HMGB1 Protein Research Articles

Involvement of TOLL-like receptors in the neuroimmunology of alcoholism

Alcohol use is a global socially significant problem that remains one of the leading risk factors for disability and premature death. One of the main pathological characteristics of alcoholism is the loss of cognitive control over the amount of consumed alcohol. Growing body of evidence suggests that alterations of neuroimmune communication occurring in the brain during prolonged alcoholization are one of the main mechanisms responsible for the development of this pathology. Ethanol consumption leads to activation of neuroimmune signaling in the central nervous system through many types of Toll-like receptors (TLRs), as well as the release of their endogenous agonists (HMGB1 protein, S100 protein, heat shock proteins, extracellular matrix breakdown proteins). Activation of TLRs triggers intracellular molecular cascades leading to increased expression of the innate immune system genes, particularly proinflammatory cytokines, subsequently causing the development of a persistent neuroinflammatory process in the central nervous system, which results in massive death of neurons and glial cells in the brain structures, which are primarily associated with the development of a pathological craving for alcohol. In addition, some subtypes of TLRs are capable of forming heterodimers with neuropeptide receptors (corticoliberin, orexin, ghrelin receptors), and may also have other functional relationships.

Effects of nalmefene hydrochloride on TLR4 signaling pathway in rats with lung ischemia-reperfusion injury.

To investigate the effect of nalmefene hydrochloride on TLR4 signaling pathway in rats with lung ischemia-reperfusion injury. Altogether 64 pure inbred male SD rats were divided into groups A, B, C, and D according to the principle of body weight similarity, with 24 rats in each group. Four groups of rats were respectively twisted on the left testis to establish unilateral testicular torsion rats. Group A was the control group, treated with normal saline, group B was the nalmefene hydrochloride high-dose group, treated with 20 μg/kg of nalmefene hydrochloride, group C was the nalmefene hydrochloride low-dose group, treated with 10 μg/kg of nalmefene hydrochloride, and group D was the sham operation group. Lung tissue was collected 60 h later. Western blotting was used to detect the expression levels of HMGB1, TLR4, CD14, and NF-κB protein, qPCR was used to detect the mRNA expression level, and enzyme-linked immunosorbent assay (ELISA) was used to detect the expression levels of inflammatory factors IL-17, IL-6, and ICAM-1. The expression levels of HMGB1, TLR4, CD14, NF-κB protein, mRNA, IL-17, IL-6, and ICAM-1 in group A were significantly higher than those in groups B, C, and D (p<0.05), while were significantly lower in group D than in groups B and C (p<0.05), and were significantly lower in group B than in group C (p<0.05). Nalmefene hydrochloride can effectively inhibit the signal pathway of TLR4, and can effectively reduce the injury caused by lung ischemia-reperfusion. The large dose is closely related to the good effect, which is worthy of promotion.

The impact of apurinic-apyrimidinic endonuclease I on hepatocyte immuno-inflammatory factors and cell apoptosis.

To explore the effect of apurinic-apyrimidinic endonuclease I (APE-1) on hepatocyte immune inflammatory factors and cell apoptosis. The gene expression profiles of peripheral blood of patients with or without immune tolerance after liver transplantation were obtained from the Gene Expression Omnibus (GEO). Differentially expressed genes were analyzed with a program in the R language, and the APE-1 gene was identified as a gene related to immune tolerance of liver transplantation. Four APE-1 shRNA vectors were constructed in parallel and verified as correct using plasmid sequencing, real-time PCR, and Western blotting. An APE-1 overexpression vector was similarly constructed and verified as correct. The STRING website predicted the protein-protein interaction network of APE-1. ELISA was used to detect the effects of APE-1 silencing and overexpression on inflammatory cytokines IL-1β, IL-10, TNFα, and INF-γ in the control group, APE-1-silenced group, and APE-1 overexpression group. Flow cytometry was used to detect apoptosis in each group. Forty differentially expressed genes related to immune tolerance after liver transplantation were screened, and the highly expressed gene APE-1 was selected. The best APE-1 shRNA_1 vector and APE-1 overexpression vector were obtained. APE-1 is predicted to interact with ANP32A, FEN1, HMGB2, LIG1, MUTYH, NTHL1, OGG1, PCNA, POLB, SET, and other proteins. APE-1 silencing resulted in a significant increase in the expression of the inflammatory factors IL-1β, IL-10, TNFα, and INF-γin L-02 cells. In contrast, the expression of APE-1 led to a significant decrease in the expression of inflammatory factors. APE-1 silencing significantly increased the rate of apoptosis of L-02 cells, and APE-1 overexpression resulted in a significant decrease in the rate of apoptosis of L-02 cells. In conclusion APE-1 affects the expression of inflammatory factors and apoptosis in L-02 cells, so it may be a key gene in immune tolerance of liver transplantation.

Diagnostic Biomarkers in Liver Injury by Drugs, Herbs, and Alcohol: Tricky Dilemma after EMA Correctly and Officially Retracted Letter of Support.

Liver injuries caused by the use of exogenous compounds such as drugs, herbs, and alcohol are commonly well diagnosed using laboratory tests, toxin analyses, or eventually reactive intermediates generated during metabolic degradation of the respective chemical in the liver and subject to covalent binding by target proteins. Conditions are somewhat different for idiosyncratic drug induced liver injury (DILI), for which metabolic intermediates as diagnostic aids are rarely available. Although the diagnosis of idiosyncratic DILI can well be established using the validated, liver specific, structured, and quantitative RUCAM (Roussel Uclaf Causality Assessment Method), there is an ongoing search for new diagnostic biomarkers that could assist in and also confirm RUCAM-based DILI diagnoses. With respect to idiosyncratic DILI and following previous regulatory letters of recommendations, selected biomarkers reached the clinical focus, including microRNA-122, microRNA-192, cytokeratin analogues, glutamate dehydrogenase, total HMGB-1 (High Mobility Group Box), and hyperacetylated HMGB-1 proteins. However, the new parameters total HMGB-1, and even more so the acetylated HMGB-1, came under critical scientific fire after misconduct at one of the collaborating partner centers, leading the EMA to recommend no longer the exploratory hyperacetylated HMGB1 isoform biomarkers in clinical studies. The overall promising nature of the recommended biomarkers was considered by EMA as highly dependent on the outstanding results of the now incriminated biomarker hyperacetylated HMGB-1. The EMA therefore correctly decided to officially retract its Letter of Support affecting all biomarkers listed above. New biomarkers are now under heavy scrutiny that will require re-evaluations prior to newly adapted recommendations. With Integrin beta 3 (ITGB3), however, a new diagnostic biomarker may emerge, possibly being drug specific but tested in only 16 patients; due to substantial remaining uncertainties, final recommendations would be premature. In conclusion, most of the currently recommended new biomarkers have lost regulatory support due to scientific misconduct, requiring now innovative approaches and re-evaluation before they can be assimilated into clinical practice.

Formation of Structurally Ordered Nanoscale Complexes of DNA with Nuclear Proteins HMGB1 and H1

The structural organization of DNA in complex with linker histone H1 and non-histone chromosomal protein HMGB1 have been studied using atomic force microscopy and vibrational spectroscopy. The possibility of formation ordered nanoscale DNA-protein complexes is reported.

Effects of Shikonin on the Bone Microenvironment and Heterogeneity of Spinal Tumor Cells by Regulating the High Mobility Group Protein B1-Toll Like Receptor 4-Nuclear Factor Kappa-B Signaling Pathway

Aim: The present study was conducted with an attempt to evaluate the potential therapeutic effect of shikonin on spinal tumors in vivo and its mechanism. Methods: The morphology of rat spinal tumor was observed by H&amp;E staining, and apoptosis of rat spinal tumor was detected by transfection method and TUNEL method. In addition, the expression of cytokine mRNA and related proteins in spinal tumors of rats were detected by real-time PCR and Western blotting assays. Results: At the same period, the BBB score in the model group was significantly lower than that in the sham operation group and the shikonin group (P &lt; 0.05). Compared with the sham operation group, the BBB score in the shikonin group was significantly lower than that in the sham operation group (P &lt; 0.05), but significantly higher than that in the model group (P &lt; 0.05). Based on the RT-PCR results, the level of HMGB1 mRNA in the shikonin group was significantly lower than that of the model group (P &lt; 0.05), and the level of TLR4 mRNA in the model group was significantly lower than that in the model group (P &lt; 0.05). Moreover, the NF-κB mRNA level in the shikonin group was significantly lower than that in the model group (P &lt; 0.05). The expression level of HMGB1, TLR4 and NF- B in the shikonin group was significantly lower than that in the model group (P &lt; 0.05). Together, the results revealed that shikonin reduced the expression of HMGB1, TLR4 and NF-κB mRNA and protein. The apoptosis rate of tumor cells in the shikonin group was significantly higher than that in the model group (P &lt; 0.05). The expression of caspase-3 in the shikonin group was significantly higher than that in the model group, which further indicated that shikonin could promote apoptosis of spinal tumor cells in rats. Conclusion : Shikonin has the potential to affect the heterogeneity of the bone microenvironment and spinal tumor cells by regulating the HMGB1-TLR4-NF-κB signaling pathway.

HMGB1/TLR4 promotes apoptosis and reduces autophagy of hippocampal neurons in diabetes combined with OSA

AimsObstructive sleep apnea (OSA) combined with type 2 diabetes (T2DM) may lead to cognitive dysfunction. We previously reported that cognitive impairment is exacerbated in KKAy mice exposed to intermittent hypoxia (IH), during which the DNA binding protein HMGB1 mediates hippocampal neuronal apoptosis by maintaining microglia-associated neuroinflammation, but the underlying mechanism remains largely unknown. Materials and methodsWe performed immunofluorescence, Western blotting, and immunohistochemistry experiments in mouse hippocampal tissues and HT22 cells. KKAy type 2 diabetes model mice and normal C57BL/6J mice were exposed to IH or intermittent normoxia. HT22 cells were cultured in high glucose medium and exposed to IH or intermittent normoxia. We transfected HMGB1 siRNA into HT22 cells and then treated them with high glucose combined with intermittent hypoxia. Key findingsIn conclusion, IH aggravated apoptosis and autophagy defects in T2DM mice, and increased the protein expression of HMGB1 and TLR4. This was also confirmed in HG + IH-treated hippocampal HT22 cells. HMGB1 siRNA can significantly reduce the protein expression of HMGB1 and TLR4, reverse neuronal apoptosis and enhance autophagy. SignificanceWe believe that HMGB1 is a key factor in the regulation of hippocampal neuronal apoptosis and autophagy defects in T2DM combined with OSA. Targeting HMGB1/TLR4 signaling as a novel approach may delay or prevent the increased apoptosis and decreased autophagy induced by T2DM combined with OSA, and may ultimately improve cognitive dysfunction.

The pathogenicity, structural and functional exploration of human HMGB1 single nucleotide polymorphisms using in silico study

The human HMGB1 gene mutations have a major impact on several immune-related diseases and cancer. The detrimental effect of non-synonymous mutations of HMGB1 has not been investigated yet, hence the present study aims to examine single nucleotide polymorphisms and their implications on the structure-function of human HMGB1. The multifaceted HMGB1 protein acts as pleiotropic cytokine and regulates essential genes for coordinated cellular functions. The mutational effect on HMGB1 was analyzed by sequence-based homology methods, supervised learning methods, and structure-based methods. The study identified 58 non-synonymous mutations in human HMGB1, out of which only 2 mutations; R10T (rs61742222) and F103C (rs61733675) were classified as the SNPs with highest deleterious and disease-causing mutants. The effect of these mutations in structure of HMGB1 was scrutinized and the R10T mutant found to have a distinct structural behaviour in the B-box domain. In addition, R10T mutant predicted that it affects the MoRF function of HMGB1 and it could disrupt the DNA binding or/and protein partner interaction activity by HMGB1. F103C mutation takes place at the TLR binding and cytokine inducing region of HMGB1, hence it could affect the protein binding activity which involves in many cellular signaling. The study identified potent mutations R10T (a cancer-causing somatic mutation) and F103C (a novel mutation) and these mutations either directly or indirectly hinder DNA binding activity and TLR and cytokine binding of HMGB1. These findings will help in understanding the molecular basis of these promising mutations and functional role of human HMGB1 in cancer and immunological diseases.AbbreviationsAGERAdvanced glycosylation end product-specific receptorCXCLChemokine (C-X-C motif) liganddbSNPThe single nucleotide polymorphism databaseHMGB1High mobility group box 1LINCSLINear Constraint SolverMDSMolecular dynamics simulationMoRFMolecular recognition featuresNPTNumber of particle, Pressure and TemperatureNVTNumber of particle, Volume and TemperaturensSNPNon-synonymous SNPPBCPartial boundary conditionPCAPrincipal component analysisPMEPartial mesh EwaldRMSDRoot mean square deviationRMSFRoot mean square fluctuationSNPSingle nucleotide polymorphismSPCSingle-point chargeTLRToll-like receptorUTRUn-translated RegionCommunicated by Ramaswamy H. Sarma

HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

High-mobility group box (HMGB)1 and HMGB2 proteins are the subject of intensive research because of their involvement in DNA replication, repair, transcription, differentiation, proliferation, cell signaling, inflammation, and tumor migration. Using inducible, stably transfected human embryonic stem cells (hESCs) capable of the short hairpin RNA-mediated knockdown (KD) of HMGB1 and HMGB2, we provide evidence that deregulation of HMGB1 or HMGB2 expression in hESCs and their differentiated derivatives (neuroectodermal cells) results in distinct modulation of telomere homeostasis. Whereas HMGB1 enhances telomerase activity, HMGB2 acts as a negative regulator of telomerase activity in the cell. Stimulation of telomerase activity in the HMGB2-deficient cells may be related to activation of the PI3K/protein kinase B/ glycogen synthase kinase-3β/β-catenin signaling pathways by HMGB1, augmented TERT/telomerase RNA subunit transcription, and possibly also because of changes in telomeric repeat-containing RNA (TERRA) and TERRA-polyA+ transcription. The impact of HMGB1/2 KD on telomerase transcriptional regulation observed in neuroectodermal cells is partially masked in hESCs by their pluripotent state. Our findings on differential roles of HMGB1 and HMGB2 proteins in regulation of telomerase activity may suggest another possible outcome of HMGB1 targeting in cells, which is currently a promising approach aiming at increasing the anticancer activity of cytotoxic agents.-Kučírek, M., Bagherpoor, A. J., Jaroš, J., Hampl, A., Štros, M. HMGB2 is a negative regulator of telomerase activity in human embryonic stem and progenitor cells.

Bioinformatics analysis of the prognosis and biological significance of HMGB1, HMGB2, and HMGB3 in gastric cancer.

High mobility group box (HMGB) consists primarily of HMGB1, HMGB2, and HMGB3 proteins. Although abnormal HMGB expression is associated with various tumors, the relationship with gastric cancer (GC) remains unclear. In this study, HMGB1, HMGB2, and HMGB3 expression was analyzed using the Oncomine and TCGA databases. Correlations between HMGB1, HMGB2, and HMGB3 and clinicopathological factors were analyzed. cBioPortal was used to analyze HMGB1, HMGB2, and HMGB3 genetic alterations and its gene regulation network in GC tissue. HMGB1, HMGB2, and HMGB3 expression was higher in tumor tissues than in normal tissues, especially in GC. High HMGB1, HMGB2, and HMGB3 expression may predict a poor prognosis among patients with GC (hazard ratios [HR] = 1.90; 95% confidence interval [CI]: [1.30-2.78]) and human digestive system neoplasm (HR = 1.85; 95% CI [1.64-2.10]). These findings suggest that HMGB1, HMGB2, and HMGB3 may be useful prognostic indicators for patients with GC.

Sophocarpine Attenuates Chronic Constriction Sciatic Nerve Injury-induced Neuropathic Pain in Mice by Inhibiting the HMGB1/TLR4/NF-κB Signaling Pathway

Background: Sophocarpine (SC) is a major alkaloid extracted from Sophora alopecuroides L. Our previous study showed that SC has analgesic effects on neuropathic pain induced by chronic constriction injury (CCI). However, the exact analgesic mechanism of SC on neuropathic pain has not yet been elucidated. Objectives: The current study aimed to examine the anti-neuropathic pain effects of SC on the HMGB1/TLR4/NF-κB signaling pathway to explore its analgesic mechanism in neuropathic pain. Methods: In this experimental study, the neuropathic pain mouse model was established by CCI of the sciatic nerve in a university-affiliated animal lab, China, 2016. Mechanical withdrawal threshold (MWT), thermal withdrawal latency (TWL), cold withdrawal threshold (CWT), and tail-curling latency (TCL) were used to assess the antinociceptive effect of SC in neuropathic pain mice. The mRNA and protein expression levels of HMGB1, TLR4, NF-κB, p-NF-κB, TNF-α, and IL-6 in the spinal cord of neuropathic pain mice were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Results: Treatment with 40 and 20 mg/kg of SC was effective in increasing MWT, lengthening TWL, reducing CWT, and prolonging TCL in mice with neuropathic pain induced by CCI. Compared to the neuropathic-pain model group, treatment with 40 mg/kg of SC could effectively down-regulate HMGB1, TLR4, NF-κB, p-NF-κB, TNF-α, and IL-6 mRNA and protein expression levels in the spinal cord of mice with neuropathic pain induced by CCI. Conclusions: Our results showed that SC has analgesic effects on neuropathic pain induced by CCI, and its analgesic mechanism may be related to down-regulating the HMGB1/TLR4/NF-κB signaling pathway.

Insights into telomeric G-quadruplex DNA recognition by HMGB1 protein.

HMGB1 is a ubiquitous non-histone protein, which biological effects depend on its expression and subcellular location. Inside the nucleus, HMGB1 is engaged in many DNA events such as DNA repair, transcription and telomere maintenance. HMGB1 has been reported to bind preferentially to bent DNA as well as to noncanonical DNA structures like 4-way junctions and, more recently, to G-quadruplexes. These are four-stranded conformations of nucleic acids involved in important cellular processes, including telomere maintenance. In this frame, G-quadruplex recognition by specific proteins represents a key event to modulate physiological or pathological pathways. Herein, to get insights into the telomeric G-quadruplex DNA recognition by HMGB1, we performed detailed biophysical studies complemented with biological analyses. The obtained results provided information about the molecular determinants for the interaction and showed that the structural variability of human telomeric G-quadruplex DNA may have significant implications in HMGB1 recognition. The biological data identified HMGB1 as a telomere-associated protein in both telomerase-positive and -negative tumor cells and showed that HMGB1 gene silencing in such cells induces telomere DNA damage foci. Altogether, these findings provide a deeper understanding of telomeric G-quadruplex recognition by HMGB1 and suggest that this protein could actually represent a new target for cancer therapy.

Effects of budesonide on bronchopulmonary bysplasia and HMGB1/TLR4/NF-κB pathway in neonatal mice induced by hyperoxia

Objective To investigate the effects of budesonide (BUN) on high mobility group protein 1 (HMGB1)/toll-like receptor 4 (TLR4) / nuclear factor κB (NF-κB) pathway in neonatal mice with hyperoxia-induced bronchopulmonary dysplasia (BPD). Methods 80 mice were randomly divided into control group, model group, low, medium and high dose BUN groups, with 16 mice in each group. Mice in the control group were exposed to air, and mice in model group and BUN low, medium and high dose groups were exposed to 60% oxygen to establish BPD model; After 24 hours of modeling, the control group and model group were inhaled with normal saline every day. The low, middle and high dose BUN groups inhaled (1, 2, 4 ml) BUN, respectively, 12 h/times, and continued to inhale until the mice were executed. On the 7th and 14th days of the model establishment, hematoxylin-eosin (HE) staining was used to observe the morphological changes of lung tissue; the levels of interleukin (IL) -1β, IL-6, tumor necrosis factor-α (TNF-α), malondialdehyde (MDA) and superoxide dismutase (SOD) in lung tissues were detected by enzyme-linked immunosorbent assay (ELISA); the levels of HMGB1, TLR4 and NF-κB in lung tissue were detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) and protein immunoblotting (WB). Results In the model group, the lung tissue structure was severely damaged and the alveolar volume was enlarged to form bullae on the 7th and 14th day. In low, middle and high dose groups, with the increase of BUN concentration, the lung tissue deformation gradually improved and the alveolar structure gradually became intact. Compared with the control group, the number of radial alveoli in the lung tissues of the model group decreased at 7th and 14th days; SOD activity decreased; the expression levels of IL-1β, IL-6, TNF-α, MDA, HMGB1, TLR4 and NF-κB mRNA and protein were increased (P<0.05). Compared with the model group, the number of radial alveoli and SOD activity in lung tissues of BUN groups increased gradually at 7th and 14th days , while the levels of IL-1β, IL-6, TNF-α, MDA, HMGB1, TLR4 and NF-κB decreased gradually at different doses (P<0.05). Conclusions BUN may inhibit the levels of inflammatory factors and oxidative stress by regulating HMGB1/TLR4/NF-κB pathway to achieve the protective effect on BPD neonatal mice. Key words: Budesonide; Hyperoxia; Bronchopulmonary dysplasia; HMGB1 protein; Toll-like receptor 4; NF-kappa B; Mice

Exosomal and extracellular HMGB1 have opposite effects on SASH1 expression in rat astrocytes and glioma C6 cells

Exosomes are a type of extracellular vesicles derived from cells and mediators of intercellular communication. Different cell types have their own unique exosomes for exchanging information. We previously found that SASH1, a tumor suppressor, was lowly expressed or absent in glioma tissues and glioma C6 cells, but the structure and function of the corresponding exosomes had been unclear. Hence, we aimed to investigate whether exosomes generated from normal glial cells and glioma cells form different protein patterns and whether those derived from normal glial cells affect SASH1 expression in glioma cells. We collected exosomes from astrocytes and C6 cells and identified their exosomal proteins through mass spectrometry. We also performed gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) analyses, whose results showed that both the total and unique exosomal proteins from each cell type were similar. Moreover, the KEGG analysis revealed different clusters of unique exosomal proteins in glial cells and glioma cells. In the normal glial cells, the top clusters were mainly involved in processes with RNA transcripts and proteins, whereas in glioma cells the clusters were attributed to PI3K-Akt signaling, cell adhesion, and cancer-related pathways. Western blot analysis showed that HMGB1 exists in exosomes derived from cultured astrocytes, although its expression was higher in glioma C6 cells. Furthermore, we found that exosomes extracted from astrocytes could increase SASH1 expression in C6 cells (P = 0.040), whereas those derived from HMGB1-depleted astrocytes could not (P = 0.6133). The expression levels of SASH1 decreased after the addition of extracellular recombinant HMGB1 protein, whereas that of TLR4 increased. Our study is the first to demonstrate that HMGB1 plays different roles depending on its form: as an extracellular protein, HMGB1 decreases SASH1 expression, but as an exosomal protein, HMGB1 increases SASH1 expression. Nevertheless, the mechanism, which partly depends on the TLR4 pathway, behind these opposing effects requires further study. Our novel findings on the structure-dependent roles of the cytokine HMGB1 in promoting or inhibiting cancer provide a fresh insight into the interactions of cancer cells with the microenvironment.

High mobility group box 1 can be used to monitor perioperative course in patients with liver cancer

ObjectiveHigh mobility group box 1 (HMGB1) is produced by inflammation. Regarding liver injuries, HMGB1 is reportedly involved in liver regeneration. The present study investigated the use of HMGB1 as a postoperative marker of surgical course in patients with liver cancer. MethodsPatients were enrolled if they had liver cancer, had undergone liver surgery, and did not develop postsurgical complications. Patients who received emergency surgery or patients with unresectable cancerous lesions were excluded. Blood samples were preoperatively obtained as well as at 1 day, 1 week, and 4 weeks following surgery; white blood cell count, serum C-reactive protein, serum albumin, and serum HMGB1 levels were measured. ResultsA total of 36 patients were included in this study. HMGB1 levels significantly changed over time, increasing from a median of 7.1 ng/ml (preoperatively) to 13.9 ng/ml at 1 week postoperatively, and then decreased to 6.3 ng/ml at 4 weeks postoperatively. Peak HMGB1 levels were delayed, and elevated HMGB1 levels persisted as compared with the changes in conventional markers. ConclusionsHMGB1 indicates a unique perioperative inflammatory state in patients with liver cancer. Serum HMGB1 may serve as a marker for monitoring surgical course in patients undergoing surgery for liver cancer.

Ginsenoside Rb1 inhibits proliferation and promotes apoptosis by regulating HMGB1 in uterine fibroid cells

To study the effects of ginsenoside Rb1 and the molecular mechanisms on proliferation and apoptosis of uterine fibroid cells, Rb1 + pc DNA3.1, Rb1 + pc DNA3.1-HMGB1, si-NC or si-HMGB1 was transfected into uterine fibroid cells by liposome method; the inhibitory rate and proliferation of human uterine fibroid cells were detected by MTT assay; apoptosis of uterine fibroid cells was detected by flow cytometry assay; HMGB1 protein expression in uterine fibroid cells was detected by Western blot assay. Compared with untreated uterine fibroid cells, the inhibitory and apoptosis rate of uterine fibroid cells treated with Rb1 were significantly up-regulated, while the expression level of HMGB1 was significantly down-regulated (p < .05). HMGB1 knockdown inhibited proliferation and promoted apoptosis of uterine fibroid cells. HMGB1 overexpression reversed the inhibitory effect on proliferation and the promotion effect on apoptosis of Rb1 in uterine fibroid cells. Ginsenoside Rb1 could inhibit uterine fibroid cells proliferation and promote apoptosis. This mechanism might be directly related to the downregulation of HMGB1, providing a basis for the treatment of uterine fibroids with ginsenoside Rb1.

Bradykinin alleviates DR retinal endothelial injury by regulating HMGB-1/NF-κB pathway.

Diabetic retinopathy (DR) is one of the most important complications of diabetes (DM) and the leading cause of blindness in adults. Bradykinin (BK) is involved in several pathophysiological processes, such as inflammation, pain, cell proliferation, and tumors. It plays a crucial role in corneal epithelial cells, corneal stromal cells, and fibroblasts. However, the role of BK in DR retinal endothelial injury remains unclear. Human retinal microvascular endothelial cells (hRECs) were cultured in vitro and randomly divided into 3 groups, control group in which hRECs were cultured in normal glucose concentration, high glucose group in which hRECs were cultured in the presence of high glucose, and BK group in which hRECs were cultured in the presence of high glucose with 1 μM BK. The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) was used to detect cell proliferation. Caspase-3 activity was adopted to detect Caspase-3 activity in hRECs. The colorimetric method was selected to determine lactate dehydrogenase (LDH) activity, superoxide dismutase (SOD) activity, and ROS content. Western blot was used to test HMGB-1/NF-κB and vascular endothelial growth factor (VEGF) expression changes. Enzyme-linked immunosorbent assay (ELISA) was performed to detect the secretion of inflammatory factor tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β). In the presence of high glucose, hRECs cells proliferation was significantly reduced, Caspase-3 activity was enhanced, LDH and ROS levels were increased, SOD activity was declined with increased expression of HMGB-1, NF-κB, VEGF, as well as secretion of TNF-α and IL-1β compared with control group (p < 0.05). BK significantly inhibited the proliferation of hRECs cells, enhanced Caspase-3 activity, decreased the content of LDH and ROS, increased SOD activity, reduced the expressions of HMGB-1 and NF-κB protein, attenuated the expression of VEGF, and restrained the secretion of TNF-α and IL-1β compared with high glucose group (p < 0.05). BK can inhibit the growth and proliferation of retinal endothelial cells by regulating HMGB-1/NF-κB signaling pathway, attenuating oxidative stress and inflammation, thereby delaying DR development and progress.

HMGB1 promotes bone fracture healing through activation of ERK signaling pathway in a rat tibial fracture model.

This work was to investigate potential roles of HMGB1-mediated ERK pathway in the healing process of bone fracture. Rat tibial fracture models were established and divided into control (rats with normal saline), HMGB1 (rats with HMGB1), and HMGB1+ PD98059 groups (rats with HMGB1 and 1 mg/kg of ERK1/2 inhibitor PD98059) with 30 rats per each. The healing of rats' fracture was observed by X-ray films, the morphological changes of bone fractures by HE staining, the callus formation by micro-CT and biomechanical test, and the expression of osteogenesis-related genes, HMGB1 and ERK-related proteins by qRT-PCR and Western blot. Rats in the HMGB1 group was increased in X-ray scores, peak torque, torsional stiffness, and the bone volume fraction (bone volume/total volume, BV/TV); meanwhile, those rats presented elevations in osteogenesis-related genes and HMGB1 expressions, as well as p-ERK/ERK ratio. However, rats in the HMGB1+ PD98059 group was significantly reduced in X-ray score, peak torque, torsional stiffness, and BV/TV, as well as the expression of osteogenesis-related genes and the ratio of p-ERK/ERK, as compared to those from HMGB1 group. HMGB1 could promote the expressions of osteogenesis-related genes and accelerate the healing process of fracture via activation of the ERK signaling pathway.

PB1699 A SCREENING OF ANTINEOPLASTIC DRUGS FOR ACUTE MYELOID LEUKEMIA REVEALS THAT FLUDARABINE AND CYTARABINE HAVE WEAK CAPACITY AS INDUCERS OF IMMUNOGENIC CELL DEATH

Background:Although antineoplastic drugs used as induction chemotherapy for patients with acute myeloid leukemia (AML) result in high rate of complete remission, disease relapse occurs in the majority of cases, unless stem cell transplant is not performed. Development of new therapies is, then, a major medical need. Among them, strategies harnessing the immune system against leukemia are under active investigation. It is known that the cancer cell death induced by some antineoplastics drugs, such as anthracyclines, is highly immunogenic and results in the maturation of dendritic cells (DCs) and in the efficient cross‐priming of anti‐tumor T cells. This process, named immunogenic cell death (ICD), is characterized by Calreticulin (CRT) and Heat Shock Proteins 70 and 90 (HSP70/90) translocation to cell membrane and release of HMGB1 protein from nucleus.Aims:In this study, we were interested in in vitro comparison of five among the most widely used drugs for AML as ICD inducers.Methods:HL‐60 AML cell lines were treated with Daunorubicin (DNR), Cytarabine (Ara‐C), Etoposide (VP‐16), Fludarabine (Fluda) and Bortezomib. After apoptosis detection by flow cytometry, traslocation of CRT, HSPs70/90 and HMGB1 release from nucleus were evaluated using flow cytometry and immunofluorescence. Successively, the treated cells were used for loading into healthy donor‐derived DCs which were used to induce T cell proliferation as evaluated by flow cytometry using CFSE.Results:All five tested drugs induced a comparable levels of apoptosis. While Bortezomib and VP16 as well as DNR, which is a well‐known ICD inducer, were capable to induce CRT and HSP70/90 traslocation and HMGB1 release from nucleus, Fluda and Ara‐C have low, if any, capacity to induce ICD‐related events. Importantly, only DNR, Bortezomib and VP16 treatment, but not Ara‐C and Fluda, induced DC maturation, thus confirming that ICD‐related events correlate with immunogenic changes in DCs. At the functional level, VP16 was comparable to DNR at eliciting T‐cell proliferation via DCs, whereas Bortezomib, despite its capacity of inducing DC maturation via ICD, had low effect on T‐cell proliferation. Again, Ara‐C and Fluda had no effect on T‐cell proliferation.Summary/Conclusion:Our results indicate a novel and poorly‐investigated feature of antineoplastic drugs, commonly used for the treatment of AML, which is based on their different immunogenic potential. Under this viewpoint, Fluda and Ara‐C have weak, if any, capacity as inducers of immunogenic cell death, whereas VP16 and DNR are very effective. Further experiments, addressing the synergistic effects of these different drugs, may help to combine their conventional cytotoxic activity with their immunogenic capacity, thus providing the platform for a novel approach to anti‐leukemia chemotherapy.

Effect of sevoflurane preconditioning on HMGB1/TLR4/NF-κB signaling pathway during lung ischemia-reperfusion in rats

Objective To evaluate the effect of sevoflurane preconditioning on high-mobility group box 1 protein (HMGB1)/Toll-like receptor 4 (TLR4)/nuclear factor kappa B (NF-κB) signaling pathway during lung ischemia-reperfusion(I/R)in rats. Methods Thirty-six clean-grade healthy male Sprague-Dawley rats, aged 8-10 weeks, weighing 200-250 g, were divided into 3 groups (n=12 each) using a random number table method: sham operation group (group S), lung I/R group (group I/R) and sevoflurane preconditioning group (group SP). The right pulmonary hilum was only isolated but not ligated in group S. Lung I/R was induced by clamping the right pulmonary hilum for 60 min followed by 120 min of reperfusion in anesthetized rats in group I/R.In group SP, 2.1% sevoflurane was inhaled for 30 min to perform sevoflurane preconditioning, and the lung I/R model was established at 10 min after the end of inhalation.The rats were sacrificed at 120 min of reperfusion, and the lungs were removed for examination of the pathological changes which were scored and for determination of wet to dry weight ratio (W/D ratio), content of tumor necrosis factor-alpha (TNF-α) in lung tissues (by enzyme-linked immunosorbent assay) and expression of HMGB1, TLR4 and NF-κB protein in lung tissues (by Western blot). Results Compared with group S, the pathological scores, W/D ratio and content of TNF-α were significantly increased, and the expression of HMGB1, TLR4 and NF-κB was up-regulated in I/R and SP groups (P<0.05). Compared with group I/R, the pathological scores, W/D ratio and content of TNF-α were significantly decreased, and the expression of HMGB1, TLR4 and NF-κB was down-regulated (P<0.05), and the pathological changes of lung tissues were significantly attenuated in group SP. Conclusion Sevoflurane preconditioning reduces lung I/R injury probably through inhibiting HMGB1/TLR4/NF-κB signaling pathway in rats. Key words: Anesthetics, inhalation; Lung; Reperfusion injury; High mobility group proteins; Toll-like receptors; NF-kappaB