miR-449a inhibits cell proliferation, migration, and inflammation by regulating high-mobility group box protein 1 and forms a mutual inhibition loop with Yin Yang 1 in rheumatoid arthritis fibroblast-like synoviocytes.

BACKGROUND: We previously found that high-mobility group box protein 1 (HMGB1) promoted cell proliferation, migration, invasion, and autophagy in rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS), but little is known about its regulatory mechanism. The aim of this study was to investigate the regulatory mechanism of HMGB1 at the posttranscription level. METHODS: Real-time qPCR, CCK-8 cell proliferation assay, transwell cell migration assay, enzyme-linked immunosorbent assay (ELISA), and western blotting were used in this study. The targeting relationship between miRNA and mRNA was presented by the luciferase reporter assay. RESULTS: MiR-449a was downregulated in RA synovial tissue and inhibited RA-FLS proliferation, migration, and IL-6 production. MiR-449a directly targeted HMGB1 and inhibited its expression. Yin Yang 1(YY1) negatively regulated miR-449a expression and formed a mutual inhibition loop in RA-FLS. MiR-449a inhibited TNFα-mediated HMGB1 and YY1 overexpression and IL-6 production. CONCLUSIONS: Our results reveal the regulatory mechanism of HMGB1 in RA and demonstrate that miR-449a is a crucial molecule in RA pathogenesis and a suitable candidate for miRNA replacement therapies in RA.

Inhibitory effects of lupane­type triterpenoid saponins from the leaves of Acanthopanax gracilistylus on lipopolysaccharide-induced TNF­α, IL­1ß and high­mobility group box 1 release in macrophages.

Acanthopanax gracilistylus (AGS) has long been used in traditional Chinese medicine for the treatment of various inflammatory diseases. 3­O­ß­D­glucopyranosyl 3α, 11α­dihydroxylup­20(29)­en­28­oic acid, acantrifoside A, acankoreoside D, acankoreoside B and acankoreoside A are major lupane­type triterpenoid saponins derived from AGS. In the present study, these five saponins were isolated from AGS by chromatography and their anti­inflammatory activities were investigated in lipopolysaccharide (LPS)­treated RAW264.7 macrophages. Cell viability was evaluated by MTT assay. Tumor necrosis factor (TNF)­α, interleukin (IL)­1ß and NF­κB p65 were measured by ELISA. The gene expression levels of TNF­α and IL­1ß was detected by reverse­transcription polymerase chain reaction. And high­mobility group box 1 (HMGB1) were analyzed by western blotting. The results demonstrated that these five saponins significantly suppressed LPS­induced expression of TNF­α and IL­1ß at the mRNA and protein level in RAW264.7 cells. Further analysis revealed that acankoreoside A and acankoreoside B were able to reduce the secretion of HMGB1 and NF­κB activity induced by LPS in RAW264.7 macrophages. Taken together, these results suggested that the anti­inflammatory activity of AGS­derived saponins may be associated with the downregulation of TNF­α and IL­1ß, and the 'late­phase' proinflammatory cytokine HMGB1, via negative regulation of the NF­κB pathway in RAW264.7 cells.

Targeting oxidative stress improves disease outcomes in a rat model of acquired epilepsy.

Epilepsy therapy is based on antiseizure drugs that treat the symptom, seizures, rather than the disease and are ineffective in up to 30% of patients. There are no treatments for modifying the disease-preventing seizure onset, reducing severity or improving prognosis. Among the potential molecular targets for attaining these unmet therapeutic needs, we focused on oxidative stress since it is a pathophysiological process commonly occurring in experimental epileptogenesis and observed in human epilepsy. Using a rat model of acquired epilepsy induced by electrical status epilepticus, we show that oxidative stress occurs in both neurons and astrocytes during epileptogenesis, as assessed by measuring biochemical and histological markers. This evidence was validated in the hippocampus of humans who died following status epilepticus. Oxidative stress was reduced in animals undergoing epileptogenesis by a transient treatment with N-acetylcysteine and sulforaphane, which act to increase glutathione levels through complementary mechanisms. These antioxidant drugs are already used in humans for other therapeutic indications. This drug combination transiently administered for 2 weeks during epileptogenesis inhibited oxidative stress more efficiently than either drug alone. The drug combination significantly delayed the onset of epilepsy, blocked disease progression between 2 and 5 months post-status epilepticus and drastically reduced the frequency of spontaneous seizures measured at 5 months without modifying the average seizure duration or the incidence of epilepsy in animals. Treatment also decreased hippocampal neuron loss and rescued cognitive deficits. Oxidative stress during epileptogenesis was associated with de novo brain and blood generation of disulfide high mobility group box 1 (HMGB1), a neuroinflammatory molecule implicated in seizure mechanisms. Drug-induced reduction of oxidative stress prevented disulfide HMGB1 generation, thus highlighting a potential novel mechanism contributing to therapeutic effects. Our data show that targeting oxidative stress with clinically used drugs for a limited time window starting early after injury significantly improves long-term disease outcomes. This intervention may be considered for patients exposed to potential epileptogenic insults.

4'-O-ß-D-glucosyl-5-O-methylvisamminol, an active ingredient of Saposhnikovia divaricata, attenuates high-mobility group box 1 and subarachnoid hemorrhage-induced vasospasm in a rat model.

BACKGROUND: High-mobility group box 1 (HMGB1) was observed to be an important extracellular mediator involved in vascular inflammation associated with subarachnoid hemorrhage (SAH). This study is of interest to examine the efficacy of 4'-O-ß-D-glucosyl-5-O-methylvisamminol (4OGOMV), C22H28O10, on the alternation of cytokines and HMGB1 in an animal model. METHODS: A rodent double hemorrhage SAH model was employed. Administration with 4OGOMV was initiated 1 h after animals were subjected to SAH. Basilar arteries (BAs) were harvested and cortexes examined for HMGB1 mRNA, protein expression (Western blot) and monocyte chemoattractant protein-1 (MCP-1) immunostaining. Cerebrospinal fluid samples were collected to examine IL-1ß, IL-6, IL-8 and MCP-1 (rt-PCR). RESULTS: Morphological findings revealed endothelial cell deformity, intravascular elastic lamina torture, and smooth muscle necrosis in the vessels of SAH groups. Correspondently, IL-1ß, IL-6 and MCP-1 in the SAH-only and SAH-plus vehicle groups was also elevated. 4OGOMV dose-dependently reduced HMGB1 protein expression when compared with the SAH groups.(p < 0.01) Likewise, 400 µg/kg 4OGOMV reduced IL-1ß, MCP-1 and HMGB1 mRNA levels as well as MCP-1(+) monocytes when compared with the SAH groups.. CONCLUSION: 4OGOMV exerts its neuro-protective effect partly through the dual effect of inhibiting IL-6 and MCP-1 activation and also reduced HMGB1 protein, mRNA and MCP-1(+) leukocytes translocation. This study lends credence to validating 4OGOMV as able to attenuate pro-inflammatory cytokine mRNA, late-onset inflammasome, and cellular basis in SAH-induced vasospasm.

Tanshinone IIA Attenuates Chronic Pancreatitis-Induced Pain in Rats via Downregulation of HMGB1 and TRL4 Expression in the Spinal Cord.

BACKGROUND: Chronic pancreatitis (CP) is a long-standing inflammation of the exocrine pancreas, which typically results in severe and constant abdominal pain. Previous studies on the mechanisms underlying CP-induced pain have primarily focused on the peripheral nociceptive system. A role for a central mechanism in the mediation or modulation of abdominal pain is largely unknown. Tanshinone IIA (TSN IIA), an active component of the traditional Chinese medicine Danshen, exhibits anti-inflammatory properties via downregulation of the expression of high-mobility group protein B1 (HMGB1), a late proinflammatory cytokine. HMGB1 binds and activates toll-like receptor 4 (TLR4) to induce spinal astrocyte activation and proinflammatory cytokine release in neuropathic pain. OBJECTIVE: In this study, we investigated the effect of TSN IIA on pain responses in rats with trinitrobenzene sulfonic acid (TNBS)-induced CP. The roles of central mechanisms in the mediation or modulation of CP were also investigated. STUDY DESIGN: A randomized, double-blind, placebo-controlled animal trial. METHODS: CP was induced in rats by intrapancreatic infusion of trinitrobenzene sulfonic acid (TNBS). Pancreatic histopathological changes were characterized with semi-quantitative scores. The abdomen nociceptive behaviors were assessed with von Frey filaments. The effects of intraperitoneally administered TSN IIA on CP-induced mechanical allodynia were tested. The spinal protein expression of HMGB1 was determined by western blot. The spinal mRNA and protein expression of proinflammatory cytokines IL-1ß, TNF-α, and IL-6 were determined by RT-PCR and western blot, respectively. The spinal expression of the HMGB1 receptor TRL4 and the astrocyte activation marker glial fibrillary acidic protein (GFAP) were determined by western blot or immunohistological staining after intraperitoneal injection of TSN IIA or intrathecal administration of a neutralizing anti-HMGB1 antibody. RESULTS: TNBS infusion resulted in pancreatic histopathological changes of chronic pancreatitis and mechanical allodynia in rats. TSN IIA significantly attenuated TNBS-induced mechanical allodynia in a dose-dependent manner. TNBS significantly increased the spinal expression of HMGB1 and proinflammatory cytokines IL-1ß, TNF-α, and IL-6. These TNBS-induced changes were significantly inhibited by TSN IIA in a dose-dependent manner. Furthermore, TSN IIA, but not the neutralizing anti-HMGB1 antibody, significantly inhibited TNBS-induced spinal TLR4 and GFAP expression. LIMITATIONS: In addition to TLR4, HMGB1 can also bind to toll-like receptor-2 (TLR2) and the receptor for advanced glycation end products (RAGE). Additional studies are warranted to ascertain whether HMGB1 contributes to CP-induced pain through activation of these receptors. CONCLUSIONS: Our results suggest that spinal HMGB1 contributes to the development of CP-induced pain and can potentially be a therapeutic target. TSN IIA attenuates CP-induced pain via downregulation of spinal HMGB1 and TRL4 expression. Therefore, TSN IIA may be a potential anti-nociceptive drug for the treatment of CP-induced pain.

Protective effects of tanshinone IIA on myocardial ischemia reperfusion injury by reducing oxidative stress, HMGB1 expression, and inflammatory reaction.

CONTEXT: Although there were reports on the protective functions of tanshinone IIA (TSA) on rat myocardial ischemia, the exerting mechanism has not been completely clarified. OBJECTIVE: An attempt was made to further verify the protective effect of TSA on myocardial ischemia reperfusion injury and elucidate its underlying mechanism. MATERIALS AND METHODS: The rats were given TSA (10, 20, and 40 mg/kg bw per day) in intraperitoneal injection for 15 d. Rami anterior descending branch of coronary artery was ligated for 30 min and then re-perfused for 120 min to establish a reperfusion model. Effects of TSA on the infarct area, creatine kinase (CK), aspartate aminotransferase (AST), high mobility group box B1 protein (HMGB1), and inflammation and oxidation were investigated. RESULTS: Compared with those in the IR group, infarct size percentages of rats' myocardium in L-TSA, M-TSA, and H-TSA groups were reduced by 1.21, 4.26, and 12.50%, respectively, CK activities by 7.4, 11.2, and 12.5%, respectively, and AST activities also declined (p < 0.05). Furthermore, compared with those in the IR group, SOD and GSH-Px activities increased, and MDA, TNF-α, IL-6, and iNOS levels decreased in L-TSA, M-TSA, and H-TSA groups (p < 0.05). Meanwhile, compared with those in the IR group, HMGB1 expressions in L-TSA, M-TSA, and H-TSA groups were lowered by 21.9, 32.4, and 35.6%, respectively. DISCUSSION AND CONCLUSION: The protective function of TSA on myocardial ischemia reperfusion injury may be possibly exerted by inhibiting the increase of ROS caused by the reperfusion to attenuate the expression of HMGB1 and inhibit inflammation.

Anti-inflammatory effects of hyperoside in human endothelial cells and in mice.

High-mobility group box 1 (HMGB1) was recently shown to be an important extracellular mediator of systemic inflammation, and endothelial cell protein C receptor (EPCR) has been shown to be involved in vascular inflammation. Hyperoside is an active compound isolated from Rhododendron brachycarpum G. Don (Ericaceae) that was reported to have anti-oxidant, anti-hyperglycemic, anti-cancer, and anti-coagulant activities. Here, we show, for the first time, the anti-septic effects of hyperoside in HMGB1-mediated inflammatory responses and on the shedding of EPCR in vitro and in vivo. The data showed that hyperoside posttreatment suppressed lipopolysaccharide (LPS)-mediated release of HMGB1 and HMGB1-mediated cytoskeletal rearrangement. Hyperoside also inhibited HMGB1-mediated hyperpermeability and leukocyte migration in septic mice and phorbol-12-myristate 13-acetate (PMA) of cecal ligation and puncture (CLP)-induced EPCR shedding. In addition, hyperoside inhibited the production of tumor necrosis factor-α (TNF-α) and the HMGB1-mediated activation of Akt, nuclear factor-κB (NF-κB), and extracellular regulated kinase (ERK) 1/2 in HUVECs. Hyperoside also reduced the CLP-induced release of HMGB1, the production of interleukin (IL)-1ß, and septic mortality. Collectively, these results suggest hyperoside as a candidate therapeutic agent for the treatment of vascular inflammatory diseases via inhibition of the HMGB1 signaling pathway.

The effects of glycyrrhizin on acute pancreatitis in mice.

OBJECTIVE: This study aimed to investigate the effects of glycyrrhizin on acute pancreatitis in mice. MATERIALS AND METHODS: Sixty Balb/c mice were randomly divided into four groups as control group, model group, low dose group and high dose group (n=15). Acute pancreatitis was induced by intraperitoneal injection of Caerulein (100 µg/kg) hourly for 6 times. Mice in low dose group and high dose group received intraperitoneal administration of 15 mg/kg and 45 mg/kg glycyrrhizin respectively 4 hours before Caerulein injection. Mice in four groups were sacrificed in three equal lots at 8, 16 and 24 hours after model construction. High Mobility Group Box-1 (HMGB1) expression and serum levels of amylase, TNF-α and IL-6 were determined. The pancreatic tissues were taken for histopathologic analysis. RESULTS: Amylase, TNF-α, IL-6 and HMGB1 levels were significantly higher and pancreas lesion was severer in model group than in control group. However, Amylase, TNF-α, IL-6 and HMGB1 levels in low dose group and high dose group decreased significantly compared with model group. The pancreas lesion was also improved after administration of glycyrrhizin. CONCLUSIONS: Glycyrrhizin could decrease the levels of pro-inflammatory cytokines and downregulate the expression of HMGB1 which finally improved the pancreas lesion in mice with acute pancreatitis.

Chemotherapy-induced immunogenic modulation of tumor cells enhances killing by cytotoxic T lymphocytes and is distinct from immunogenic cell death.

Certain chemotherapeutic regimens trigger cancer cell death while inducing dendritic cell maturation and subsequent immune responses. However, chemotherapy-induced immunogenic cell death (ICD) has thus far been restricted to select agents. In contrast, several chemotherapeutic drugs modulate antitumor immune responses, despite not inducing classic ICD. In addition, in many cases tumor cells do not die after treatment. Here, using docetaxel, one of the most widely used cancer chemotherapeutic agents, as a model, we examined phenotypic and functional consequences of tumor cells that do not die from ICD. Docetaxel treatment of tumor cells did not induce ATP or high-mobility group box 1 (HMGB1) secretion, or cell death. However, calreticulin (CRT) exposure was observed in all cell lines examined after chemotherapy treatment. Killing by carcinoembryonic antigen (CEA), MUC-1, or PSA-specific CD8(+) CTLs was significantly enhanced after docetaxel treatment. This killing was associated with increases in components of antigen-processing machinery, and mediated largely by CRT membrane translocation, as determined by functional knockdown of CRT, PERK, or CRT-blocking peptide. A docetaxel-resistant cell line was selected (MDR-1(+), CD133(+)) by continuous exposure to docetaxel. These cells, while resistant to direct cytostatic effects of docetaxel, were not resistant to the chemomodulatory effects that resulted in enhancement of CTL killing. Here, we provide an operational definition of "immunogenic modulation," where exposure of tumor cells to nonlethal/sublethal doses of chemotherapy alters tumor phenotype to render the tumor more sensitive to CTL killing. These observations are distinct and complementary to ICD and highlight a mechanism whereby chemotherapy can be used in combination with immunotherapy.

Sivelestat improves outcome of crush injury by inhibiting high-mobility group box 1 in rats.

Severe crush injury is associated with high mortality because of resulting hyperkalemia in early phase and multiorgan dysfunction in later phase. In this study, we investigated the effects of sivelestat administration 1 h before reperfusion on the outcome of crush injury. Crush injury was induced by 6 h of direct compression to both hindlimbs of anesthetized rats with blocks weighing 3.5 kg each side, followed by 3 h of reperfusion. Rats were randomly assigned to three groups. In the control group, rats were infused with normal saline at 1 mL/kg per hour throughout the experiment without compression. Rats in the positive control group were compressed for 6 h, followed by fluid resuscitation initiated 1 h before release with normal saline. The infusion rate was increased from 1 to 10 mL/kg per hour and continued for 4 h. Rats in the treated group underwent the same procedures as in the positive control group, but sivelestat was added to normal saline (concentration was adjusted to infuse 10 mg/kg per hour) during fluid resuscitation (for 4 h). Treatment with sivelestat significantly improved survival rate with P = 0.032. This was accompanied by lower serum high-mobility group box 1 (HMGB1) levels after 3-h reperfusion, attenuated lung injury (assessed using hematoxylin-eosin stain), and suppression of HMGB1 expression in the lung and the liver. These results suggest that treatment with sivelestat improves the outcome of crush injury, likely by inhibiting HMGB1 in rats.

Cardioprotection of Asperosaponin X on experimental myocardial ischemia injury.

BACKGROUND: Asperosaponin X was isolated from the roots of Dipsacus asper. In this study, we investigated the anti-myocardial ischemia and reperfusion (I/R) injury effects of Asperosaponin X in vivo and elucidated the potential mechanism in vitro. RESULTS: Asperosaponin X significantly attenuated hypoxia-induced cytotoxicity in a concentration-dependent manner in H9c2 cells. Treatment of H9c2 cells with Asperosaponin X 5 µM or 10 µM blocked TNF-α-induced nuclear factor kappaB (NF-κB) phosphorylation by blocking HMGB1 expression. Treatment of rats with Asperosaponin X 10mg/kg, (i.v.) protected the animals from myocardial I/R injury as indicated by a decrease in infarct volume, improvement in hemodynamics and reduction of myocardial damage severity. Treatment with Asperosaponin X also lowered serum levels of pro-inflammatory factors and reduced High mobility group box-1 protein (HMGB1), phosphorylated IκB-α and NF-κB expression in ischemic myocardial tissue. Additionally, continuous i.v. of Asperosaponin X 14 days attenuated cardiac remodeling. CONCLUSIONS: These protective effects suggested that Asperosaponin X may be due to block of myocardial inflammatory cascades through an HMGB1-dependent NF-κB signaling pathway.

Hyperthermia in combination with X-irradiation induces inflammatory forms of cell death.

Autoimmune diseases and cancer can be treated by influencing the immune system. Apo and nec cells are strong modulators of the immune system contributing to anti-inflammatory and pro-inflammatory responses, respectively. We examined which form of cell death was induced by HT and X-irradiation. Nec was the prominent form of cell death after combined treatment and the amount of dead cells was higher when exposing the cells to radiation before HT. Combined applications further led to an increased percentage of cells in a more radioresponsive G2 cell cycle phase. The danger signal HMGB1 is released when combining HT with radiation, a further hint that those treatments may induce inflammation and immune activation. We conclude that immune responses are appropriately adapted to the damage that has occurred and may contribute to anti-cancer immunity or chronic autoimmunity, respectively.

[Effect of Xuebijing injection on renal high mobility group box-1 protein expression and acute kidney injury in rats after scald injury].

OBJECTIVE: To investigate the change in renal high mobility group box-1 protein (HMGB1) levels, and the effect of Chinese traditional medicine-Xuebijing injection on HMGB1 expression as well as acute kidney injury in rats after scald injury. METHODS: Wistar rats were subjected to 30% full-thickness scald injury followed with delayed resuscitation. Totally 78 animals were divided into sham scald group (n=18), scald injury group (n=30), and Xuebijing injection treatment group (n=30). All animals were sacrificed at 8, 24, and 72 hours postburn. Renal tissue and blood samples were harvested to determine HMGB1 mRNA as well as protein expression and organ functional parameters. HMGB1 mRNA level was semi-quantitatively measured by the reverse transcription polymerase chain reaction taking GAPDH as an internal standard, and protein expressions of HMGB1 were detected by both Western blot and immunohistochemistry. Serum creatinine (Cr) contents were measured by automatic biochemistry analyzer. In addition, pathological lesions in kidney were observed under light microscope using HE staining. RESULTS: Compared with sham scald group, both mRNA and protein expressions of HMGB1 were significantly enhanced in the kidney at 8, 24, and 72 hours after scald injury (P<0.05, P<0.01), meanwhile serum Cr contents were markedly increased following acute insults (P<0.05, P<0.01). Treatment with Xuebijing injection could markedly down-regulated renal HMGB1 mRNA expression and protein release at 24 hours and 72 hours (P<0.05, P<0.01), and significantly reduced serum Cr content following scald injury (P<0.05). Many inflammatory cells in renal tissues were observed using light microscope following scald. The histological morphology of kidney lesions was a-HMGB1, a late mediator, appears to be inmeliorated after treatment with Xuebijing injection. CONCLUSIONS: volved in the pathogenesis of excessive inflammatory response and acute kidney damage. Treatment with Xuebijing injection can inhibit HMGB1 synthesis and release in renal tissues, and may prevent the development of acute kidney injury induced by serious scald injury.

Receptor for advanced glycation end products plays a more important role in cellular survival than in neurite outgrowth during retinoic acid-induced differentiation of neuroblastoma cells.

The receptor for advanced glycation end products (RAGE), a member of the immunoglobulin superfamily, is known to interact with amphoterin. This interaction has been proposed to play a role in neurite outgrowth and process elongation during neurodifferentiation. However, there is as yet no direct evidence of the relevance of this pathway to neurodifferentiation under physiological conditions. In this study we have investigated a possible role of RAGE and amphoterin in the retinoic acid-induced differentiation of neuroblastoma cells. The functional inactivation of RAGE by dominant negative and antisense strategies showed that RAGE is not required for process outgrowth or differentiation, although overexpression of RAGE accelerates the elongation of neuritic processes. Using the antisense strategy, amphoterin was shown to be essential for process outgrowth and differentiation, suggesting that amphoterin may interact with other molecules to exert its effect in this context. Interestingly, the survival of the neuroblastoma cells treated with retinoic acid was partly dependent on the expression of RAGE, and inhibition of RAGE function partially blocked the increase in anti-apoptotic protein Bcl-2 following retinoic acid treatment. Based on these results we propose that a combination therapy using RAGE blockers and retinoic acid may prove as a useful approach for chemotherapy for the treatment of neuroblastoma.