HMGB1 Protein Expression Research Articles

Different effects of arginine vasopressin on high-mobility group box 1 expression in astrocytes isolated from stroke-prone spontaneously hypertensive rats and congenic SHRpch1_18 rats.

Stroke-prone spontaneously hypertensive rats (SHRSP/Izm) develop severe hypertension and astrocytic oedema following ischaemic stimulation. During ischaemic stress high-mobility group box 1 (Hmgb1) expression in astrocytes is induced, and subsequently potentiates deterioration of the brain due to ischaemic injury, which manifests as both cerebral inflammation and astrocytic oedema. Arginine vasopressin (AVP) induces brain injury and increases astrocytic swelling. After stroke, Hmgb1 and peroxiredoxin (Prx) are released at different times and activate macrophages in the brain via Toll-like receptors (Tlr2s). The purpose of this study was to examine whether AVP and/or hypoxia and reoxygenation (H/R) contribute to Hmgb1 regulation following ischaemic stroke. Thus, Hmgb1, Prx2 and Tlr2 expression levels in astrocytes isolated from Wistar Kyoto rats (WKY/Izm), spontaneously hypertensive rats (SHR/Izm), SHRSP/Izm and congenic rat strain SHRpch1_18 treated with AVP and/or H/R were compared. Gene and protein expression levels were determined by reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR, and Western blot. mRNA expression of Hmgb1, Prx2 and Tlr2 induced by AVP was dose-dependent, and Hmgb1 and Prx2 expression was higher in SHR/Izm, SHRSP/Izm and SHRch1_18 than in WKY/Izm. Tlr2 expression with AVP was reduced in SHR/Izm compared to WKY/Izm. In SHRpch1_18, Hmgb1 expression increased after AVP plus H/R. AVP-modulated expression of Hmgb1 protein was reduced by the addition of the antioxidant N-acetylcysteine (NAC). These results suggest that oxidative stress by AVP enhanced expression of Hmgb1, Prx2 and Tlr2 in astrocytes. We hypothesize that regulation of Hmgb1 by AVP during H/R might be related to induction of inflammation and stroke in SHRSP/Izm and SHRpch1_18 rats.

Regulation of autophagy by systemic admission of microRNA-141 to target HMGB1 in l-arginine-induced acute pancreatitis invivo.

MicroRNAs are endogenous, non-coding RNAs of approximately 20-22 nucleotides that regulate gene expression by binding to the 3' untranslated region (UTR) of target mRNAs and can be applied in gene therapy. Acute pancreatitis is an inflammatory pancreatic disease that carries considerable morbidity and mortality. The purpose of this study was to explore the therapeutic potential of microRNA-141 (miR-141) for acute pancreatitis invivo. AP was induced by two hourly intra-peritoneal (i.p.) injections of l-arginine (200mg×2/100g.BW). Control mice received normal saline injections. In a separate group, normal saline, empty adenoviral vector and miR-141 adenoviral vector were given to the mice via tail vein hydrodynamically at 72h before the first l-arginine injection. All the mice were euthanized at 24h after the last l-arginine injection, and the pancreatic tissues were assessed by qRT-PCR, Western blotting and electron microscopy. miR-141 directly inhibited HMGB1 expression in mouse hepal-6 cell. Furthermore, systemic administration of the miR-141 knock-downthe expression of HMGB1 protein and further antagonized the downstream protein Beclin-1, leading to the reduction of autophagosomes and autolysosomes, blockade of the LC3-II level and the increased levels of p62 protein after injection of l-arginine. In addition, the level of Lamp-2 was not significantly different. For the first time miR-141 was applied in acute pancreatitis treatment invivo. Inhibition of HMGB1 by miR-141 may block the process of autophagosome formation through the HMGB1/Beclin-1 pathway. The miR-141 appears to be a promising candidate for the gene therapy of acute pancreatitis.

Betulin attenuates kidney injury in septic rats through inhibiting TLR4/NF-κB signaling pathway.

This study aims to evaluate the renoprotective effect of betulin and the possible mechanisms in septic rats. In the animal study, rats underwent cecal ligation and puncture (CLP) were used as septic models. Betulin 4 mg/kg or 8 mg/kg was administrated intraperitoneally immediately after the model establishment. In the cell study, lipopolysaccharide (LPS) stimulated rat HBZY-1 mesangial cells were used. Cells were pretreated with betulin before the LPS stimulation. Histological change of kidney was examined using periodic acid-schiff staining. Serum creatinine and blood urea nitrogen were detected using biochemical assay kits, and proinflammatory factors in kidney and culture medium were detected using ELISA kits. Immunohistochemistry analysis was used to measure F4/80, HMGB-1 and TLR4 expression in renal tissue of rats. mRNA and protein expressions of HMGB-1, TLR4, IκBα, p65 and p-p65 were measured using Real-time PCR and Western blot, respectively. Nuclear factor kappa-B (NF-κB) nuclear translocation was observed using immunofluorescence staining. Results showed that betulin attenuated CLP-induced renal damage, reduced levels of serum creatinine and blood urea nitrogen, and decreased the proinflammatory cytokines secretion in the kidney of septic rats and culture medium of LPS-stimulated cells. Betulin could also downregulated HMGB-1 and TLR4 mRNA and protein expression in the kidney of septic rats, as well as inhibited NF-κB signal activation. Our finding suggests that betulin attenuates kidney injury in septic rats through anti-inflammation and TLR4/NF-κB signaling inhibition.

Significance of P53 and high mobility group box 1 protein in different levels of liver fibrosis in chronic hepatitis B

To investigate the role of P53 and high mobility group protein 1 (HMGB1) protein expression in liver fibrosis stages in chronic hepatitis B patients. According to the pathological grades, 103 patients were divided into 3 groups: no fibrosis group (n=18), low fibrosis group (n=49) or high fibrosis group (n=36). Serum HMGB1 levels were determined and receiver operating characteristic (ROC) curve was made based on the HMGB1 level and liver fibrosis score. Liver fibrosis model was developed by CCl4 in 60 male SD rats, which were sacrificed 6 or 12 weeks later. The degree of fibrosis was examined by Masson staining; HMGB1 and P53 protein expression were analyzed by Western blot; histone deacetylase (HDAC) activity, TNF-α, IL-1β and IL-6 levels in serum were measured. The serum levels of HMGB1 level in low and high fibrosis groups were significantly higher than that in no fibrosis group (P<0.01, respectively). ROC curve showed that serum HMGB1 in the diagnosis of hepatic fibrosis with cut off at 74 pg/mL, specificity at 65% and sensitivity at 87%. Compared with the control group, HMGB1 expression in both low and high fibrosis group was decreased in nucleus but was increased in cytoplasm, accompanied by the elevated P53 expression, increased HDAC activity and inflammatory cytokine levels (all P<0.01, respectively). P53 and HMGB1 expression was significantly increased in chronic hepatitis B patients with liver fibrosis; serum HMGB1 level was positively correlated with the degree of liver cirrhosis and HMGB1 could be used as a sensitive and specific index for liver fibrosis prognosis.

High mobility group B1 and N1 (HMGB1 and HMGN1) are associated with tumor-infiltrating lymphocytes in HER2-positive breast cancers.

Although the prognostic and predictive significance of tumor-infiltrating lymphocytes (TILs) in HER2-positive breast cancers has been established, the drivers of TIL influx remain unclear. We tested whether potential triggers for this response could include high mobility group B1 and N1 (HMGB1 and HMGN1) proteins, which are immunogenic damage-associated molecular pattern molecules. We evaluated TILs and the immunohistochemical expression of HMGB1 and HMGN1 in 447 HER2-positive breast cancer tissues. Normal luminal cells exhibited nuclear expression of HMGB1 and HMBN1. The nuclear and cytoplasmic expression levels of HMG proteins showed a significant inverse correlation (rho=-0.150, p=0.001 for HMGB1; rho=-0.247, p<0.001 for HMGN1). Low levels of HMGB1 and HMGN1 nuclear expression were identified in 185 (41.4%) and 208 (46.5%) cases, respectively. High levels of cytoplasmic HMGB1 and HMGN1 expression were identified in 107 (23.9%) and 49 (11.0%) cases, respectively. High cytoplasmic expression of HMG proteins was significantly associated with a high histological grade, high levels of TILs, peritumoral lymphocytic infiltration, and tertiary lymphoid structures in HER2-positive breast cancer tissues. Tumors with low levels of cytoplasmic HMGB1 and HMGN1 showed significantly lower levels of TILs than those with high levels of each or both HMG proteins. However, the nuclear or cytoplasmic expression of either HMG protein was not found to be significantly associated with survival. High levels of cytoplasmic HMGB1 and HMGN1 protein expression correlated with high levels of TILs in HER2-positive breast cancers. The manipulation of HMGB1 and HMGN1 could represent an immunotherapeutic approach to promote TIL influx into a tumor.

Rhinacanthin-C, A Fat-Soluble Extract from Rhinacanthus nasutus, Modulates High-Mobility Group Box 1-Related Neuro-Inflammation and Subarachnoid Hemorrhage-Induced Brain Apoptosis in a Rat Model

High-mobility group box 1 (HMGB1) was shown to be a major extracellular mediator involved in relayed neuro-inflammation in animals after subarachnoid hemorrhage (SAH). It is of interest to examine the effect of rhinacanthin-C (RCT-C, C25H30O5) on pro-inflammatory cytokines/HMGB1 in an SAH-related early brain injury model. A rodent double SAH model was used. RCT-C was administered orally at 100, 200, and 400 μmol/kg/day. Cerebral spinal fluid samples were obtained to assess interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor α using a real-time polymerase chain reaction. Basilar arteries were harvested and cerebral cortex was examined for HMGB1 mRNA and protein expression (western blot) and caspases (real-time polymerase chain reaction). An intrathecal injection of 1 ng of HMGB-1 recombinant protein was given in the 400 μmol/kg/day RCT-C plus SAH groups. The levels of IL-1β, IL-6, and tumor necrosis factor α mRNA were significantly increased in animals subject to SAH, compared with the healthy controls, but were absent in the RCT-C groups. Cleaved caspase-9a as well as HMGB-1 mRNA and protein were significantly reduced in the 400 μmol/kg/day RCT-C treatment groups. Similarly, administration of RCT-C reduced HMGB-1 mRNA and protein expression (P <0.01). RCT-C exerts a neuroprotective effect by reducing cleaved caspase-3- and caspase-9a-related apoptosis. Decreased HMGB-1 mRNA and protein expression in the RCT-C groups corresponds to its anti-inflammatory effect. HMGB-1 recombinant protein administration impaired the neuroprotective and immunosuppressive effect of RCT-C. This finding lends credence that RCT-C modulates the HMGB-1-related pathway and attenuates brain apoptosis in the pathogenesis of SAH.

Tanshinone IIA Attenuates Chronic PancreatitisInduced 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-1β, 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-1β, 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 CPinduced 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. Key words: Chronic pancreatitis, HMGB1, proinflammatory cytokine, Tanshinone IIA, spinal cord, astrocyte, TLR4

Correlation of High Mobility Group Box-1 Protein (HMGB1) with Clinicopathological Parameters in Primary Retinoblastoma.

HMGB1 is considered to be DNA chaperone as it binds without any specificity. It is the structural protein which alters nuclear homeostasis and genomic stability of chromatin. Its role in retinoblastoma (Rb) remains unclear. The aim of the present study was to evaluate the expression of HMGB1 protein in primary enucleated retinoblastomas. Expression of HMGB1 in 69 prospective cases of primary retinoblastoma were assessed by immunohistochemistry and reverse transcriptase PCR (RT-PCR) technique and correlated with clinicopathological parameters. Immunohistochemical staining revealed expression of HMGB1 in 55.07 % (38/69) cases. Semi-quantitative RT-PCR was performed on 31 fresh tumor tissues. mRNA expression was observed in 77.41 % (24/31) cases. Expression of HMGB1 was statistically significant with poor tumor differentiation (p = 0.0440) & optic nerve invasion (p = 0.0128). HMGB1 expression was frequently seen in poorly differentiated tumors and those with histopathological high risk factors. Therefore, HMGB1 may contribute to tumor invasiveness and could serve as a poor prognostic marker in Rb.

Effect of HMGB1 on the VEGF-C expression and proliferation of esophageal squamous cancer cells

To explore the effect of HMGB1 on the VEGF-C expression and proliferation of esophageal squamous cancer cells as well as its possible mechanism. A cassette encoding siRNA targeting HMGB1 mediated by rAAV was constructed, the rAAV-siHMGB1-hrGFP, and a vector encoding siRNA mismatching HMGB1 was constructed, the rAAV-miHMGB1-hrGFP. This experiment in vitro included three groups, namely, the blank control group (group A) of KYSE150 cells transfected by rAAV-hrGFP, negative mismatch control group (group B) of KYSE150 cells transfected with rAAV-miHMGB1-hrGFP, and RNA interference group (group C) of KYSE150 cells transfected with rAAV-siHMGB1-hrGFP. We examined the expression of HMGB1 mRNA and protein in the three group cells by real-time PCR and Western blot after 24 h and 48 h, respectively. Then, VEGF-C expression and cell proliferation in the three group cells with or without sRAGE, as an inhibitor of RAGE signal pathway, were assayed by ELISA and MTT after 24 h. The expression of HMGB1 mRNA and protein in KYSE150 cells in vitro in the group C transfected with rAAV-siHMGB1-hrGFP at the final concentration of 2×10(6) v.g/cell was significantly lower than that of the group A or B after 24 h and 48 h (P < 0.01). The VEGF-C expression of KYSE150 cells was (502.43 ± 13.10) pg/ml in the group C, significantly reduced in comparison with that of the group A (686.40 ± 10.94) pg/ml or group B (682.31 ± 9.61) pg/ml after 24 h (P < 0.05). At the same time, the proliferation of KYSE150 cells in the group C was significantly inhibited compared with that of groups A and B after 24 h (P < 0.01). Moreover, sRAGE at the final concentration of 0.2 µg/ml inhibited the VEGF-C expression and proliferation of KYSE150 cells compared with the corresponding group without sRAGE after 24 h (P < 0.01 or P < 0.05). However, there was no significant difference of the VEGF-C expression and proliferation of KYSE150 cells with sRAGE in the group C compared with that of cells with sRAGE of the group A or group B after 24 h (P > 0.05). In esophageal squamous cell carcinoma, HMGB1 can promote the VEGF-C expression and proliferation of the cancer cells through RAGE signal pathway, and HMGB1-RAGE may become a potential target for cell proliferation and lymph node metastasis of this cancer.

HMGB1 and RAGE in skeletal muscle inflammation: Implications for protein accumulation in inclusion body myositis

Inflammation is associated with protein accumulation in IBM, but precise mechanisms are elusive. The “alarmin” HMGB1 is upregulated in muscle inflammation. Its receptor RAGE is crucial for β-amyloid-associated neurodegeneration. Relevant signaling via HMGB1/RAGE is expected in IBM pathology. By real-time-PCR, mRNA-expression levels of HMGB1 and RAGE were upregulated in muscle biopsies of patients with IBM and PM, but not in muscular dystrophy or non-myopathic controls. By immunohistochemistry, both molecules displayed the highest signal in IBM, where they distinctly co-localized to intra-fiber accumulations of β-amyloid and neurofilament/tau. In these fibers, identification of phosphorylated Erk suggested that relevant downstream activation is present upon HMGB1 signaling via RAGE. Protein expressions of HMGB1, RAGE, Erk and phosphorylated Erk were confirmed by Western blot. In a well established cell-culture model for pro-inflammatory cell-stress, exposure of human muscle-cells to IL-1β+IFN-γ induced cytoplasmic translocation of HMGB1 and subsequent release as evidenced by ELISA. Upregulation of RAGE on the cell surface was demonstrated by immunocytochemistry and flow-cytometry. Recombinant HMGB1 was equally potent as IL-1β+IFN-γ in causing amyloid-accumulation and cell-death, and both were abrogated by the HMGB1-blocker BoxA. The findings strengthen the concept of unique interactions between degenerative and inflammatory mechanisms and suggest that HMGB1/RAGE signaling is a critical pathway in IBM pathology.

Angiotensin II-induced hypertensive renal inflammation is mediated through HMGB1-TLR4 signaling in rat tubulo-epithelial cells

Background and purposeAngiotensin II is a vaso-constrictive peptide that regulates blood pressure homeostasis. Even though the inflammatory effects of AngII in renal pathophysiology have been studied, there still exists a paucity of data with regard to the mechanism of action of AngII-mediated kidney injury. The objective of this study was to elucidate the mechanistic role of HMGB1-TLR4 signaling in AngII-induced inflammation in the kidney. Experimental approachRat tubular epithelial cells (NRK52E) were treated with AngII over a preset time-course. In another set of experiments, HMGB1 was neutralized and TLR4 was knocked down using small interfering RNA targeting TLR4. Cell extracts were subjected to RT-PCR, immunoblotting, flow cytometry, and ELISA. Key resultsAngII-induced inflammation in NRK52E cells increased gene and protein expression of TLR4, HMGB1 and key proinflammatory cytokines (TNFα and IL1β). Pretreatment with Losartan (an AT1 receptor blocker) attenuated the AngII-induced expression of TLR4 and inflammatory cytokines. TLR4 silencing was used to elucidate the specific role played by TLR4 in AngII-induced inflammation. TLR4siRNA treatment in these cells significantly decreased the AngII-induced inflammatory effect. Consistent observations were made when the Ang II treated cells were pretreated with anti-HMGB1. Downstream activation of NFκB and rate of generation of ROS was also decreased on gene silencing of TLR4 and exposure to anti-HMGB1. Conclusions and implicationsThese results indicate a key role for HMGB1-TLR4 signaling in AngII-mediated inflammation in the renal epithelial cells. Our data also reveal that AngII-induced effects could be alleviated by HMGB1-TLR4 inhibition, suggesting this pathway as a potential therapeutic target for hypertensive renal dysfunctions.

Effect of toll-like receptor/myeloid differentiation factor 88 signaling pathway on invasiveness of breast cancer cells

Objective To study the expression and significance of toll- like receptors 4/myeloid differentiation factor 88(TLR4/MyD88)in human high invasive breast cancer cells MDA- MB - 231 and poor invasive breast cancer cells MCF- 7. Methods TLR4, MyD88 and high mobility group protein B1(H MGB1) mRNA expression levels were detected in MDA- MB- 231 cells and MCF- 7 cells by reverse transcription- polymerase chain reaction(RT- PCR).TLR4, MyD88 and HMGB1 protein expression levels were detected by Western blotting and immunofluorescence methods. Results MDA- MB- 231 and MCF- 7 cells expressed TLR4, MyD88 and HMGB1 at both mRNA and protein levels.The mRNA expression of TLR4 and MyD88 in MDA- MB- 231 cells was obviously higher than in MCF- 7 cells respectively,and the expression in MDA- MB- 231 cells was (10.43±4.00)and(2.09±1.50)times as much as that in MCF- 7 cells(P 0.05). Conclusion The invasive level and the expression level of TLR4 and MyD88 in breast cancer cells were consistent.The high expression of TLR4/MyD88 may be used as predictive molecular markers in evaluating outcome and guiding therapeutic strategy of breast cancer. Key words: Breast cancer; Toll-like receptors 4; Myeloid differentiation factor 88; Invasiveness

Angiotensin‐II Induced Hypertensive Renal Injury is Mediated through HMGB1‐TLR4 Signaling in Rat Tubulo‐Epithelial Cells

Angiotensin II is a vaso-constrictive peptide that regulates blood pressure homeostasis. Even though the inflammatory effects of AngII in renal pathophysiology have been studied, there still exists a paucity of data with regard to the mechanism of action of AngII-mediated kidney injury. The objective of this study was to elucidate the mechanistic role of HMGB1-TLR4 signaling in AngII-induced inflammation in the kidney. Rat tubular epithelial cells (NRK52E) were treated with AngII over a preset time-course. In another set of experiments, HMGB1 was neutralized and TLR4 was knocked down using small interfering RNA targeting TLR4. Cell extracts were subjected to RT-PCR, immunoblotting, flow cytometry, ELISA. AngII induced inflammation in NRK52E cells increased gene and protein expression of TLR4, HMGB1 and proinflammatory cytokines. Pretreatment with Losartan attenuated the AngII-induced expression of TLR4 and inflammatory molecules. TLR4 silencing was used to elucidate the specific role played by TLR4 in Ang...

Tu1858 The Establishment of Intestinal Goblet-Like Cells and Its Utilization

Aims: The alarmin HMGB1 is released during cell damage and acts as an early or late stage inflammatory cytokine at multiple levels of target cells. The Na+/H+ exchanger Isoform 3 (NHE3) is the major intestinal salt and fluid absorptive transporter and its function is defective in intestinal inflammation. Aim of the study: We wanted to know if HMGB1 can affect NHE3 mRNA or protein expression as well as its localization in mucosal tissue form patients with ulcerative colitis and in NHE3-expressing Caco2bbe colonic cells. Methods and Results: We studied the mRNA and protein expression and localization of HMGB1 and NHE3 in rectal biopsies from the inflamed and noninflamed mucosa of patients with ulcerative colitis by quantitative qPCR and immunohistochemistry. TNF-α and IFN-γmRNA level was significantly elevated in lesions compared to that in its adjacent normal tissue samples of UC patient's rectal, suggesting a reliable endoscopic sampling of inflamed vs noninflamed tissue. HMGB1 mRNA and protein expression was increased in the inflamed as compared to adjacent normal tissues, and it was mostly distributed in cytoplasm and extracellular region. NHE3 protein was markedly reduced in inflamed compared to adjacent normal tissues, though its mRNA level was not significantly different between inflamed and noninflamed mucosa. When Caco2bbe cells overexpressing human NHE3 (Caco2bbe/ hNHE3) were incubated with high concentration of rh-HMGB1 (10μg/ml), NHE3 mRNA was found to be decreased at 1h (0.385±0.015 vs. 1.011±0.011, p 0.05). NHE3 protein, measured byWestern analysis, was not significantly changed when co-cultured with 10μg/ml rhHMGB1 for 1h or 6h, however, it was dramatically decreased at 24h of co-culture with 10μg/ml rhHMGB1. IFN-gamma levels were markedly increased when Caco2bbe/hNHE3 cells were incubated with 10 μg/ml rhHMGB1 for 1h as compared with normal group (29.855±0.055 vs. 29.525±0.050, p 0.05) and 24h (29.066±0.181 vs. 29.525±0.050, p>0.05) of co-culture with 10μg/ml rhHMGB1 as compared to normal group. This suggests that incubation with rhHMGB1 may down-regulate NHE3 mRNA expression via elevated IFN-gamma level at early stage (1h), resulting in a decrease in NHE3 protein expression at late stage (24h). Conclusion: Decreased NHE3 protein level was accompanied by elevated cytoplasm and extracellular HMGB1 in human ulcerative rectitis tissue. This may result from the regulatory effect of cytokines such as IFNgamma induced by exogenous HMGB1 at late stage of inflammation.

Targeting HMGB1 Increased the Sensitivity of MM Cells to Chemotherapy

Background ：Multiple myeloma (MM) is an malignant disease of clonal plasma cell hyperplasia, which incidence is increasing in recent years. Chemotherapy is the main method to treat MM, but because of drug resistance, MM is still an incrurable disease. High-mobility group box 1 protein (HMGB1) is a chromatin associated nuclear protein, which participated in DNA damage repair , rearrangement, and it also as an extracellular damage associated molecular pattern molecule(DAMP) involved in inflammation, neoplastic progression, angiogenesis, invasion and metastasiss and drug resistance of kinds of tumor cells. There’s no research about the role of HMGB1 in multiple myeloma before.Methods: First, We detected HMGB1 mRNA and protein expression level in MM cell lines and primary MM cells by semiquantitative real time-polymerase chain (qRT-PCR)and western blot respectively. The concentration of HMGB1 in the culture supernatants of MM cell lines and primary MM cells was detected by ELISA. Second, we constructed Lentivirus vector with HMGB1 shRNA, infected the lentivirus to MM cells to knockdown HMGB1 expression. Then Chemo-drugs (ADM, quercetin and bortezomib) were added to MM cells, flow cytometry was used for the detection of cell apoptosis.Results:MM cell line and primary MM cell all express HMGB1 with high level. The concentration of HMGB1 in the MM cell supernatants was significantly elevated after chemotherapy treatment compared with untreated cells(p<0.05), and HMGB1 in MM patients’ bone marrow supernatants was very high. Knockdown of HMGB1 increased 8226 and ARP-1 cell apoptosis induced by ADM and quercetin (NC shRNA VS HMGB1 shRNA, p<0.05), but there’s no significant difference induced by bortezomib.Conclusions: Our study showed that the concentration of HMGB1 in the MM cell supernatants was significantly elevated after chemotherapy treatment. Further more, knockdown of HMGB1 by shRNA increased the sensitivity of MM cells to chemotherapy. DisclosuresNo relevant conflicts of interest to declare.

Anti-inflammative effect of glycyrrhizin on rat thermal injury via inhibition of high-mobility group box 1 protein

AimGlycyrrhizin (Gly) has been reported as an inhibitor of extracellular HMGB1 (high-mobility group box 1 protein) cytokine's activity, and protects spinal cord, liver, heart and brain against ischemia-reperfusion-induced injury in rats. The purpose of this study was to investigate the protective effect of Gly in rat skin thermal injury model and to elucidate the underlying mechanisms. MethodsTwenty-four male Sprague-Dawley rats (200–250g) were randomly divided into control group, vehicle-treated and Gly-treated burn groups, each group contained eight animals. In the latter two groups, rats were subjected to 30% TBSA (Total Body Surface Area) full-thickness scald injury. In Gly-treated burn group, glycyrrhizin (60mg/kg) was administered intraperitoneally immediately after and at 24th hour burn; in vehicle-treated burn group, Ringer's solution (4ml/kg, as a vehicle) was administered intraperitoneally immediately after and at 24th hour burn. The animals were sacrificed at 48h after injury. Aortic blood samples were obtained to detect tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) with ELISA (Enzyme-Linked Immuno Sorbent Assay) kits. Lung, liver and kidney tissue samples were collected to determine the expression of HMGB1 mRNA and protein. HMGB1 mRNA level was semiquantitatively measured by Real-Time PCR using β-actin as an internal standard, and protein expression of HMGBI was determined by Western blot. ResultsSevere skin scald injury caused a significant increase in plasma TNF-α and IL-1β versus the control group (P<0.001) in 48h after burns. Intraperitoneal administration of Gly (60mg/kg) significantly reduced the levels of serum TNF-α and IL-1β (P<0.01). Gly treatment reduced these biochemical indices accompanied by lower level of HMGB1 protein (P<0.05) and mRNA expression (P<0.01). ConclusionThese results demonstrate that Gly possesses an anti-inflammation effect to protect the remote organs from burn-induced injury.

Resveratrol attenuates HMGB1 signaling and inflammation in house dust mite-induced atopic dermatitis in mice.

Resveratrol is a polyphenol abundantly found in red grape skin and is effective against antiaging and anti-inflammation associated with immune responses. In this study, we have investigated the effect of resveratrol on skin lesion, high mobility group box (HMGB)1 and inflammation pathway in an atopic dermatitis (AD) mouse model. AD-like lesion was induced by the application of house dust mite extract to the dorsal skin of NC/Nga mouse. After AD induction, resveratrol (20 mg/kg, p.o.) was administered daily for 2 weeks. We evaluated dermatitis severity, histopathological changes, serum levels of T helper (Th) cytokines (interferon (IFN)γ, interleukin (IL)-4) and changes in protein expression by Western blotting for HMGB1, receptor for advanced glycation end products (RAGE), toll like receptor (TLR)4, nuclear factor (NF)κB, phosphatidylinositide 3-kinase (PI3K), extracellular signal-regulated kinase (ERK)1/2, cyclooxygenase (COX)2, tumor necrosis factor (TNF)α, IL-1β, IL-2Rα and other inflammatory markers in the skin of AD mice. Treatment of resveratrol inhibited the development of the AD-like skin lesions. Histological analysis showed that resveratrol inhibited hypertrophy, intracellular edema, mast cells and infiltration of inflammatory cells. Furthermore, resveratrol treatment down-regulated HMGB1, RAGE, p-NFκB, p-PI3K, p-ERK1/2, COX2, TNFα, IL-1β, IL-2Rα, IFNγ and IL-4. Considering all these findings together, the HMGB1 pathway might be a potential therapeutic target in skin inflammation, and resveratrol treatment could have beneficial effects on AD by modulating the HMGB1 protein expression.

The involvement of high mobility group 1 cytokine and phospholipases A2 in diabetic retinopathy

BackgroundDiabetic retinopathy, the main microvascular complications of diabetes and one of the leading causes of blindness worldwide. Interesting reports on the role of inflammatory/proangiogenic high mobility group 1 (HMGB-1) cytokine and phospholipases A2 (PLA2) in neovascularization have diverted our concentration to reveal whether HMGB-1 and PLA2 plays role in diabetic retinopathy.MethodsWe performed our study in streptozotocin (STZ)-induced diabetic rat model. The expression levels of the cytokines, chemokines, and cell adhesion molecules in retinal tissues were evaluated by quantitative RT-PCR. HMGB-1 and PLA2 protein levels along with VEGF, TNF-α, IL-1β and ICAM-1 levels were also measured.ResultsWe observed the retinal pericytes, endothelial injury/death and breakdown of blood–retinal barrier (BRB). The protein expression of HMGB-1, PLA2 and IL-1β were significantly increased in micro vessels from retina of diabetic rats. Diabetic rats had also high retinal levels of VEGF, ICAM-1 and TNF-α. Further investigation revealed that pericyte death is mediated by HMGB-1-induced cytotoxic activity of glial cells, while HMGB-1 can directly mediate endothelial cell death. Similarly, increased expression of PLA2 represents the diabetic mediated alteration of BRB, perhaps up regulating the VEGF.ConclusionsOur data suggest that HMGB-1 and PLA2 involved in retinal pericyte and endothelial injury and cell death in diabetic retinopathy. From this study, we suggest that HMGB-1 and PLA2 may be interesting targets in managing diabetic retinopathy.

Effect of simvastatin on expression of IL17, HMGB1 and TLR4 in LN kidney tissues of rats

ObjectivesTo observe the intervention influence and effect of simvastatin on the expression of interleukin 17 (LI17), high mobility group protein 1 (HMGB1) and TLR4 path in Lupus nephritis (LN) rats. MethodsA total of 28 BSXSB male mice with LN (16 weeks) were randomly divided into observation group and the comparison group, observation group was given 6 mg•kg−1•d−1 simvastatin in 0.1% PBS lavage for 4 weeks, the comparison group was not given any treatment. Blood urea nitrogen (BUN) level and urine trace albumin (Scr) level of two groups were determined. The expression of IL17, HMGB1 and TLR4 protein was detected using immune histochemical method, and the kidney histological damage was observed. ResultsBNU, LI17, HMGB1, TLR4 protein and HMGB1 mRNA in observation group was significantly lower than that in control group (P<0.05); There was no statistical difference of Scr level between two groups (P>0.05). Histological observation showed glomerular lesions integral of observation group was obviously lower than that of control group. ConclusionsSimvastatin can reduce the expression of IL17, HMGB1 and TLR4 protein in LN mice, thereby can inhibit the autoimmune response as a potential treatment function of LN.

Inhibition on the proliferation of human endometrial cancer cells by RNAi inhibiting HMGB1 gene expression and its possible molecular mechanism

To investigate the effect of HMGB1 small interfering RNA (siRNA) on the proliferation, cell cycle and apoptosis of human endometrial cancer cell line HEC-1A, and its possible molecular mechanism. Lentivirus vector with HMGB1 shRNA was constructed and infected the endometrial cancer cell line HEC-1A. After viral infection for 72 h, real time PCR and Western blot were performed to investigate HMGB1 mRNA and protein expression. The cell proliferation was determined with methyl thiazolyl tetrazolium (MTT) method. Flow cytometry was performed to analyze the cell cycle progression of propidium iodide (PI)-stained HEC-1A cells and the apoptotic rate of annexinV/PI-stained cells. Western blot was used to detect the protein expression of AKT, pAKT and CyclinD1. Lentivirus vector with HMGB1 shRNA inhibited the mRNA (P<0.05) and protein (P<0.01) expression of HMGB1 in the cell line HEC-1A. The MTT assay demonstrated that HMGB1 knockdown significantly reduced the cell proliferation. FCM results showed that HMGB1 knockdown significantly resulted in the disruption of the cell cycle at G0/G1 phase and the induction of apoptosis. The apoptotic rate was (17.89±0.23)%, (4.69±0.20)% and (4.62±0.17)% in the HMGB1 knockdown group, the blank group and the negative group respectively. There was significance difference between the 3 groups (P<0.01). The protein expressions of pAKT and cyclinD1 were down-regulated after the HMGB1 knockdown for 72 h. Knockdown of HMGB1 expression can significantly inhibit the proliferation and induce the cell cycle arrest and apoptosis in the endometrial cancer cell line HEC-1A. PI3K/ AKT pathway and down-regulation of the protein expression of cyclinD1 may be involved in its therapeutic mechanism.