Hang Sun

LPS induces HMGB1 relocation and release by activating the NF-κB-CBP signal transduction pathway in the murine macrophage-like cell line RAW264.7.

BACKGROUND High mobility group protein B1 (HMGB1) is an important late inflammatory mediator in sepsis. Understanding the mechanisms that regulate HMGB1 release from cells and their downstream signal transduction pathways may lead to the ability to develop anti-HMGB1 therapies to treat inflammation. MATERIALS AND METHODS We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS+ ethylpyruvate (EP) and examined the resulting HMGB1 expression and release. We also studied the expression of related signal transduction factors (NF-κB, p38 MAPK, and CBP). RESULTS AND CONCLUSION Gene expression of HMGB1 mRNA in RAW264.7 cell showed no significant change at 0-18 h after stimulation with LPS, but increased significantly at 24, 36, and 48 h. HMGB1 mRNA expression in the LPS+EP group was significantly lower than in LPS alone. HMGB1 was distributed mainly in the nucleus; the cytoplasmic level was low before LPS stimulation. After stimulation with LPS, cytoplasmic HMGB1 increased gradually and plateaued at a high level at 12-48 h. Nuclear HMGB1 decreased gradually at 12-24 h, then increased, maintaining a comparatively high level at 36-48 h. EP prevented this pattern significantly. LPS induced p38 MAPK activation and NF-κB signal pathways first, followed by CBP activation. Activated CBP acetylated HMGB1 was stored in a crino-lysosome and secreted activated NF-κB resulted in increased transcription and synthesis of HMGB1, but the expression of up-regulated HMGB1 mRNA was delayed. Extracellular HMGB1 originated from early synthetic reserves present in the nucleus. New HMGB1 protein was synthesized in the nucleus and transferred into the cytoplasm, causing an increase in HMGB1 in the nucleus and cytoplasm. EP inhibits HMGB1 mRNA up-regulation and release from LPS- stimulated macrophages. The molecular function of EP is to attenuate the activation p38 MAPK, NF-κB, and CBP signaling pathways.

Augmenter of liver regeneration protects kidneys from ischaemia/reperfusion injury in rats

BACKGROUND Augmenter of liver regeneration (ALR), which was identified originally for its crucial role in promoting hepatocyte proliferation, is expressed in both the liver and kidney. Protective effects of ALR have been demonstrated in experimental models of acute liver failure. In the present study, we investigated the effect of ALR on renal ischaemia/reperfusion (I/R) injury and the possible mechanisms of its action. METHODS Male Sprague-Dawley rats were subjected to renal ischaemia for 60 min and then administered with either saline or recombinant human ALR (rhALR). A sham-operated group served as control. The expression of ALR in the sham-operated and acute kidney injury (AKI) groups was detected by immunohistochemistry and western blotting. Renal dysfunction and injury were assessed by measurement of serum biochemical markers and histological grading. Expression of proliferating cell nuclear antigen (PCNA) was determined by immunohistochemistry. RESULTS Renal ALR expression increased significantly in rats with ischaemic AKI compared with the sham-operated rats. Serum biochemical parameters showed that renal dysfunction was improved by administration of rhALR. Histological analysis revealed that treatment with rhALR also reduced the extent of kidney injury. Intraperitoneal injection of rhALR enhanced the proliferation of renal tubular cells. Conclusions. Administration of rhALR effectively reduces tubular injury and ameliorates the impairment of renal function. The protective effect of rhALR is associated with enhancement of renal tubular cell regeneration.

Augmenter of liver regeneration attenuates tubular cell apoptosis in acute kidney injury in rats: the possible mechanisms.

Augmenter of liver regeneration (ALR), the expression of which increased in rat kidneys after renal ischemia/reperfusion (I/R) injury, enhances renal tubular cell regeneration in vivo and in vitro. We aimed to investigate the effects of ALR on apoptosis of renal tubular cells after renal I/R injury in vivo and consider the possible mechanisms. Rats that were subjected to bilateral renal ischemia for 60 min followed by reperfusion were administered with either vehicle or recombinant human ALR (rhALR). Renal dysfunction and histologic injury were assessed by the measurement of serum biochemical markers and histological grading. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labeling (TUNEL). Caspase-3 activity was measured using a colorimetric protease assay. Expression of Bcl-2, Bax Fas, phosphorylated-Akt (p-Akt), and phosphorylated-p53 (p-p53) was determined by western blotting. Compared with vehicle-treated rats, renal dysfunction and histologic injury were significantly attenuated by administration of rhALR. The number of TUNEL-positive tubular cells and caspase-3 activity were decreased, Bcl-2 and p-Akt expression was up-regulated, and Bax and p-p53 expression was down-regulated by administration of rhALR. However, administration of rhALR had no effect on Fas protein expression. These results indicate that the protective effect of rhALR on renal I/R injury is associated with its anti-apoptotic action in renal tubular cells. RhALR inhibits apoptosis by increasing the ratio of Bcl-2 to Bax and by decreasing the activity of caspase-3. The activation of Akt and inactivation of p53 are involved in the rhALR anti-apoptosis process.

Augmenter of liver regeneration improves therapeutic effect of hepatocyte homotransplantation in acute liver failure rats.

Hepatocyte transplantation (HCT) is an available option on treatment for acute liver failure (ALF). However, short-term survival of engraftment and immunological rejections of recipient are major obstacles. Augmenter of liver regeneration (ALR) has cytoprotective and immunoregulatory effects in liver injury, and has been used in many experimental applications. In the present study, we investigated the potential effect and mechanism of recombinant human ALR (rhALR) on ALF rats treated with intraperitoneal HCT. ALF rats induced by d-galactosamine (GalN) were studied in vivo, and were intraperitoneal injected with or without hepatocytes and rhALR 24h after the induction. Animal survival, serum and ascites liver enzymes, tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were assessed. Histological examination was performed, and liver regeneration, apoptosis and immunological responses were identified by immunohistochemistry assay. Our results showed that rhALR promoted hepatocytes regeneration, attenuated liver injury and suppressed immunological responses. The ascites liver enzyme, serum and ascites pro-inflammatory cytokines (TNF-α, IL-1β), liver histological injury, apoptotic hepatocytes and activated immunocytes were significantly reduced in ALF rats treated with rhALR and HCT compared with those without rhALR. The proliferative and mitotic hepatocytes were markedly increased, and overall survival improved with rhALR. The administration of rhALR improved survival and promoted liver recovery in HCT treatment for ALF, which was associated with the role of proliferative promoter and immunosuppressor. This study suggests that co-treated with rhALR and HCT can provide a promising strategy for the treatment of ALF.

Evaluation of high mobility group box 1 protein as a presurgical diagnostic marker reflecting the severity of acute appendicitis

ObjectivesTo validate the role of high mobility group box-1(HMGB1) in diagnosis of acute appendicitis (AA) with different pathological severity.MethodsAccording to the pathologically diagnosis, 150 patients underwent appendectomies between Jan. 2007 and Dec, 2010 were divided into acute simple, acute suppurative and acute gangrenous appendicitis as group 1, 2 and 3, respectively. Each patient group contains 50 sex and age matched cases to make comparison with 50 healthy volunteers. The mRNA and protein expression levels of serum HMGB1 were determined by real-time quantitative PCR and enzyme linked immunosorbent assay (ELISA). Serum High-sensitivity C-reactive protein (hs-CRP) levels were determined by rate nephelometric immunoassay.ResultsIn comparison with health volunteers, relative HMGB1 mRNA levels in group 1, 2 and 3 were significantly increased 3.05 ± 0.51,8.33 ± 0.75 and 13.74 ± 1.09 folds, reflecting a tendency of augmented severity. In accordance, serum protein levels of HMGB1 were 10.97 ± 1.64, 14.42 ± 1.56 and 18.08 ± 2.41 ng/ml in 3 patient groups, which are significantly higher than that of healthy volunteers’ 5.47 ± 0.73 ng/ml. hs-CRP levels were 12.85 ± 3.41, 21.04 ± 1.98 and 31.07 ± 5.46 ng/ml in 3 patients groups compared with 2.06 ± 0.77 ng/ml in controls. The concentrations of HMGB1 and hs-CRP were both positively correlated with disease severity.ConclusionSerum HMGB1 constitutes as a valuable marker in diagnosis of AA. Positively correlated with hs-CRP level, mRNA and protein expression of HMGB1 to a certain extent reflected the severity of AA.

Inhibition effect of glycyrrhizin in lipopolysaccharide-induced high-mobility group box 1 releasing and expression from RAW264.7 cells.

Introduction High-mobility group box 1 (HMGB1) is a therapeutic target for sepsis. Glycyrrhizin (GL) is the aglycone of glycyrrhizin derived from licorice. We clarified the anti-inflammatory effects of GL. We explored the anti-HMGB1 effect of GL and elucidated its molecular mechanism, which will be of benefit for sepsis treatment. Methods We stimulated murine macrophage-like RAW 264.7 cells with lipopolysaccharide (LPS) and LPS + GL, then measured the expression and release of HMGB1. The expression of related signal transduction factors was detected. Results High-mobility group box 1 was distributed mainly in the nucleus with lower cytoplasmic levels in RAW 264.7 cells before LPS stimulation. After stimulation, cytoplasmic HMGB1 levels increased gradually, whereas in nuclear fluctuation a trend of HMGB1 expression was observed. Significant upregulation of HMGB1 mRNA occurred 12 h after LPS stimulation. Glycyrrhizin prevented the transfer of HMGB1 from the nucleus to the cytoplasm and inhibited upregulation of HMGB1 mRNA induced by LPS. Phospho–p38 mitogen-activated protein kinase and activated activating protein 1 increased significantly 8 h after LPS stimulation. Tumor necrosis factor &agr; and interleukin 6 increased 4 h after LPS stimulation and peaked at 48 h, and HMGB1 increased at 8 h. The Toll-like receptor 4/MD2/nuclear factor &kgr;B signaling pathway was activated 4 h after LPS stimulation. Glycyrrhizin inhibited this pathway. Conclusions Glycyrrhizin inhibited the expression and release of HMGB1 through blocking the p38 mitogen-activated protein kinase/activating protein 1 signaling pathway then inhibited the massive release of tumor necrosis factor &agr; and interleukin 6.

The role of high mobility group box chromosomal protein 1 expression in the differential diagnosis of hepatic actinomycosis: a case report

IntroductionPrimary hepatic actinomycosis is a rare disease, but is important in the differential diagnosis of hepatoma in endemic areas. As high mobility group box chromosomal protein 1 plays an important role in the pathogenesis of both acute and chronic inflammatory conditions, we postulate that high mobility group box chromosomal protein 1 may have a possible pathogenic role in hepatic actinomycosis. To the best of our knowledge, our report is the first to detect an association between highly elevated high mobility group box chromosomal protein 1 expression and hepatic actinomycosis.Case presentationA 67-year-old Chinese man was admitted to our hospital with a three-month history of epigastric pain, anorexia, and subjective weight loss. Ultrasonography and computed tomography of the patient’s abdomen confirmed a hypodense mass measuring seven cm in diameter in the left lateral segment of his liver. A hepatic tumor was suspected and surgical resection was scheduled. Histopathologic examination revealed that the overall features of the hepatic tissues were consistent with hepatic actinomycosis. Whole blood and hepatic tissue samples of the patient, of patients who had hepatocellular carcinoma and of healthy donors were collected. Serum high mobility group box chromosomal protein 1 concentration in actinomycosis was 8.5ng/mL, which was higher than the hepatocellular carcinoma level of 5.2ng/mL and the normal level of <three ng/mL. High mobility group box chromosomal protein 1 messenger ribonucleic acid levels and high mobility group box chromosomal protein 1 protein content in the affected tissues of this patient with hepatic actinomycosis were higher than those of the control and hepatocellular carcinoma tissues. The results of immunohistochemistry showed the following: in the control tissues, high mobility group box chromosomal protein 1 was distributed mainly in the cytoplasm; in the hepatocellular carcinoma tissues, high mobility group box chromosomal protein 1 was distributed primarily in the nucleus; and in the actinomycosis tissues, high mobility group box chromosomal protein 1 was increased in both the cytoplasm and nucleus.ConclusionHigh mobility group box chromosomal protein 1 may have a potent biological effect on the pathogenesis of hepatic actinomycosis as a novel cytokine and may be a useful marker in the differential diagnosis of hepatic actinomycosis.

Endoplasmic reticulum stress preconditioning antagonizes low-density lipoprotein-induced inflammation in human mesangial cells through upregulation of XBP1 and suppression of the IRE1α/IKK/NF‑κB pathway

Elevated plasma low‑density lipoprotein (LDL) is associated with systemic inflammation, and is an important factor in the pathogenesis of chronic kidney disease. The aim of the present study was to investigate the effects of endoplasmic reticulum (ER) stress preconditioning on LDL‑induced inflammatory responses, in human mesangial cells (HMCs). HMCs were exposed to LDL (200 nm), with or without pretreatment with tunicamycin, an ER stress inducer, and tested for changes to gene expression levels. Small interfering RNA technology was used to knockdown the expression of inositol‑requiring enzyme‑1α (IRE1α) and X‑box‑binding protein‑1 (XBP‑1), in order to determine their effects on LDL‑treated HMCs. LDL treatment resulted in a significant, and time‑dependent, increase in the relative mRNA expression levels of proinflammatory cytokines and CD40, which was coupled with enhanced phosphorylation of IRE1α, IκB kinase (IKK), and nuclear factor (NF)‑κB p65 and p65 nuclear translocation. The LDL‑induced inflammatory responses were significantly reduced in the IRE1α‑depleted HMCs. Furthermore, pretreatment with tunicamycin significantly attenuated the induction of proinflammatory cytokines and CD40, by LDL. Whereas, silencing XBP1 expression significantly restored the production of proinflammatory cytokines, in the LDL‑treated HMCs with ER stress preconditioning. The phosphorylation levels of IRE1α, IKK, and NF‑κB p65 were markedly increased in the XBP1‑depleted HMCs. Conversely, overexpression of XBP1 blocked LDL‑induced inflammation in the HMCs. The results of the present study demonstrate that ER stress preconditioning antagonizes LDL‑induced inflammatory responses in HMCs, which may be mediated through upregulation of XBP1, and subsequent inactivation of the IRE1α/IKK/NF‑κB pathway.

Exogenous augmenter of liver regeneration (ALR) attenuates inflammatory response in renal hypoxia re-oxygenation injury.

Abstract Recent studies have highlighted the role of the innate immune system in initiating the inflammatory cascade which leads to detrimental changes in renal ischemia reperfusion (I/R) injury. The augmenter of liver regeneration (ALR) is an anti-apoptosis factor which is highly expressed in renal tubulars of renal cortex and medulla after inducing renal I/R injury in rats. It has been shown that exogenous ALR can enhance renal tubular regeneration. However, whether ALR’s protective effect against renal I/R injury results from its immune regulatory function remains unknown. Using rat renal tubular epithelial cell (NRK-52E), we investigate the effect of recombinant rat ALR (rrALR) on immune inflammatory response in hypoxia re-oxygenation (H/R) injury in vitro, and further discuss the possible mechanisms. Cultured NRK-52E cells subjected to hypoxia for 6 h followed by re-oxygenation for 12, 24 and 72 h are administered with different doses of rrALR. Expression of Toll-like receptor 4 (TLR4) and transcription nuclear factor-κB (NF-κB) is assessed by reverse-transcriptase polymerase chain reaction (RT-PCR) and western blot. Expression of interleukin (IL)-6 and IL-1β are determined by enzyme-linked immunosorbent assay (ELISA). In rrALR intervened H/R cells, TLR4 and NF-κB are down regulated at both mRNA and protein levels compare with those in control cells. Also, rrALR appears to downregulate IL-6 and IL-1β expression in concentration-dependent manners. In conclusion, rrALR protects NRK-52E cells from H/R injury possibly by relieving the inflammatory response through regulation of TLR4-NF-κB signaling pathway.

Augmenter of liver regeneration inhibits TGF-β1-induced renal tubular epithelial-to-mesenchymal transition via suppressing TβR II expression in vitro

Tubular epithelial-to-mesenchymal transition (EMT) plays a crucial role in the progression of renal tubular interstitial fibrosis (TIF), which subsequently leads to chronic kidney disease (CKD) and eventually, end-stage renal disease (ESRD). We propose that augmenter of liver regeneration (ALR), a member of the newly discovered ALR/Erv1 protein family shown to ameliorate hepatic fibrosis, plays a similar protective role in renal tubular cells and has potential as a new treatment option for CKD. Here, we showed that recombinant human ALR (rhALR) inhibits EMT in renal tubular cells by antagonizing activation of the transforming growth factor-β1 (TGF-β1) signaling pathway. Further investigation revealed that rhALR suppresses the expression of TGF-β receptor type II (TβR II) and significantly alleviates TGF-β1-induced phosphorylation of Smad2 and nuclear factor-κB (NF-κB). No apparent adverse effects were observed upon the addition of rhALR alone to cells. These findings collectively suggest that ALR plays a role in inhibiting progression of renal tubular EMT, supporting its potential utility as an effective antifibrotic strategy to reverse TIF in CKD.