SOCS3 mRNA Expression Research Articles

Comparisons of SOCS mRNA and protein levels in Xenopus provide insights into optic nerve regenerative success

In vertebrates from fishes to mammals, optic nerve injury induces increased expression ofSuppressor of Cytokine Signaling 3(SOCS3) mRNA, a modulator of cytokine signaling that is known to inhibit CNS axon regeneration. Unlike amniotes, however, anamniotes successfully regenerate optic axons, despite this increase. To address this seeming paradox, we examined the SOCS3 response to optic nerve injury in the frog,Xenopus laevis, at both the mRNA and protein levels. Far from being only transiently induced, SOCS3 mRNA expression increased throughout regeneration in retinal ganglion cells, but immunostaining and Western blots indicated that this increase was reflected at the protein level in regenerating optic axons but not in ganglion cell bodies. Polysome profiling provided additional evidence that SOCS3 protein levels were regulated post-translationally by demonstrating that the mRNA was efficiently translated in the injured eye. In tumor cells, another member of theSOCS gene family,SOCS2, is known to mediate SOCS3 degradation by targeting it for proteasomal degradation. Unlike the SOCS2 response in mammalian optic nerve injury, SOCS2 expression increased inXenopusretinal ganglion cells after injury, at both the mRNA and protein levels; it was, however, largely absent from both uninjured and regenerating optic axons. We propose a similar degradation mechanism may be spatially restricted inXenopusto keep SOCS3 protein levels sufficiently in check within ganglion cell bodies, where SOCS3 would otherwise inhibit transcription of genes needed for regeneration, but allow them to rise within the axons, where SOCS3 has pro-regenerative effects.

Heat Shock Factor 1 Inhibits the Expression of Suppressor of Cytokine Signaling 3 in Cerulein-Induced Acute Pancreatitis.

Heat shock factor 1 (HSF1), an important transcriptional molecule in the heat shock process, can regulate the expression of a lot of inflammatory mediators in addition to heat shock proteins. This study evaluated the inhibitive function of HSF1 on the expression of suppressor of cytokine signaling 3 in cerulein-induced acute pancreatitis. After HSF1 mice, HSF1 mice, and AR42J cells were treated with cerulein, histopathological score, expression of SOCS3 mRNA, and protein levels were analyzed by using RT-PCR, quantitative real-time RT-PCR, and western blotting, respectively. DNA binding and transcription activity of HSF1 to the SOCS3 promoter were detected by chromatin immunoprecipitation and luciferase reporter assays. The histopathological scores of the pancreas decreased significantly in the cerulein-induced HSF1 mice compared with the cerulein-induced HSF1 mice. SOCS3 mRNA and protein level decreased in the pancreas of the unstimulated HSF1 and HSF1 mice, whereas increased in the pancreas of the cerulein-induced HSF1 and HSF1 mice, with higher in the pancreas of cerulein-induced HSF1mice. In the pcDNA3.1-transfected AR42J cells, SOCS3 protein decreased and was upregulated after the cerulein stimulation, whereas HSF1 overexpression inhibited the upregulation. In the scramble-transfected AR42J cells, SOCS3 protein decreased and was upregulated after the cerulein stimulation, whereas HSF1-RNAi further promoted the upregulation. EMSA and chromatin immunoprecipition showed that HSF1 could directly bind to SOCS3 promoter region. Reporter assays showed that HSF1 could inhibit the transcriptional activity on SOCS3 promoter. HSF1 can protect AR42J cells from cerulein-induced pancreatitis through inhibiting the expression of SOCS3.

Control of progression towards liver fibrosis and hepatocellular carcinoma by SOCS3 polymorphisms in chronic HCV-infected patients

Background & aimsChronic Hepatitis C is one of the most important risk factors of liver cirrhosis and hepatocellular carcinoma. Before reaching these ultimate steps, insulin resistance triggered by hepatitis C virus infection is known to participate in the progression of liver disease. The present study aims to investigate the influence of two functional polymorphisms on SOCS3 mRNA expression and on the outcomes of CHC progression in a North African context. Patients & methodsIn this case-control study, 601 Moroccan subjects composed of 200 healthy controls, 101 resolvers and 300 patients with persistent HCV infection including 95 mild chronic hepatitis, 131 Advanced Liver Diseases and 74 HCC were enrolled. They were genotyped for the 4874 A/G (rs4969170) and A + 930– > G (rs4969168) SOCS3 variants using TaqMan SNPs assays. SOCS3 mRNA expression was assessed using Real Time PCR technique. ResultsLogistic regression analysis showed that variation at rs4969168 was associated with spontaneous clearance of HCV (P < 0.05). In addition, minor allele frequencies were significantly higher in AdLD patients when compared to the mCHC group both for rs4969168 (P = 7.0 E-04) and rs4969170 (P = 4.0 E-05). A significant association between haplotype and liver disease progression was also found. Moreover, SOCS3 mRNA was significantly more expressed in peripheral leukocytes from patients with HCC than in those from mCHC. Finally, rs4969170 was significantly associated with LDL-lipoprotein (P = 0.04), total cholesterol (P = 5.0 E-04), and higher fasting glucose levels (P = 0.005) in patients with persistent HCV infection. ConclusionsOur results underline the importance of the functional SOCS3 polymorphisms in the modulation of CHC progression and suggest their contribution to HCC development by affecting its mRNA expression and perturbing key metabolic parameters.

Intervention effects of Compound Houttuyniae Herba to diabetic renal damage based on SOCS-JAK/STAT negative feedback regulation

ObjectiveTo research the protective effects of different extracts from Compound Houttuyniae Herba (CHH) and its mechanism through JAK/STAT-SOCS-1 signaling pathway. MethodsThe normal group comprised db/m mice (n = 8). db/db mice were randomly divided into seven groups with eight mice in each group according to the applied treatment method: model group, metformin hydrochloride (MH) group, AG490 group, water extract (WE) group, ethanol extract (EE) group, volatile oil (VO) group, and mixture (MG) group (mixture of above three extract) of CHH. After 8 weeks, the general status, biochemical indicators, and renal histological changes in the mice were evaluated, the total RNA and protein were collected and RT-PCR method was used to examine the effect on mRNA expression of JAK2, STAT3, SOCS1, and Western blot method was used to detect the protein expression of JAK2, P-JAK2, STAT3, P-STAT3, SOCS1, c-fos, and c-jun. Immunofluorescence was used to observe the protein expression of c-fos and c-jun in kidney tissue. ResultsCompared with normal group, the serum level of TGF-β1, FN of EE, VO, and MG groups were decreased (P < 0.05). Renal function was also improved but not significantly. Also, the renal histology was improved especially in the mixture group. The protein expression of JAK2, STAT3, P-JAK2, P-STAT3, and the genetic expression of JAK2 and STAT3 in kidney tissue were significantly increased in the model group (P < 0.05). Compared with the model group, the expression of P-JAK2 and P-STAT3 was down-regulated in treatment groups; The expression of SOCS-1 of VO and mixture groups were elevated (P < 0.05). ConclusionCHH has beneficial effects on diabetic renal injury, which may protect and improve the kidney function and reduce urinary protein, maintain the integrity of the kidney structure and function.

The Expression Levels of IL-4/IL-13/STAT6 Signaling Pathway Genes and SOCS3 Could Help to Differentiate the Histopathological Subtypes of Non-Small Cell Lung Carcinoma.

BackgroundThe interleukin (IL)-4/IL-13/signal transducer and activator of transcription (STAT) 6 signaling pathway and the SOCS3 gene, one of its main regulators, constitute an important link between the inflammation process in the epithelial cells and inflammatory-related tumorigenesis. The present study is the first to evaluate IL-4, IL-13, STAT6, and SOCS3 mRNA expression in non-small cell lung carcinoma (NSCLC) histopathological subtypes.MethodsGene expression levels were assessed using TaqMan® probes by quantitative reverse transcription PCR (qRT-PCR) in lung tumor samples and unchanged lung tissue samples.ResultsIncreased expression of IL-4, IL-13, and STAT6 was observed in all histopathological NSCLC subtypes (squamous cell carcinoma [SCC], adenocarcinoma [AC], and large cell carcinoma [LCC]). Significantly higher expression of IL-13 and STAT6 (p = 0.019 and p = 0.008, respectively) was found in SCC than in LCC. No statistically significant differences were found for IL-4. Significantly higher SOCS3 expression was found in LCC than in AC (p = 0.027). A negative correlation (rho = –0.519) was observed for the STAT6 and SOCS3 genes in SCC (p = 0.005). No associations were found between gene expression and tumor staging (post-operative Tumor Node Metastasis [pTNM], American Joint Committee on Cancer [AJCC]), patients’ age, sex, or history of smoking.ConclusionsAs the number of LCC cases in our study was quite low, the statistically significant results obtained should be confirmed in a larger group of patients, particularly as the relationships identified between increased IL-4, IL-13, and STAT6 mRNA expression and decreased SOCS3 expression suggest that these genes may serve as potential diagnostic markers for differentiating between NSCLC histopathological subtypes.

Abstract 4321: Up-regulation of METTL3 promoted m6A modification and epigenetic silencing of SOCS2 in human liver cancer

Abstract Epigenetic alteration is a common trait of human cancers. Chemical modifications on DNA and core histone proteins are the major mechanisms for epigenetic regulation. Recently, emerging evidence suggested that reversible chemical modifications on RNA also play a critical role in epigenetic control of gene expression. N6-methyladenosine (m6A) is the most abundant modification found in mammalian mRNA. m6A is involved in regulating mRNA stability, splicing, and translation. However, the implications of m6A modification in human carcinogenesis remain poorly understood. In this study, we investigated the expression of m6A methyltransferases and demethylases in human hepatocellular carcinoma (HCC). We found the major m6A methyltransferase METTL3 was remarkably up-regulated in HCC. High METTL3 expression was associated with poor overall and disease-free survival in HCC patients. Consistent with the METTL3 overexpression, we found that mRNA m6A level was significantly elevated in human HCC. To investigate the roles of METTL3 in human HCC, we employed lentiviral-based shRNA and CRISPR/Cas9 systems to inactivation METTL3 in HCC cell lines. We showed that knockdown and knockout of METTL3 drastically suppressed HCC proliferation and migration in vitro and abolished HCC tumorigenicity and lung metastasis in vivo. In contrast, overexpression of METTL3 by CRISPR/dCas9 SAM system promoted HCC growth. Using RNA-seq and m6A-Seq, we identified tumor suppressor gene SOCS2 as a novel target of METTL3. We detected m6A modification in SOCS2 mRNA and the m6A modification was diminished upon METTL3 knockdown. m6A modification promoted SOCS2 mRNA degradation. We demonstrated that knockdown of METTL3, mutation of m6A modification sites, and treatment of demethylating agent augmented SOCS2 mRNA expression in HCC cells. In addition, knockdown of YTHDF2, an m6A reader protein, also rescued SOCS2 mRNA expression in HCC cells. The above findings together demonstrated that up-regulation of METTL3 lead to m6A modification of SOCS2, which in turn promotes SOCS2 mRNA degradation through YTHDF2 dependent mechanism. Our findings provided a proof-of-concept model to demonstrate the importance of aberrant m6A modification in epigenetic silencing of tumor suppressor genes. Citation Format: Mengnuo Chen, Lai Wei, Cheuk-Ting Law, Felice H. Tsang, Jialing Shen, Carol L. Cheng, Long-Hin Tsang, Daniel W. Ho, David K. Chiu, Joyce M. Lee, Carmen C. Wong, Irene O. Ng, Chun-Ming Wong. Up-regulation of METTL3 promoted m6A modification and epigenetic silencing of SOCS2 in human liver cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4321.

Role of Adenylyl Cyclase-Associated Protein 1 (CAP1) in Mediating Resistin Actions in Mouse Liver Cells

Resistin is a pro-inflammatory adipokine produced by the white adipose tissue (WAT) adipocytes and macrophages. Obesity results in chronic inflammation of the WAT, marked by an increase in resistin and other inflammatory cytokines, and by infiltrating leukocytes. Elevated resistin levels are believed to play a major role in the development of insulin resistance in the peripheral tissues. Adenylyl cyclase-associated protein 1 (CAP1) was recently identified as a receptor for resistin. In the present study we aimed to investigate whether CAP1 mediates resistin actions which may affect insulin sensitivity in the liver. As a model we used BNL CL.2 mouse liver cell line. Concentration- and time-dependent experiments demonstrated that resistin upregulated TNFα, SOCS3, IL-1α, and IL-6 mRNA expression maximally when used in concentration of 12.5 ng/ml for 6 hours. In order to determine the CAP1 involvement in mediating resistin actions in the liver, we transfected BNL CL.2 cells with CAP1 siRNA and performed a real-time PCR array measuring the expression of 84 key genes involved in insulin signaling, adipokine signaling, and inflammation. Results demonstrated that resistin upregulated mRNA expression of IL-6; this effect was ameliorated when CAP1 was downregulated. Knock-down of CAP1 facilitated mRNA expression of genes involved in insulin signaling and adipokine signaling pathways, while it resulted in downregulation of infiltrating leukocyte markers expression. Taken together these results indicate that CAP1 is a mediator of resistin actions in the liver. Disclosure D. Avtanski: None. A. Garcia: None. P. Thangeswaran: None. B. Caraballo Bordon: None. L. Poretsky: None.

Correlation of SOCS-1 gene with onset and prognosis of breast cancer.

The aim of the present study is to study the expressions of suppressor of cytokine signaling (SOCS)-1 in the tumor tissues and adjacent normal tissues of patients with breast cancer. The study was also planned to investigate the association of SOCS-1 gene expression with patients' clinical pathology, molecular subtype and prognosis. A total of 60 cases of frozen and paraffin-embedded specimens of tumor tissues and corresponding adjacent normal tissues of patients with breast cancer were selected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of SOCS-1 messenger RNA (mRNA) in the patients' tumor tissues and adjacent normal tissues. The immunohistochemical method was applied to detect the expressions of SOCS-1 proteins in the patients' breast cancer tissues and adjacent normal tissues. Moreover, the correlations of SOCS-1 protein expressions in breast cancer tissues with patients' pathological parameters, molecular subtypes and prognosis were analyzed in combination with the clinical data. The results of RT-qPCR showed that the SOCS-1 mRNA expression in breast cancer tissues was significantly lower than that in adjacent normal tissues (p<0.01). The immunohistochemical results indicated that the positive expression rate of the SOCS-1 proteins in breast cancer tissues (23.33%) was remarkably lower than that in adjacent normal tissues (88.33%) (p<0.01). The low expression of SOCS-1 in breast cancer tissues was related to lymph node metastasis and clinical staging. The positive expression rates of the luminal A SOCS-1 proteins were the highest (47.62%) (p<0.01). The 5-year overall survival rate of the breast cancer patients was 63.33% (38/60). The univariate survival analysis revealed that the patients with low expression of SOCS-1 had poorer prognosis. In conclusion, the low expression of SOCS-1 plays a key role in the pathogenesis of breast cancer; in particular, it is associated with the lymph node metastasis and clinical staging of the tumor; so, the SOCS-1 expression in breast cancer tissues can be regarded as an important reference for the prognostic estimation of breast cancer.

IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from human skin mast cells by a STAT3-dependent mechanism.

Abstract Mast cells are tissue resident immune cells that cause allergies. The agents responsible for allergic reactions are pre-formed mediators like histamine and inflammatory lipids such as Prostaglandin D2 (PGD2) that are rapidly biosynthesized following crosslinking of FcɛRI with allergen. In humans, increased IL-6 levels are consistently correlated with allergic diseases like asthma and urticaria in which mast cells play a vital role. However, the exact role of IL-6 in allergic disease is not known. In this study, we show that human in situ-matured skin mast cells (huSMCs) express functional membrane-bound receptors for IL-6, and demonstrate our novel finding that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis. Treatment of huSMCs with IL-6 alone induced the phosphorylation of STAT3 at the activating tyrosine 705, and the expression of SOCS3 and VEGF mRNA. IL-6 alone did not induce PGD2 secretion, but significantly and dose-dependently enhanced PGD2 secretion from FcɛRI-activated huSMCs. Accordingly, IL-6 enhanced the FcɛRI-induced expression of COX-2, which is directly involved in PGD2 biosynthesis. In addition, phosphorylation of STAT3 at both tyrosine 705 and serine 727, which is required for maximal transcriptional activity, was increased in huSMCs treated with IL-6 + FcɛRI crosslinking. Moreover, the IL-6-mediated increase in FcɛRI-induced COX-2 expression was inhibited with C188-9, the small molecule inhibitor of STAT3 tyrosine phosphorylation. Together, these data demonstrate that IL-6 potentiates FcɛRI-induced PGD2 biosynthesis from huSMCs by a STAT3-dependent mechanism. Thus, we hypothesize that IL-6 contributes to allergic disease by enhancing the production of inflammatory PGD2 from mature mast cells.

One-Week Exposure to a Free-Choice High-Fat High-Sugar Diet Does Not Interfere With the Lipopolysaccharide-Induced Acute Phase Response in the Hypothalamus of Male Rats

Obesity has been associated with increased susceptibility to infection in humans and rodents. Obesity is also associated with low-grade hypothalamic inflammation that depends not only on body weight but also on diet. In the present study, we investigated if the bacterial endotoxin [lipopolysaccharide (LPS)]-induced acute phase response is aggravated in rats on a 1-week free-choice high-fat high-sugar (fcHFHS) diet and explained by diet-induced hypothalamic inflammation. Male Wistar rats were on an fcHFHS diet or chow for 1 week and afterwards intraperitoneally injected with LPS or saline. Hypothalamic inflammatory intermediates and plasma cytokines were measured after LPS. Both LPS and the fcHFHS diet altered hypothalamic Nfkbia mRNA and nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha (NFKBIA) protein levels, whereas Il1β, Il6, and Tnfα mRNA expression was solely induced upon LPS. We observed an interaction in hypothalamic Nfkbia and suppressor of cytokine signaling (SOCS) 3 mRNA upon LPS; both were higher in rats on a fcHFHS diet compared with chow animals. Despite this, plasma cytokine levels between fcHFHS diet-fed and chow-fed rats were similar after LPS administration. Consuming a fcHFHS diet but not LPS injections increased hypothalamic Atf4 (a cellular stress marker) mRNA expression, whereas Tlr4 mRNA was decreased only upon LPS. Our study does not support a role for diet-induced mild hypothalamic inflammation in the increased susceptibility to infection despite altered Nfkbia and Socs3 mRNA expression after the diet. Additional factors, related to increased fat mass, might be involved.

An Increase in Expression of SOCS1 Gene with Increase in Hepatitis C Virus Viral Load.

Hepatitis C virus (HCV) is a global health problem, with an estimated prevalence of >185 million infections worldwide. About 399,000 people die every year because of HCV-associated liver complications such as liver cancer, liver cirrhosis, and hepatocellular carcinoma. Pakistan has the second-highest prevalence of HCV. The treatment of this life-threatening disease has always been a challenge, but the recent development of direct acting antivirals (DAAs) has offered hope to so many patients with this infection. Although DAAs have dramatically improved virologic clearance, their cost is prohibitive in low economic settings, which is why interferon (IFN) is still used in Pakistan and other developing countries. Many genes-specifically, interferon stimulating genes-alter treatment response. This study recruited 93 participants and used quantitative real-time polymerase chain reaction for the quantification of SOCS1 gene messenger RNA (mRNA) in the peripheral blood mononuclear cells of 5 different groups: IFN treated, IFN resistant, IFN nonresponders treated with DAAs, untreated, and healthy controls. A moderate positive correlation (rs = 0.492) was observed between viral load and SOCS1 mRNA expression level. Our results suggest the over-expression of SOCS1 in PBMCs of IFN non-responders as compared to responders and DAA treated IFN non-responders.

IGF-1 induces SOCS-2 but not SOCS-1 and SOCS-3 transcription in juvenile Nile tilapia (Oreochromis niloticus).

Insulin-like growth factor-1 (IGF-1) plays a crucial role in regulating growth in vertebrates whereas suppressors of cytokine signaling (SOCS) act as feedback inhibitors of the GH/IGF-1 axis. Although SOCS-2 binds the IGF-1 receptor and inhibits IGF-1-induced STAT3 activation, presently there is no clear evidence as to whether IGF-1 could induce SOCS gene expression. The current study aimed to determine whether IGF-1 could induce the transcription of SOCS in juvenile Nile tilapia (Oreochromis niloticus). We show that there is a common positive relationship between the mRNA expression of IGF-I and SOCS-2 under different nutritional statuses and stimulants, but not the mRNA expression of SOCS-1 and SOCS-3 Furthermore, rhIGF-1 treatment and transcriptional activity assay confirmed the hypothesis that IGF-1 could induce SOCS-2 expression, whereas it had no effect or even decreased the expression of SOCS-1 and SOCS-3 Overall, we obtained evidence that the transcription of SOCS-2, but not SOCS-1 or SOCS-3, could be induced by IGF signaling, suggesting that SOCS-2 serves as a feedback suppressor of the IGF-1 axis in juvenile Nile tilapia.

The role and gene expression profile of SOCS3 in colorectal carcinoma.

SOCS3 has been postulated to play a role in the occurrence and progression of malignancies. However, the relationship of SOCS3 with colorectal carcinoma remains poorly understood. The purpose of the study was to explore the role of SOCS3 in colorectal carcinoma and its underlying mechanisms. Protein and mRNA expression of SOCS3 in colorectal carcinoma and normal colorectal mucosa was detected using immunohistochemistry and real-time quantitative PCR. SOCS3 expression was significantly lower in colorectal carcinoma tissue than in normal colorectal mucosa, and was negatively correlated with tumor invasion depth, lymph node metastasis, differentiation degree, and TNM stage. A stably transfected colorectal carcinoma cell line (8348SOCS3) with high expression of SOCS3 was established. The effects of SOCS3 overexpression on the growth, proliferation, invasion and tumor formation of colorectal carcinoma cells were examined by CCK-8 assay, transwell method and tumorigenicity assays in nude mice. Then we found SOCS3 overexpression significantly decreased proliferation and invasion capability of 8348 cells in vitro and in vivo. Furthermore, the effect of SOCS3 overexpression on the gene expression profile of colorectal carcinoma cells was analyzed using human genome arrays. The results revealed 369 genes that were differentially expressed in 8348SOCS3 cells. 193 genes was significantly increased and 176 genes was significantly decreased. Bioinformatics analysis demonstrated that high SOCS3 expression affected multiple signaling pathways in colorectal carcinoma including TGF-β/Smads, NF-κB, and HIF-MAPK pathways. Especially for the TGF-β/Smads pathways, high SOCS3 expression could inhibit TGF-β1 expression and activate Smad4 expression. These data suggested that low expression of SOCS3 was associated with the occurrence and progression of colorectal carcinoma. SOCS3 protein may be a useful indicator for malignancy and prognosis of colorectal carcinoma and also a new target for gene therapy.

Effects of Sucrosomial® iron in Pre-Clinical and Clinical Conditions of Inflammation

Introduction: Proteins such as Ferritin (Ft), Hemoglobin (Hb) and Transferrin Receptor (TfR1) are involved in tissue iron distribution while systemiciron metabolism is regulated by Hepcidin. Interleukin-6 (IL-6) release is induced in response to an inflammatory status and enhances Hepcidin expression, as consequence, dietary iron absorption and macrophage iron recycling are inhibited. Anemia of Chronic Disease (ACD) is a form of anemia associated with many different chronic disorders. It is characterized by high hepcidin and CRP or IL-6 expression and low serum iron transferrin concentration while ferritin can be normal or elevated. Oral iron delivery is especially attractive thanks to the ease of administration, but commonly used iron salts are poorly absorbed and ineffective to increase Hb concentration in particular in patients with ACD. We have developed an oral Iron formulation named Sucrosomial® Iron, preparation of ferric pyrophosphate, covered by a phospholipids plus sucrester matrix, with high bioavailability and tolerability, which promotes ferric iron absorption thanks to gastro-resistant and matrix composition properties and showed Hb concentration improvement not inferior to intravenous iron.Objective: to showpre-clinical and clinical evidences of absorption, effectiveness and tolerability of Sucrosomial Iron using in vivo and in vitro models and in patients with ACD too.Methods: To study the effects of oral supplementation on Iron metabolism and inflammatory status, Hb, Ft, TfR1, Socs3 and Hepcidin expression were analyzed. We have supplemented wild-type (wt) and iron deficient (ID) mice, maintained in iron-free diet for 8 weeks, with 1mg/kg/die of Sucrosomial® Iron or Iron Sulfate or Placebo by oral gavage, for 2 weeks. The role of Sucrosomial® Iron on the inflammatory response, was studied in LPS induced HepG2 hepatoma cells. Cells were treated with Sucrosomial® Iron and the levels of hepcidin were assessed 6, 18 and 24 hours after treatment. To show the efficacy and compliance of Sucrosomial® Iron a multi-centric randomized study on 300 patients with ACD were performed. Patients were supplemented with iron sulfate (65 mg o.i.d.), microencapsulated iron, micronized iron, Sucrosomial® Iron, heminic-chelated bisglycinated and iron chelated bisglycinated iron (30 mg t.i.d.). Median Hb value at the beginning was 8.2 g/dl. In group of patients with high CRP value median Hb value was 7.8 g/dl.Results: In mice treated with Sucrosomial® Iron, Hb level mean increase was 4,9 g/dl while TSAT value is not significantly higher than animals treated with Sulfate Iron. Furthermore mice treated with Sucrosomial® Iron showed a preferential accumulation of iron in spleen. Hepcidin and Socs3 mRNA expression was not induced during Sucrosomial® Iron treatment while was up-regulated in mice treated with Iron Sulfate (Figure 1). In vitro experiments on HepG2 LPS-induced cells displayed that treatment with Sucrosomial Iron® is able to reduce hepcidin concentration at different time points (Figure 2).Data from the clinical study showed that Sucrosomial® Iron is well tolerate and effective ACD patients. Only patients treated with Sucrosomial® Iron showed constant increase in Hb concentration over time. In all groups Hb level achieves a plateau phase after three months and ferritin level starts to increase. At three months higher levels of Hb are present in Sucrosomial® Iron (13.2 g/dl), heminic chelated bisglycinated iron (11.7 g/dl), chelated bisglycinated iron (11.3 g/dl) treated patients. In patients with high CRP level (>30 ng/ml) only those receiving Sucrosomial® Iron showed Hb increase from the tenth week and this continue up to the sixth month (Hb 12.5 g/dl) and seems to be correlated to a significantly decrease of CRP (5 mg/L) (Figure 3). Side effects are higher in ferrous sulfate (15/50) and sunactive treated groups (6/60).Conclusions: In summary, results showed that Sucrosomial® Iron treatment could regulate iron homeostasis through an increase in Hb concentration and better tolerability during inflammatory status. These evidences may suggest that this new oral iron formulation behaves differently than other oral iron salts, perhaps due to different absorption pathways. Besides we have observed a decrease in CRP and Hepcidin concentration, these results should be further investigate. [Display omitted] DisclosuresBrilli:Pharmanutra S.p.A.: Consultancy. Pera:Pharmanutra S.p.A.: Employment. Tarantino:Pharmanutra S.p.A.: Employment.

Insulin resistance in 3T3-L1 adipocytes by TNF-α is improved by punicic acid through upregulation of insulin signalling pathway and endocrine function, and downregulation of proinflammatory cytokines

Insulin resistance (IR) has become a major threat to public health due to its role in metabolic syndrome. Inflammation associated with IR is an interesting area of biomedical research in recent years and is expected to affect insulin signalling pathway via downregulating glucose transporters. In the present study, we evaluate the potential of punicic acid (PA), a nutraceutical found in pomegranate seed oil, against TNF-α induced alteration in 3T3-L1 adipocytes on glucose metabolism, endocrine function and inflammation. IR was induced in 3T3-L1 adipocytes by treating with TNF-α (10 ng/mL) and various concentrations of PA (5, 10, 30 μM) were incubated simultaneously. After 24 h, we found that TNF-α treatment increased mRNA expression of SOCS3, PTP1B and a decrease in IRS1 causing diminished glucose uptake. Further, it showed significantly increased transcriptional activity of NFκB and leptin secretion while PA maintained leptin levels normal. Additionally, PA prevented the over-expression of phosphorylated JNK in a dose dependent manner during IR. PA also ameliorated significantly the upregulation of proinflammatory cytokines. From the results, we conclude that PA is effective to ameliorate TNF-α induced IR and also we recommend the intake of PA for control and management of IR and its associated complications.

Corilagin Counteracts IL-13Rα1 Signaling Pathway in Macrophages to Mitigate Schistosome Egg-Induced Hepatic Fibrosis.

The IL-13Rα1 signaling pathway and M2 macrophages play crucial roles in schistosome egg-induced hepatic fibrosis via the expression of pro-fibrotic molecules. This study aims to investigate the inhibitory effect and mechanism of action of corilagin on schistosome egg-induced hepatic fibrosis via the IL-13Rα1 signaling pathway in M2 macrophages in vitro and in vivo. The mRNA and protein expression of IL-13Rα1, PPARγ, KLF4, SOCS1, STAT6, p-STAT6, and TGF-β was measured in vitro with corilagin treatment after IL-13 stimulation and in vivo corilagin treatment after effectively killing the adult schistosomes in schistosome-infected mice. Histological analysis of liver tissue was assessed for the degree of hepatic fibrosis. The results revealed that corilagin significantly reduced the expression of PPARγ, KLF4, SOCS1, p-STAT6, and TGF-β compared with model group and praziquantel administration (p < 0.01 or p < 0.05) in vivo and in vitro, which indicated a strong inhibitory effect of corilagin on IL-13Rα1 signaling pathway. As well, the inhibitory effect of corilagin showed a significant dose-dependence (p < 0.05). The area of fibrosis and distribution of M2 macrophages in mouse liver tissue were reduced significantly and dose-dependently with corilagin treatment compared to model group or praziquantel administration (p < 0.01 or p < 0.05), indicating that corilagin suppressed IL-13Rα1 signaling pathway and M2 macrophage polarization effectively in vivo. Furthermore, the anti-fibrogenic effect persisted even when IL-13Rα1 was up- or down-regulated in vitro. In conclusion, corilagin can suppress schistosome egg-induced hepatic fibrosis via inhibition of M2 macrophage polarization in the IL-13Rα1 signaling pathway.

Carboxymethyl-chitosan attenuates inducible nitric oxide synthase and promotes interleukin-10 production in rat chondrocytes.

Osteoarthritis (OA) is a common age-related degenerative joint disease, which is caused by the breakdown of joint cartilage and the underlying bone. Carboxymethyl (CM)-chitosan is a soluble derivative of chitosan that has similar physicochemical properties to the extracellular proteoglycans identified in hyaline cartilage. Previous studies have demonstrated that CM-chitosan serves a protective role in a rabbit OA model. The aim of the present study was to investigate the effect of CM-chitosan on NO production and inflammation through its upregulation of interleukin (IL)-10, and the activation of the janus kinase (JAK)/signal transducer and activator of transcription (STAT)/suppressor of cytokine signaling (SOCS) signaling pathway. In the present study primary rat chondrocytes were induced to inflammation with 2 µg/ml lipopolysaccharide. The cells were subsequently subjected to increasing concentrations of CM-chitosan (50, 100 and 200 µg/ml) and the relative mRNA and protein expression of inducible nitric oxide synthase (iNOS), IL-10, JAK1, STAT3 and SOCS3 were measured by RT-qPCR and western blot analysis respectively. The results revealed that CM-chitosan attenuated inflammation by significantly reducing iNOS expression and upregulating the anti-inflammatory cytokine IL-10 in a dose-dependent manner (P<0.05). The expression of JAK1, STAT3 and SOCS3 were also significantly upregulated by CM-chitosan (all P<0.05). The protective role of CM-chitosan against NO production was due to its upregulation of IL-10 and its activation of the JAK/STAT/SOCS signaling pathway.

Correlation of SOCS3 mRNA Expression Level with Clinical Characteristics and Early Response to Treatment in Pediatric Acute Lymphoblastic Leukemia

The expression levels of SOCS3 mRNA in bone marrow mononuclear cells from 45 cases of newly diagnosed ALL at initial diganosis and induction remission and 13 normal children as controls were detected by SYBR Green fluorescence quantitative PCR; the correlation of SOCS3 mRNA expression level with risk and clinical characteristics of ALL was analyzed by means of statistical method; the immunofluorescence histochemistry method and laser confocal microscopy were used to detect the sites and expression level of SOCS3 mRNA in bone marrow samples of ALL patients. The SOCS3 mRNA expression level at initial diagnosis of 45 ALL patients was significantly lower than that of normal controls (P<0.05), and that in induction remission of 45 patients was not significant different from normal controls (P<0.05); the SOCS3 mRNA expression level at initial diagnosis of patients with standasd and high risk was higher than that of patients with low risk (P<0.05). The 45 patients were divided into high expression and low expression groups according to SOCS3 mRNA expression level at initial diagnosis by median method, comparison of clinical characteristics in 2 groups was found that the SOCS3 mRNA high expression group had even more higher leukocyte count in peripheral blood, even more higher LDH level and much more poor prognostic genes; in addition, the high expression group showed more higher risk in comparison between 2 group. The SOCS3 mRNA expression is down-regulated at initial diagnosis, while recovered to normal level after induction remission of disease, thus the SOCS3 can be used as an indicator for evaluation of disease status. The high expression of SOCS3 mRNA up-regulates the disease risk, therefore the SOCS3 mRNA can be used as a factor for evaluation of ALL risk. The treatment of ALL via regulation of SOCS3 mRNA expression level maybe can become a new way. The regulation of SOCS3 mRNA expression level maybe can become a new way for treatment of ALL.

Effect of nitric oxide-releasing derivative of indomethacin on Prevotella intermedia lipopolysaccharide-induced production of proinflammatory mediators in murine macrophages

The purpose of this study was to investigate the influences of NCX 2121, a nitric oxide (NO)-releasing derivative of indomethacin, upon the generation of proinflammatory mediators using murine macrophages activated by lipopolysaccharide (LPS) isolated from Prevotella intermedia, which is one of the pathogens implicated in periodontal diseases. Inducible NO synthase (iNOS)-derived NO, IL-1β and IL-6 as well as their relevant mRNA were significantly attenuated by NCX 2121 in RAW264.7 cells activated by P. intermedia LPS. NCX 2121 was much more effective than the parental compound indomethacin in reducing these proinflammatory mediators. NCX 2121 triggered induction of heme oxygenase-1 (HO-1) in cells exposed to P. intermedia LPS, and its inhibitory influence upon P. intermedia LPS-elicited NO generation was notably blocked by SnPP treatment. NCX 2121 attenuated NF-κB-dependent SEAP release induced by P. intermedia LPS. NCX 2121 did not display inhibitory action towards IκB-α degradation triggered by LPS. Instead, it significantly diminished nuclear translocation as well as DNA-binding action of NF-κB p50 subunit elicited by P. intermedia LPS. Further, NCX 2121 significantly up-regulated SOCS1 mRNA expression in cells challenged with P. intermedia LPS. In summary, NCX 2121 down-regulates P. intermedia LPS-elicited generation of NO, IL-1β and IL-6 in murine macrophages in a mechanism that involves anti-inflammatory HO-1 induction as well as decrement of NF-κB activation, which may be associated with SOCS1 expression. NCX 2121 may have potential benefits as a host immunomodulatory agent for the therapy of periodontal disease.

Specific Features of the Hypothalamic Leptin Signaling Response to Cold Exposure Are Reflected in Peripheral Blood Mononuclear Cells in Rats and Ferrets.

Objectives: Cold exposure induces hyperphagia to counteract fat loss related to lipid mobilization and thermogenic activation. The aim of this study was investigate on the molecular mechanisms involved in cold-induced compensatory hyperphagia.Methods: We analyzed the effect of cold exposure on gene expression of orexigenic and anorexigenic peptides, and of leptin signaling-related genes in the hypothalamus of rats at different ages (1, 2, 4, and 6 months), as well as in ferrets. We also evaluated the potential of peripheral blood mononuclear cells to reflect hypothalamic molecular responses.Results: As expected, cold exposure induced hypoleptinemia in rats, which could be responsible for the increased ratio of orexigenic/anorexigenic peptides gene expression in the hypothalamus, mainly due to decreased anorexigenic gene expression, especially in young animals. In ferrets, which resemble humans more closely, cold exposure induced greater changes in hypothalamic mRNA levels of orexigenic genes. Despite the key role of leptin in food intake control, the effect of cold exposure on the expression of key hypothalamic leptin signaling cascade genes is not clear. In our study, cold exposure seemed to affect leptin signaling in 4-month-old rats (increased Socs3 and Lepr expression), likely associated with the smaller-increase in food intake and decreased body weight observed at this particular age. Similarly, cold exposed ferrets showed greater hypothalamic Socs3 and Stat3 gene expression. Interestingly, peripheral blood mononuclear cells (PBMC) mimicked the hypothalamic increase in Lepr and Socs3 observed in 4-month-old rats, and the increased Socs3 mRNA expression observed in ferrets in response to cold exposure.Conclusions: The most outstanding result of our study is that PBMC reflected the specific modulation of leptin signaling observed in both animal models, rats and ferrets, which points forwards PBMC as easily obtainable biological material to be considered as a potential surrogate tissue to perform further studies on the regulation of hypothalamic leptin signaling in response to cold exposure.