Epithelial microRNA-30a-3p targets RUNX2/HMGB1 axis to suppress airway eosinophilic inflammation in asthma.

BACKGROUND: Type 2-high asthma is a prominent endotype of asthma which is characterized by airway eosinophilic inflammation. Airway epithelial cells play a critical role in the pathogenesis of asthma. Our previous miRNA profiling data showed that miR-30a-3p was downregulated in bronchial epithelial cells from asthma patients. We hypothesize that epithelial miR-30a-3p plays a role in asthma airway inflammation. METHODS: We measured miR-30a-3p expression in bronchial brushings of asthma patients (n = 51) and healthy controls (n = 16), and analyzed the correlations between miR-30a-3p expression and airway eosinophilia. We examined whether Runt-related transcription factor 2 (RUNX2) was a target of miR-30a-3p and whether RUNX2 bound to the promoter of high mobility group box 1 (HMGB1) by using luciferase reporter assay and chromatin immunoprecipitation (ChIP)-PCR. The role of miR-30a-3p was also investigated in a murine model of allergic airway inflammation. RESULTS: We found that miR-30a-3p expression were significantly decreased in bronchial brushings of asthma patients compared to control subjects. Epithelial miR-30a-3p expression was negatively correlated with parameters reflecting airway eosinophilia including eosinophils in induced sputum and bronchial biopsies, and fraction of exhaled nitric oxide in asthma patients. We verified that RUNX2 is a target of miR-30a-3p. Furthermore, RUNX2 bound to the promoter of HMGB1 and upregulated HMGB1 expression. RUNX2 and HMGB1 expression was both enhanced in airway epithelium and was correlated with each other in asthma patients. Inhibition of miR-30a-3p enhanced RUNX2 and HMGB1 expression, and RUNX2 overexpression upregulated HMGB1 in BEAS-2B cells. Intriguingly, airway overexpression of mmu-miR-30a-3p suppressed Runx2 and Hmgb1 expression, and alleviated airway eosinophilia in a mouse model of allergic airway inflammation. CONCLUSIONS: Epithelial miR-30a-3p could possibly target RUNX2/HMGB1 axis to suppress airway eosinophilia in asthma.

The Concentration of Selected Inflammatory Cytokines (IL-6, IL-8, CXCL5, IL-33) and Damage-Associated Molecular Patterns (HMGB-1, HSP-70) Released in an Early Response to Distal Forearm Fracture and the Performed Closed Reduction With Kirschner Wire Fixation in Children.

The evaluation of trauma after surgery through objective analysis of biochemical markers can help in selecting the most appropriate therapy. Thus the aim of the study was the evaluation of the concentration of selected inflammatory cytokines (IL-6, IL-8, CXCL5, IL-33), C-reactive protein (CRP), and damaged-associated molecular patterns (DAMPs): HMGB-1, HSP-70 in the plasma of children in response to bone fracture and 12-14 hours after subsequent surgery performed by closed reduction with percutaneous Kirschner wire fixation (CRKF). The study will answer the question if the CRFK procedure leads to excessive production of inflammatory and damage markers. Blood samples from 29 children with distal forearm fractures were collected 30 min. before CRKF procedure and 12-14 hours after performance of the procedure. The control group was composed of 17 healthy children. IL-6 and CRP concentrations were analyzed using routinely performed in vitro diagnostics tests; the remaining proteins were analyzed with the use of the ELISA method. Increased values of IL-6, CRP, and HSP-70 represented an early inflammatory response to distal forearm fractures classified as SH-II type according to the Salter-Harris classification system. However, the median CRP concentration was within the reference values not indicative of inflammation. The CRKF procedure may be a good solution for the treatment of bone fractures, as damaged associated molecular patterns - HMGB-1 and HSP-70 - did not significantly differ 12-14 hours after the approach was applied as compared to the control group. Moreover, the increase in IL-6 concentration after the CRKF procedure was 1.5-fold to the level before CRKF, while the increase of this marker in response to the distal forearm fracture was 4.3-fold compared to the control group. Based on this data, it appears reasonable to suggest that the CRKF approach caused less damage and inflammatory response in comparison to the response to the fracture itself.

IGF-1 alleviates CCL4-induced hepatic cirrhosis and dysfunction of intestinal barrier through inhibition TLR4/NF-κB signaling mediated by down-regulation HMGB1.

INTRODUCTION AND OBJECTIVES: Cirrhosis has gradually become a serious public health issue, especially the national prevalence of cirrhosis was 29.2% in northwest China. Recent evidence has revealed that intestinal barrier (IB) dysfunction results from and contributes to cirrhosis. Our previous results have indicated that insulin-like growth factors (IGF-1) improved the impaired IB function and downregulated high mobility group protein box-1 (HMGB-1). Nevertheless, the role of the IGF-1/HMGB1 axis in cirrhosis remains largely unknown. MATERIALS AND METHODS: Western blotting and qRT-PCR were used to detect protein and mRNA levels of related genes. The levels of AST, ALT, IL-1ß, and TNF-α were examined using commercial kits. Immunofluorescence was used to evaluate the expression of HMGB1 in tissues. RESULTS: In carbon tetrachloride (CCl4)-treated rat, the levels of AST (380.12 vs. 183.97), ALT (148.12 vs. 53.56), IL-1ß (155.94 vs. 55.60), and TNF-α (155.00 vs. 48.90) were significantly increased compared with the control group, while IGF-1 treatment significantly alleviated CCL4-induced inflammatory response and IB dysfunction by downregulating HMGB1-mediated the TLR4/MyD88/NF-κB signaling pathway. In vitro experiments, HMGB1 treatment promoted inflammatory cytokines secretion and reduced cell viability and tight junctions by activating the TLR4/MyD88/NF-κB signaling pathway in Caco-2 cells, but IGF-1 alleviated these effects. CONCLUSION: Our findings suggest that IGF-1 might serve as a potential therapeutic target for cirrhosis and IB dysfunction via inactivation of the TLR4/MyD88/NF-κB pathway through down-regulation HMGB1.

Glycyrrhizic acid, as an inhibitor of HMGB1, alleviates bleomycin-induced pulmonary toxicity in mice through the MAPK and Smad3 pathways.

AIM: High-mobility group box 1 (HMGB1) protein has been noticed particularly for its pivotal role in several pathologies. However, the relevance between HMGB1 and pathological progress in lung toxicity still remains unclear. In the study, we evaluated the effect of glycyrrhizic acid as an HMGB1 inhibitor on the early inflammation and late fibrosis in bleomycin-induced pulmonary toxicity in mice. METHODS: We established a bleomycin-induced pulmonary toxicity model to detect the relevance between HMGB1 and pathological changes in the early inflammatory and late fibrotic stages. RESULTS: We found that bleomycin-induced increase in inflammatory cytokines interleukin (IL)-ß1, tumor necrosis factor (TNF)-α, monocyte chemotactic protein (MCP)-1, and inflammatory lesions in lung tissue in the early stage of the model. However, markers of fibrosis such as transforming growth factor (TGF)-ß1 and α-smooth muscle actin (α-SMA) were significantly elevated on day 7 after bleomycin instillation. Interestingly, HMGB1 also began to rise on day 7, rather than in the early inflammatory phase. However, early (from day 0 to 14 after bleomycin instillation) or late (from day 14 to 28) intervention with HMGB1 neutralizing antibody or glycyrrhizic acid alleviated inflammation and fibrosis through down-regulating the inflammatory signaling mitogen-activated protein kinase (MAPK) and fibrotic signaling Smad3 pathway. CONCLUSION: Our results suggested that HMGB1 mediates both inflammation and fibrosis in this model. The development of high-potency and low-toxicity HMGB1 inhibitors may be a class of potential drugs for the treatment of pulmonary fibrosis.

MicroRNA-181b Inhibits Inflammatory Response and Reduces Myocardial Injury in Sepsis by Downregulating HMGB1.

MicroRNAs (miRNAs) are short endogenous noncoding RNAs regulating protein translation. However, the specific mechanism by which miR-181b influences sepsis via high-mobility group box-1 protein (HMGB1) still remains unknown. Thus, the aim of this study is to investigate the mechanism of miR-181b in regulating inflammatory response in sepsis-induced myocardial injury through targeting high-mobility group box-1 protein (HMGB1). Through cecal ligation and puncture (CLP), the rat model of sepsis was established. Then, the effect of altered expression of miR-181b and HMGB1 on cardiomyocytes was investigated. The positive expression rate of HMGB1, concentration of inflammatory factors, and serum myocardial enzyme of myocardial tissues were determined. Besides, the binding site between miR-181b and HMGB1 was determined by bioinformatics information and dual-luciferase reporter gene assay. The expression of related genes in cells of each group was determined by RT-qPCR and western blot analysis, and the apoptosis rate of transfected cells in each group was determined by TUNEL assay. HMGB1 expression and inflammatory factors were significantly increased in myocardial tissue of rats with sepsis. Cell morphology and the infiltration of inflammatory cells were significantly improved by overexpression of miR-181b. miR-181b directly targeted HMGB1, and downregulation of HMGB1 reduced inflammatory factors and myocardial injury and inhibited cardiomyocyte apoptosis in sepsis. This present study suggests that miR-181b decreased inflammatory factors and reduced myocardial injury in sepsis through downregulation of HMGB1. Thus, a better understanding of this process may aid in the development of novel therapeutic agents in sepsis.

MiR-216a-5p ameliorates learning-memory deficits and neuroinflammatory response of Alzheimer's disease mice via regulation of HMGB1/NF-κB signaling.

OBJECTIVE: The objective of this study was to explore whether miR-216a-5p could affect the learning-memory ability and inflammatory response of Alzheimer's disease (AD) mice via regulation of the HMGB1/NF-κB pathway. METHODS: Mice were divided into the normal (wild-type C57BL/6 mice), AD (APP/PS1 double-transgenic mice), AD + miR-216a-5p, and AD + vector groups. The Morris water maze test was used to examine learning and memory ability. Nissl staining and TUNEL staining were performed to observe the survival and apoptosis of hippocampal neurons. In addition, Aß deposition and the expression of inflammatory cytokines were determined, while miR-216a-5p expression and HMGB1/NF-κB pathway-related proteins were detected by qRT-PCR and Western blotting, respectively. RESULTS: AD mice exhibited decreased miR-216a-5p expression but increased HMGB-1 protein expression in the hippocampus, and these mice had a prolonged escape latency, fewer number of times crossing the platform location and shortened time in the target quadrant compared to those in normal mice. AD mice also had an elevated number of TUNEL-positive cells, increased deposition of Aß, increased expression of inflammatory cytokines and decreased number of Nissl-positive cells. In addition, AD mice presented with downregulated expression of cytoplasmic NF-κB p65 protein but upregulated expression of nuclear NF-κB p65 protein. However, AD mice treated with miR-216a-5p exhibited significant improvements of the abovementioned parameters. The dual-luciferase reporter assay confirmed that HMGB1 is a target gene of miR-216a-5p. CONCLUSION: MiR-216a-5p can improve learning-memory ability and attenuate the inflammatory response of AD mice through targeted inhibition of the HMGB1/NF-κB pathway.

IFN-τ Attenuates LPS-Induced Endometritis by Restraining HMGB1/NF-κB Activation in bEECs.

Endometritis is a common inflammatory disease in uterine tissues that leads to animal infertility. Among the causes, Escherichia coli infection is one of the main reasons. Interferon-tau (IFN-τ) is the initial pregnancy signal for ruminant embryos and can induce immune tolerance in humans and other species. However, there are scarce reports on whether IFN-τ has a regulatory effect on endometrial inflammatory damage through HMGB1-NF-κB signalling. The purpose of this study was to investigate the regulatory mechanism of IFN-τ in HMGB1-NF-κB signalling in LPS-induced endometritis. ELISA and qPCR were used to detect the expression of LPS-induced pro-inflammatory cytokines in bovine endometrial epithelial cells (bEECs or BEND) under IFN-τ intervention, and the levels of HMGB1, p-IKK and p-p65 were detected by Western blotting. The nuclear translocation of NF-κB p65 was determined through immunofluorescence. In addition, bEECs were transfected with si-HMGB1 to elucidate the key role of HMGB1 and IFN-τ in the endometrial inflammatory cascade. The results indicated that IFN-τ inhibits the expression of related pro-inflammatory cytokines in an inflammatory injury model of bovine endometrial epithelial cells induced by LPS. Furthermore, experiments have proven that IFN-τ has protective effects on E. coli endotoxin-induced endometritis in mice in vivo. IFN-τ inhibited the HMGB1-NF-κB axis and significantly reduced the secretion of pro-inflammatory cytokines, the expression of HMGB1 protein and the levels of IKK and NF-κB p65 phosphorylation. In summary, our results showed that IFN-τ resists E. coli endotoxin-induced endometritis by attenuating HMGB1/NF-κB signalling.

Lactoferrin suppresses LPS-induced expression of HMGB1, microRNA 155, 146, and TLR4/MyD88/NF-кB pathway in RAW264.7 cells.

OBJECTIVE: This current study evaluated the underlying mechanisms of LF against the inflammatory microRNAs (miRNAs), HMGB1 expression, and TLR4-MyD88-NF-кB pathway in LPS-activated murine RAW264.7 cells. METHODS: MTT assay was used to assess cell metabolism and the cell culture levels of the cytokines (TNF-α, IL-6) were evaluated by Enzyme-linked immunosorbent assay (ELISA). The expression of miRNAs was quantified by using qPCR and the expression of HMGB1, TLR4, MyD88, and phosphorylated NF-κB (P-p65) were determined with Western blot and qPCR, respectively. RESULTS: The results indicated that LF downregulates IL-6 and TNF-α expression. LF exhibited the degradation of P-p65 and reduced the production of HMGB1, TLR4, and MyD88 in LPS-induced inflammatory response. Importantly, in parallel with the suppression of cytokines and HMGB1-TLR4-MyD88-NF-кB pathway, LF could induce a decrease in inflammatory selected miRNAs, mmu-mir-155, and mmu-mir-146a expression. CONCLUSIONS: Altogether, these findings provide LF as a prominent anti-inflammatory agent that could modulate HMGB1, mmu-mir-155, mmu-mir-146a, and TLR4/MyD88/NF-кB pathway.

Chronic stress promotes acute myeloid leukemia progression through HMGB1/NLRP3/IL-1ß signaling pathway.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor prognosis and overall survival. Clinical investigations show that chronic stress is commonly present in the course of AML and associated with adverse outcome. However, the underlying molecular mechanisms are elusive. In the present study, a chronic restraint stress mouse model was established to evaluate the effect of stress on AML. We found that mice under chronic stress exhibited significantly increased liver and spleen infiltration of leukemic cells and poorer overall survival. This was accompanied by elevated cellular NLR family pyrin domain containing 3 (NLRP3) and interleukin-1ß (IL-1ß) in the liver or bone marrow, and secreted IL-1ß in the plasma, indicating the activation of inflammasomes under chronic restraint stress. High mobility group box 1 (HMGB1) expression was markedly increased in newly diagnosed AML patients, but reduced in complete remission AML patients. The expression level of HMGB1 was positively correlated with NLRP3 mRNA in AML patients. Knockdown of HMGB1 significantly decreased NLRP3 and IL-1ß expression in AML cell lines, and secreted IL-1ß in supernatant of AML cell culture, while HMGB1 stimulation caused contrary effects. These results implied that HMGB1 could be involved in the regulation of inflammasome activation in AML development. Mice model showed that chronic restraint stress-facilitated proliferation and infiltration of AML cells were largely abrogated by knocking down HMGB1. Knockdown of HMGB1 also ameliorated overall survival and remarkably neutralized NLRP3 and IL-1ß expression under chronic restraint stress. These findings provide evidences that chronic stress promotes AML progression via HMGB1/NLRP3/IL-1ß dependent mechanism, suggesting that HMGB1 is a potential therapeutic target for AML. KEY MESSAGES: • Chronic restraint stress promoted acute myeloid leukemia (AML) progression and mediated NLRP3 inflammasome activation in xenograft mice. • HMGB1 mediated NLRP3 inflammasome activation in AML cells. • Knockdown of HMGB1 inhibited AML progression under chronic stress in vivo.

Lidocaine Alleviates Neuropathic Pain and Neuroinflammation by Inhibiting HMGB1 Expression to Mediate MIP-1α/CCR1 Pathway.

High mobility group box 1 (HMGB1) released from sensory nerve tissues can induce neuropathic pain. Whether HMGB1 is implicated in the mechanism underlying the effect of lidocaine in pain management remains to be determined. This study aims to explore the effect of lidocaine in a rat model of spared nerve injury (SNI) and the underlying mechanism. An SNI model was established via nerve ligation. Two weeks after the SNI model was established, rats were intrathecally injected with lidocaine, an HMGB1 antibody (HMG Ab), an MIP-1α antibody (MIP-1α Ab), a CCR1 inhibitor (CCR1-RS) or a CCR5 antagonist (CCR5-Mar). Pain behaviors were assessed before and after model establishment to calculate the number of spontaneous flinches (NSF), paw withdrawal threshold (PWT), paw withdrawal thermal latency (PWL) and sciatic function index (SFI). Cell apoptosis and the inflammatory response in the cerebrospinal fluid (CSF) were detected by TUNEL staining and ELISA. The mRNA and protein expression levels of MIP-1α, CCR1 and CCR5 were determined by RT-PCR and Western blotting. The expression levels of HMGB1, MIP-1α, CCR1 and CCR5 were measured by Western blotting and immunofluorescence. Pain behavior testing in SNI rats showed that SNI rats exhibited an increased NSF and a decreased PWT, PWL and SFI. Cell apoptosis in the spinal dorsal horn and the generation of inflammatory cytokines were enhanced in SNI rats, and the expression levels of HMGB1, MIP-1α, CCR1 and CCR5 were upregulated. HMGB1 cytoplasmic translocation, the coexpression of MIP-1α with NeuN, and the coexpression of CCR1 and CCR5 with OX42 were also observed in SNI rats. Neuropathic pain and neuroinflammation were suppressed by the intrathecal injection of lidocaine, HMG Ab, MIP-1α Ab, CCR1-RS or CCR5-Mar. Lidocaine inhibited the expression levels of HMGB1, MIP-1α, CCR1 and CCR5, and the HMGB1 antibody suppressed the expression of MIP-1α, CCR1 and CCR5. Lidocaine attenuates neuropathic pain and neuroinflammation by inhibiting HMGB1 to regulate the MIP-1α/CCR1/CCR5 pathway. Graphical Abstract.

Downregulation of lncRNA FIRRE relieved the neuropathic pain of female mice by suppressing HMGB1 expression.

Long non-coding RNAs are novel regulators in neuropathic pain. In this study, we aimed to explore the role and the mechanism of lncRNA FIRRE in regulating the secretion of microglial cells-derived proinflammatory cytokines in neuropathic pain. The female mouse model of neuropathic pain was established by bilateral chronic constriction injury (CCI) surgery. The mouse primary microglial cells were induced by lipopolysaccharide (LPS). The interaction between FIRRE and high mobility group box 1 (HMGB1) was assessed by RNA immunoprecipitation, RNA pull-down, and ubiquitination assays. FIRRE expression was upregulated in the spinal cord tissue of female CCI mice and LPS-induced microglial cells. The concentrations of IL-1ß, TNF-α, and IL-6 from LPS-induced microglial cells were reduced by FIRRE knockdown. FIRRE bound to HMGB1 and negatively regulated its protein level. The ubiquitination degradation of HMGB1 was promoted by FIRRE silence. The HMGB1 over-expression reversed the inhibitory effect of FIRRE silence on the secretion of IL-1ß, TNF-α, and IL-6 from LPS-induced microglial cells. The in vivo experiment showed that FIRRE knockdown alleviated neuropathic pain of CCI female mice. Our findings indicated that lncRNA FIRRE downregulation inhibits the secretion of microglial cells-derived proinflammatory cytokines by decreasing HMGB1 expression, thereby relieving neuropathic pain of female mice.

Expression profiles and potential corneal epithelial wound healing regulation targets of high-mobility group box 1 in diabetic mice.

As a damage-associated molecular pattern molecule, high-mobility group box 1 protein (HMGB1) is involved in diabetes and its complications. However, the role of HMGB1 in diabetic keratopathy is not yet understood. The purpose of this study was to investigate the potential roles of HMGB1 in the development of diabetic keratopathy as well as potential strategies to block HMGB1 in order to prompt epithelial wound healing and nerve regeneration in diabetic corneas. The results demonstrated that diabetic keratopathy developed in mice over the duration of the diabetic condition with typical symptoms, including damaged ocular surfaces and corneal nerves. The diabetic corneas had significantly increased protein expression levels of HMGB1 and its receptors-the receptor for advanced glycation end products (RAGE) and toll-like receptor 4 (TLR4)-compared to the age-matched normal corneas (P < 0.05). Corneal HMGB1 levels significantly increased during the corneal wound healing process of the diabetic mice, peaking on the first day after the wound was created and then decreasing to the unwounded level on the seventh day. Exogenous HMGB1 peptide significantly retarded wound and nerve healing, while glycyrrhizin (an HMGB1 inhibitor) significantly prompted wound and nerve healing. Further, the western blot results confirmed that RAGE and TLR4 were also involved in corneal wound and nerve healing. In conclusion, these data showed that HMGB1 and its related receptors are highly involved in the development of diabetic keratopathy. This finding indicates that the blockage of HMGB1 might serve as a strategy to prompt diabetic corneal and nerve wound healing.

Epigallocatechin Gallate (EGCG) Improves Anti-Angiogenic State, Cell Viability, and Hypoxia-Induced Endothelial Dysfunction by Downregulating High Mobility Group Box 1 (HMGB1) in Preeclampsia.

BACKGROUND Preeclampsia (PE) is a serious complication of pregnancy with no effective therapy. This study assessed whether epigallocatechin gallate (EGCG) could reduce the production of anti-angiogenic factors, improve cell viability, and suppress endothelial dysfunction in vitro via regulating high mobility group box 1 (HMGB1) in preeclampsia. MATERIAL AND METHODS Human umbilical vein endothelial cells (HUVECs) grown in conditioned medium from hypoxic JEG-3 cells were used to investigate the effects of EGCG on anti-angiogenic state, cell viability, and markers of endothelial dysfunction. To confirm that EGCG exerted its effects via HMGB1, we also examined the impact of EGCG on anti-angiogenic state, cell viability, and endothelial dysfunction following HMGB1 treatment in vitro. RESULTS EGCG inhibited HMGB1 expression in hypoxic trophoblast cells in a dose-dependent manner. In addition, EGCG relieved anti-angiogenic state and endothelial dysfunction in hypoxic trophoblast cells by downregulating HMGB1. Moreover, EGCG dose-dependently promoted cell proliferation by downregulating HMGB1. CONCLUSIONS Taken together, our data show the protective role of EGCG in preeclampsia and revealed EGCG-mediated effects on the production of anti-angiogenic factors, cell viability, and endothelial dysfunction through downregulating HMGB1. These observations suggest that EGCG is a novel therapeutic candidate for preeclampsia.

Inhibiting HMGB1-RAGE axis prevents pro-inflammatory macrophages/microglia polarization and affords neuroprotection after spinal cord injury.

BACKGROUND: Spinal cord injury (SCI) favors a persistent pro-inflammatory macrophages/microglia-mediated response with only a transient appearance of anti-inflammatory phenotype of immune cells. However, the mechanisms controlling this special sterile inflammation after SCI are still not fully elucidated. It is known that damage-associated molecular patterns (DAMPs) released from necrotic cells after injury can trigger severe inflammation. High mobility group box 1(HMGB1), a ubiquitously expressed DNA binding protein, is an identified DAMP, and our previous study demonstrated that reactive astrocytes could undergo necroptosis and release HMGB1 after SCI in mice. The present study aimed to explore the effects and the possible mechanism of HMGB1on macrophages/microglia polarization, as well as the neuroprotective effects by HMGB1 inhibition after SCI. METHODS: In this study, the expression and the concentration of HMGB1 was determined by qRT-PCR, ELISA, and immunohistochemistry. Glycyrrhizin was applied to inhibit HMGB1, while FPS-ZM1 to suppress receptor for advanced glycation end products (RAGE). The polarization of macrophages/microglia in vitro and in vivo was detected by qRT-PCR, immunostaining, and western blot. The lesion area was detected by GFAP staining, while neuronal survival was examined by Nissl staining. Luxol fast blue (LFB) staining, DAB staining, and western blot were adopted to evaluate the myelin loss. Basso-Beattie-Bresnahan (BBB) scoring and rump-height Index (RHI) assay was applied to evaluate locomotor functional recovery. RESULTS: Our data showed that HMGB1 can be elevated and released from necroptotic astrocytes and HMGB1 could induce pro-inflammatory microglia through the RAGE-nuclear factor-kappa B (NF-κB) pathway. We further demonstrated that inhibiting HMGB1 or RAGE effectively decreased the numbers of detrimental pro-inflammatory macrophages/microglia while increased anti-inflammatory cells after SCI. Furthermore, our data showed that inhibiting HMGB1 or RAGE significantly decreased neuronal loss and demyelination, and improved functional recovery after SCI. CONCLUSIONS: The data implicated that HMGB1-RAGE axis contributed to the dominant pro-inflammatory macrophages/microglia-mediated pro-inflammatory response, and inhibiting this pathway afforded neuroprotection for SCI. Thus, therapies designed to modulate immune microenvironment based on this cascade might be a prospective treatment for SCI.

Cav3.2 overexpression in L4 dorsal root ganglion neurons after L5 spinal nerve cutting involves Egr-1, USP5 and HMGB1 in rats: An emerging signaling pathway for neuropathic pain.

Overexpression of Cav3.2 T-type Ca2+ channels in L4 dorsal root ganglion (DRG) participates in neuropathic pain after L5 spinal nerve cutting (L5SNC) in rats. The L5SNC-induced neuropathic pain also involves high mobility group box 1 (HMGB1), a damage-associated molecular pattern protein, and its target, the receptor for advanced glycation end-products (RAGE). We thus studied the molecular mechanisms for the L5SNC-induced Cav3.2 overexpression as well as neuropathic pain in rats by focusing on; 1) possible involvement of early growth response 1 (Egr-1), known to regulate transcriptional expression of Cav3.2, and ubiquitin-specific protease 5 (USP5) that protects Cav3.2 from proteasomal degradation, and 2) possible role of HMGB1/RAGE as an upstream signal. Protein levels of Cav3.2 as well as Egr-1 in L4 DRG significantly increased in the early (day 6) and persistent (day 14) phases of neuropathy after L5SNC, while USP5 protein in L4 DRG did not increase on day 6, but day 14. An anti-HMGB1-neutralizing antibody or a low molecular weight heparin, a RAGE antagonist, prevented the development of neuropathic pain and upregulation of Egr-1 and Cav3.2 in L4 DRG after L5SNC. L5SNC increased macrophages accumulating in the sciatic nerves, and the cytoplasm/nuclear ratio of immunoreactive HMGB1 in those macrophages. Our findings suggest that L5SNC-induced Cav3.2 overexpression in L4 DRG and neuropathic pain involves Egr-1 upregulation downstream of the macrophage-derived HMGB1/RAGE pathway, and that the delayed upregulation of USP5 might contribute to the persistent Cav3.2 overexpression and neuropathy.

Downregulation of lncRNA TUG1 attenuates inflammation and apoptosis of renal tubular epithelial cell induced by ischemia-reperfusion by sponging miR-449b-5p via targeting HMGB1 and MMP2.

We aimed to evaluate the functions of long non-coding RNA taurine upregulated gene 1 (lncRNA TUG1) in renal ischemia-reperfusion (I/R) injury and identify the potential mechanisms. Pathological changes of renal tissues were examined using H&E staining after mimic renal I/R injury in vivo. The contents of serum renal functional parameters and inflammatory factors were measured. The expression of TUG1 and miR-449b-5p in renal tissues and HK-2 cells stimulated by I/R were detected. Then, the effects of TUG1 silencing on inflammation and apoptosis of cells were evaluated. Dual luciferase reporter assays were executed for determining the correlation between miR-449b-5p and TUG1, high mobility group box 1 (HMGB1), or matrix metalloproteinase 2 (MMP2). Subsequently, cells were co-transfected with miR-449b-5p mimic and pcDNA3.1 TUG1. The levels of inflammation, apoptosis, and the expression of HMGB1 and MMP2 were detected. The results revealed that renal tissues were obviously damaged after I/R accompanied by changes in renal functional markers and inflammatory factors. TUG1 was highly expressed whereas miR-449b-5p was lowly expressed. TUG1 silencing reduced the inflammation and apoptosis. Dual luciferase reporter assays confirmed that miR-449b-5p was a target of TUG1 as well as HMGB1 and MMP2 were direct targets of miR-449b-5p. Meanwhile, miR-449b-5p mimic presented the same results with TUG1 silencing, which were reversed after TUG1 overexpression. Moreover, MMP2 and HMGB1 expression was decreased after miR-449b-5p overexpression while that of was increased after TUG1 overexpression. These findings demonstrated that TUG1 silencing attenuates I/R-induced inflammation and apoptosis via targeting miR-449b-5p and regulating HMGB1 and MMP2 expression.

Propofol upregulates miR-320a and reduces HMGB1 by downregulating ANRIL to inhibit PTC cell malignant behaviors.

BACKGROUND: Our previous study states that propofol suppresses proliferation and migration of papillary thyroid cancer (PTC) cells by downregulation of lncRNA ANRIL. This study intended to probe the downstream mechanism of ANRIL in PTC with potential microRNAs (miR) and genes. METHODS: ANRIL expression was detected in normal thyroid epithelial cells (Nthy-ori 3-1) and PTC cells (TPC-1, FTC-133, K1 and BCPAP). ANRIL expression was inhibited in TPC-1 and BCPAP cells to explore the effects of si-ANRIL in PTC malignant behaviors. The gain-and loss-of functions of ANRIL/miR-320a were performed to measure their roles in PTC. Levels of ANRIL, miR-320a, HMGB1, apoptosis- and Wnt/ß-catenin and NF-κB pathways-related proteins were measured. Dual-luciferase reporter gene assay and RNA pull-down assay were applied to verify ANRIL/miR-320a/HMGB1 relation. si-ANRIL was transplanted into xenograft tumors in nude mice. RESULTS: ANRIL was upregulated in TPC-1 and BCPAP cells. miR-320a targeted HMGB1, and ANRIL bound to miR-320a. In TPC-1 and BCPAP cells, si-ANRIL prevented PTC cell malignant behaviors, and inactivated the Wnt/ß-catenin and NF-κB pathways; while si-ANRIL + miR-320a inhibition showed opposite trends. Overexpressing miR-320a promoted malignant behaviors of TPC-1 cells. In 6 µg/mL propofol-treated TPC-1 cells, miR-320a inhibition weakened propofol's inhibitory effects on PTC cell growth. After ANRIL inhibition, the volume and weight of xenograft tumors were decreased. CONCLUSION: Propofol upregulated miR-320a and reduced HMGB1 by downregulating ANRIL and inactivating the Wnt/ß-catenin and NF-κB pathways, thus preventing PTC cell malignant behaviors. This study may offer new insights in PTC prevention and treatment.

Novel biomarkers for risk stratification of Barrett's oesophagus associated neoplastic progression-epithelial HMGB1 expression and stromal lymphocytic phenotype.

BACKGROUND: The incidence of oesophageal adenocarcinoma is increasing globally. Barrett's oesophagus (BO) is a pre-malignant condition with no biomarker to risk stratify those at highest risk of dysplasia and malignant transformation. METHODS: Subcellular epithelial protein (HMGB1, p53, RUNX3) expression, alongside expression of CD20, CD4, CD8 and Foxp3 to characterise stromal B lymphocyte, and helper, cytotoxic and regulatory T-lymphocyte cell infiltrate, respectively, was assessed by immunohistochemistry in 218 human tissue samples including normal oesophageal/gastric biopsies (n = 39), BO (non-dysplasia, dysplasia, non-dysplastic background from progressors to dysplasia or cancer, n = 121) and oesophageal adenocarcinoma (n = 58). RESULTS: There is a dynamic subcellular epithelial expression of HMGB1 (loss of nuclear, emergence of cytoplasmic), associated with epithelial p53 expression and differential immune cell phenotype in oesophageal neoplastic progression. We identify a protein signature and lymphocyte infiltrate in non-dysplastic BO when progressive disease (dysplasia or adenocarcinoma) is present but not histologically represented in the biopsied field. There is a dynamic stromal lymphocytic infiltrate in oesophageal neoplastic progression. CONCLUSIONS: This data reveals novel insights into the microenvironment of BO and progression towards cancer and identifies a novel high-risk biomarker of disease progression to aid surveillance strategies to identify early progression and impact future incidence of oesophageal cancer.

MicroRNA-548 regulates high mobility group box 1 expression in patients with preterm birth and chorioamnionitis.

High mobility group box 1 (HMGB1) is a prototypic alarmin and plays an important role in the pathogenesis of inflammatory process in spontaneous preterm birth. This study was conducted to compare the levels of HMGB1 in amniotic fluid and amnion membranes in women with chorioamnionitis/intra-amniotic inflammation to the levels in healthy controls. We also aimed to elucidate the involvement of microRNA-548 (miR-548) in regulating HMGB1 expression and its function in human amniotic epithelial cells (hAECs). A bioinformatics analysis predicted the binding of HMGB1 by the miR-548 cluster. A repressed and forced expression assay in hAECs was performed to investigate the causal relationship between the miR-548 cluster and HMGB1. The levels of HMGB1 in amniotic fluid and amnion membranes were significantly higher in patients with intra-amniotic inflammation/chorioamnionitis than in those without inflammation. The miR-548 was significantly under-expressed in amnion membranes from patients with chorioamnionitis than in normal term controls. Repressed expression of miR-548 up-regulated HMGB1 expression in hAECs and increased its release from hAECs. Moreover, forced expression of miR-548 suppressed HMGB1 and inflammatory cytokines in hAECs, which increased when treated with lipopolysaccharide. These results suggest miR-548 can alter the inflammatory responses in hAECs, and might be involved in the pathogenesis of preterm birth by regulating HMGB1.

Protective mechanism of GPR30 agonist G1 against ultraviolet B-induced injury in epidermal stem cells.

The regeneration of the skin is vital to our wound healing and skin repair abilities. Adult epidermal stem cells (ESCs) have been shown to have the potential to renew old and dead skin cells, and ESCs have been implemented in stem cell-based therapies. GPR30 is a G protein-coupled membrane receptor for oestrogen, which has been shown to regulate cell proliferation and programmed cell death. Here, we examined the biological function of GPR30 in isolated adult murine ESCs. We show that GPR30 is fairly expressed in ESCs and is repressed upon ultraviolet B (UV-B) treatment in a dose-dependent manner. The activation of GPR30 by its agonist G1 ameliorates UV-B induced cellular oxidative stress and induction of IL-6 and IL-8. Furthermore, G1 protects against UV-B-induced cell death and improves the viability of ESCs. G1 also suppresses UV-B-induced HMGB-1 expression and protects the capacity of ESCs from the harm by UV-B radiation. Mechanistically, we show that co-treatment with G1 rescues UV-B-induced reduced Wnt1, cyclin D1 and ß-catenin production, indicating the involvement of conical Wnt/ß-catenin. Collectively, our data indicate that the activation of GPR30 has a protective role in ESCs, and GPR30 agonist G1-mediated ESC protection has potential implications in stem cell-based therapies for skin diseases.