Expression Of High Mobility Group Protein B1 Research Articles

Mechanism of Huayu Jiedu Decoction in Inhibiting Malignant Biological Characteristics of Multiple Myeloma

To analyze and explore the effects of Huayu Jiedu Decoction on the malignant biological characteristics of multiple myeloma (MM) cells and its molecular mechanism, so as to provide experimental basis and theoretical basis for the alternative therapy of anti-MM in traditional Chinese medicine. Different concentrations of Huayu Jiedu Decoction were used to intervene myeloma U266 cells. The changes of cell proliferation activity were detected by CCK-8 assay, apoptosis was detected by Annexin V/PI double staining flow cytometry, and apoptosis and protein expression of related signaling pathways were detected by Western blot. Real-time quantitative PCR was used to detect mRNA expression changes of high mobility group protein B1 (HMGB1), CXC chemokine receptor 4 (CXCR4) and interleukin-6 (IL-6). Huayu Jiedu Decoction inhibited the proliferative activity of U266 cells and induced their apoptosis in a concentration and time dependent manner ( r =-0.713, r =-0.827). After treatment with Huayu Jiedu Decoction for 48 h, the expressions of anti-apoptotic protein Bcl-2 and survivin were down-regulated, while the expression of pro-apoptotic protein Bax was up-regulated, and the phosphorylation level of TLR4/NF-κB signaling pathway was inhibited. After intervention of Huayu Jiedu decoction, the expressions of HMGB1 and IL-6 mRNA were significantly decreased, while the expression of CXCR4 was not significantly decreased. Huayu Jiedu Decoction can inhibit the proliferative activity of U266 cells and induce programmed death. Its molecular mechanism may be related to regulating the expression of apoptotic proteins, inhibiting the activation of TLR4/NF-κB pathway and down-regulating the expression of HMGB1 and IL-6 mRNA.

Mechanism of analgesic effect of electroacupuncture on rats with cervical spondylosis radiculopathy based on activation of astrocytes and HMGB1/TLR4/MyD88 signaling pathway

To observe effects of electroacupuncture (EA) on the activation of astrocytes and high mobility group protein B1(HMGB1)/Toll-like receptor 4 (TLR4)/myeloid differentiation factor 88 (MyD88) signaling pathway, as well as related cytokines in rats with cervical spondylosis radiculopathy(CSR), so as to explore the analgesic mechanism of EA in treating CSR. Twenty-four male SD rats were randomly divided into blank, sham surgery, model, and EA groups, with 6 rats in each group. CSR rat model was established by using cervical spinal cord canal puncture method. On the 7th day after successful modeling, EA was applied to rats in the EA group at bilateral "Hegu"(LI4) and "Taichong"(LR3) for 20 minutes(1.5 Hz, 1 mA), once daily for 7 consecutive days. Before and after intervention, gait impairment scores and mechanical pain thresholds were assessed. HE staining was used to observe pathological changes in spinal cord tissue. Western blot was used to detect the expression of HMGB1, TLR4, MyD88, and glial fibrillary acidic protein (GFAP) in the spinal cord. ELISA was used to measure the contents of CXC chemokine ligand 1 (CXCL1), chemokine ligand 2 (CCL2), tumor necrosis factor (TNF)-α, and interleukin (IL)-1β in spinal cord. Immunofluorescence staining was used to observe GFAP protein positive expression in spinal cord tissue. There was no significant difference of all indexes between the blank group and the sham surgery group. Compared with the sham surgery group, mechanical pain threshold of rats in the model group was decreased(P<0.01), while gait impairment score, the contents of CXCL1, CCL2, TNF-α, IL-1β, protein expressions of HMGB1, TLR4, MyD88 and GFAP, and positive expression of GFAP in spinal cord tissue were increased (P<0.01)；HE staining indicated severe overall morphological damage in the spinal cord of rats in the model group, with significant shrinkage of gray matter neurons, reduced number of Nissl bodies, and increased inflammatory cell infiltration. Compared with the model group, mechanical pain threshold in the EA group was increased (P<0.01), while gait impairment score, the contents of CXCL1, CCL2, TNF-α, IL-1β, protein expressions of HMGB1, TLR4, MyD88 and GFAP, and positive expression of GFAP in spinal cord were reduced (P<0.01)；HE staining showed more intact neuronal cell bodies, increased number of Nissl bodies, and reduced shrinkage of gray matter neurons, inflammatory cell infiltration, and microvascular dilation in the spinal cord of rats in the EA group. EA can effectively alleviate pain in CSR rats, which is possibly by inhibiting astrocyte activation, HMGB1/TLR4/MyD88 signaling pathway, and reducing the release of related inflammatory cytokines, thus alleviating central sensitization in spinal segments.

Targeting endoplasmic reticulum associated degradation pathway combined with radiotherapy enhances the immunogenicity of esophageal cancer cells

ABSTRACT Immunogenic cell death (ICD) is essential for the activation of immune system against cancer. We aimed to investigate the efficacy of endoplasmic reticulum (ER)-associated protein degradation (ERAD) inhibitors (EerI and NMS-873) in enhancing radiation-induced ICD in esophageal cancer (EC). EC cells were administered with ERAD inhibitors, radiation therapy (RT), and the combination treatment. ICD hallmarks including calreticulin (CALR), adenosine triphosphate (ATP), and high mobility group protein B1 (HMGB1) were detected. The efficacy of ERAD inhibitors combined with RT in stimulating ICD was analyzed. Additionally, the role of ICD hallmarks in immune cell infiltration and patient survival was investigated. Inhibiting ERAD pathways was able to stimulate ICD component emission from dying EC cells in a dose-dependent pattern. Radiation-induced ICD was significantly increased after high doses RT (≥10 Gy). ERAD inhibitor combined with moderate dose RT (≥6 Gy) was capable of stimulating increased ICD in EC cells. Dual therapy could elicit the antitumor immune response by enhancing dendritic cells maturation and phagocytosis. Further investigation revealed a significant correlation between CALR and tumor-infiltrating immune cells. Low expression of ATP and HMGB1 and high expression of CALR were associated with favorable survival in patients with EC. The immunogenicityof EC can be enhanced by ERAD inhibitors combined with moderate doses of RT. ICD hallmark genes, especially CALR, are correlated to immune cell infiltration and clinical outcomes in EC. The present results demonstrated an important method to improve the immunogenicity of EC cells for enhanced antitumor immune response.

HMGB1 promotes the development of castration‑resistant prostate cancer by regulating androgen receptor activation.

AR signalling pathway reactivation plays a key role in the development of castration-resistant prostate cancer (CRPC). High-mobility group protein B1 (HMGB1) is an important factor involved in the occurrence and development of a variety of tumours by regulating gene transcription. In the present study, the association between HMGB1 and prostate cancer (PCa) and the effects of HMGB1 on androgen receptor (AR) transcription and signalling pathway reactivation in PCa cells in vitro and in vivo were evaluated. A bioinformatics method was used to determine the mRNA expression level of HMGB1 in PCa specimens and its correlation with the mRNA expression of AR. Immunohistochemical staining was used to detect the expression of these proteins in clinical PCa samples. Reporter gene and ChIP assays were performed to determine the activity of AR and the effect of HMGB1 on the ability of AR to bind to the promoters of prostate specific antigen and transmembrane protease, serine 2. A bioluminescence resonance energy transfer assay was employed to observe the direct interaction between HMGB1 and AR protein. Additionally, a castrated nude mouse xenograft tumour model was established to verify the effect of HMGB1 on PCa. The results revealed that HMGB1 expression was significantly increased in PCa specimens, which may have a strong correlation with AR expression. Moreover, HMGB1 could reactivate the AR signalling pathway, directly interact with AR, and promote the development of CRPC in an androgen-independent manner. The results of the present study indicated that HMGB1 promoted the development of CRPC by interacting with AR, which inferred that decreasing the expression of HMGB1 may be a potential effective method for CRPC prevention and treatment.

LncRNA HOTAIR Inhibition by Regulating HMGB1/ROS/NF-κB Signal Pathway Promotes the Recovery of Spinal Cord Function

Spinal cord ischemia-reperfusion injury (SCII) is one of the most serious complications of clinical aortic aneurysm and vascular malformation surgery. Long noncoding RNA (lncRNA) is involved in the progression of SCII, whereas long noncoding RNA HOX transcript antisense RNA (lncRNA HOTAIR) is unclear in SCII. This study is aimed at confirming the role and related mechanism of HOTAIR in SCII. Later on, a model of SCII was established by clamping the aortic arch for 14 minutes. RNA expression of HOTAIR was detected via qRT-PCR at 12 h, 24 h, 36 h, and 48 h after SCII. The Tarlov scoring system and TUNEL assay were used to evaluate neurological function and neuronal apoptosis. Oxidative stress factor levels were assessed according to the instructions of the kit. Inflammatory cytokines were assessed by ELISA. Western blot was used to detect levels of p65, p-p65, I-κBα, and p-I-κBα. We found HOTAIR was raised in SCII rats. si-HOTAIR was able to reverse SCII-induced oxidative stress in SCII rats. The HMGB1 expression was upregulated in SCII tissues and negatively correlated with HOTAIR. HMGB1 was able to partially reverse si-HOTAIR inhibition of oxidative stress, inflammatory injury, and neuronal cell apoptosis in SCII. In addition, the ROS/NF-κB signaling pathway is involved in HOTAIR/HMGB1 regulation of SCII. In a word, HOTAIR inhibition is able to inhibit oxidative stress, inflammatory injury, and neuronal apoptosis in SCII through downregulation of the high mobility group protein B1(HMGB1), which is achieved by inhibiting the ROS/NF-κB signaling pathway. The HOTAIR/HMGB1/ROS/NF-κB molecular pathway may be a new mechanism for the treatment of SCII.

HMGB1-Promoted Neutrophil Extracellular Traps Contribute to Cardiac Diastolic Dysfunction in Mice.

BackgroundHeart failure with preserved ejection fraction (HFpEF) remains an increasing public health problem with substantial morbidity and mortality but with few effective treatments. A novel inflammatory mechanism has been proposed, but the inflammatory signals promoting the development of HFpEF remain greatly unknown.Methods and ResultsSerum of patients with HFpEF was collected for measurement of circulating neutrophils and markers of neutrophil extracellular traps (NETs). To induce HFpEF phenotype, male C57BL/6 mice underwent uninephrectomy, received a continuous infusion of d‐aldosterone for 4 weeks, and maintained on 1.0% sodium chloride drinking water. Heart tissues were harvested, immune cell types determined by flow cytometry, NETs formation by immunofluorescence, and western blotting. Differentiated neutrophils were cultured to investigate the effect of HMGB1 (high mobility group protein B1) and SGLT2 (sodium‐glucose cotransporter‐2) inhibitor on NETs formation in vitro. Circulating neutrophils and NETs markers are elevated in patients with HFpEF, as are cardiac neutrophils and NETs formation in HFpEF mice. NETs inhibition with deoxyribonuclease 1 in experimental HFpEF mice reduces heart macrophages infiltration and inflammation and ameliorates cardiac fibrosis and diastolic function. Damage‐associated molecular pattern HMGB1 expression is elevated in cardiac tissue of HFpEF mice, and HMGB1 inhibition reduces heart neutrophil infiltration and NETs formation and ameliorates diastolic function. Lastly, SGLT2 inhibitor empagliflozin down‐regulates heart HMGB1 expression, attenuates NETs formation and cardiac fibrosis, and improves diastolic function in HFpEF mice.ConclusionsNETs contribute to the pathogenesis of HFpEF, which can be ameliorated by HMGB1 inhibition and SGLT2 inhibitors. Thus, HMGB1 and NETs may represent novel therapeutic targets for the treatment of HFpEF.

MiR-505 inhibits replication of Borna disease virus 1 via inhibition of HMGB1-mediated autophagy.

Borna disease virus 1 (BoDV-1) is a highly neurotropic RNA virus which was recently demonstrated to cause deadly human encephalitis. Viruses can modulate microRNA expression, in turn modulating cellular immune responses and regulating viral replication. A previous study indicated that BoDV-1 infection down-regulated the expression of miR-505 in rats. However, the underlying mechanism of miR-505 during BoDV-1 infection remains unknown. In this study, we found that miR-505 can inhibit autophagy activation by down-regulating the expression of its target gene HMGB1, and ultimately inhibit the replication of BoDV-1. Specifically, we found that the expression of miR-505 was significantly down-regulated in rat primary neurons stably infected with BoDV-1. Overexpression of miR-505 can inhibit the replication of BoDV-1 in cells. Bioinformatics analysis and dual luciferase reporter gene detection confirmed that during BoDV-1 infection, the high-mobility group protein B1 (HMGB1) that mediates autophagy is the direct target gene of miR-505. The expression of HMGB1 was up-regulated after BoDV-1 infection, and overexpression of miR-505 could inhibit the expression of HMGB1. Autophagy-related detection found that after infection with BoDV-1, the expression of autophagy-related proteins and autophagy-related marker LC3 in neuronal cells was significantly up-regulated. Autophagy flow experiments and transmission electron microscopy also further confirmed that BoDV-1 infection activated HMGB1-mediated autophagy. Further regulating the expression of miR-505 found that overexpression of miR-505 significantly inhibited HMGB1-mediated autophagy. The discovery of this mechanism may provide new ideas and directions for the prevention and treatment of BoDV-1 infection in the future.

Magnesium sulfate reduces vascular endothelial cell apoptosis in rats with preeclampsia via the miR-218-5p/HMGB1 pathway

ABSTRACT Objective This study aims to investigate the mechanism by which magnesium sulfate regulates the miR-218-5p/HMGB-pathway-mediated apoptosis of vascular endothelial cells (VECs) in rats with preeclampsia (PE). Methods Twenty pregnant rats were randomly divided into four groups: normal, PE, MgSO4, and high-mobility group protein B1 (HMGB1)-agomir groups. On the 14th day of each rat’s pregnancy, endotoxin was used to establish a PE model in the PE, MgSO4, and HMGB1-agomir groups. Then, the MgSO4 and HMGB1-agomir groups were treated with magnesium sulfate. Finally, HMGB1 overexpression was performed only in the HMGB1-agomir group. The rats’ urinary protein content and systolic blood pressure at 24 h were detected on the 11th, 13th, 15th, 17th, and 19th day of pregnancy. Results Compared with the PE group, 24-h urinary protein content, blood pressure, VEC apoptosis rate, apoptosis marker levels, and HMGB1 expression decreased while miR-218-5p levels increased in the MgSO4 group. The dual-luciferase assay revealed that HMGB1 can be targeted and regulated by miR-218-5p. Compared with the MgSO4 group, 24-h urinary protein content, blood pressure, VEC apoptosis rate, apoptosis marker levels, and HMGB1 expression increased while miR-218-5p levels decreased in the HMGB1-agomir group. Conclusion MgSO4 reduces VEC apoptosis in PE rats via the miR-218-5p/HMGB1 pathway and thus plays a role in treating PE.

Protective role and molecular mechanism of action of Nesfatin-1 against high glucose-induced inflammation, oxidative stress and apoptosis in retinal epithelial cells.

Diabetic retinopathy (DR) is a major complication of diabetes mellitus that may cause severe visual impairment. It has been reported that the levels of nesfatin-1 in the serum and vitreous humor were negatively correlated with DR; however, its role in DR has not been fully elucidated. Therefore, the present study was performed to investigate the effect of nesfatin-1 on high glucose-treated human retinal epithelial cells (ARPE-19) and explore the underlying mechanism. The effects of nesfatin-1 on cell viability, inflammation, oxidative stress and apoptosis were examined under high glucose conditions. The Cell Counting Kit-8 assay was used to determine cell viability. The levels of inflammatory cytokines were evaluated using ELISA kits. The reactive oxygen species and malondialdehyde content was estimated using commercial assay kits. Flow cytometry was performed to detect apoptotic cells and western blot analysis was employed to evaluate the expression of apoptosis-associated proteins. Moreover, the levels of NF-κB, NACHT, LRR and PYD domains-containing protein 3 (NLRP3) and high-mobility group protein B1 (HMGB1) were determined via western blot analysis. The results revealed that nesfatin-1 enhanced cell viability and suppressed inflammation, oxidative stress and apoptosis in the presence of high glucose concentration. Moreover, the activation of the NF-κB/NLRP3 inflammasome signaling and the expression of HMGB1 were inhibited by nesfatin-1. Furthermore, HMGB1 overexpression partially abrogated the inactivation of the NF-κB/NLRP3 inflammasome pathway caused by nesfatin-1. Taken together, these findings demonstrated that nesfatin-1 inhibited the activation of the NF-κB/NLRP3 inflammasome signaling via modulating HMGB1 and exerted a protective effect on ARPE-19 cells against high glucose-induced inflammation, oxidative stress and apoptosis.

MiR-142-3p reduces the viability of human cervical cancer cells by negatively regulating the cytoplasmic localization of HMGB1.

High mobility group protein B1 (HMGB1) is a nuclear protein that has been reported to contribute to tumor growth in humans. The present study identified a microRNA (miR/miRNA) that targets the 3' untranslated region (3'UTR) of the HMGB1 gene and assessed its effects on the proliferation of human cervical cancer cells and associated molecular mechanism. Western blotting was performed to determine HMGB1 levels in HeLa cells. TargetScan software was used to identify miRNA binding sites adjacent to the HMGB1. The viability of HeLa cells transfected with miR-142-3p mimics or inhibitors was determined using an MTT assay. The subcellular distribution (cytoplasmic or nuclear) of HMGB1 in HeLa cells was observed by western blotting. HMGB1 expression in HeLa and CaSKi cells was significantly higher compared with normal control cervical cells. TargetScan analysis indicated that miR-142-3p binds to the 3'UTR of HMGB1. Transfection with a miR-142-3p inhibitor increased cytoplasmic HMGB1 expression in HeLa cells, as shown by western blot analysis, while transfection with miR-142-3p mimics decreased the cytoplasmic expression of HMGB1 in HeLa cells. Therefore, miR-142-3p negatively regulated HMGB1 levels in cervical cancer cells. These findings indicated that miR-142-3p inhibited the proliferation of human cervical cancer cells, at least in part, by negatively regulating the cytoplasmic localization of HMGB1.

Ethyl Pyruvate Alleviates Pulmonary Hypertension through the Suppression of Pulmonary Artery Smooth Muscle Cell Proliferation via the High Mobility Group Protein B1/Receptor for Advanced Glycation End-Products Axis.

Purpose: Pulmonary arterial hypertension (PAH) is a formidable disease with no effective treatment at present. With the goal of developing potential therapies, we attempted to determine whether ethyl pyruvate (EP) could alleviate PAH and its mechanism.Methods: Pulmonary smooth muscle cells were cultured in conventional low-oxygen environments, and cellular proliferation was monitored after treatment with either EP or phosphate-balanced solution (PBS). Expression of high mobility group protein B1 (HMGB1) and receptor for advanced glycation end-products (RAGE) protein were detected by western blot. After hyperkinetic PAH rat models were treated with EP, hemodynamic data were collected. Right ventricular hypertrophy and pulmonary vascular remodeling were evaluated. Expression of HMGB1 and RAGE protein was also detected.Results: In vitro, proliferative activity increased in low-oxygen environments, but was inhibited by EP treatment. Furthermore, Western blotting showed the decreased expression of HMGB1 and RAGE protein after EP treatment. In vivo, pulmonary artery pressures were attenuated with EP. Right ventricular hypertrophy and pulmonary vascular remodeling were also reversed. Additionally, the expression levels of HMGB1 and RAGE were reduced in lung tissues.Conclusions: EP can alleviate PAH by suppressing the proliferation of pulmonary artery smooth muscle cells via inhibition of HMGB1/RAGE expression.

Hydrogen plays a protective role in intestinal injury of mice with severe sepsis by regulating the release of heme oxygenase-1 and high mobility group protein B1

To investigate the protective effect of the hydrogen (H2) inhalation on intestinal injury in severe sepsis mice and its relationship with nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)/high mobility group protein B1 (HMGB1) pathway. The 280 male wild-type (WT) and Nrf2 knockout (Nrf2-KO) ICR mice (6-8 weeks old, 20-25 g weight) were assigned into 8 groups randomly (WT and Nrf2-KO mice, 4 groups respectively, n = 35): Sham group, H2 inhalation group (Sham+H2 group), sepsis model group which was induced by cecal ligation and puncture (CLP group) and H2 treatment group (CLP+H2 group). In Sham group, except cecal ligation and perforation, the same surgery was used. The mice in Sham group and CLP group inhaled only air for 1 hour and 6 hours, respectively, while the mice in Sham+H2 group and CLP+H2 group inhaled 2% H2 for 60 minutes. After modeling, 20 mice randomly in each group were selected to measure 7-day natural survival rates; the remaining mice(15 mice) were killed in 24 hours after modeling, and the bacterial load was determined by collecting the peritoneal lavage fluid (PLF) of each mouse to measure the colony forming unit (CFU); and the intestinal tissue was taken after perfusion, and the intestinal tissue of 3 mice was used to extract protein for HO-1 and HMGB1 levels by using Western blotting. Intestinal tissue of 3 mice was extracted to prepare histomorphology sections for the identification of HO-1. Intestinal tissue of 3 mice was used to detect the expression of HMGB1 by immunofluorescence. The 7-day survival rate of WT and Nrf2-KO mice in CLP group was zero; in WT mice, the 7-day survival rate of CLP+H2 group was significantly higher than that of CLP group (45% vs. 0%, P < 0.05), while no difference of the 7-day survival rate in Nrf2-KO mice was noted between CLP and CLP+H2 groups (0% vs. 0%, P > 0.05). Compared with Sham group, the CFU in the PLF of both mice in CLP group increased significantly. Compared with CLP group, 2% H2 treatment could significantly improve the bacterial clearance rate of WT mice with severe sepsis (CFU, ×103: 34.7±6.3 vs. 74.2±8.1, P < 0.01), but had no significant effect on Nrf2-KO mice with severe sepsis (CFU, ×103: 73.3±7.5 vs. 75.8±8.6, P > 0.05). The results of Western blotting, immunohistochemical and immune-fluorescence showed that the expression of HO-1 and HMGB1 in the CLP group were higher than those in the Sham group. Compared with CLP group, the expression of HO-1 in WT mice of CLP+H2 group was significantly higher [HO-1 protein expression (HO-1/β-actin): 0.716±0.035 vs. 0.460±0.045, HO-1 positive expression (A value): 0.703±0.135 vs. 0.430±0.116, both P < 0.01], while the expression of HMGB1 was significantly lower [HMGB1 protein expression (HMGB1/β-actin): 0.052±0.038 vs. 0.082±0.008, HMGB1 positive expression (A value): 24.530±9.317 vs. 41.830±2.458, both P < 0.01]. In Nrf2-KO mice, compared with CLP group, there was no statistical difference in the above indexes in CLP+H2 group [HO-1 protein expression (HO-1/β-actin): 0.148±0.004 vs. 0.164±0.043, HO-1 positive expression (A value): 0.109±0.020 vs. 0.113±0.025, HMGB1 protein expression (HMGB1/β-actin): 0.109±0.020 vs. 0.113±0.025, HMGB1 positive expression (A value): 39.570±12.660 vs. 42.790±9.680, all P > 0.05]. H2 can inhibit the intestinal injury of mice with severe sepsis through Nrf2/HO-1/HMGB1 pathway, and Nrf2 plays an important role in the treatment of intestinal injury with H2.

Knockdown of exosome‑mediated lnc‑PVT1 alleviates lipopolysaccharide‑induced osteoarthritis progression by mediating the HMGB1/TLR4/NF‑κB pathway via miR‑93‑5p.

Osteoarthritis is a chronic degenerative joint disease. Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) is involved in the progression of osteoarthritis and exosomes serve a central role in intercellular communication. However, whether PVT1 can be mediated by exosomes in osteoarthritis has not been reported. Whole blood was drawn from osteoarthritis patients and healthy volunteers. Lipopolysaccharide (LPS) was used to stimulate human normal chondrocytes (C28/I2) to construct a cell damage model in vitro. Protein levels were examined via western blot analysis. eThe expression of PVT1, microRNA (miR)-93-5p and high mobility groupprotein B1 (HMGB1) was evaluated through reverse transcription-quantitative PCR. Cell viability and apoptosis were determined through CCK-8 or flow cytometric assay. Inflammatory cytokines were measured via ELISA. The relationship between PVT1 or HMGB1 and miR-93-5p was confirmed by dual-luciferase reporter assay. PVT1, HMGB1 and exosomal PVT1 were upregulated while miR-93-5p was downregulated in osteoarthritis patient serum and LPS-induced C28/I2 cells. Exosomes from osteoarthritis patient serum and LPS-treated C28/I2 cells increased PVT1 expression in C28/I2 cells. PVT1 depletion reversed the decrease of viability and the increase of apoptosis, inflammation responses and collagen degradation of C28/I2 cells induced by LPS. PVT1 regulated HMGB1 expression via sponging miR-93-5p. miR-93-5p inhibition abolished PVT1 silencing-mediated viability, apoptosis, inflammation responses and collagen degradation of LPS-stimulated C28/I2 cells. HMGB1 increase overturned miR-93-5p upregulation-mediated viability, apoptosis, inflammation responses and collagen degradation of LPS-stimulated C28/I2 cells. Furthermore, PVT1 modulated the Toll-like receptor 4/NF-κB pathway through an miR-93-5p/HMGB1 axis. In summary, exosome-mediated PVT1 regulated LPS-induced osteoarthritis progression by modulating the HMGB1/TLR4/NF-κB pathway via miR-93-5p, providing a new route for possible osteoarthritis treatment.

Long Noncoding RNA HNF1A-AS1 Regulates Osteosarcoma Advancement Through Modulating the miR-32-5p/HMGB1 Axis.

Background: Osteosarcoma (OS) is a primary malignant tumor in children and adolescents. Long noncoding RNA HNF1A antisense RNA 1 (HNF1A-AS1) is connected with OS development. However, there are few reports on the role and mechanism of HNF1A-AS1 in OS. Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of HNF1A-AS1, miR-32-5p, and high-mobility group protein B1 (HMGB1). Western blot analysis was performed to detect the protein level of HMGB1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, transwell, or flow cytometer assays were applied to determine the proliferation, migration, invasion, and apoptosis of OS cells. The interaction between HNF1A-AS1 and miR-32-5p or HMGB1 was predicted by the starBase database and confirmed by dual-luciferase reporter assay. Enzyme-linked immunosorbent assay was employed to analyze levels of HMGB1 in the OS cell supernatant. Results: HNF1A-AS1 and HMGB1 were upregulated, while miR-32-5p was downregulated, in OS tissues and cells. Functionally, HNF1A-AS1 depletion induced apoptosis and impeded proliferation, migration, and invasion of OS cells. Interestingly, HNF1A-AS1 bound to miR-32-5p to regulate the expression of HMGB1. Furthermore, miR-32-5p knockdown overturned the effects of HNF1A-AS1 knockdown on apoptosis, proliferation, migration, and invasion of OS cells. In addition, the effects of HNF1A-AS1 silencing on the malignant behaviors of OS cells were reserved by HMGB1 overexpression. In addition, HNF1A-AS1 regulated the HMGB1 level in the OS cell supernatant through the miR-32-5p/HMGB1 axis. Conclusion: Downregulation of HNF1A-AS1 blocked OS progression through the miR-32-5p/HMGB1 axis, which provides a possible target and prognostic biomarker for treatment of OS.

Glycyrrhizic Acid Attenuates Balloon-Induced Vascular Injury Through Inactivation of RAGE Signaling Pathways

Percutaneous coronary intervention is a well-established technique used to treat coronary artery disease, but the risk of coronary artery in-stent restenosis following percutaneous coronary intervention is still high. Previous studies revealed that high mobility group protein B1 (HMGB1) plays a critical role in neointima formation. In this study, we aimed to investigate the role of glycyrrhizic acid (GA), an HMGB1 inhibitor, in the process of neointima formation and the potential mechanisms. We investigated the role of GA in neointima formation through an iliac artery balloon injury model in rabbits. Proliferation, migration, and phenotype transformation of human vascular smooth muscle cells (VSMCs) were observed. Besides, inflammation and receptor for advanced glycosylation end products (RAGE) signaling pathways were studied. The results indicate that GA attenuated neointima formation and downregulated HMGB1 expression in injured artery in rabbits. HMGB1 promoted proliferation, migration, and phenotype transformation through the activation of RAGE signaling pathways in VSMCs, and blockade of HMGB1 by GA (1, 10, and 100 μM) could attenuate those processes and reduce proliferation of human VSMCs. In conclusion, the HMGB1 inhibitor GA might be useful to treat proliferative vascular diseases by downregulating RAGE signaling pathways. Our results indicate a new and promising therapeutic agent for restenosis.

HMGB1 regulates SNAI1 during NSCLC metastasis, both directly, through transcriptional activation, and indirectly, in a RSF1-IT2-dependent manner.

High‐mobility group protein B1 (HMGB1) has important functions in cancer cell proliferation and metastasis. However, the mechanisms of HMGB1 function in non‐small‐cell lung cancer (NSCLC) remain unclear. This study aimed to investigate the underlying mechanism of HMGB1‐dependent tumor cell proliferation and NSCLC metastasis. Firstly, we found high HMGB1 expression in NSCLC and showed that HMBG1 promoted proliferation, migration, and invasion of NSCLC cells. HMGB1 could bind to SNAI1 promoter and activate the expression of SNAI1. In addition, HMGB1 could transcriptionally regulate the lncRNA RSF1‐IT2. RSF1‐IT2 was found to function as ceRNA, sponging miR‐129‐5p, which targets SNAI1. Notably, HMGB1 was also identified as a target of miR‐129‐5p, which indicates the establishment of a positive feedback loop. Consequently, high expression of RSF1‐IT2 and SNAI1 was found to closely correlate with tumor progression in both HMGB1‐overexpressing xenograft nude mice and patients with NSCLC. Taken together, our findings provide new insights into molecular mechanisms of HMGB1‐dependent tumor metastasis. Components of the HMGB1–RSF1‐IT2–miR‐129‐5p–SNAI1 pathway may have a potential as prognostic and therapeutic targets in NSCLC.

MiR-129-5p inhibits proliferation of gastric cancer cells through targeted inhibition on HMGB1 expression.

The aim of this study was to investigate the effects of micro ribonucleic acid (miR)-129-5p on the proliferation and apoptosis of gastric cancer cells via targeted repression on the expression of high mobility group protein B1 (HMGB1). Expression levels of miR-129-5p and HMGB1 in gastric cancer tissues (n=25) and adjacent normal tissues were measured via reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The regulatory effect of miR-129-5p on the proliferation of gastric cancer MGC-803 and SGC7901 cells was determined through Cell Counting Kit-8 (CCK-8) assay. Flow cytometry was employed to analyze the apoptosis rate of gastric cancer cells. To further discover the mechanism of miR-129-5p in regulating malignant behaviors of gastric cancer cells, the miRDB database was employed to predict the binding targets of miR-129-5p. Finally, binding sites of HMGB1 3'-untranslated region (3'-UTR) to miR-129-5p were discovered. Subsequently, HMGB1 wild-type or mutant 3'-UTR Luciferase reporter vectors were constructed, and transfected to MGC-803 and SGC7901 cells together with miR-129-5p or negative control miRNA. Next, Western blotting was adopted to measure the protein expression level of HMGB1 in MGC-803 and SGC7901 cells transfected with miR-129-5p or negative control miRNA, so as to investigate whether miR-129-5p affected HMGB1 protein expression. Additionally, to determine whether HMGB1 mediated the regulatory effect of miR-129-5p on the proliferation of gastric cancer cells, MGC-803 and SGC7901 cells were transfected with pcDNA-HMGB1 or pcDNA-vector, respectively. The expression level of HMGB1 was measured via RT-qPCR, and cell proliferation was determined by CCK-8 assay. The expression level of miR-129-5p in gastric cancer tissues was significantly lower than that in adjacent normal tissues (p<0.001). Meanwhile, the level of miR-129-5p was overtly lower in gastric cancer MGC-803 and SGC7901 cell lines than that in normal gastric mucosal epithelial GES-1 cells (p<0.001). These results indicated that miR-129-5p was lowly expressed in gastric cancer tissues and cell lines. Subsequent results demonstrated that the expression of HMGB1 increased remarkably in gastric cancer tissues compared with normal adjacent tissues (p<0.05). The proliferation ability of MGC-803 (p<0.001) and SGC7901 (p<0.01) cells with over-expressed miR-129-5p was remarkably weakened. Overexpression of miR-129-5p distinctly promoted the apoptosis rate of gastric cancer MGC-803 (p<0.01) and SGC7901 (p<0.001) cells. Moreover, miR-129-5p up-regulation significantly reduced the Luciferase activity of wild-type HMGB1 (p<0.001). However, no significant effect was observed on that of mutant HMGB1. The results suggested that overexpression of miR-129-5p significantly down-regulated the level of HMGB1 in gastric cancer cells. In addition, the messenger RNA (mRNA) level of HMGB1 in cells transfected with miR-129-5p also decreased significantly (p<0.001). HMGB1 overexpression overtly reversed the inhibitory effect of miR-129-5p on the proliferation of gastric cancer cells (p<0.05). All these results demonstrated that the miR-129-5p/HMGB1 axis played a key role in regulating the growth of gastric cancer cells. MiR-129-5p suppresses the progression of gastric cancer through targeted inhibition on the expression of HMGB1.

Mechanism of glycyrrhizin on ferroptosis during acute liver failure by inhibiting oxidative stress

The present study aimed to investigate the anti-ferroptosis effects of the HMGB1 inhibitor glycyrrhizin (GLY). The present study used a cell and animal model of acute liver failure (ALF), induced using tumor necrosis factor-α, lipopolysaccharide and D-galactosamine, to investigate the effects of GLY. The expression of glutathione peroxidase 4 (GPX4) and high mobility group protein B1 (HMGB1), heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor 2 (Nrf2) were detected were detected by western blotting in L02 hepatocytes and mouse liver. The expression of GPX4 and HMGB1 in L02 hepatocytes and mouse liver was detected by immunofluorescence. The pathological changes to liver tissues were determined by hematoxylin and eosin staining. The levels of lactate dehydrogenase (LDH), Fe2+, reactive oxygen species (ROS) and glutathione (GSH) were tested using kits. Compared with the normal group, the degree of liver damage and liver function in the model animal group was severe. The protein levels of HMGB1 in L02 cells and liver tissues were significantly increased. The expression of NRF2, HO-1 and GPX4 was significantly decreased. The levels of LDH, Fe2+, malondialdehyde (MDA) and ROS were increased, whereas the level of GSH was decreased. Treatment with GLY reduced the degree of liver damage, the expression of HMGB1 was decreased, and the levels of Nrf2, HO-1 and GPX4 were increased. The levels of LDH, Fe2+, MDA, ROS were decreased, while the level of GSH was increased by GLY treatment. The results of the present study indicated that HMGB1 is involved in the process of ferroptosis. The HMGB1 inhibitor GLY significantly reduced the degree of ferroptosis during ALF by inhibiting oxidative stress.

MiR-129-5p Alleviates Neuropathic Pain Through Regulating HMGB1 Expression in CCI Rat Models.

Recently, microRNAs are reported to be participated in the development of pain and persistence of neuropathic and inflammatory pain in animal models. Here, we characterized the functional role of miR-129-5p in pain processing in chronic constriction injury (CCI) rat models. Bilateral CCI operation was used to generate neuropathic pain rat model. Paw withdrawal threshold (PWT) and paw withdrawal latency (PWL) were used to assess pain-related behaviors. Gene expression was evaluated using qRT-PCR, luciferase assay, western blotting, and enzyme-linked immunosorbent assay. Compared with the control rats, expression level of miR-129-5p was downregulated significantly over time in CCI rats post operation. Interestingly, downregulation of miR-129-5p in CCI rats was correlated with increased proinflammatory cytokine expression and pain-related behaviors. Furthermore, we found that miR-129-5p alleviated neuropathic pain through downregulating high mobility group protein B1 (HMGB1) expression in CCI rats as overexpression of miR-129-5p suppressed expression of both HMGB1 and proinflammatory cytokine and alleviated pain sensation in CCI rats. In summary, our results show that alteration in miR-129-5p expression contributes to pain processing in our CCI pain rat model, suggesting miR-129-5p could be a causal factor in neuropathic pain and serve as a promising potential biomarker and therapeutic target for neuropathic pain.

Stress overload-induced periodontal remodelling coupled with changes in high mobility group protein B1 during tooth movement: an in-vivo study.

High mobility group protein B1 (HMGB1), a bone-active cytokine and an osteocyte alarmin, might have dual functions in bone metabolism that could benefit bone formation and accelerate osteoclastogenic activity. High mobility group protein B1 was recently shown to be involved in tooth movement. Here, we investigated the expression of HMGB1, which remains poorly elucidated, under stress overload-induced periodontal remodelling conditions in vivo. Thirty-six Sprague-Dawley rats (male, 180-200g) were randomly divided into three groups: two experimental groups, in which 50 or 100g of force was applied to the first molars for 7d to induce movement; and one control group, in which no force was applied. These stresses induced tooth movement over significantly different distances, and marked morphological changes were consistently observed in the periodontal tissues of the experimental rats, as demonstrated by histological staining. A real-time PCR analysis showed upregulation of the receptor activator of nuclear factor-kappaB ligand-to-osteoprotegerin ratio and downregulation of the Hmgb1 gene. Changes in both location and expression of the HMGB1 were observed through immunofluorescence analysis. Our data suggest that HMGB1 expression during orthodontic tooth movement might be regulated in a time- and force-dependent manner that is substantially more complex than anticipated.