Different Concentrations Of IL-1β Research Articles

Blocking TXNIP reduced IL-1β Induced chondrocyte cell inflammation.

Osteoarthritis (OA) is the most common joint disease in the elderly and is characterized by progressive and irreversible degeneration of articular cartilage, particularly cartilage loss and callus formation. This study would like to investigate the important role and the molecular mechanism of OA progression following interleukin 1β (IL-1β)-induced chondrocyte injury regulated by TXNIP. In this study, high-purity mouse chondrocytes were obtained by enzymatic two-step digestion for primary culture. Toluidine blue staining and type II collagen immunofluorescence were used to identify cells through histochemical staining after slide mounting. The relative expression of TXNIP was detected by immunohistochemical staining and qRT-PCR.Aiming at the shRNA sequence of the TXNIP gene, the shRNA expression vector was constructed and packaged with lentivirus to form the lentiviral vector shTXNIP. After inhibiting the expression of TXNIP by transfecting shTXNIP into normal mouse chondrocytes, the CCK-8 kit was used for detecting its effect on cell proliferation after transfection, and the effect on chondrocyte apoptosis was detected by flow cytometry. The staining kit was used to detect the effect of TXNIP knockout on chondrocyte aging, and the differential expression of TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen genes in chondrocytes was detected by RT-PCR and Western-bolt. Western blot was used to detect the expression of upstream-related protein P-ERK, downstream-related protein NLRP3 and Caspase1 after inflammatory injury of mouse articular chondrocytes. Results showed that the expression level of TXNIP in chondrocytes induced by different concentrations of il-1β was proportional to the concentration. After silencing TXNIP by shRNA, cell proliferation increased, chondrocyte apoptosis was weakened, and chondrocyte aging was weakened. The differential expression of genes such as TNF, IL-6, MMP3, MMP13, ADAMTS-5 and type II collagen and the differential expression of protein levels were relatively decreased. In addition, the expression of the upstream-related protein P-ERK did not change much when TXNIP was silenced, and the expression levels of the downstream-related proteins NLRP3 and Caspase1 were slightly reduced. In conclusion, silencing TXNIP can inhibit il-1β-induced chondrocyte apoptosis and aging, and has a positive effect on cell proliferation. However, this study has not clarified the molecular mechanism involved in TXNIP and the process of its signaling expression pathway.

Elucidating the mechanism of IL-1β-Mediated Piezo1 expression regulation of chondrocyte autophagy and apoptosis via the PI3K/AKT/mTOR signaling Pathway

For the pathogenesis of osteoarthritis (OA), the classical view is that chondrocyte apoptosis is associated with and may cause age-related joint degeneration. Recent observations indicate that Piezo1, a mechanical stress channel expressed in articular cartilage, plays a crucial role in this process. We wanted to investigate whether other conditions activate the expression of Piezo1 in chondrocytes. Therefore, we simulated OA to investigate whether Piezo1 gene expression and channel function were affected by the inflammatory factor,interleukin-1β, and the role of Piezo1 in the regulation of autophagy and apoptosis of chondrocytes. After the primary culture of human chondrocytes, the primary chondrocytes were treated with different concentrations of IL-1β. It was found that IL-1β upregulated the expression of Piezo1 in human chondrocytes. After Piezo1 activation, we analyzed the expression of autophagy and apoptosis of chondrocytes and investigated whether the downstream PI3K/AKT/mTOR pathway mediated the autophagy and apoptosis of chondrocytes. IL-1β activates Piezo1 to inhibit chondrocyte autophagy and promote chondrocyte apoptosis partially, represented by up-regulation of related proteins c-caspase 3, Bax expression, and down-regulation of Bcl2, LC3, p62 expression. Piezo1-siRNA inverted this step partially. Inhibition of the PI3K/AKT/mTOR pathway reduces Piezo1 inhibition of chondrocyte autophagy and activation of chondrocyte apoptosis. Therefore, IL-1β-mediated Piezo1 inhibition of chondrocyte autophagy and promotion of chondrocyte apoptosis partially through the PI3K/AKT/mTOR pathway is considered a novel pathogenesis of osteoarthritis.

YKL-40 Promotes the Expression of Inflammatory Factors in Type Ⅱ Alveolar Epithelial Cell Model of A549 Cell Line

YKL-40, also known as chitinase-3-like-1 (CHI3L1), is a human cartilage glycoprotein-39, with its N-terminus consisting of tyrosine (Y), lysine (K), and leucine (L), hence the name YKL-40. In this study, we explored whether YKL-40 could promote the expression of inflammatory factors in type Ⅱ alveolar epithelial cells. A549 cells were cultured in vitro with interleukin (IL)-1β (20 ng/mL), IL-6 (20 ng/mL), tumor necrosis factor-alpha (TNF-α) (20 ng/mL), and interferon-gamma (IFN-γ) (20 ng/mL). The expression of YKL-40 transcription was determined by RT-qPCR. A549 cells were cultured with IL-1β at 5, 10, and 20 ng/mL and the expression of YKL-40 protein was determined by Western blot. A549 cells were cultured with recombinant YKL-40 protein at 0, 100, 500, and 1 000 ng/mL and the expression levels of IL-6 and IL-8 were measured by RT-qPCR. Three pairs of small interfering RNAs targeting YKL-40 (si- YKL-40-1/2/3) and the negative control (NC) were designed and used to transfect A549 cells, respectively, and the expression of YKL-40 was determined by RT-qPCR and Western blot. si- YKL-40-3 was screened out for subsequent experiments. In A549 cells, si- YKL-40-3 and si-NC were transfected and, then, IL-1β (20 ng/mL) was added in for culturing. The expression of YKL-40, IL-6, and IL-8 was determined by RT-qPCR and the expression of multiple factors in the supernatant was measured with the QAH-INF-1 kit. RT-qPCR results showed that IL-1β could up-regulate YKL-40 protein transcription level compared with that of the control group and the difference was statistically significant ( P<0.01), but IL-6, TNF-α, and IFN-γ could not up-regulate YKL-40 protein transcription level. Western blot results showed that IL-1β (20 ng/mL) could significantly promote the expression of YKL-40 and, compared with that of the control group, the differences showed by groups treated with different concentrations of IL-1β were all statistical significant ( P<0.01). After adding human recombinant YKL-40 protein to A549 cells, the results showed that the expression of inflammatory factors IL-6 and IL-8 was significantly increased and the difference was statistically significant compared with that of the control group ( P<0.05). After the expression of YKL-40 was decreased by si- YKL-40-3 transfection, the expression of IL-6 ( P<0.05), IL-8 ( P<0.05), and other inflammatory factors was inhibited compared with that of the control group. YKL-40 can promote the expression and secretion of IL-6, IL-8, and other acute inflammatory factors in A549 cell line, a type Ⅱ alveolar epithelial cell model, thus aggravating the inflammatory response. Targeted inhibition of YKL-40 expression may effectively inhibit inflammatory response.

Ubiquitin-specific protease 49 attenuates IL-1β-induced rat primary chondrocyte apoptosis by facilitating Axin deubiquitination and subsequent Wnt/β-catenin signaling cascade inhibition.

Osteoarthritis (OA) is an age-related chronic joint degenerative disease. Interleukin 1 beta (IL-1β) is considered a marker for the progression of OA. In this study, we found that Ubiquitin-Specific Peptidase 49 (USP49) was significantly less expressed in OA patients compared with healthy individuals. Treating primary rat chondrocytes with different concentrations of IL-1β resulted in decreased Usp49 expression, while Usp49 overexpression could attenuate IL-1β-induced chondrocyte apoptosis by promoting Axin deubiquitination. The deubiquitination of Axin led to the accumulation of the protein, which in turn resulted in β-catenin degradation and Wnt/β-catenin signaling cascade inhibition. Interestingly, we also found that [6]-gingerol, an anti-OA drug, could upregulate the protein level of Usp49 and suppress the Wnt/β-catenin signaling cascade in primary rat chondrocytes. Taken together, our study not only demonstrates that Usp49 can negatively regulate the progression of OA by inhibiting the Wnt/β-catenin signaling cascade, but also elucidates the underlying molecular mechanisms.

VO-OHpic attenuates intervertebral disc degeneration via PTEN/Akt pathway.

Intervertebral disc degeneration (IVDD) is mainly associated with a chronic process of the nucleus pulposus (NP) cells disabled. Also, it is accepted to be the basic result of low back pain. The role of phosphatase and tensin homolog (PTEN) in negatively regulating the Akt/PKB signaling, which is the major cell survival pathway, has been documented in previous studies. The present work aimed to investigate the role of PTEN inhibitor VO-OHpic (VO) in the protection of IVDD and to explore its potential mechanisms. NP cells isolated from patients' lumbar discs were subjected to different concentrations of IL-1β or H2O2 to establish NP cells degenerated model. Cell proliferation was analyzed by the Cell Counting Kit-8 (CCK-8) assay. The expression levels of collagen II, aggrecan, PTEN, PI3K, Akt, SOD1, SOD2, p16, and β-galactosidase (β-gal) were detected by Western blotting, immunofluorescence staining or RT-PCR. Flow cytometry was used to measure the ROS level and cell cycle distribution. Our research showed that collagen II, aggrecan, PI3K, and Akt markedly decreased in IL-1β- or H2O2-induced degenerated NP cells. VO could reverse the effects of IL-1β and H2O2 by the PTEN inhibition. Also, we found that VO increased the antioxidant enzymes SOD1, SOD2, CAT, GSH, POD production, and suppressed the ROS in the disc. Besides, data showed VO promoted NP cells proliferation by cell cycle mediation. These results suggest that VO treatment prevents NP degradation via restraining oxidative stress and increasing cell proliferation through the PTEN/Akt pathway in vitro. VO may become a novel cytokine for the therapy of IVDD in the future.

MiR-101-3p alleviates IL-1β-induced chondrocyte injury by targeting stanniocalcin 1

To explore the effects of miR-101-3p on IL-1β-induced chondrocyte injury and its underlying mechanisms. Methods: Chondrocytes were divided into 4 groups: a control group (NC group), a IL-1β group, a negative control group (IL-1β+miR-NC group), and a miR-101-3p group (IL-1β+miR-101-3p group), which were treated with IL-1β after transfecting with miR-101-3p mimic or negative mimic. The expressions of miR-101-3p-5p and stanniocalcin 1 (STC1) at different concentrations of IL-1β (1, 5, 10 ng/mL)-induced chondrocytes were detected by Western blotting and real-time PCR. MTT assay was used to detect cell proliferation rate, while caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein, such as matrix metalloproteinase 9 (MMP9) and collagen Type II. In addition, 3'-untranslated regions (UTR) of wild-type STC1 (STC1-3'-UTR-WT) or 3'-UTR of mutant STC1 (STC1-3'-UTR-MUT) were co-transfected with miR-101-3p mimic or miR-NC, respectively, while luciferase reporter assay was used to examine the regulative role of miR-101-3p in STC1. In order to detect whether STC1 was involved in the effect of miR-101-3p on chondrocytes, miR-NC (miR-NC group), miR-101-3p (miR-101-3p group), anti-NC (anti-NC group) and anti-miR-101-3p (anti-miR-101-3p group) were respectively transfected into the cells, and the expression of STC1 protein was detected by Western blotting. Subsequently, the cells were randomly divided into a miR-101-3P group (IL-1β+miR-101-3p group), an over-expression control group (IL-1β+miR-101-3p+ad-GFP group), and an over-expression STC1 group (IL-1β+miR-101-3p+ad-STC1 group) to investigate whether STC1 was involved in the role of miR-101-3p in chondrocyte. Similarly, MTT assay was used to detect cell proliferation rate, caspases assay kits and flow cytometry were used to measure the cell caspase and apoptosis level. Western blotting assay was used to detect the expression levels of pro-inflammatory and ECM-related protein MMP9 and collagen Type II. Results: Compared with the 0 ng/mL IL-1β, the expression of miR-101-3p was decreased in chondrocyte at different concentration of IL-1β (1, 5, 10 ng/mL) (all P<0.05), while the level of STC1 was increased (P<0.05). Compared with the NC group, the chondrocyte proliferation rate was down-regulated (P<0.05), while the apoptosis rate, the levels of caspases, IL-6 and TNF-α were increased in the IL-1β group (P<0.05). Moreover, the MMP9 levels were increased obviously, and the protein levels of collagen Type II were decreased in the IL-1β group compared with the NC group (both P<0.05). Compared with the IL-1β+miR-NC group, the proliferation rate was increased (P<0.05), whereas the apoptosis rates, the caspase-3/9 levels, the IL-6 and TNF-α levels were increased in the IL-1β+miR-101-3p group (all P<0.05). Then MMP9 levels were decreased obviously (P<0.05), and the protein levels of collagen Type II were increased in IL-1β+miR-101-3p group compared with the IL-1β+miR-NC group (both P<0.05). In addition, the double luciferase assay showed that the STC1 levels could be inhibited in the miR-101-3p group compared with the miR-NC group (P<0.05). STC1 levels were decreased in the miR-101-3p group compared with the miR-NC group (P<0.05), and the STC1 levels were increased in the anti-miR-101-3p group compared with those in the anti-NC group (P<0.05). The results of miR-101-3p+ad-STC1 group showed that compared with the miR-101-3p+ad-GFP group, the STC1 could reverse the effects of miR-101-3p on IL-1β-induced proliferation, apoptosis, inflammatory responses and ECM protein of chondrocytes. Conclusion: The regulation of miR-101-3p/STC1 signal pathway may have a role in reducing the IL-1β-induced chondrocyte injury.

IL-1β promotes the migration of olfactory epithelium neural stem cells through activating matrix metalloproteinase expressions

BackgroundTo investigate the effects of IL-1β on the migration of olfactory epithelium neural stem cells (OENSCs), and to assess the mechanisms. MethodsThe effects of different concentrations of IL-1β on cell proliferation, apoptosis and migration were evaluated by cell counting assay, flow cytometry and transwell migration assay, respectively. Matrix metalloproteinase (MMP)-2 and MMP-9 expression in both protein and mRNA levels were detected. Small interfering RNA (siRNA) technique was employed to knockdown MMP-2 and MMP-9 expression. Additionally, c-Jun N-terminal kinase (JNK) and nuclear factor-κB (NF-κB) inhibitors were applied to assess the potential signaling pathways involved in the effects of IL-1β on cell migration. ResultsIL-1β promoted cell migration of OENSCs in a concentration-dependent manner at the concentration range of 0–80 ng/ml, but did not affect cell proliferation and apoptosis. Mechanically, IL-1β promoted MMP-2 and MMP-9 expressions. Knockdown of MMP-2 or MMP-9 could significantly reduce IL-1β-induced cell migration. IL-1β activated JNK, NF-κB, Extracellular Signal-Regulated Kinase (ERK) and p-65 phosphorylation. Finally, we evidenced that inhibition of JNK or NF-κB significantly inhibited cell migration. ConclusionOur study demonstrated that IL-1β promoted the migration of OENSCs through activating MMP expression. Moreover, JNK and NF-κB signaling pathways were involved in the regulation. This study provides important experimental evidence for the application of OENSCs in the transplantation therapy.

Baicalin alleviates IL-1β-induced inflammatory injury via down-regulating miR-126 in chondrocytes

Baicalin is a flavonoid extracted from Scutellaria baicalensis Georgi, with anti-inflammatory and anti-apoptotic activities. The objective of this study was to explore the effect and mechanism of baicalin on chondrocyte inflammatory response in OA. Different concentrations of IL-1β (0, 0.1, 2, 5 and 10 ng/mL) were used to simulate inflammatory injury in CHON-001 cells. The expression of miR-126 was altered by transfection with miR-126 mimic. Thereafter, cells were treated with baicalin, and cell viability, apoptosis, the expressions of apoptosis-related protein and pro-inflammatory factors were respectively detected using CCK-8 assay, flow cytometry, qRT-PCR and western blot analysis. We found that IL-1β induced a significantly inflammatory injury in CHON-001 cells. Baicalin alleviated IL-1β-induced inflammatory injury, as it increased cell viability, decreased cell apoptosis and repressed the production of IL-6, IL-8 and TNF-α. miR-126 was up-regulated by IL-1β treatment while was down-regulated by baicalin. More interestingly, the protective actions of baicalin on IL-1β-injured CHON-001 cells were partially eliminated by miR-126 overexpression. Further, NF-κB signaling pathway was activated by IL-1β, and deactivated by addition of baicalin. The deactivation of NF-κB signaling pathway induced by baicalin upon IL-1β exposure was recovered by miR-126 overexpression. In conclusion, this study demonstrated that baicalin protected CHON-001 cells against IL-1β-induced inflammatory injury possibly via down-regulation of miR-126 and thereby deactivation of NF-κB signaling pathway.

Nicotinamide Phosphoribosyltransferase Inhibitor APO866 Prevents IL-1β-Induced Human Nucleus Pulposus Cell Degeneration via Autophagy

Background/Aims: Intervertebral discs consist of an extracellular matrix (ECM) with a central gelatinous nucleus pulposus (NP) enclosed in an outer layer known as the annulus fibrosus. ECM metabolic disorders result in loss of boundary between the annulus fibrosus and NP, which can lead to intervertebral disc degeneration (IDD). Proinflammatory cytokines, such as interleukin (IL)-1β, mediate the progression of IDD. Nicotinamide phosphoribosyltransferase (Nampt) catalyzes the first step in the biosynthesis of nicotinamide adenine dinucleotide (NAD) and is known to be induced by IL-1β. APO866 is an inhibitor of NAD biosynthesis and is involved in autophagy. LC3 (microtubule-associated protein 1 light chain 3) is a key regulator of autophagy and is used as an indicator of increased autophagy. Herein, we investigate the role of APO866 in regulating autophagy in NP cells and IL-1β mediated NP cell degeneration and apoptosis. Methods: NP cells were extracted from IDD tissues and cultured in DMEM/F12 medium. Nampt was induced by different concentrations of IL-1β (0, 0.5, 1, 5, 10 ng/mL) for 24 h or NP cells were treated with 10 ng/mL IL-1β for 0, 6, 12, 48 h. QRT-PCR and western blots were used to detect Nampt and ECM-related protein expression in NP tissue of patients with IDD and in NP cells. Confocal analysis was used to detect membrane-bound LC3, Aggrecan, and Collagen II. Results: Nampt is expressed in NP tissue at higher levels in severe grades of IDD (Grade IV and V) compared with low grades (Grade II and III). In NP cells, 10 ng/mL IL-1β induced Nampt expression for 48 h, increased expression of the degradative-associated proteins, ADAMTS4/5 and MMP-3/13, and decreased expression of ECM-related proteins, Aggrecan and Collagen II. However, the Nampt inhibitor APO866 blocked IL-1β induction, and the knockdown of Nampt expression increased the expression of ECM proteins that were inhibited by IL-1β. Moreover, evidence provided by the autophagic markers LC3 and Beclin-1 indicated that APO866 induced NP cell autophagy. Furthermore, although APO866 inhibited the downregulated expression of ECM-related proteins by IL-1β, this function was blocked by autophagy inhibitor, 3-methyladenine. Conclusion: APO866 protects NP cells and induces autophagy by inhibiting IL-1β-induced NP cell degeneration and apoptosis, which may have therapeutic potential in IDD.

Expression of signaling pathway of mammalian target of rapamycin in articular cartilage cell induced by interleukin 1β

Objective: To observe effect of interleukin(IL)-1β on the expression of signaling pathway of mammalian target of rapamycin(mTOR) of articular cartilage. Methods: Articular cartilage of rats was isolated under sterile technique, cells were digested by type Ⅱ collagenase and trypsin and cultured in vitro, pre-culture the Ⅱ cells for three days, different concentrations of IL-1β were added for 24 hours.The cells were stained with toluidine blue and HE, to observe morphological changes of cells.RT-PCR was used to detect the mRNA expression of typeⅡcollagen gene, aggrecan gene, mTOR gene and P70S6K gene, Western blotting was used to detect the expression of protein related to mTOR. Results: With increasing concentrations of IL-1β, the phenotype of cells appeared polygon into a spindle, the mRNA expression of gene of type Ⅱ collagen (the control group: 0.821±0.014; 1 ng/ml: 0.614±0.014; 10 ng/ml: 0.549±0.009; 100 ng/ml: 0.520±0.008), aggrecan(0.867±0.005; 0.857±0.001; 0.554±0.008; 0.538±0.004) and mTOR(0.845±0.015; 0.785±0.009; 0.569±0.025; 0.518±0.014) reduced, but P70S6K(0.465±0.024; 0.566±0.022; 0.663±0.022; 0.896±0.015) increased by PCR .Expression of protein detected by Western blotting was similar to the trend of PCR. Conclusion: mTOR signaling pathway may play an important role on the degeneration of articular cartilage, regulating mTOR signaling pathway may provides a new idea of delaying the degeneration process of cells.

Morphological, Immunocytochemical, and Biochemical Studies of Rat Costal Chondrocytes Exposed to IL-1β and TGF-β1.

This study was undertaken to determine the effects of IL-1β and TGF-β1 on the expression of differentiation-associated genes in chondrocytes in vitro. Rat costal chondrocytes were exposed to different concentrations of IL-1β and TGF-β1 for 48 h and tested for gene expression. IL-1β increased the expression of aggrecanase-1 and aggrecanase-2 and decreased the content of aggrecan and collagen II. Low concentration of TGF-β1 decreased the expression of aggrecan and collagen II and increased the expression of aggrecanase-2. However, the level of aggrecanase-1 was significantly elevated in the presence of high concentration of TGF-β1. IL-1β and TGF-β1 show the ability to modulate the production of aggrecan and collagen II in chondrocytes in vitro.

Crucial Role of Complement Activation and IL-1β in Bone Marrow Niche of Immune Thrombocytopenia

It is well known that complement activation and cytokine dysregulation are critical in the pathogenesis of immune thrombocytopenia (ITP). We previously showed impaired proliferative capacity and reduced ability to inhibit T-cell proliferation of mesenchymal stem cells (MSCs) from ITP patients (Zhang JM, et al. Stem Cell Transl Med 2016), indicating that the bone marrow (BM) niche might be involved in ITP etiology. Recent studies have defined sympathetic nerve fibers and ensheathing Schwann cells as regulators in the BM niche (Arranz L, et al. Nature 2014). Given that the initial pathogenic mechanisms underlying ITP have not yet been identified, we hypothesized that complement activation played a role in the ITP BM niche including nestin+MSCs and Schwann cells.Thirty-one consecutive patients with primary ITP and 19 healthy donors were enrolled in this prospective study. Samples from patients with ITP were divided into the two groups according to whether complement activation product C1q, C3b, C4d or C5b-9 was detectable on the surface of nestin+MSCs: the complement+nestin+MSC-ITP group and the complement-nestin+MSC-ITP group. Here we found that the complement+nestin+MSC-ITP group had fewer nestin+MSCs, lower proliferative capacity, less Cxcl12 production and a reduced capacity for inhibiting T cell proliferation, as well as higher levels of the pro-inflammatory cytokines interleukin-1β (IL-1β) and TNF-α in bone marrow supernatant compared with the complement-nestin+MSC-ITP group and the nestin+MSC-control group, indicating that activation of the complement cascade and enhancement of the inflammatory response are involved in the ITP BM niche.To further define the possible mechanism for the vulnerability of nestin+MSC-ITP to the complement system, messenger RNA (mRNA) from 5 samples from each group respectively were analyzed by PrimeView™ Human Gene Expression Array (Affymetrix). Diminished levels of complement factor H mRNA consistent with factor H secretion was revealed in the complement+nestin+MSC-ITP group. Furthermore, the mRNA levels of C3aR, C5aR, IL-1R and caspase1 were up-regulated, whereas mRNA levels of CD46, CD55 and CD59 showed no statistically significant differences in this group. Upon further exposure to TNF-α, the complement+nestin+MSC-ITP group insignificantly up-regulated factor H compared with the other two groups.Interestingly, in the complement+nestin+MSC-ITP group, complement activation products were detected on the outer surface of Schwann cells. Immunostaining of glial fibrillary acidic protein (Gfap) to visualize Schwann cells showed undesirable innervation in bone marrow of the complement+nestin+MSC-ITP group. GFAP mRNA expression was concurrently reduced in this group. To characterize the supporting role of Schwann cells, Schwann cells and nestin+MSCs derived from neonatal BMCD45-CD31-Ter119-Nes-GFP+Pdgfrα-/+ cells were co-cultured in vitro with different concentrations of IL-1β for 24 h. The results indicated that sympathetic neuropathy could sensitize nestin+MSCs to cell death triggered by IL-1β. Schwann cells were additionally incubated with an antagonist of interleukin-1 receptor (IL-1Ra), which consequently reversed IL-1β mediated cell damage. Together, these data suggested that IL-1β contributes to neuroglial damage, which compromised nestin+MSC survival.To further define potential regulators that can reverse the alternations of the ITP BM niche, we developed an ITP murine model. ITP mice were randomly assigned to two groups and treated with either prednisone or IL-1Ra. Both of these two groups exhibited improved proliferative capacity and inhibition of T cell proliferation of nestin+MSCs and recovered Schwann cells innervations. No statistically differences were observed between the two groups.In conclusion, the impaired nestin+MSCs and Schwann cells by complement activation and IL-1β in the BM niche play a role in the pathogenesis of ITP. The effect of IL-1Ra in correcting the dysfunction of nestin+MSCs and Schwann cells suggests a potential novel therapeutic approach of IL-1Ra for patients with ITP. DisclosuresNo relevant conflicts of interest to declare.

Matrix Metalloproteinase-3 in Odontoblastic Cells Derived from Ips Cells: Unique Proliferation Response as Odontoblastic Cells Derived from ES Cells

We previously reported that matrix metalloproteinase (MMP)-3 accelerates wound healing following dental pulp injury. In addition, we reported that a proinflammatory cytokine mixture (tumor necrosis factor-α, interleukin (IL)-1β and interferon-γ) induced MMP-3 activity in odontoblast-like cells derived from mouse embryonic stem (ES) cells, suggesting that MMP-3 plays a potential unique physiological role in wound healing and regeneration of dental pulp in odontoblast-like cells. In this study, we tested the hypothesis that upregulation of MMP-3 activity by IL-1β promotes proliferation and apoptosis of purified odontoblast-like cells derived from induced pluripotent stem (iPS) and ES cells. Each odontoblast-like cell was isolated and incubated with different concentrations of IL-1β. MMP-3 mRNA and protein expression were assessed using RT-PCR and western blotting, respectively. MMP-3 activity was measured using immunoprecipitation and a fluorescence substrate. Cell proliferation and apoptosis were determined using ELISA for BrdU and DNA fragmentation, respectively. siRNA was used to reduce MMP-3 transcripts in these cells. Treatment with IL-1β increased MMP-3 mRNA and protein levels, and MMP-3 activity in odontoblast-like cells. Cell proliferation was found to markedly increase with no changes in apoptosis. Endogenous tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 were constitutively expressed during all experiments. The exocytosis inhibitor, Exo1, potently suppressed the appearance of MMP-3 in the conditioned medium. Treatment with siRNA against MMP-3 suppressed an IL-1β-induced increase in MMP-3 expression and activity, and also suppressed cell proliferation, but unexpectedly increased apoptosis in these cells (P<0.05). Exogenous MMP-3 was found to induce cell proliferation in odontoblast-like cells derived from iPS cells and ES cells. This siRNA-mediated increase in apoptosis could be reversed with exogenous MMP-3 stimulation (P<0.05). Taken together, IL-1β induced MMP-3-regulated cell proliferation and suppressed apoptosis in odontoblast-like cells derived from iPS and ES cells.

The induction expression of human β-defensins in gingival epithelial cells and fibroblasts

ObjectiveThe gingival epithelial cells and fibroblasts can produce antimicrobial peptides when stimulated by inflammatory cytokines. The purpose of the present study was to test whether gingival keratinocytes and gingival fibroblasts respond differently to inflammatory cytokine activation. This will enable us to understand the chronic inflammatory response in the process of periodontal disease. DesignGingival keratinocytes and fibroblasts were isolated and treated with different concentrations of IL-1β and quantitative real-time PCR was performed to evaluate the induced expressions of hBD-1, hBD-2 and hBD-3. The induced response was compared between the gingival epithelial cells and fibroblasts. The inhibitors of p38 protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) were applied to explore the molecular mechanism during the induction of hBDs in both cells. ResultsThe results showed that the hBDs expressions were found to be induced by different concentrations of IL-1β, but with several differences between gingival epithelial cells and fibroblasts. The hBDs mRNA expression in gingival fibroblasts was more sensitive compared with keratinocytes to different concentrations of IL-1β. The hBD-1 and hBD-3 expressions in these two cells were down-regulated by IL-1β and hBD-2 expression was up-regulated. The inflammatory cytokine IL-1β had dual effect on hBDs expression. ConclusionsThe gingival epithelial cells and fibroblasts respond differently to the inflammatory cytokine IL-1β which indicated different roles played by the two cells in the host defense. The dual effect of IL-1β on hBDs expression may contribute to the defensins down-regulation in periodontal disease.

F-actin may play an important role in IL-1β-stimulated hippocampal neurons

ObjectivesThe aim of this study was to investigate whether IL-1β treatment affects F-actin in hippocampal neurons. Materials and methodsPrimary culture of hippocampal neurons were prepared from rat embryos, and the effect of different concentrations of IL-1β on the cell phenotype was investigated. Cell viability was monitored by CCK-8 activity analysis. Western blotting and immunofluorescence were performed to analyze protein levels and morphological changes in cells treated with IL-1β. ResultsIL-1β increased viability at low doses and these increases were severely blunted at higher concentrations. Similarly, the expression of F-actin was increased at low concentrations and decreased at high concentrations. ConclusionsIL-1β has a critical role in regulating hippocampal neuron viability by stimulating F-actin expression. These data suggest that IL-1β secretion at low doses is beneficial for the expression of F-actin, which can improve the survival of hippocampal neurons during stress. However,these effect were severely blunted at higher concentrations.

Interleukin-1β regulates cell proliferation and activity of extracellular matrix remodelling enzymes in cultured primary pig heart cells

After myocardial infarction, elevated levels of interleukins (ILs) are found within the myocardial tissue and IL-1β is considered to play a major role in tissue remodelling events throughout the body. In the study presented, we have established a cell culture model of primary pig heart cells to evaluate the effects of different concentrations of IL-1β on cell proliferation as well as expression and activity of enzymes typically involved in tissue remodelling. Primary pig heart cell cultures were derived from three different animals and stimulated with recombinant pig IL-1β. RNA expression was detected by RT-PCR, protein levels were evaluated by Western blotting, activity of matrix metalloproteinases (MMPs) was quantified by gelatine zymography and cell proliferation was measured using colorimetric MTS assays. Pig heart cells express receptors for IL-1 and application of IL-1β resulted in a dose-dependent increase of cell proliferation ( P < 0.05 vs. control; 100 ng/ml; 24 h). Gene expression of caspase-3 was increased by IL-1β ( P < 0.05 vs. control; 100 ng/ml; 3 h), and pro-caspase-3 but not active caspase was detected in lysates of pig heart cells by Western blotting. MMP-2 gene expression as well as enzymatic activities of MMP-2 and MMP-9 were increased by IL-1β ( P < 0.05 vs. control; 100 ng/ml; 3 h for gene expression, 48 and 72 h for enzymatic activities of MMP-2 and MMP-9, respectively). Our in vitro data suggest that IL-1β plays a major role in the events of tissue remodelling in the heart. Combined with our recently published in vivo data (Meybohm et al., PLoS One, 2009), the results presented here strongly suggest IL-1β as a key molecule guiding tissue remodelling events after myocardial infarction.