecal injection of SOCE antagonist SKF96365 remarkably alleviated nerve injury- and formalin-induced pain and suppressed c-Fos expression in response to innocuous mechanical stimulation. RNA sequencing revealed that SKF96365 altered the expression of spinal transcription factors, including Fos, Junb, and Socs3, during neuropathic pain. In order to identify the genes critical for SKF96365-induced effects, we performed weighted gene co-expression network analysis (WGCNA) to identify the genes most correlated with paw withdrawal latency phenotypes. Of the 16 modules, MEsalmon module was the most highly correlated with SKF96365 induced effects. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the enriched genes of MEsalmon module were significantly related to Toll-like receptor signaling, steroid biosynthesis, and chemokine signaling, which may mediate the analgesic effect caused by SKF9636 treatment. Additionally, the SOCE antagonist YM-58483 produced similar analgesic effects in nerve injury- and formalin-induced pain. Our results suggest that manipulation of spinal SOCE signaling might be a promising target for pain relief by regulating neurotransmitter production and spinal transcription factor expression.

Integrated Bioinformatic Analyses Reveal Immune Molecular Markers and Regulatory Networks for Cerebral Ischemia-Reperfusion.

Cerebral ischemia-reperfusion injury (CIR) following a stroke results in secondary damage and is a leading cause of adult disability. The present study aimed to identify hub genes and networks in CIR to explore potential therapeutic agents for its treatment. Differentially expressed genes based on the GSE23163 dataset were identified, and weighted gene co-expression network analysis was performed to explore co-expression modules associated with CIR. Hub genes were identified by intersecting immune gene profiles, differentially expressed genes, and modular genes. Gene Ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and transcription factor-microRNA-gene regulatory network analyses were then conducted in selected crucial modules. Subsequently, their expression levels in animal models were verified using real-time quantitative polymerase chain reaction and Western blotting. Finally, potential drug molecules were screened for, and molecular docking simulations were performed to identify potential therapeutic targets. Seven hub genes-namely, Ccl3, Ccl4, Ccl7, Cxcl1, Hspa1a, Cd14, and Socs3-were identified. Furthermore, we established a protein interaction network using the STRING database and found that the core genes selected through the cytohubba plugin remained consistent. Animal experiments showed that at the transcriptional level, all seven genes showed significant differences (p < 0.001, fold change vs sham, 5-200). At the translational level, however, only Ccl3, Ccl4, Ccl7, Hspa1a, and Socs3 showed significant differences, while Cxcl1 and Cd14 did not. Nifedipine, with the highest predicted score, was identified as a therapeutic agent and successfully docked with the protein encoded by the hub genes. The expression of Ccl3, Ccl4, Ccl7, Hspa1a, and Socs3 was significantly different in CIR tissues compared to normal tissues both at the transcriptional and translational levels. Systems biology approaches indicated that these could be possible CIR marker genes, providing a stepping stone for further experimental studies.

Gene expression analysis of SOCS, STAT and PIAS genes in lung cancer patients

One of the most devastating diseases with the highest prevalence and mortality rate worldwide is lung cancer. Non-small cell lung cancer (NSCLC) is the subtype of lung cancer in 85% of cases. In this work, the expression levels of the STAT, SOCS and PIAS family genes involved in angiogenesis, proliferation and differentiation were examined. Using QRT-PCR technique, the expression level of STAT3 gene was assessed and tumor tissue samples had higher expression than normal tissue. In addition, the histological grade of adenocarcinoma was associated with the increase in STAT3 gene expression. The expression of the SOCS1 and SOCS2 genes in tumors was measured to be 0.58-fold and 0.36-fold lower than in healthy samples adjacent to the tumor, but this reduction in expression was not significant. In addition, when examining the relationship between the expression of SOCS1 and 2 and the clinical features of tumor samples, there was a significant decrease in the expression of the SOCS1 and 2 genes in the adenocarcinoma subtype. Compared to neighboring tumor samples, the expression of PIAS1 in the tumors was not different with controls. Our research revealed that tissue samples from adenocarcinoma had higher levels of STAT3 expression. Taken together, the mentioned genes can be suggested as possible targets for further studies in NSCLC.

Blood exosome marker miRNA-30d-5p: Role and regulation mechanism in cell stemness and gemcitabine resistance of hepatocellular carcinoma

BackgroundCancer 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. MethodsThe 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). ResultsBioinformatics 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. ConclusionsBlood 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.

Ozone attenuates chemotherapy-induced peripheral neuropathy via upregulating the AMPK-SOCS3 axis.

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. 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. 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. 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.

The miR-17∼92 miRNAs promote plasma cell differentiation by suppressing SOCS3-mediated NIK degradation

The miR-17∼92 family microRNAs (miRNAs) play a key role in germinal center (GC) reaction through promoting T follicular helper (TFH) cell differentiation. It remains unclear whether they also have intrinsic functions in B cell differentiation and function. Here we show that mice with B cell-specific deletion of the miR-17∼92 family exhibit impaired GC reaction, plasma cell differentiation, and antibody production in response to protein antigen immunization and chronic viral infection. Employing CRISPR-mediated functional screening, we identify Socs3 as a key functional target of miR-17∼92 in regulating plasma cell differentiation. Mechanistically, SOCS3, whose expression is elevated in miR-17∼92 family-deficient B cells, interacts with NIK and promotes its ubiquitination and degradation, thereby impairing NF-κB signaling and plasma cell differentiation. This moderate increase in SOCS3 expression has little effect on IL-21-STAT3 signaling. Our study demonstrates differential sensitivity of two key signaling pathways to alterations in the protein level of an miRNA target gene.

Bioinformatics analysis and key gene verification of sepsis myocardial macrophage microarray data based on GEO database

Objective: Bioinformatics analysis was used to screen differentially expressed genes (DEGs) in macrophages of sepsis myocardial injury and to verify key genes. Methods: Experiment 1 (gene chip and bioinformatics analysis): The gene chip data GSE104342 of cardiac macrophages in septic mice was downloaded from Gene Expression Omnibus database. DEGs were obtained by R language analysis. DAVID online database was used to obtain gene ontology and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of DEGs. STRING online database was used for protein-protein interaction network analysis of DEGs, and then key genes were screened by using Cytoscape software and molecular complex detection (MCODE) plug-ins. Experiment 2 (sepsis model construction and related protein verification): Ten male C57BL/6 mice, aged 8-14 weeks. Five mice were randomly selected as control group, and 5 mice were selected as the sepsis group by building a mice sepsis model in vivo. Echocardiography was used to detect the cardiac function. Hematoxylin-eosin staining was used to assess the cardiac morphology. TUNEL staining was used to evaluate cardiomyocyte apoptosis. Immunofluorescence staining was used to detect the expression of differentiation antigen cluster 206 (CD206),inducible nitric oxide synthases (iNOS),F4/80,suppressor of cytokine signaling 3 (Socs3) ,interleukin 1 receptor antagonist (Il1rn) and chemokine C-C motif ligand 7 (Ccl7) protein. RAW264.7 macrophages were cultured in vitro and divided into 2 groups: LPS groupstimulated by lipopolysaccharide (LPS, 1 mg/L) and blank control group treated with equal-volume phosphate buffer solution. Reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression of Socs3, Il1rn and Ccl7 in vitro. Results: Experiment 1: 24 647 genes were screened in GSE104342 dataset and 177 genes (0.72%) were differential expression, including 120 up-regulated genes and 57 down-regulated genes. Gene ontology enrichment analysis showed that DEGs were mainly involved in inflammatory response, immune response, apoptosis regulation and antigen processing and presentation. KEGG signaling pathway analysis showed that DEGs in cardiac macrophages of septic mice were mainly enriched in cytokine-cytokine receptor interaction, tumor necrosis factor signaling pathway, NOD like receptor signaling pathway. Three hub genes were obtained by STRING and Cytoscape analysis, including Socs3, Il1rn and Ccl7. Experiment 2: In vivo, it was found that compared with the control group, the cardiac function of the sepsis mice decreased significantly, the myocardial cells were significantly edema, inflammatory cell infiltration, myocardial fiber rupture, some myocardial nuclei dissolved and disappeared, and the cardiomyocyte apoptosis increased, suggesting that the sepsis myocardial injury model of mice was successfully constructed. Compared with the control group, the expression of CD206 in the myocardium of septic mice was down-regulated, the expression of iNOS, F4/80, Socs3, Il1rn and Ccl7 were up-regulated. In addition, there was co-localization between Socs3, Il1rn, Ccl7 and F4/80 protein. Compared with the blank control group, the expression of Socs3, Il1rn and Ccl7 significantly upregulated after LPS intervention in vitro by RT-PCR. Conclusions: The selected key genes Socs3, Il1rn and Ccl7 were up-regulated in myocardial macrophages of septic mice. Socs3, Il1rn and Ccl7 are expected to become new targets for the diagnosis and treatment of sepsis cardiac injury.