mRNA Levels Of HMGB1 Research Articles

Effects of iris xanthin on airway inflammation, airway remodeling, and the HMGB1/TLR4/NF-κB pathway in asthmatic young mice

To explore the effects of iris xanthin on airway inflammation, airway remodeling, and the high mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) pathway in asthmatic young mice. Sixty male BALB/c young mice were randomly assigned into six groups: a blank group, a model group, a dexamethasone group, and low, medium, and high dose groups of iris xanthin, with ten mice per group. Asthma models were induced through intraperitoneal injections of a sensitizing agent [ovalbumin (OVA) 20 μg + aluminum hydroxide gel 2 mg], followed by 4% OVA aerosol inhalation. Lung function was measured using a pulmonary function tester to determine lung volume (LV), resting ventilation per minute (VE), and airway reactivity (Penh value). Hematoxylin-eosin (HE) staining was employed to examine and analyze airway remodeling. The contents of interleukin (IL)-1β, IL-6, and tumor necrosis factor alpha (TNF-α) in bronchoalveolar lavage fluid were quantified using ELISA. Real-time fluorescence quantitative polymerase chain reaction and Western blot analysis were used to assess the expression of HMGB1/TLR4/NF-κB pathway-related mRNA and proteins in lung tissues. Compared to the model group, the dexamethasone and iris xanthin-treated groups (low, medium, and high doses) exhibited significant increases in LV and VE (P<0.05), with incremental dose-dependent increases observed in the iris xanthin groups. Additionally, Penh values, IL-1β, IL-6, TNF-α, and airway remodeling indicators, along with mRNA levels of HMGB1, TLR4, and NF-κB p65 and protein levels of HMGB1, TLR4, and p-NF-κB p65, were all reduced (P<0.05) in a dose-dependent manner. When compared to the dexamethasone group, the low and medium dose iris xanthin groups showed decreases in LV and VE (P<0.05), whereas Penh values, IL-1β, IL-6, TNF-α, and airway remodeling indicators, along with mRNA levels of HMGB1, TLR4, NF-κB p65 and protein levels of HMGB1, TLR4, and p-NF-κB p65, were increased (P<0.05). No significant differences were noted in these indices between the high dose iris xanthin group and the dexamethasone group (P>0.05). Iris xanthin can effectively alleviates airway inflammation and inhibits airway remodeling in asthmatic young mice, possibly through the suppression of the HMGB1/TLR4/NF-κB pathway.

HMGB1 Expression Levels Correlate with Response to Immunotherapy in Non-Small Cell Lung Cancer.

High-mobility group box 1 protein (HMGB1) is subject to exportin 1 (XPO1)-dependent nuclear export, and it is involved in functions implicated in resistance to immunotherapy. We investigated whether HMGB1 mRNA expression was associated with response to immune checkpoint inhibitors (ICI) in non-small cell lung cancer (NSCLC). RNA was isolated from pretreatment biopsies of patients with advanced NSCLC treated with ICI. Gene expression analysis of several genes, including HMGB1, was conducted using the NanoString Counter analysis system (PanCancer Immune Profiling Panel). Western blotting analysis and cell viability assays in EGFR and KRAS mutant cell lines were carried out. Evaluation of the antitumoral effect of ICI in combination with XPO1 blocker (selinexor) and trametinib was determined in a murine Lewis lung carcinoma model. HMGB1 mRNA levels in NSCLC patients treated with ICI correlated with progression-free survival (PFS) (median PFS 9.0 versus 18.0 months, P=0.008, hazard ratio=0.30 in high versus low HMGB1). After TNF-α stimulation, HMGB1 accumulates in the cytoplasm of PC9 cells, but this accumulation can be prevented by using selinexor or antiretroviral drugs. Erlotinib or osimertinib with selinexor in EGFR-mutant cells and trametinib plus selinexor in KRAS mutant abolish tumor cell proliferation. Selinexor with a PD-1 inhibitor with or without trametinib abrogates the tumor growth in the murine Lewis lung cancer model. An in-depth exploration of the functions of HMGB1 mRNA and protein is expected to uncover new potential targets and provide a basis for treating metastatic NSCLC in combination with ICI.

Ligusticum cycloprolactam inhibits IL-1β-induced apoptosis and inflammation of rat chondrocytes via HMGB1/TLR4/NF-κB signaling pathway

Chondrocytes are unique resident cells in the articular cartilage, and the pathological changes of them can lead to the occurrence of osteoarthritis(OA). Ligusticum cycloprolactam(LIGc) are derivatives of Z-ligustilide(LIG), a pharmacodynamic marker of Angelica sinensis, which has various biological functions such as anti-inflammation and inhibition of cell apoptosis. However, its protective effect on chondrocytes in the case of OA and the underlying mechanism remain unclear. This study conducted in vitro experiments to explore the molecular mechanism of LIGc in protecting chondrocytes from OA. The inflammation model of rat OA chondrocyte model was established by using interleukin-1β(IL-1β) to induce. LIGc alone and combined with glycyrrhizic acid(GA), a blocker of the high mobility group box-1 protein(HMGB1)/Toll-like receptor 4(TLR4)/nuclear factor-kappa B(NF-κB) signaling pathway, were used to intervene in the model, and the therapeutic effects were systematically evaluated. The viability of chondrocytes treated with different concentrations of LIGc was measured by the cell counting kit-8(CCK-8), and the optimal LIGc concentration was screened out. Annexin V-FITC/PI apoptosis detection kit was employed to examine the apoptosis of chondrocytes in each group. The enzyme-linked immunosorbent assay(ELISA) was employed to measure the expression of cyclooxygenase-2(COX-2), prostaglandin-2(PGE2), and tumor necrosis factor-alpha(TNF-α) in the supernatant of chondrocytes in each group. Western blot was employed to determine the protein levels of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), caspase-3, HMGB1, TLR4, and NF-κB p65. The mRNA levels of HMGB1, TLR4, NF-κB p65, and myeloid differentiation factor 88(MyD88) in chondrocytes were determined by real-time fluorescent quantitative PCR(RT-qPCR). The safe concentration range of LIGc on chondrocytes was determined by CCK-8, and then the optimal concentration of LIGc for exerting the effect was clarified. Under the intervention of IL-1β, the rat chondrocyte model of OA was successfully established. The modeled chondrocytes showed increased apoptosis rate, promoted expression of COX-2, PGE2, and TNF-α, up-regulated protein levels of Bax, caspase-3, HMGB1, TLR4, and NF-κB p65 and mRNA levels of HMGB1, TLR4, NF-κB p65, and MyD88, and down-regulated protein level of Bcl-2. However, LIGc reversed the IL-1β-induced changes of the above factors. Moreover, LIGc combined with GA showed more significant reversal effect than LIGc alone. These fin-dings indicate that LIGc extracted and derived from the traditional Chinese medicine A. sinensis can inhibit the inflammatory response of chondrocytes and reduce the apoptosis of chondrocytes, and this effect may be related to the HMGB1/TLR4/NF-κB signaling pathway. The pharmacological effect of LIGc on protecting chondrocytes has potential value in delaying the progression of OA and improving the clinical symptoms of patients, and deserves further study.

Puerarin Alleviates Oxidized Oil-Induced Oxidative Injury and Inflammation via Inhibition of the Nrf2/Keap1 and HMGB1/TLR4/MAPK Signaling Pathways: An Investigation in a Chicken Model.

Puerarin has possessed a wide range of pharmacological activities. However, little is known about the protective effects of puerarin on the oxidized oil-induced injury. Here, the antioxidant and anti-inflammatory effects of puerarin are described using a chicken model. A total of 360 broilers are arranged in four treatments. Diets include two types of soybean oil (fresh or oxidized) and two levels of puerarin (0 or 750 mgkg-1 ). Results show that puerarin alleviates oxidized soybean oil-induced hepatic and thymic oxidative injury. This effect is observed by increasing the SOD activity and the expressions of Nrf2 signaling pathway-related genes and reducing the MDA content in the liver and thymus. Moreover, puerarin supplementation decreases the concentrations and mRNA levels of pro-inflammatory factors in the liver and thymus. The potential mechanism responsible for this is the decrease in the mRNA or protein levels of HMGB1, TLR4, MyD88, and p65 in the liver or thymus. Western blotting results indicate that puerarin also decreases the phosphorylation of JNK1/2, ERK1/2, and p38 in the liver and thymus. This study demonstrates puerarin may be a potential nutrient supplement in the treatment of oxidized oil-induced damage, and the Nrf2/Keap1 and HMGB1/TLR4/MAPK signaling pathways might be its important target.

Unraveling the protective mechanisms of Chuanfangyihao against acute lung injury: Insights from experimental validation.

Chuanfangyihao (CFYH) is an effective treatment for acute lung injury (ALI) in clinical practice; however, its underlying mechanism of action remains unclear. Therefore, the aim of the present study was to elucidate the pharmacological mechanism of action of CFYH in ALI through experimental validation. First, a rat model of ALI was established using lipopolysaccharide (LPS). Next, the pathological changes in the lungs of the rats and the pathological damage were scored. The wet/dry weight ratios were measured, and ROS content was detected using flow cytometry. ELISA was used to examine IL-6, TNF-α, IL-1β, IL-18, and LDH levels. Immunohistochemistry was used to detect Beclin-1 and NLRP3 expression. Western blotting was performed to analyze the expression of HMGB1, RAGE, TLR4, NF-κB p65, AMPK, p-AMPK, mTOR, p-mTOR, Beclin-1, LC3-II/I, p62, Bcl-2, Bax, Caspase-3, Caspase-1, and GSDMD-NT. The mRNA levels of HMGB1, RAGE, AMPK, mTOR, and HIF-1α were determined using reverse transcription quantitative PCR. CFYH alleviated pulmonary edema and decreased the expression of IL-6, TNF-α, TLR4, NF-κB p65, HMGB1/RAGE, ROS, and HIF-1α. In addition, pretreatment with CFYH reversed ALI-induced programmed cell death. In conclusion, CFYH alleviates LPS-induced ALI, and these findings provide a preliminary clarification of the predominant mechanism of action of CFYH in ALI.

Fibroblast growth factor 21 attenuates ventilator-induced lung injury by inhibiting the NLRP3/caspase-1/GSDMD pyroptotic pathway

BackgroundVentilator-induced lung injury (VILI) is caused by overdistension of the alveoli by the repetitive recruitment and derecruitment of alveolar units. This study aims to investigate the potential role and mechanism of fibroblast growth factor 21 (FGF21), a metabolic regulator secreted by the liver, in VILI development.MethodsSerum FGF21 concentrations were determined in patients undergoing mechanical ventilation during general anesthesia and in a mouse VILI model. Lung injury was compared between FGF21-knockout (KO) mice and wild-type (WT) mice. Recombinant FGF21 was administrated in vivo and in vitro to determine its therapeutic effect.ResultsSerum FGF21 levels in patients and mice with VILI were significantly higher than in those without VILI. Additionally, the increment of serum FGF21 in anesthesia patients was positively correlated with the duration of ventilation. VILI was aggravated in FGF21-KO mice compared with WT mice. Conversely, the administration of FGF21 alleviated VILI in both mouse and cell models. FGF21 reduced Caspase-1 activity, suppressed the mRNA levels of Nlrp3, Asc, Il-1β, Il-18, Hmgb1 and Nf-κb, and decreased the protein levels of NLRP3, ASC, IL-1β, IL-18, HMGB1 and the cleaved form of GSDMD.ConclusionsOur findings reveal that endogenous FGF21 signaling is triggered in response to VILI, which protects against VILI by inhibiting the NLRP3/Caspase-1/GSDMD pyroptosis pathway. These results suggest that boosting endogenous FGF21 or the administration of recombinant FGF21 could be promising therapeutic strategies for the treatment of VILI during anesthesia or critical care.

Aloin inhibits gastric cancer cell proliferation and migration by suppressing the STAT3/HMGB1 signaling pathway

To investigate the molecular mechanism underlying the inhibitory effect of aloin on the proliferation and migration of gastric cancer cells.Human gastric cancer MGC-803 cells treated with 100, 200 and 300 μg/mL aloin were examined for changes in cell viability, proliferation and migration abilities using CCK-8, EdU and Transwell assays. HMGB1 mRNA level in the cells was detected with RT-qPCR, and the protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 were determined using Western blotting. JASPAR database was used to predict the binding of STAT3 to HMGB1 promoter. In a BALB/c-Nu mouse model bearing subcutaneous MGC-803 cell xenograft, the effect of intraperitoneal injection of aloin (50 mg/kg) on tumor growth was observed. The protein expressions of HMGB1, cyclin B1, cyclin E1, E-cadherin, MMP-2, MMP-9 and p-STAT3 in the tumor tissue was examined using Western blotting, and tumor metastasis in the liver and lung tissues was detected using HE staining.Treatment with aloin concentration-dependently inhibited the viability of MGC-803 cells (P < 0.05), significantly reduced the number of EdU-positive cells (P < 0.01), and attenuated the migration ability of the cells (P < 0.01). Aloin treatment dose-dependently down-regulated HMGB1 mRNA expression (P < 0.01), lowered the protein expressions of HMGB1, cyclin B1, cyclin E1, MMP-2, MMP-9 and p-STAT3, and up-regulated E-cadherin expression in MGC-803 cells. Prediction based on JASPAR database suggested that STAT3 could bind to the promoter region of HMGB1. In the tumor-bearing mice, aloin treatment significantly reduced the tumor size and weight (P < 0.01), lowered the protein expressions of cyclin B1, cyclin E1, MMP-2, MMP-9, HMGB1 and p-STAT3 and increased the expression of E-cadherin in the tumor tissue (P < 0.01).Aloin attenuates the proliferation and migration of gastric cancer cells by inhibiting the STAT3/HMGB1 signaling pathway.

Expression of HMGB1-TLR4 in Placentas from Preeclamptic Pregnancies and Its Effect on Proliferation and Invasion of HTR-8/SVneo Cells

Objectives: Placental inflammation possibly underlies preeclampsia (PE) pathogenesis. This study aimed to investigate the expression of the high mobility box group 1 (HMGB1)-toll-like receptor 4 (TLR4) signalling pathway in preeclamptic placentas and determine whether HMGB1 regulates the biological behaviour of trophoblasts in vitro. Design: Placental biopsies were taken from 30 preeclamptic patients and 30 normotensive controls. In vitro experiments were carried out in HTR-8/SVneo human trophoblast cells. Participants/Materials, Setting, Methods: HMGB1, TLR4, and nuclear factor kappa B (NF-κB) mRNA and protein were quantified to compare their expression in human placentas from preeclamptic and normotensive pregnancies. HTR-8/SVneo cells were stimulated with HMGB1 (50–400 μg/L) for 6–48 h, and proliferation and invasion of HTR-8/SVneo cells were measured via Cell Counting Kit-8 and transwell assays. HTR-8/SVneo cells were also transfected with HMGB1 and TLR4 small interfering RNA (siRNA) to investigate the effect of knocking down these proteins. The mRNA and protein expression of TLR4, NF-κB, and matrix metalloproteinase 9 (MMP-9) were determined using quantitative real-time PCR and Western blotting, respectively. Data were analysed with either a t-test or one-way analysis of variance. Results: The mRNA and protein levels of HMGB1, TLR4, and NF-κB were significantly higher in the placentas from preeclamptic pregnancies than from normal pregnancies (p < 0.05). HMGB1 stimulation (at concentrations up to 200 μg/L) of HTR-8/SVneo cells significantly increased invasion and proliferation over time. However, at an HMGB1 stimulation concentration of 400 μg/L, the invasion and proliferation ability of HTR-8/SVneo cells decreased. Compared to controls, mRNA and protein expression levels of TLR4, NF-κB, and MMP-9 increased (mRNA level fold change: 1.460, 1.921, 1.667; protein level fold change: 1.600, 1.750, 2.047) when stimulated with HMGB1 (p < 0.05) but decreased when HMGB1 was knocked down (p < 0.05). TLR4 siRNA transfection combined with HMGB1 stimulation reduced the mRNA (fold change: 0.451) and protein (fold change: 0.289) expression of TLR4 (p < 0.05), while NF-κB and MMP-9 were unaffected (p > 0.05). Limitations: Only one trophoblast cell line was used in this study, and the findings were not confirmed in animal studies. Conclusions: This study explored the pathogenesis of PE from two aspects: inflammation and trophoblast invasion. The overexpression of HMGB1 in placentas from preeclamptic pregnancies suggests this protein may be involved in PE pathogenesis. In vitro, HMGB1 was found to regulate the proliferation and invasion of HTR-8/SVneo cells by activating the TLR4-NF-κB-MMP-9 pathway. These findings have implications for targeting HMGB1 could be a therapeutic strategy for treating PE. In the future, we will further verify this in vivo and in other trophoblast cell lines, further exploring the molecular interactions of the pathway.

Clinical Biomarker of Sterile Inflammation, HMGB1, in Patients with Chronic Non-Specific Low Back Pain: A Pilot Cross-Sectional Study

The present study explores whether the inflammatory biomarker of sterile inflammation, high mobility box 1 (HMGB1), contributes to the inflammatory/nociceptive pathophysiology that characterizes chronic non-specific low back pain (LBP). Patients with chronic LBP (N = 10, >3 pain score on a 11-point Visual Analogue Scale, VAS) and asymptomatic participants (N = 12) provided peripheral blood (PB) samples. The proportion of classical CD14++ monocytes within PB leukocytes was determined by flow cytometry. The plasma and extracellular HMGB1 levels in unstimulated adherent cell (AC) cultures were measured using specific immunoassays. HMGB1 localization in ACs was assessed by immunofluorescent staining. The relative gene expression levels of tumor necrosis factor α (TNFα), interleukin-1 beta (IL-1β) and HMGB1 were determined by quantitative polymerase chain reaction (qRT-PCR) in relation to the pain intensity (11-point VAS scores) in patients with LBP. The extracellular release of HMGB1 in the LBP patient AC cultures was significantly elevated (p = 0.001) and accompanied by its relocation into the cytoplasm from the nuclei. The number of CD14++ monocytes in the patients' PB was significantly (p = 0.03) reduced, while the HMGB1 plasma levels remained comparable to those of the controls. The mRNA levels of TNFα, IL-1β and HMGB1 were overexpressed relative to the controls and those of HMGB1 and IL-1β were correlated with the VAS scores at a significant level (p = 0.01-0.03). The results suggest that HMGB1 may play an important role in the pathophysiology of chronic non-specific LBP.

Prolonged alcohol consumption influences microRNA expression in the nucleus accumbens of the rat brain.

The microRNA (miR) species analyzed in this study are involved in molecular mechanisms of TLR4 and TLR7 signaling, mediating the development of neuroinflammation and neurodegeneration. We have investigated the expression levels of miR-let7b, miR-96, miR-182, miR-155, and the mRNA content of HMGB1, TLR3, TLR4 in the nucleus accumbens (NAc) of the brain of rats exposed to long-term alcoholization. The long-term alcoholization caused a decrease in miR-let7b, miR-96, miR-182, and TLR7 mRNA levels; this was accompanied by an increase in miR-155, TLR4, and Hmgb1 mRNA levels in the NAc of rat brain. TLR7 is functionally linked to miR-let7b. The data of a simultaneous decrease in miR-let7b and TLR7 mRNA are of interest for further studies; they may indicate on the lack of functionally significant links between Hmgb1 and the miR-let7b-TLR7 system in NAc. The existing evidence of a functional relationship between TLR4 with miR-155 and miR-182 and our observations on their expression changes during chronic alcoholization are very interesting and require further investigation. The suggestion about the development of neuroinflammatory process in NAc under prolonged alcohol exposure are relevant for studying the level of TLR gene expression in NAc, as well as the expression of miR species, which may have a functional relationship with the TLR system.

Baiying qingmai formulation ameliorates thromboangiitis obliterans by inhibiting HMGB1/RAGE/NF-κB signaling pathways.

Baiying Qingmai Formulation (BF) is a classical clinical prescription used for decades to treat thromboangiitis obliterans (TAO). Although it effectively relieves pain and ischemic ulcers in patients with TAO, its anti-TAO mechanisms remain unclear. The chemical components of BF were analyzed using high-performance liquid chromatography and the potential targets of the compounds identified in BF were analyzed using molecular docking. Further, the signaling pathways and molecular mechanism of BF in treating TAO were studied using a rat model of TAO. Seven compounds (gallic acid, catechin, chlorogenic acid, caffeic acid, paeoniflorin, quercetin, and paeonol) were identified in BF, and molecular docking predicted their high affinities with HMGB1/RAGE/NF-κB proteins. In in vivo studies, BF not only inhibited the protein expression of HMGB1, RAGE, ICAM-1, and VCAM-1; mRNA levels of HMGB1 and RAGE; and the phosphorylation of NF-κB, ERK, Janus kinase (JNK) and p38 MAPK in the femoral artery, but also reduced the levels of inflammatory cytokines (IL-6, TNF-α, IL-1β, HMGB1) and stable metabolite (TXB2) of cytokine promoting thrombosis (TXA2) in the plasma. Moreover, BF stimulated the secretion of stable metabolite (6-keto-PGF1α) of cytokine inhibiting thrombosis (PGI2) in the plasma. BF inhibited the inflammatory response and thrombosis in the femoral artery, thus reducing the degree of vascular occlusion, which alleviated the symptoms in rats with TAO. Our findings suggest that BF ameliorates TAO by inhibiting the activation of the ERK, JNK, p38 MAPK and HMGB1/RAGE/NF-κB signaling pathways, thereby providing novel ideas for the treatment of TAO and essential information for the further development and utilization of BF as a promising drug to treat TAO.

Autophagy-based unconventional secretion of HMGB1 in glioblastoma promotes chemosensitivity to temozolomide through macrophage M1-like polarization

BackgroundGlioblastoma (GB) is the most common and highly malignant brain tumor characterized by aggressive growth and resistance to alkylating chemotherapy. Autophagy induction is one of the hallmark effects of anti-GB therapies with temozolomide (TMZ). However, the non-classical form of autophagy, autophagy-based unconventional secretion, also called secretory autophagy and its role in regulating the sensitivity of GB to TMZ remains unclear. There is an urgent need to illuminate the mechanism and to develop novel therapeutic targets for GB.MethodsCancer genome databases and paired-GB patient samples with or without TMZ treatment were used to assess the relationship between HMGB1 mRNA levels and overall patient survival. The relationship between HMGB1 protein level and TMZ sensitivity was measured by immunohistochemistry, ELISA, Western blot and qRT-PCR. GB cells were engineered to express a chimeric autophagic flux reporter protein consisting of mCherry, GFP and LC3B. The role of secretory autophagy in tumor microenvironment (TME) was analyzed by intracranial implantation of GL261 cells. Coimmunoprecipitation (Co-IP) and Western blotting were performed to test the RAGE-NFκB-NLRP3 inflammasome pathway.ResultsThe exocytosis of HMGB1 induced by TMZ in GB is dependent on the secretory autophagy. HMGB1 contributed to M1-like polarization of tumor associated macrophages (TAMs) and enhanced the sensitivity of GB cells to TMZ. Mechanistically, RAGE acted as a receptor for HMGB1 in TAMs and through RAGE-NFκB-NLRP3 inflammasome pathway, HMGB1 enhanced M1-like polarization of TAMs. Clinically, the elevated level of HMGB1 in sera may serve as a beneficial therapeutic-predictor for GB patients under TMZ treatment.ConclusionsWe demonstrated that enhanced secretory autophagy in GB facilitates M1-like polarization of TAMs to enhance TMZ sensitivity of GB cells. HMGB1 acts as a key regulator in the crosstalk between GB cells and tumor-suppressive M1-like TAMs in GB microenvironment and may be considered as an adjuvant for the chemotherapeutic agent TMZ.

Trifluoro-icaritin alleviates chronic inflammatory pain through α7nAChR-mediated suppression of HMGB1/NF-κB signaling in the spinal cord of rats

Inflammatory pain is a chronic, persistent and serious disease that greatly impacts public health, which is often accompanied by allodynia, hyperalgesia, and spontaneous pain. It is evident that α7 nicotinic acetylcholine receptor (α7nAChR) plays a key role in cholinergic anti-inflammatory pathway and exhibits the inhibition of neuroinflammation in chronic pain. Trifluoro-icaritin (ICTF), a derivative of icaritin from the extract of a genus of Epimedium plant, is identified to possess profound anti-inflammatory activity. However, whether ICTF has anti-nociceptive effect on inflammatory pain and its potential mechanisms remain poorly elucidated. Intraperitoneal injection (i.p.) of ICTF to complete Freund’s adjuvant (CFA)-induced inflammatory pain rats once daily for 21 consecutive days. Pain-related behaviors were evaluated with paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and CatWalk gait analysis. Expression of pain-related signaling molecules in the spinal cord were detected using qRT-PCR, western blot assay, and immunofluorescence staining. This results showed that ICTF (3.0 mg/kg, i.p.) effectively alleviated mechanical allodynia and thermal hyperalgesia not 0.3 and 1.0 mg/kg in CFA rats. Subsequently, we further observed that ICTF (3.0 mg/kg) dramatically decreased the mRNA and protein levels of HMGB1, NF-κB p65, and IL-1β but markedly enhanced α7nAChR and IL-10 expression in the spinal cord of CFA rats, and Immunofluorescence staining also showed that ICTF (3.0 mg/kg) significantly increased the expression of α7nAChR and reduced IBA1 in the spinal cord of CFA rats, along with suppressing the alterations of gait parameters induced by CFA. Moreover, Intrathecal injection (i.t.) of α7nAChR antagonist alpha-bungarotoxin (α-Bgtx, 1.0 μg/kg) not only reversed the anti-nociceptive effect of ICTF on pain hypersensitivity, but also inhibited the down-regulation of HMGB1, NF-κB p65, and IL-1β as well as the up-regulation of α7nAChR and IL-10 protein expression induced by ICTF treatment. Altogether, our results illustrate that ICTF enables to ameliorate CFA-induced inflammatory pain through α7nAChR-mediated inhibition of HMGB1/NF-κB signaling pathway in the spinal cord of rats, suggesting that ICTF may be exploited as a potential painkiller against chronic inflammatory pain.

Effects of miR-1305 on Proliferation and Apoptosis of Non-Small Cell Lung Cancer Cells via Regulating High Mobility Group Box-1 Level

Abnormal expression of HMGB1 is closely related to non-small cell lung cancer (NSCLC). miR-1305 regulates HMGB1 level by MiRDB analysis. Therefore, we investigated whether miR-1305 affects NSCLC cell proliferation and apoptosis by regulating HMGB1. The control group (NC group), miR-1305 Mimics group and miR-1305 Mimics+pcDNA-HMGB1 group were set followed by analysis of miR-1305 and HMGB1 mRNA level real time-PCR, relationship between miR-1305 and HMGB1 by dual fluorescein reporter assay, HMGB1 and Tubulin level by Western blot, cell proliferation by clone formation assay, cell apoptosis by Annexin V-FITC/PI staining. Compared with normal tissues, miR-1305 was significantly downregulated in NSCLC tissues (P &lt;0.01), while HMGB1 mRNA was upregulated (P &lt;0.01). HMGB1 was the target gene of miR-1305. Compared to NC group, HMGB1 level in miR-1305 Mimics group was significantly reduced (P &lt;0.01). Compared with miR-1305 Mimics group, HMGB1 level was significantly increased in miR-1305 Mimics+pcDNA-HMGB1group (P &lt;0.05). HMGB1 mRNA level was not significantly changed. In addition, the number of cell clones and proliferation ability was decreased in miR-1305 Mimics group, which were reversed in miR-1305 Mimics+pcDNA-HMGB1 group. miR-1305 can bind HMGB1 3′-UTR, reduce its protein level, thereby inhibiting NSCLC cell proliferation and promoting cell apoptosis. HMGB1 overexpression can prevent the effect of miR-1305.

Luteolin Relieved DSS-Induced Colitis in Mice via HMGB1-TLR-NF-κB Signaling Pathway.

The aim of this study was to investigate the effect of luteolin (Lu) on dextran sodium sulfate (DSS)-induced colitis in mice. Mice spleen was weighed. The length of colon was measured. H&E staining was used to observe the pathological changes of colon in mice. Superoxide dismutase (SOD) and malondialdehyde (MDA) in serum and intestine of mice were detected by commercial kits. Serum and intestinal cytokines were detected by ELISA kits. The expression of HMGB1 mRNA was detected by real-time PCR. The expression of HMGB1-TLR-NF-κB pathway was detected by Western blot. The level of HMGB1 was detected by immunohistochemistry. The results showed that Lu significantly increased the colon length/body weight ratio and significantly decreased the spleen weight/body weight ratio. Lu significantly increased serum and intestinal SOD levels and decreased MDA levels. Lu significantly increased serum and intestinal cytokine levels in mice. qPCR and immunohistochemistry results showed that Lu significantly reduced HMGB1 mRNA level and protein level. In addition, Lu significantly reduced the expression of HMGB1-TLR-NF-κB signaling pathway protein of intestine in mice. In conclusion, Lu significantly reduced and alleviated DSS-induced colitis in mice, and the mechanism was related to the regulation of intestinal HMGB1-TLR-NF-κB signaling pathway in mice.

Melatonin Ameliorates Lipopolysaccharide-Induced Microglial Inflammation via Triggering SIRT1/HMGB1 Signaling Axis.

Stroke is one of the highest incidence neurological disorder with great morbidity and mortality rate. The secondary neuroinflammation contributed by microglial activation is a consequential response observed in the pathogenesis of stroke. High-mobility group box1, a non-histone nuclear protein, interacts with immune cells, such as microglia, and leads to a cascade amplification of the secondary neuroinflammatory responses, which are related to neuronal damage later. Melatonin is a neurohormone, well-known as its anti-oxidative and anti-inflammatory effects. However, until now, more findings are required for better understanding about anti-inflammatory effect of melatonin on HMGB1 and HMGB1-triggered pathway in LPS-induced microglial activation. Melatonin effect on the viability of BV2 microglial cells was measured by CCK-8 assay; mRNA levels of HMGB1 and other inflammatory cytokines were determined by quantitative real-time polymerase chain reaction assay or enzyme-linked immunosorbent assays; the protein expression levels of TLR4/MyD88/NF-κB and SIRT1 were detected by Western blot, and HMGB1 translocation and release from BV2 microglial cells were examined by immunofluorescence assay. The results of this study demonstrated that melatonin suppressed LPS-triggered BV2 microglial activation-mediated inflammation by inhibiting high expression and release of HMGB1 and moderating the activation of subsequent TLR4/MyD88/NF-κB signaling pathway, which was activated by SIRT1 elevation. Furthermore, LPS-induced expression of pro-inflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β) was notably reversed by melatonin pre-treatment. In summary, our findings suggest that melatonin may act as a promising therapeutic agent for reducing post-stroke neuroinflammation by targeting HMGB1 and the subsequent signaling axis.

Ketamine alleviates HMGB1-induced acute lung injury through TLR4 signaling pathway.

Acute lung injury (ALI) is a common critical respiratory disease that seriously threatens human health. Ketamine has good anti-inflammatory and immune-regulating properties that can delay the lung injury process. High mobility group box protein 1 (HMGB1) plays an important role in the occurrence, development and treatment of ALI. Toll-like receptor 4 (TLR4) is the receptor for HMGB1. The aim of this study was to determine the role of the HMGB1 TLR4 signaling pathway in the treatment of ALI using ketamine. A total of 30 healthy, male, 8-week-old Sprague-Dawley rats were randomly, equally divided into a control group, an lipopolysaccharide (LPS) group and a ketamine group. In order to establish a rat ALI model, 15 mg/kg of LPS was injected into the femoral veins. Ketamine was intravenously injected (10 mg/kg) into the experimental group rats. The rats were euthanized 24 h after modeling and lung tissue samples were collected. Western blot was used to test TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 protein expression in the lung tissue. Real-time polymerase chain reaction (RT-PCR) was performed to detect TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA levels. Compared with the controls, the LPS group had significantly higher TLR4, MyD88, TRAF-6, LOX-1, and HMGB1 mRNA and protein levels (p < 0.05). These levels were significantly lower after ketamine intervention in comparison with the LPS group (p < 0.05). A positive correlation was found between TLR4 and HMGB1 expression in the LPS and ketamine groups (r = 0.952, p < 0.001; r = 0.941, p < 0.001). Ketamine attenuates HMGB1-induced ALI, possibly by regulating the TLR4 signaling pathway.

MiR-200a promotes the survival of cardiac cells and improves cardiac injury in chronic heart failure rats

IntroductionmiRNAs play an important role in cardiovascular abnormalities such as heart failure. In the present work we evaluated the role of miR-200a in the condition of chronic heart failure and also the mechanism involved.Material and methodsIn the study 180 subjects, among whom 100 were reported for chronic heart failure and 80 as normal, were included. ELISA and qRT-PCR was done to evaluate levels of HMGB1 and miR-200a in subjects. The cardiac hemodynamics and functioning, oxidative stress and expression of mediators of inflammation were studied in rats with chronic heart failure induced after transfecting them with miR-200a or HMGB1. Luciferase activity was measured to establish any correlation between HMGB1 and miR-200a.ResultsThe chronic heart failure patients included in the study showed suppressed levels of miR-200a and elevated HMGB1 compared to normal subjects. In chronic heart failure rats, the transfection of miR-200a attenuated the cardiac function and other hemodynamic parameters. In addition, improvement in oxidative stress as well as inflammatory mediators was observed. The outcomes also confirmed that HMGB1 was the potential target of miR-200a. It was also noted that upon transfection miR-200a resulted in suppression of protein as well as mRNA levels of HMGB1 in the cardiac tissue of chronic heart failure rats. Also overexpression of HMGB1 decreased the effects of miR-200a.ConclusionsThe outcomes indicate that miR-200a exerts a protective effect on cardiac cell injury via the HMGB1 pathway.

Role of the DNA repair genes H2AX and HMGB1 in human fat distribution and lipid profiles

IntroductionRegional fat distribution strongly relates to metabolic comorbidities. We identified the DNA repair genes H2AX and HMGB1 to be differentially expressed between human subcutaneous (SAT) and omental visceral adipose tissue...

MiR-129-5p improves cardiac function in rats with chronic heart failure through targeting HMGB1.

Increasing evidence shows that miRNAs play pivotal roles in cardiovascular diseases, including heart failure (HF). The aim of this study was to investigate the role of miR-129-5p in chronic heart failure and the underlying mechanisms. The levels of miR-129-5p and HMGB1 in chronic heart failure patients (CHF) and normal controls were examined by RT-qPCR and ELISA. Cardiac function, hemodynamics parameters, oxidative stress, and inflammation factors were analyzed in CHF rat model after transfection of miR-129-5p or HMGB1. Dual-luciferase activity reporter assay was conducted to validate the interaction between miR-129-5p and HMGB1. Downregulation of miR-129-5p and upregulation of HMGB1 were observed in the serum of CHF patients, respectively. Transfection of miR-129-5p improved heart function and hemodynamic parameters, as well as attenuated oxidative stress and inflammation factors in CHF rats. We further confirmed that HMGB1 is a direct target of miR-129-5p. Transfection of miR-129-5p also decreased the mRNA and protein levels of HMGB1 in myocardial tissues of CHF rats. Overexpression of HMGB1 diminished the effects of miR-129-5p on ameliorating oxidative stress and inflammatory response in rats with CHF. Our findings suggest that miR-129-5p protects the heart by targeting HMGB1.