High Mobility Group Protein B1 Research Articles

Bullatacin triggers immunogenic cell death of colon cancer cells by activating endoplasmic reticulum chaperones

BackgroundIt is well accepted that the immune system efficiently contributes to positive outcomes of chemotherapeutic cancer treatment by activating immunogenic cell death (ICD). However, only a limited number of ICD-inducing compounds are well characterized at present; therefore, identification of novel ICD inducers is urgently needed for cancer drug discovery, and the need is becoming increasingly urgent.MethodsHerein, we assessed the antitumour activity of bullatacin by MTS assay and apoptosis assay. ICD biomarkers, such as calreticulin (CRT), high-mobility group protein B1 (HMGB-1), heat shock protein (HSP)70, HSP90 and ATP, were assessed by Western blotting, ELISA and flow cytometry. Western blot and qPCR assays were performed to explore the underlying mechanisms of bullatacin-induced ICD. Flow cytometry was used to detect macrophage phagocytosis.ResultsFirst, bullatacin induced apoptosis in both SW480 cells and HT-29 cells in a time-dependent manner at 10 nM, as assessed by flow cytometry. Moreover, Western blot and flow cytometry assays showed that CRT and HSP90 (biomarkers of early ICD) significantly accumulated on the cell membrane surface after approximately 6 h of treatment with bullatacin. In addition, ELISAs and Western blot assays showed that the second set of hallmarks required for ICD (HMGB1, HSP70 and HSP90) were released in the conditioned media of both SW480 and HT-29 cells after 36 h of treatment. Furthermore, qPCR and Western blot assays indicated that bullatacin triggered ICD via activation of the endoplasmic reticulum stress (ERS) signalling pathway. Finally, bullatacin promoted macrophage phagocytosis.ConclusionThis study documents that bullatacin, a novel ICD inducer, triggers immunogenic tumour cell death by activating ERS even at a relatively low concentration in vitro.

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.

ADAM10 inhibits the interaction between IL-17 and HMGB1 in Buerger's disease.

Buerger's disease is a rare disease that causes critical limb ischemia; however, the underlying pathophysiological mechanism remains unclear. Therefore, we investigated the interaction between interleukin (IL)-17 and high-mobility group protein B 1 (HMGB1) and determined whether A disintegrin and metalloproteinase 10 (ADAM10) inhibit this interaction. The study population included 15 patients with Buerger's disease and 10 healthy donors without a history of giving peripheral blood samples. Cytokine levels were measured using a luminex multiplex assay in plasma. Flow cytometry was used to analyze the subtypes of helper T (Th) cells among peripheral blood mononuclear cells (PBMCs). The effect of ADAM10 on PBMCs was analyzed in vitro. The levels of inflammatory cytokines and production of pathogenic Th cells were found to be higher in Korean patients with Buerger's disease. IL-17 treatment induced HMGB1 associated molecules. HMGB1 also induced IL-17 and Th17 associated transcription factors in Buerger's patients. We observed that ADAM10 regulates the interaction between IL-17 and HMGB1 via advanced glycation end products (RAGE)/nuclear factor-kappa B (NF-kB) pathway in patients with Buerger's disease. This study suggests that IL-17 and HMGB1 cytokines contribute to the pathogenesis of Buerger's disease. These results indicate that ADAM10 alleviates inflammation in Buerger's disease via the HMGB1 and RAGE/NF-κB signaling pathway and provides insights into the molecular basis of and a potential therapeutic strategy for Buerger's disease.

Dexmedetomidine Preconditioning Mitigates Myocardial Ischemia/Reperfusion Injury via Inhibition of Mast Cell Degranulation

Background: The degranulation of cardiac mast cells is associated with the occurrence and development of myocardial ischemia/reperfusion (I/R) injury (MIRI). Dexmedetomidine (DEX) has a cardioprotective effect from I/R injury. The present study investigated whether DEX preconditioning-induced cardioprotection was related to suppression of the degranulation of cardiac mast cells. Methods: The ligation of the left anterior descending coronary artery for 30 min of ischemia and 120 min of reperfusion was used to establish a rat MIRI model in vivo. The MIRI Langendorff model in vitro of rat isolated heart was constructed by global ischemia for 30 min and reperfusion for 60 min. The effect of compound 48/80 (C48/80) and DEX pretreatment on MIRI was then evaluated. Hemodynamics and electrocardiogram were assessed for each rat/heart. TTC staining, hematoxylin-eosin staining, and toluidine blue staining, transmission electron microscope detection, TUNEL staining, ELISA, IHE staining, qRT-PCR, and western blot were detected. Further work was performed using a cell coculture test to verify. Findings: In vivo and in vitro experimental results revealed that hemodynamic disorder, arrhythmia, infarct size, histopathological score, and mast cell degranulation were dramatically increased in the I/R injury groups compared to the non-I/R groups. Mastocyte secretagogue compound 48/80 (C48/80) aggravated these damages, and DEX preconditioning mitigated these damages. C48/80 increased the levels of I/R injury-induced cardiac troponin-1 (cTnI) and tryptase, cardiomyocytes apoptosis, and the expression of high mobility group protein B1 (HMGB1), Toll-like receptor 4 (TLR4), and nuclear factor kappa B (NF-κB) p65 in cardiac tissues, and DEX pretreatment partially reduced these changes. C48/80 inhibited the proliferation of H9C2(2-1) and RBL-2H3, exacerbated apoptosis of H9C2(2-1), and increased the levels of cTnI and tryptase, and DEX pretreatment abolished these changes. Interpretation: Our data suggest that DEX preconditioning alleviates the MIRI-induced degranulation of mast cells and the apoptosis of cardiomyocytes, and inhibits the activation of inflammatory-related factors HMGB1, TLR4, and NF-κB p65. Funding Information: This research was supported by the National Natural Science Foundation of China (81860050 & 82060060), Yunnan Health Training Project of High Levels Talents (L-2018007), Program for Innovative Research Team of Kunming Medical University (CXTD201802), Yunnan Provincial Ten Thousand-Talent Program-Famous Doctor (YNWR-MY-2018-043), Innovative fund of Kunming Medical University (2020D013), and Yunnan Provincial Education Department (2020Y0120). Declaration of Interests: The authors declare no conflict of interest. Ethics Approval Statement: All experimental procedures were performed in accordance with the guidelines formulated by the Institutional Animal Care and Use Committee of Kunming Medical University (No. KMU20191119).

Glaucocalyxin A Attenuates Allergic Responses by Inhibiting Mast Cell Degranulation through p38MAPK/NrF2/HO-1 and HMGB1/TLR4/NF-κB Signaling Pathways.

Glaucocalyxin A (GLA) has various pharmacological effects like antioxidation, immune regulation, and antiatherosclerosis. Here, in this study, the effect and mechanism of GLA on mast cell degranulation were studied. The results of the anti-DNP IgE-mediated passive cutaneous anaphylaxis (PCA) showed that GLA dramatically inhibited PCA in vivo, as evidenced by reduced Evans blue extravasation and decreased ear thickness. In addition, GLA significantly reduced the release of histamine and β-hexosaminidase, calcium influx, cytokine (IL-4, TNF-α, IL-1β, IL-13, and IL-8) production in the RBL-2H3 (rat basophilic leukemia cells), and RPMCs (peritoneal mast cells) in vitro. Moreover, we further investigated the regulatory mechanism of GLA on antigen-induced mast cells by Western blot, which showed that GLA inhibited FcεRI-mediated signal transduction and invalidated the phosphorylation of Syk, Fyn, Lyn, Gab2, and PLC-γ1. In addition, GLA inhibited the recombinant mouse high mobility group protein B1- (HMGB1-) induced mast cell degranulation through limiting nuclear translocation of NF-κBp65. Treatment of mast cells with siRNA-HMGB1 significantly inhibited HMGB1 levels, as well as MyD88 and TLR4, decreased intracellular calcium levels, and suppressed the release of β-hexosaminidase. Meanwhile, GLA increased NrF2 and HO-1 levels by activating p38MAPK phosphorylation. Consequently, these data suggest that GLA regulates the NrF2/HO-1 signaling pathway through p38MAPK phosphorylation and inhibits HMGB1/TLR4/NF-κB signaling pathway to reduce mast cell degranulation and allergic inflammation. Our findings could be used as a promising therapeutic drug against allergic inflammatory disease.

Oleandrin, a cardiac glycoside, induces immunogenic cell death via the PERK/elF2\u03b1/ATF4/CHOP pathway in breast cancer

Chemotherapeutic agents have been linked to immunogenic cell death (ICD) induction that is capable of augmenting anti-tumor immune surveillance. The cardiac glycoside oleandrin, which inhibits Na+/K+-ATPase pump (NKP), has been shown to suppress breast cancer growth via inducing apoptosis. In the present study, we showed that oleandrin treatment triggered breast cancer cell ICD by inducing calreticulin (CRT) exposure on cell surface and the release of high-mobility group protein B1 (HMGB1), heat shock protein 70/90 (HSP70/90), and adenosine triphosphate (ATP). The maturation and activation of dendritic cells (DCs) were increased by co-culturing with the oleandrin-treated cancer cells, which subsequently enhanced CD8+ T cell cytotoxicity. Murine breast cancer cell line EMT6 was engrafted into BALB/c mice, and tumor-bearing mice were administered with oleandrin intraperitoneally every day. Oleandrin inhibited tumor growth and increased tumor infiltrating lymphocytes including DCs and T cells. Furthermore, the differential mRNA expression incurred by oleandrin was investigated by mRNA sequencing and subsequently confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. Mechanistically, oleandrin induced endoplasmic reticulum (ER) stress-associated, caspase-independent ICD mainly through PERK/elF2α/ATF4/CHOP pathway. Pharmacological and genetic inhibition of protein kinase R-like ER kinase (PERK) suppressed oleandrin-triggered ICD. Taken together, our findings showed that oleandrin triggered ER stress and induced ICD-mediated immune destruction of breast cancer cells. Oleandrin combined with immune checkpoint inhibitors might improve the efficacy of immunotherapy.

The role of Toll-like receptors in neurobiology of alcoholism

Alcoholism is a global socially significant problem and still remains one of the leading causes of disability and premature death. One of the main signs of the disease is the loss of cognitive control over the amount of alcohol consumed. Among the mechanisms of the development of this pathology, changes in neuroimmune mechanisms occurring in the brain during prolonged alcohol consumption and its withdrawal have recently become the focus of numerous studies. Ethanol consumption leads to the activation of neuroimmune signaling in the central nervous system through many subtypes of Toll-like receptors (TLRs), as well as release of their endogenous agonists (high-mobility group protein B1 (HMGB1), S100 protein, heat shock proteins (HSPs), and extracellular matrix degradation proteins). TLR activation triggers intracellular molecular cascades of reactions leading to increased expression of genes of the innate immune system, particularly, proinflammatory cytokines, causing further development of a persistent neuroinflammatory process in the central nervous system. This leads to death of neurons and neuroglial cells in various brain structures, primarily in those associated with the development of a pathological craving for alcohol. In addition, there is evidence that some subtypes of TLRs (TLR3, TLR4) are able to form heterodimers with neuropeptide receptors, thereby possibly playing other roles in the central nervous system, in addition to participating in the activation of the innate immune system.

Angiotensin II promotes EMT of hepatocellular carcinoma cells through high mobility group protein B1 mediated by E4F1

To screen for specific transcription factors (TFs) that induce expression of the HMGB1 promoter in response to stimulation by Ang-II. A HMGB1 overexpressing vector and small interfering (si)RNA were constructed and used to transfect the three HCC cell lines used in scratched monolayer wound healing and Transwell assays. Chromatin immunoprecipitation (ChIP) assays were used to confirm the relationship between a specific TF and the HMGB1 promoter. Invasion and migration by HMGB1 overexpressing HCC cells after treatment with Ang-II were significantly increased compared to negative controls (NC); E-cadherin was down-regulated while vimentin was up-regulated. However, compared with NC, invasion and migration by HMGB1 siRNA HCC cells stimulated by Ang-II were not altered; the expression of E-cadherin and vimentin was also unaltered. Nineteen TFs were predicted by Promoter 2.0 Prediction Server and TFsitescan. Real-time qPCR was used to evaluate TF expression levels. E4F1 was the only TF abnormally elevated in all three HCC cell lines when stimulated by Ang-II. WB and ChIP assays revealed high expression of E4F1 compared to other TFs in cells stimulated by Ang-II. E4F1 is activated by Ang-II and binds to the HMGB1 promoter region to promote HMGB1 expression; it then enhances Ang-II to induce HCC cell invasion and migration, and EMT.

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.

Alternol triggers immunogenic cell death via reactive oxygen species generation

ABSTRACT Alternol is a naturally occurring compound that exerts antitumor activity in several cancers. However, whether Alternol induces antitumor immune response remains unknown. In this study, we investigated whether Alternol induced immunogenic cell death (ICD) in prostate cancer cells. Alternol triggered ICD in prostate cancer cells, as evidenced by the release of damage-associated molecular patterns (DAMPs) (i.e., calreticulin, CALR; high mobility group protein B1, HMGB1; and adenosine triphosphate, ATP) and pro-inflammatory cytokine (i.e., interleukin [IL]-1α, IL-1β, IL-6, and IL-8) expression. Alternol facilitated tumor-associated antigen uptake and cross-presentation, CD8 + T-cell priming, and T-cell infiltration in tumor-draining lymph nodes (LNs) and tumors. The presence of Alternol fostered antitumor immune response in vivo, resulting in delayed tumor growth and prolonged survival. Moreover, inhibition of reactive oxygen species (ROS) generation blocked Alternol-induced upregulation of pre-inflammation cytokines, endoplasmic reticulum (ER) stress, and consequent antitumor immune response. Overall, our data indicate that Alternol triggers ICD in prostate cancer cells, which is mediated by ROS generation.

16202 Effect of high-mobility group box 1 on keratinocyte and fibroblast and its involvement in the development of psoriasis

Background: Psoriasis is a chronic immune-mediated inflammatory skin disease, in which keratinocyte and fibroblast play an important role. High-mobility group protein B1 (HMGB1) is a nuclear protein that routinely participates in the maintenance of genomic stability and the regulation of gene transcription. HMGB1 is also a physiological activator of immune responses that can be released from keratinocyte and fibroblast nuclei in psoriatic lesions. Although it is implicated in the pathogenesis of autoimmune diseases and cutaneous disorders, the precise role of HMGB1 in psoriasis has yet to be studied.

Naringin attenuates renal interstitial fibrosis by regulating the TGF-β/Smad signaling pathway and inflammation.

Interstitial fibrosis is a typical feature of all progressive renal diseases. The process of fibrosis is frequently coupled with the presence of pro-fibrotic factors and inflammation. Naringin is a dihydroflavone compound that has been previously reported to exhibit anti-fibrotic effects in the liver, where it prevents oxidative damage. In the present study, a rat model of renal interstitial fibrosis and fibrosis cell model were established to evaluate the effects of naringin on inflammatory proteins and fibrosis markers in kidney of rats and NRK-52E cells, and to elucidate the role of the TGF-β/Smad signaling pathway in this mechanism. Compared with those in fibrotic NRK-52E cells that were stimulated by transforming growth factor-β (TGF-β), gene expression levels of α-smooth muscle actin (α-SMA), collagen 1 (COL1A1), collagen 3 (COL3A1), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α) were all found to be significantly decreased in fibrotic NRK-52E cells following treatment with naringin (50, 100 and 200 ng/ml). Results from the histopathological studies showed that naringin treatment preserved the renal tissue structure and reduced the degree of fibrosis in the kidney tissues of rats that underwent unilateral ureteral obstruction (UUO). In addition, naringin administration reduced the expression of α-SMA, COL1A1, COL3A1, IL-1β, IL-6 and TNF-α in the kidneys of rats following UUO. The current study, using western blot analysis, indicated that naringin also downregulated the activation of Smad2/3 and the expression of Smad4, high-mobility group protein B1, activator protein-1, NF-κB and cyclooxygenase-2 whilst upregulating the expression of Smad7 in fibrotic NRK-52E cells and rats in the UUO group. In conclusion, naringin could antagonize renal interstitial fibrosis by regulating the TGF-β/Smad pathway and the expression of inflammatory factors.

MicroRNA-574-5p Attenuates Acute Respiratory Distress Syndrome by Targeting HMGB1.

Acute respiratory distress syndrome (ARDS) is a critical condition with high mortality. HMGB1 (high-mobility group protein B1) is one of the key proinflammatory factors in the ARDS “inflammatory storm.” According to previous studies, some microRNAs (miRNAs) play important roles in this process. We aimed to determine the contributing miRNAs targeting the expression and release of HMGB1. miRNA expression in the peripheral blood of patients with ARDS was measured by miRNA microarray. miRNAs targeting HMGB1 were screened and explored for further study. In LPS-induced cell and mouse ARDS models, we explored the effect of this miRNA on the expression and secretion of HMGB1 by Western blot, real-time qPCR, and ELISA. The effects of this miRNA on the NF-κB signaling pathway, proinflammatory cytokines, and NLRP3 (nod-like receptor protein 3) inflammasome were detected by Western blot and real-time qPCR. In ARDS models, microRNA-574-5p (miR-574-5p) expression could be induced by the TLR4/NF-κB pathway upon LPS stimulation. It could suppress the inflammatory response by targeting HMGB1. Enforcing the expression of miR-574-5p or HMGB1 siRNA silencing inhibits the activation of NF-κB signaling pathway and the NLRP3 inflammasome. Moreover, overexpression of HMGB1 reversed the antiinflammatory effect of miR-574-5p. In ARDS mice, overexpression of miR-574-5p suppresses alveolar leukocytes infiltration, interstitial edema, protein effusion, and inflammation. This study demonstrated that miR-574-5p provided negative feedback to LPS-induced inflammation and relieved ARDS. It may provide new therapeutic strategies for ARDS.

Pristimerin attenuates sepsis-induced lung injury by regulating nuclear factor kappaB/high-mobility group box 1 pathway

Purpose: To determine the effect of pristimerin on sepsis-induced lung injury, and the underlying mechanism of action.Methods: Lung injury was established in mice via induction of sepsis through cecal ligation and puncture (CLP). The effect of pristimerin was evaluated based on lung wet/dry weight and PaO2/FiO2 ratios. Lung tissue was subjected to immunohistochemical and histopathological analyses, as well as Western blotting. Furthermore, the serum levels of inflammatory mediators were determined.Results: Pristimerin reversed the altered lung wet/dry weight ratio and PaO2/FiO2 ratio in the lung, and also reduced lung injury score, relative to CLP group (p < 0.05). Moreover, it suppressed nucleocytoplasmic translocation of high mobility group protein B1 (HMGB1) in lung tissue. Serum levels of inflammatory mediators and expression levels of inducible nitric oxide synthase and nuclear factorkappaB p65 were significantly reduced by pristimerin (p < 0.05).Conclusion: Pristimerin ameliorates sepsis-induced lung injury by inhibiting HMGB1/NF-κB. Thus, this compound has a potential for clinical application in the management of lung injury.
 Keywords: Pristimerin, Sepsis, Lung injury, Inflammatory mediators, HMGB1

Licorice Ameliorates Cisplatin-Induced Hepatotoxicity Through Antiapoptosis, Antioxidative Stress, Anti-Inflammation, and Acceleration of Metabolism.

Cisplatin (CP) is one of the most effective antitumor drugs in the clinic, but has serious adverse reactions, and its hepatotoxicity has not been fully investigated. Licorice (GC), a traditional herbal medicine, has been commonly used as a detoxifier for poisons and drugs, and may be an effective drug for CP-induced hepatotoxicity. However, its mechanism and the effector molecules remain ambiguous. Therefore, in this study, a network pharmacology and proteomics-based approach was established, and a panoramic view of the detoxification of GC on CP-induced hepatotoxicity was provided. The experimental results indicated that GC can recover functional indices and pathological liver injury, inhibit hepatocyte apoptosis, upregulate B-cell lymphoma/leukemia 2 (Bcl-2) and superoxide dismutase (SOD) levels, and downregulate cellular tumor antigen p53 (p53), caspase-3, malondialdehyde high mobility group protein B1 (HMGB1), tumor necrosis factor alpha (TNF-α), and interleukin 1β (IL-1β) levels. Proteomics indicated that GC regulates phosphatidylcholine translocator ABCB1 (ABCB1B), canalicular multispecific organic anion transporter 1 (ABCC2), cytochrome P450 4A2 (CYP4A2), cytochrome P450 1A1 (CYP1A1), cytochrome P450 1A2 (CYP1A2), estrogen receptor (ESR1), and DNA topoisomerase 2-alpha (TOP2A), inhibits oxidative stress, apoptosis, and inflammatory responses, and accelerates drug metabolism. In this study, we provide the investigation of the efficacy of GC against CP-induced hepatotoxicity, and offer a promising alternative for the clinic.

TLR, RAGE, and HMGB1 in the Inflammatory Response in Ph(-) Myeloproliferative Neoplasm

Recent evidence of increased constitutional symptoms and inflammatory cytokines in Philadelphia chromosome negative (Ph (-)) MPN suggests that an inflammatory response is important in the pathogenesis of Ph (-) MPN. Toll-like receptors (TLR), Receptor for Advanced Glycation End products (RAGE) and High mobility group protein B1 (HMGB1) are the important pathways for the inflammatory response. All these three important pathway proteins were studied in MPN diseases in the current studies. Materials and Methods: TLR assay. TLR 2,3, 4, 7, 9 quantification was performed by immuno-staining of 1×106 mononuclear cells (peripheral blood) which were incubated with fluorescence-conjugated anti-TLR-2,3, 4, 7, 9 antibodies and assayed by flow cytometry. HMGB1assay:HMGB1 ELISA kit from Immuno-Biological Laboratories, Inc. (IBL-America) were used. The plasma samples were diluted four times with the provided sample dilution buffer, and assayed in duplicate according to the manufacturer's suggestion. RAGE (RT-PCR) Assay: Total RNA was extracted from normal control or patient mononuclear cells. Predesigned primers for RAGE, and internal control genes were ordered from Qiagen (Germantown, MD). Real-time PCR was performed using SsoAdvanced™ Universal SYBR® Green Supermix (Bio-Rad, Hercules, CA) on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System. At least three house-keeping genes (ribosomal protein L4, TATA box binding protein, and tubulin-α 1b) were used as normalization controls. The expression of RAGE were compared with each internal control. Average of three was used to calculate the ratio of final patient to normal Results: Total of 97 patients with MPN were studied 1) TLR: TLR 3,7,9 was not significantly different from controls. But TLR 2 was significantly increased in both PV, as well as in the MPN group when PV, ET and MF were grouped together as MPN (Fig A). TLR 4 was not significantly increased in PV, ET, MF individually but was found to be significantly increased than the controls, when they are grouped together as MPN (Fig B). 2) RAGE: No significant difference was found between ET, PV, MF individually or when they were grouped together as MPN than the controls (Fig C). 3) HMGB1: No significant difference was seen between ET, PV, MF or when they were grouped as MPN (Fig D). Conclusion: Current study suggests that TLR pathway especially TLR2, and to a lesser extent TLR4 are the important pathways for inflammatory response with increased inflammatory cytokines in MPN, while HMGB1 and RAGE pathways were not different from controls. Figure Disclosures No relevant conflicts of interest to declare.

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.

Isomangiferin Attenuates Renal Injury in Diabetic Mice via Inhibiting Inflammation.

AimRenal injury induced by diabetes is reported to be associated with inflammation. Isomangiferin (ISO), a xanthone C-glucoside from the Cyclopia subfamily, exhibits many pharmacological properties. This study aimed to evaluate the protection of ISO against renal damage in diabetic mice.MethodsSerum glucose, insulin, uric acid, creatinine, total cholesterol (TC), triglyceride (TG), and inflammatory cytokines in serum and the kidney of db/db diabetes model mice were detected. The components of high mobility group protein B1 (HMGB1)/NACHT leucine-rich repeat- and PYD-containing 3 (NLRP3)/nuclear factor kappa-B (NF-κB) pathway in the kidney were detected by Western blot and immunohistochemical analysis.ResultsISO improved lipid profile and glucose tolerance, and inhibited the production of inflammatory cytokines in a db/db model mice. Moreover, ISO decreased biochemical indexes in the serum and inhibited the activation of HMGB1/NLRP3/NF-κB signaling in the kidney of db/db model mice.ConclusionISO provides protection against renal injury via inhibiting HMGB1/NLRP3/NF-κB signaling in a diabetic mouse model.

Paeonol promotes the phagocytic ability of macrophages through confining HMGB1 to the nucleus

Phagocytosis is a basic immune response to the pathogens invading. Immunosuppression may occur in diseases like sepsis and cancer, and cause a low phagocytic ability of phagocytes. High mobility group protein B1 (HMGB1) is a DNA chaperone which is closely related to the phagocytosis. Nonetheless, its influence on phagocytosis is still controversial. We found that paeonol could inhibit the translocation of HMGB1 from the nucleus to the cytoplasm, it may have an impact on phagocytosis. In the present study, we performed in vivo and in vitro experiments to investigate the influence of paeonol on phagocytosis. Zymosan was used to test the phagocytic function of macrophages. Our results showed that paeonol promotes the phagocytosis of macrophages through confining HMGB1 to the nucleus. Through interacting with P53, the nuclear HMGB1 keep it in the nucleus and decrease the negative influence of P53 on the phosphorylation of Focal Adhesion Kinase (FAK). The increasing of phosphorylated FAK promotes the formation of pseudopod and enhances the phagocytic ability of macrophages.