Mechanism of HMGB1 in scarring after glaucoma drainage valve implantation / 国际眼科杂志(Guoji Yanke Zazhi)

AIM: To explore the dynamic expression of high mobility group box 1(HMGB1)in scar tissues after glaucoma drainage valve implantation, and to further reveal the role and possible mechanism of HMGB1 in scarring after glaucoma surgery.METHODS: A total of 60 New Zealand white rabbits were randomly divided into control group(n=20), model group(n=20, silicone implantation under conjunctival sac)and model with drug administration group(n=20, silicone implantation under conjunctival sac combined with 5-fluorouracil injection). The conjunctival tissues were collected at 4 and 8 wk after surgery. HE staining and Masson staining were used to detect the proliferation and distribution of fibroblasts and collagen fibers in conjunctival tissues. Immunohistochemistry was utilized to detect the distribution and changes of HMGB1, transforming growth factor(TGF)-β1, Smad3 and α-smooth muscle actin(SMA)in conjunctival tissues. RT-PCR and Western blot were adopted to detect the mRNA and protein expression of HMGB1, TGF-β1, Smad3 and α-SMA in conjunctival tissues.RESULTS: HE staining and Masson staining showed that the proliferation of inflammatory cells, fibroblasts and collagen fibers in the model group was significantly higher than that in the control group at both 4 and 8 wk. Meanwhile, the proliferation of fibroblasts and collagen fibers in the model with drug administration group was significantly lower than that in the model group. Immunohistochemical staining showed that the expression of HMGB1, TGF-β1, Smad3 and α-SMA protein was observed in the conjunctival tissues of the model group both 4 and 8 wk, with brown and significantly deeper staining of the model group at 8 wk. Meanwhile, the positive staining in the model with drug administration group at both 4 and 8 wk was significantly lower than that in the model group. There was positive correlations between the number of fibroblasts stained with HE and the expression of HMGB1 in the conjunctival tissue of the model group at both 4 and 8 wk(r=0.602, 0.703, all P<0.05). RT-PCR and Western blot revealed that the mRNA and protein expression levels of HMGB1, TGF-β1, Smad3 and α-SMA in the model group were significantly higher than those in the control group at both 4 and 8 wk(all P<0.05). Meanwhile, the mRNA and protein expression levels of HMGB1, TGF-β1, Smad3 and α-SMA in the model with drug administration group were significantly lower than those in the model group(all P<0.05). There was positive correlations between mRNA expressions of HMGB1 and TGF-β1, Smad3 in the model group and the model with drug administration group(all P<0.05).CONCLUSION: The expression of HMGB1 increased at a time-dependent manner after glaucoma valve implantation. HMGB1 acts an indispensable role in the initiation and progression of scar formation after glaucoma surgery, which may be involved in the regulation of TGF-β/Smad signaling pathway.

Compound Tinglizi Decoction intervenes COPD-associated pulmonary hypertension through regulation of HMGB1-mediated pyroptosis and immune imbalance / 中国中药杂志

This paper aimed to investigate the effects of high mobility group box 1(HMGB1)-mediated pulmonary artery smooth muscle cell pyroptosis and immune imbalance on chronic obstructive pulmonary disease-associated pulmonary hypertension(COPD-PH) in rats and the intervening mechanism of Compound Tinglizi Decoction. Ninety rats were randomly divided into a normal group, a model group, low-dose, medium-dose, and high-dose Compound Tinglizi Decoction groups, and a simvastatin group. The rat model of COPD-PH was established by fumigation combined with lipopolysaccharide(LPS) intravascular infusion, which lasted 60 days. Rats in the low, medium, and high-dose Compound Tinglizi Decoction groups were given 4.93, 9.87, and 19.74 g·kg~(-1) Compound Tinglizi Decoction by gavage, respectively. Rats in the simvastatin group were given 1.50 mg·kg~(-1) simvastatin by gavage. After 14 days, the lung function, mean pulmonary artery pressure, and arterial blood gas of rats were analyzed. Lung tissues of rats were collected for hematoxylin-eosin(HE) staining to observe the pathological changes. Real-time fluorescent quantitative polymerase chain reaction(qRT-PCR) was used to determine the expression of related mRNA in lung tissues, Western blot(WB) was used to determine the expression of related proteins in lung tissues, and enzyme linked immunosorbent assay(ELISA) was used to determine the levels of inflammatory factors in the lung tissues of rats. The ultrastructure of lung cells was observed by transmission electron microscope. The forced vital capacity(FVC), forced expiratory volume in 0.3 second(FEV_(0.3)), FEV_(0.3)/FVC, peek expiratory flow(PEF), respiratory dynamic compliance(Cdyn), arterial partial pressure of oxygen(PaO_2), and arterial oxygen saturation(SaO_2) were increased, and resistance of expiration(Re), mean pulmonary arterial pressure(mPAP), right ventricular hypertrophy index(RVHI), and arterial partial pressure of carbon dioxide(PaCO_2) were decreased by Compound Tinglizi Decoction in rats with COPD-PH. Compound Tinglizi Decoction inhibited the protein expression of HMGB1, receptor for advanced glycation end products(RAGE), pro caspase-8, cleaved caspase-8, and gasdermin D(GSDMD) in lung tissues of rats with COPD-PH, as well as the mRNA expression of HMGB1, RAGE, and caspase-8. Pulmonary artery smooth muscle cell pyroptosis was inhibited by Compound Tinglizi Decoction. Interferon-γ(IFN-γ) and interleukin-17(IL-17) were reduced, and interleukin-4(IL-4) and interleukin-10(IL-10) were incresead by Compound Tinglizi Decoction in lung tissues of rats with COPD-PH. In addition, the lesion degree of trachea, alveoli, and pulmonary artery in lung tissues of rats with COPD-PH was improved by Compound Tinglizi Decoction. Compound Tinglizi Decoction had dose-dependent effects. The lung function, pulmonary artery pressure, arterial blood gas, inflammation, trachea, alveoli, and pulmonary artery disease have been improved by Compound Tinglizi Decoction, and its mechanism is related to HMGB1-mediated pulmonary artery smooth muscle cell pyroptosis and helper T cell 1(Th1)/helper T cell 2(Th2), helper T cell 17(Th17)/regulatory T cell(Treg) imbalance.

Dahuang Mudantang Alleviates Intestinal Injury in Rat Model of Acute Pancreatitis by Regulating HMGB1/RAGE/NF-κB Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the mechanism of Dahuang Mudantang in alleviating the intestinal injury in the rat model of acute pancreatitis via the high-mobility group box 1 （HMGB1）/receptor for advanced glycation endproduct （RAGE）/nuclear factor-κB （NF-κB） signaling pathway. MethodOne hundred and twenty SPF-grade Wistar rats received retrograde injection of 5% sodium taurocholate into the biliopancreatic duct for the modeling of intestinal injury in acute pancreatitis. The rats were randomized into blank， model， low-， medium-， and high-dose （3.5， 7， 14 g·kg-1， administrated by gavage） Dahuang Mudantang， and octreotide （1×10-5 g·kg-1， subcutaneous injection） groups （n=20）. The rats in blank and model groups received equal volume of distilled water by gavage. Drugs were administered 1 h before and every 12 h after modeling， and samples were collected 24 h after modeling. The general status of the rats was observed. The biochemical methods were employed to measure the levels of amylase （AMS） and C-reactive protein （CRP） in the serum. The enzyme-linked immunosorbent assay （ELISA） was employed to measure the levels of tumor necrosis factor （TNF）-α， interleukin （IL）-1β， and IL-6 in the colon tissue. The morphological changes of pancreatic and colon tissues were observed by hematoxylin-eosin （HE） staining. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot were employed to measure the expression levels of HMGB1， RAGE， inhibitor of NF-κB kinase （IKK）， and NF-κB suppressor protein α（IκBα）in the colon tissue. ResultThe rats in the model group showed poor general survival， writhing response， reduced frequency of defecation， and dry stool. The symptoms of rats in the model group were mitigated in each treatment group， and the high-dose Dahuang Mudantang showed the most significant effect. Compared with the normal group， the model group had elevated AMS and CRP levels （P<0.05）， which were lowered by Dahuang Mudantang （P<0.05）， especially that at the high dose （P<0.05）. Compared with the normal group， the modeling elevated that levels of TNF-α， IL-1β， and IL-6 （P<0.05）. Such elevations were lowered by Dahuang Mudantang （P<0.05）， and the high-dose group and the octreotide group showed better performance （P<0.05）. The modeling caused necrotic， congested， and destructed pancreatic and colonic tissues， which were ameliorated by the drugs， especially high-dose Dahuang Mudantang. Compared with the normal group， the modeling up-regulated the mRNA levels of HMGB1， RAGE， IKK， IκBα， and NF-κB （P<0.05）. Compared with the model group， Dahuang Mudantang and octreotide down-regulated the mRNA levels of HMGB1， RAGE， IKK， IκBα， and NF-κB （P<0.05）， and the high-dose Dahuang Mudantang demonstrated the best performance （P<0.05）. Western blot results showed a trend consistent with the results of Real-time PCR. ConclusionDahuang Mudantang can improved the general status， reduce inflammation， and alleviate histopathological changes in the pancreatic and colon tissues in the rat model of acute pancreatitis by inhibiting the HMGB1/RAGE/NF-κB signaling pathway.

Mechanism of regulatory effect of recombinant human HMGB1 on endothelial cell angiogenesis / 器官移植

Objective To unravel the possible mechanism of the role of recombinant human high mobility group box 1 (rhHMGB1) protein in regulating the angiogenesis of endothelial cells. Methods Endothelial cells were divided into the control group, bone marrow mesenchymal stem cells (MSC) supernatant group and rhHMGB1 group. The proliferation and survival of endothelial cells were detected by cell counting kit(CCK)-8 assay. The relative expression levels of vascular endothelial growth factor (VEGF), Yes-associated protein (YAP), CD31 and hypoxia inducible factor (HIF)-1α proteins were determined by Western blot. The relative expression levels of VEGF, YAP, CD31 and HIF-1α messenger RNA (mRNA) were detected by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR). The migration ability of endothelial cells was assessed by Transwell chamber test. The localization of YAP was detected by immunofluorescence staining. Results Compared with the control group, the migration rate of endothelial cells was increased in the rhHMGB1 group (P < 0.05), and the cell migration rate was enhanced over time. Compared with the control group, the relative expression levels of VEGF and p-YAP proteins were up-regulated in the MSC supernatant group, and the differences were statistically significant (both P < 0.05). Compared with the control group, the relative expression levels of VEGF and HIF-1α proteins, VEGF and CD31 mRNA and YAP and p-YAP proteins were up-regulated, and YAP/p-YAP ratio was increased in the rhHMGB1 group, and the differences were statistically significant (all P < 0.05). Compared with the MSC supernatant group, the relative expression levels of CD31 mRNA and YAP protein were up-regulated, and the YAP/p-YAP ratio was increased in the rhHMGB1 group, and the differences were statistically significant (all P < 0.05). Conclusions Exogenous high-concentration rhHMGB1 may promote the migration ability of endothelial cells and up-regulate the expression levels of angiogenesis-related proteins by regulating the recruitment of YAP to the nucleus.

The mechanism of the receptors for advanced glycation end products affecting pericyte detachment from blood vessels after traumatic brain injury / 西安交通大学学报(医学版)

【Objective】 To observe the reactive change of cortical perivascular cells after craniocerebral injury and explore its mechanism. 【Methods】 The controllable cortical impact animal model was used to simulate craniocerebral injury, the expressions of cortical pericyte markers at different time points after trauma were studied by Western blotting, and the biological behavior of vascular pericytes after craniocerebral injury was determined by transmission electron microscopy. Post-traumatic high mobility group box 1 (HMGB1), receptor for advanced glycation end product (RAGE), and nuclear factor κB (NF-κB) were detected by Western blotting. The experimental animals were divided into FPS-ZM1 (a specific RAGE receptor blocker) injection group and wild-type group. Wet and dry brain weight and transmission electron microscopy were used to study the post-traumatic effects of HMGB1-RAGE on pericytes. The primary mouse brain microvascular pericytes were cultured and supplemented with HMGB1 recombinant protein; the cultured pericytes supplemented with FPS-ZM1 were used as the control to explore the effect of HMGB1-RAGE pathway on vascular pericytes in vitro. 【Results】 The expression levels of early post-traumatic cortical pericyte markers platelet-derived growth factor receptor beta (PDGFR-β) and NG2 proteoglycan (NG2) decreased (PDGFR-β, Control vs. CCI 3D P<0.05; NG2, Control vs. CCI 6H P<0.05; Control vs. CCI 1D P<0.05). We found that pericytes were detached from blood vessels, accompanied by local blood-brain barrier opening. The expression of HMGB1-RAGE-NF-κB signaling pathway was increased in the early cortex after trauma (HMGB1, Control vs. CCI 6H P<0.05, Control vs. CCI 1D P<0.05; RAGE, Control vs. CCI 6H P<0.05, Control vs. CCI 1D P<0.05, Control vs. CCI 3D P<0.05, Control vs. CCI 5D P<0.05, Control vs. CCI 7D P<0.05; NF-κB, Control vs. CCI 6H P<0.05, Control vs. CCI 1D P<0.05). After blocking the binding of RAGE with the ligand, cortical edema was reduced (CCI 6H P<0.05, CCI 1D P<0.05), and neurovascular unit damage was reduced. HMGB1 recombinant protein could increase the migration ability of cultured pericytes (Control vs. HMGB1 P<0.05, Control vs. HMGB1+FPS-ZM1 P<0.05), and could be reversed by FPS-ZM1 (HMGB1 vs. HMGB1+FPS-ZM1 P<0.05). 【Conclusion】 High-level HMGB1 after traumatic brain injury mediates pericytes’ detachment from blood vessels through RAGE on pericytes and leads to the occurrence of local cerebral edema.

High mobility group box-1 inhibition reduces expression of matrix metalloproteinase-9 in astrocytes in rats after spinal cord injury / 中华创伤骨科杂志

Objective:To investigate the role and underlying mechanisms of inhibiting high mobility group box-1 (HMGB1) in the expression of matrix metalloproteinase-9 (MMP-9) in spinal cord astrocytes (AS) in rats after spinal cord injury (SCI).Methods:After an SCI model was established in Sprague-Dawley (SD) rats using a modified Allen's Weight-Dropping method and ethyl pyruvate (EP) or glycyrrhizin (GL) was used to inhibit the effect of HMGB1, the rats were divided into a sham group, an SCI group, an SCI+EP (50 mg/kg) group, and an SCI+GL (100 mg/kg) group. The expression levels of glial fibrillary acid protein (GFAP) and MMP-9 in spinal cord AS were observed. After the spinal cord AS in SD rats was cultured and incubated by the oxygen-glucose deprivation/reoxygenation (OGD/R) procedure, the expression of MMP-9 protein was detected at 6 h/R 6 h, 12 h, 24 h, and 48 h after OGD. The time point with the highest expression was chosen in the subsequent experiments as an OGD/R group. HMGB1 was inhibited by HMGB1 shRNA or EP to observe the effect of HMGB1 on the expression of MMP-9 protein in AS treated with OGD/R. Then, toll-like receptor 4 (TLR4) inhibitor, TIR-domain-containing adaptor inducing interferon- β (TRIF) inhibitor, and nuclear factor-kappa B (NF- κB) inhibitor were used to investigate the effects of TLR4/TRIF/NF- κB signaling pathway during the regulation of HMGB1 on MMP-9 in vitro. Results:Western blot showed that the expression of MMP-9 protein in the spinal cord was significantly increased in rats at 1 d after SCI, and the expression of MMP-9 protein in the SCI+EP group and the SCI+GL group was significantly lower than that in the SCI group ( P<0.001). Immunofluorescence showed that GFAP and MMP-9 proteins were co-localized in the spinal cord after SCI, and the expression of GFAP and MMP-9 proteins in the SCI+EP and SCI+GL groups was significantly lower than that in the SCI group ( P<0.05). Since the expression of MMP-9 protein in the spinal cord AS cultured in vitro was significantly higher in the OGD 6h/R 12h group than that in the normal group and the OGD 6h/R 6h, 24, and 48 h groups, the OGD 6h/R 12h was taken as the OGD/R group. The MMP-9 protein expression in AS in the OGD/R+HMGB1 shRNA group and the OGD/R+EP group was significantly lower than that in the OGD/R group ( P<0.001). In the cultured AS, moreover, inhibiting TLR4, TRIF, and NF- κB reduced MMP-9 protein expression after OGD 6 h/R 12 h when compared with that in the OGD/R group ( P<0.001). Conclusions:HMGB1 inhibition may result in a reduction in MMP-9 expression both in the spinal cord AS in SCI rats and in AS after OGD/R treatment in vitro. HMGB1 may regulate MMP-9 protein expression in AS after OGD/R treatment via the TLR4/TRIF/NF- κB signal pathway.

Recent progress on the roles of DAMP and NET in organ ischemia-reperfusion injury / 器官移植

Ischemia-reperfusion injury (IRI) is a common pathophysiological phenomenon, secondary to multiple pathological processes, such as organ transplantation, acute kidney injury and myocardial infarction. IRI may significantly aggravate the severity of diseases and increase the fatality of patients. Aseptic inflammation is one of the critical mechanisms of IRI. Damage-associated molecular pattern (DAMP) is a pivotal substance, which mediates aseptic inflammation. After released into extracellular space, it could effectively activate the immune system, and initiate and maintain the inflammatory responses by binding with pattern recognition receptor (PRR). Neutrophil extracellular trap (NET) is a DNA-based network structure released by neutrophils during the process of inflammatory responses, which contains histones and multiple granular proteins. Recent studies have demonstrated that DAMP and NET may aggravate IRI via aseptic inflammation. In this article, relevant studies of DAMP, NET and their relationship in IRI were reviewed, which was of great significance for understanding the pathophysiological mechanism of IRI and studying the corresponding prevention and treatment strategies.

Inhibitory effect of silencing HMGB1 gene on growth of human epithelial ovarian cancer xenografts in nude mice / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effect of HMGB1 gene on the growth of human epithelial ovarian cancer xenografts in nude mice, and to lay a foundation for finding new targets for the treatment of ovarian cancer. Methods: Human epithelial ovarian cancer SKOV3 cells in logarithmic growth phase were selected to establish a human epithelial ovarian cancer xenograft model in nude mice. Nude mice with successful model establishment were randomly divided into control group and HMGB1-siRNA group. On the 7th, 9th, 11th, 14th, and 16th days after cell inoculation, the same amount of saline and HMGB1-siRNA were respectively injected into two groups of mice under the armpit.After 3 weeks, the nude mice were sacrificed by cervical dislocation, the tumor tissues were separated, and the volume of the tumor was measured. The apoptosis of transplanted tumor cells was detected by Tunnel staining. The expressions of HMGB1, STAT3 and p-STAT3 were detected by Western blotting. The expression of vascular endothelial growth factorA(VEGF-A) and microvascularization were detected by immunohistochemistry. Results: Compared with the control group, the growth of tumor volume slowed down in HMGB1 siRNA group, and on the 21st day, the tumor volume of HMGB1-siRNA group was significantly smaller than that of the control group (P<0.05). HMGB1-siRNA successfully knocked down the expression of HMGB1 mRNA in transplanted tumor tissue. The apoptosis rate of tissue cells in HMGB1-siRNA group was significantly increased ([34±8]% vs [6±2]%, P=0.04), and the expressions of HMGB1 and p-STAT3 were significantly reduced (P<0.05). The expression of VEGF-Aand the number of microvessels were significantly lower than those of the control group (both P<0.05). Conclusion: Knockdown of HMGB1 gene reduces the expression of VEGF-A and microvessel formation possibly by inhibiting the HMGB1/STAT3 signaling pathway, thereby promoting the apoptosis of tumor tissues and slowing the growth of xenografts.

Circulating high mobility group box-1 and toll-like receptor 4 expressions increase the risk and severity of epilepsy

This study aimed to investigate the association of serum high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) expressions with the risk of epilepsy as well as their correlations with disease severity and resistance to anti-epilepsy drugs. One hundred and five epilepsy patients and 100 healthy controls (HCs) were enrolled in this case-control study, and serum samples were collected from all participants to assess the HMGB1 and TLR4 expressions by enzyme-linked immunosorbent assay (ELISA). Both serum HMGB1 (P<0.001) and TLR4 (P<0.001) expressions were higher in epilepsy patients than in HCs, and they displayed good predictive values for risk of epilepsy. Moreover, HMGB1 was positively correlated with TLR4 level (r=0.735, P<0.001). HMGB1 and TLR4 levels were both elevated in patients with an average seizure duration >5 min compared to patients with a seizure duration ≤5 min (P=0.001 and P=0.014, respectively). Also, HMGB1 and TLR4 were increased in patients with seizure frequency >3 times per month compared to patients with seizure frequency ≤3 times per month (both P=0.001). In addition, HMGB1 and TLR4 expressions were higher in intractable cases compared to drug-responsive cases (P<0.001). In conclusion, both HMGB1 and TLR4 expressions were correlated with increased risk and severity of epilepsy and their level was higher in patients resistant to anti-epilepsy drugs.

Effect of Capsaicin on Proliferation and HMGB1 and IL-6 Expressions in Hepatoma SMMC-7721 Cells / 中国实验方剂学杂志

Objective: To investigate the effect of capsaicin on proliferation in human hepatoma SMMC-7721 cells and its possible molecular mechanism. Method: Capsaicin (50,100,150,200,250,300 μmol·L-1) groups and blank group were set up. The cell viability was detected by cell counting kit-8 (CCK-8) assay after SMMC-7721 cells were treated with capsaicin (50,100,150,200,250,300 μmol·L-1) for 24, 48, 72 h. The morphological changes were observed under an inverted microscope after SMMC-7721 cells were treated with capsaicin (150,200,250 μmol·L-1) for 24 h. The mRNA expression levels of high mobility group box 1 (HMGB1) and interleukin-6(IL-6) were measured by Real-time PCR after SMMC-7721 cells were treated with capsaicin (150,200,250 μmol·L-1) for 24 h. The levels of HMGB1 and IL-6 in cell culture supernatant were detected by enzyme-linked immunosorbent assay(ELISA) after SMMC-7721 cells were treated with capsaicin (150,200,250 μmol·L-1) for 24 h. Result: Compared with the blank group, there was no significant difference between 50 and 100 μmol·L-1 capsaicin groups treated for 24, 48, 72 h; after treated with the other concentrations of capsaicin (150, 200, 250, 300 μmo·L-1) at different time points, the proliferation inhibition rate was statistically significant (P-1) groups showed different degrees of morphological changes in SMMC-7721 cells, which became round and wrinkled, with a poor attachment and more exfoliation; compared with the blank group, the mRNA expressions of HMGB1 and IL-6 in SMMC-7721 cells of capsaicin (150, 200, 250 μmol·L-1) groups were significantly down-regulated (PPConclusion: Capsaicin inhibits cell proliferation of SMMC-7721 cells, and the possible mechanism may be related to the down-regulation of HMGB1 and IL-6 at the mRNA and protein levels.

Inhibitory Effect of Capsaicin on Growth of Breast Cancer MDA-MB-231 Cells Transplanted Tumor in Nude Mice / 中国实验方剂学杂志

Objective: To investigate the inhibitory effect of capsaicin on the growth of breast cancer MDA-MB-231 cells transplanted tumour in nude mice and its possible molecular mechanism. Method: Transplanted tumor model of breast cancer MDA-MB-231 cells in nude mice were established. Then the tumor-bearing mice were randomly divided into 4 groups:model group, and low, medium and high-dose capsaicin groups (5, 10, 20 mg·kg-1). Mice of low, medium and high-dose capsaicin groups (5, 10, 20 mg·kg-1) were intraperitoneally injected with the corresponding dose of capsaicin, and the model group was injected with the same volume of phosphate buffer saline (PBS), once every 3 days, for a total of 8 times in succession. Body weight of mice and transplantation tumor volume were measured before each injection of capsaicin. Mice of each group were put to death 24 h after the last administration, and then the tumor volume, mass and the tumor inhibitory rate were calculated. The protein expression levels of high mobility group box 1 (HMGB1) and Toll-like receptors 4(TLR4) were measured by immunohistochemistry and Western blot. Result: No significant difference was observed between each group in body weight. However, compared with the model group, capsaicin (5, 10, 20 mg·kg-1) remarkably inhibited the tumor volume and mass (PPP-1) also markedly inhibited the protein expression levels of HMGB1 and TLR4 (PConclusion: Capsaicin remarkably inhibits the growth of breast cancer MDA-MB-231 cells transplanted tumour in nude mice, and the possible mechanism may be related to the down-regulation of HMGB1 and TLR4 at the protein level.

Effect of Sanhuang Yinchi Decoction in Preventing Acute Liver Injury by HMGB1 Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the effect and mechanism of Sanhuang Yinchi decoction (SHYCD) in preventing carbon tetrachloride (CCl4)-induced acute liver injury by regulating high mobility group box1(HMGB1) signaling pathway. Method:A total of 48 KM mice were randomly divided into blank control group, model group, low, middle and high-dose SHYCD groups and positive control group. The model of acute liver injury induced by CCl4 in mice was established. The low, middle and high-dose SHYCD groups were intragastrically administered with drugs (16, 32, 48 g·kg-1·d-1) respectively, and the positive control group was given cell growth stimulating hormone (20 mg·kg-1·d-1) through intraperitoneal injection. Pathological changes of mouse liver tissue sections were observed by hematoxylin-eosin staining (HE); relevant enzyme kits were used to determine liver function indexes in mice serum-alanine aminotransferase (AST) and aspartate aminotransferase (ALT); the expression level of interleukin-6 (IL-6) in mouse serum was determined by enzyme-linked immunosorbent assay (ELISA); Western blot was used to detect the expressions of high mobility group box-1(HMGB1), cysteine aspartic acid protease(Caspase-3), apoptosis-related molecules B cell lymphoma(Bcl-2), Bcl-2 associated x protein(Bax), and Toll-like receptor 4 (TLR4). Result:Compared with the normal group, the model group significantly increased serum AST, ALT (PPPPPConclusion:SHYCD can prevent liver injury by regulating HMGB1/TLR4/NF-κB signaling pathway, reducing cellular inflammatory response and inhibiting apoptosis, so as to prevent acute liver injury in mice. This indicates that HMGB1 may become a new target to prevent acute liver injury.

Effects of combined administration of Radix Angelicae Sinensis and hydrocortisone on the therapeutic action in the murine asthma model / 中国应用生理学杂志

OBJECTIVE@#To investigate the effects of Radix Angelicae Sinensis (RASI) and hydrocortisone combination on the murine asthma model and the mechanism.@*METHODS@#BALB/c mice were randomly divided into control group, blood stasis model group, asthma model group, HSS group, RASI group and RASI+HSS group (=12). Ovalbumin (OVA) was used to replicate mice asthma model and hydrocortisone sodium succinate (HSS) to copy blood stasis model. Effects of RASI, HSS and their combination on hemorheology, anti-asthma (asthmatic behaviors, lung function, lung index and water content in lung tissue) were observed. and anti-asthma mechanisms The expression of relative cytokines, high-mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) was detected by ELISA and immunohistochemistry respectively.@*RESULTS@#Eight g/kg RASI, 0.05 g/kg HSS and their combination could significantly relieve asthma behavioral indicators, improve lung function, reduce lung index and water content in lung tissue, decrease the levels of TNF-α, IL-1β and IL-6 in broncho-alveolar lavage fluid (BALF), and inhibit the high expression of HMGB1, TLR4 and NF-κB in lung tissue. The improvement of lung function and the decrease in level of relative cytokines (TNF-α、IL-1βIL-6) were better in RASI+HSS group than those in RASI group and HSS group, and the inhibition of protein expression of TLR4 and NF-κB was also too. Combined administration of RASI and hydrocortisone could decrease serum thromboxane B2 (TXB2) content and blood viscosity, which were increased induced by hydrocortisone.@*CONCLUSIONS@#Combined administration of RASI and hydrocortisone have obvious anti-asthma effects and one of the mechanisms is to inhibit protein synthetization of HMGB1, TLR4 and NF-κB.The combined administration of RASI and hydrocortisone has stronger improvement of lung function than that of RASI and hydrocortisone alone, and it may be related to the inhibition of TLR4 and NF-κB synthetization. The combined administration of RASI can alleviate abnormal changes of hemorheology induced by hydrocortisone in treatment of asthma.

Effect of TTTY10 regulating miR-490-3p on migration and invasion of cervical cancer cell via HMGB1 signaling pathway / 中国肿瘤生物治疗杂志

@# Objective：To investigate the effect of long-chain non-coding RNATTTY10 (lncRNATTTY10) on the migration and invasion of cervical cancer cells, and to explore its regulatory effect on miR-490-3p and HMGB1 (high mobility group box 1) signaling pathways. Methods: Fourteen paris of cervical cancer tissues and corresponding paracancerous tissues resected at the Department of Obstetrics and Gynecology,Affiliated Wuhan Central Hospital of Tongji Medical College fromAugust 2013 to December 2014 were collected for this study. The expression of TTTY10 in cervical cancer tissue and different cervical cancer cell lines were detected by qPCR. Plasmids encoding TTTY10-siRNA or empty plasmids were transfected into cervical cancer CasKicells, and the transfection efficiency was detected by qPCR. Transwell migration assay and Transwell invasion assay were used to detect the migration and invasion abilities of cervical cancer cells after TTTY10 silencing. qPCR was used to detect the expression of miR-490-3p and HMGB1 mRNA after TTTY10 silencing. Dual luciferase reporter assay validated the interaction between miR-490-3p and HMGB1. Western blotting was used to detect the expression of HMGB1 signaling pathway related proteins after TTTY10 silencing. Results: The expression of TTTY10 in cervical cancer tissues was significantly higher than that in paracancerous tissues (P<0.01), the expression of TTTY10 in cervical cancer cell lines was significantly higher than that in cervical epithelial cells (P<0.01). TTTY10-siRNAplasmids could efficiently transfectCasKicells to knockdown TTTY10 expression (P<0.01). Silencing of TTTY10 inhibited the migration and invasion of cervical cancer CasKi cells (P<0.05), promoted the expression of miR-490-3p (P<0.01) and inhibited the expression of HMGB1 mRNAin cervical cancer (P<0.05 or P<0.01). miR-490-3p could specifically bind to the 3'-UTR of HMGB1 mRNA(P<0.01). HMGB1 signaling pathway related proteins were down-regulated after TTTY10 silencing. Conclusion: TTTY10 can target regulate the expression of miR-490-3p and affect the migration and invasion ability of cervical cancer CasKi cells through the HMGB1 signaling pathway; TTTY10 can be used as a diagnostic marker and potential treatment target of cervical cancer.

Relationship of HMGB1 and TLR4 with airway inflammation in asthma and the role of vitamin D / 西安交通大学学报(医学版)

Objective To evaluate the relationship of high mobility group box 1 (HMGB1) and TLR4 with airway inflammation and the role of vitamin D.Methods Totally 24 BALB/c mice were randomly divided into control group,asthma group,and 1,25-(OH)2D3 group,each having 8 mice.The pathological changes in lung tissue of the mice were observed by hematoxylin-eosin (HE) staining,bronchial wall thickness was measured with computer pathological image analysis system software.The expressions of HMGB1 and TLR4 in lung tissue were detected by immunohistochemical method.Bronchoalveolar lavage fluid (BALF) was collected for cytological examination;the contents of HMGB1,TLR4,IL-4 and IFN-γ in BALF and the peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA).Results The expressions of HMGB1 and TLR4 in lung tissue were stronger in asthma group,but weaker in intervention group.The total number of leukocytes as well as the percentages of eosinophils,neutrophils and lymphocytes increased significantly in BALF in asthma group,but significantly decreased in intervention group (all P ＜ 0.05).The ratio of monocyte/macrophage significantly decreased in asthma group,but increased significantly in intervention group (P＜0.05).The contents of HMGB1,TLR4 and IL-4 in BALF and the peripheral blood were significantly higher in asthma group than in control and intervention groups,whereas IFN-γ level was significantly lower than that in control and intervention groups (all P＜0.05).HMGB1 and TLR4 contents had a positive correlation with the total number of cells and IL-4 concentration in BALF,respectively (r1=0.796,0.730;r2=0.695,0.648;all P＜0.05).Conclusion HMGB1 and TLR4 were associated with airway inflammation and immune disorders.An appropriate amount of 1,25-(OH)2D3 can relieve airway inflammation,which may be associated with regulating Th1/Th2 cells balance.

The correlation of HMGB-1 with the halitosis and chronic periodontitis / 实用口腔医学杂志

Objective:To assess the correlation of high mobility group box 1 (HMGB-1) with Organoleptic rating(OR),dental plaque index(PLI) and halitosis with chronic periodontitis.Methods:Serum samples from 20 halitosis patients,20 chronic periodontitis patients with oral malodor and 8 healthy controls were collected.The level of HMGB-1 in the serum was detected by ELISA.The OR and PLI were estimated by organoleptic method and periodontal examination respectively.The data were analyzed with SPSS 17.0.Results:The levels of HMGB1 in the serum from 2 groups of patients was significantly higher than that of the controls(P ＜0.01).The level of HMGB-1 in the patients of halitosis with chronic periodontitis was significantly higher than that of the halitosis patients(P ＜ 0.01).There was no statistical difference of OR between the 2 groups of patients(P ＞ 0.05).The PLI of the chronic periodontitis patients with oral malodor was significantly higher than that of halitosis patients (P ＜ 0.01).The correlation of HMGB1 with OR and PLI of 2 groups of patients were positively correlated(P ＜ 0.05 and 0.01 respectively),the OR of halitosis patients showed no relevant(P ＞0.05).Conclusion:HMGB-1 may be one of the important factors that result in chronic periodontal inflammation and stubborn bad breath.

High-mobility group box protein1 increases tumor necrosis factorαexpression in human bronchial epithelial cells / 实用医学杂志

Objective To investigate the effect of high-mobility group box protein1 (HMGB1) on the expression of TNF-αand its mechanism in 16HBE in vitro. Methods groups with different HMGB1 (0, 100, 500, 2 000 ng/mL) concentration was set; RAGE antagonizing groups were as control, HMGB1-2000ng, anti-RAGE and anti-RAGE+HMGB1. The changes of TNF-αmRNA and secretion were determined by quantitative PCR and ELISA. RAGE protein level was measured by western blotting. Results HMGB1 intervention and TNF-α expression of 16HBE presented a positive dose-dependent relationship. Thechanges of RAGE was HMGB1positively concentration dependent. In comparison with HMGB1 2 000 ng/mL group, anti-RAGE+HMGB1showed a remarkable reduction of TNF-α secretion. Conclusion In vitro, HMGB1 increases TNF-α expression in 16HBE with a dose-dependent manner through RAGE.

Cloning and function analysis of high mobility group box 1(HMGB1)protein of Schistosoma japonicum(Mainland strain) / 中国血吸虫病防治杂志

Objective To clone and express a high mobility group box 1(HMGB1)protein of Schistosoma japonicum(Main-land strain)and analyze its function. Methods The DNA fragment of open reading frame encoding Sj HMGB1 protein was ampli-fied by RT-PCR from the mRNA of S. japonicum worms,then it was subcloned into the expression vector pET28a(+)to form the recombinant expression plasmid SjHMGB1-pET28a. The recombinant expression plasmid was transformed into the component E. coli BL21(DE3),and the tranformant containing recombinant expression plasmid was induced with IPTG to express the recombi-nant protein SjHMGB1. The recombinant SjHMGB1 protein was purified by affinity chromatography with nickel chelating affinity chromatography agarose gel. The Gel retard experiment and animal immunization were performed to analyze the DNA binding ca- pacity and the immunologic property of recombinant SjHMGB1. The expression levels of HMGB1 in different life cycle stages of S. japonicum were analyzed by Western bloting and RT-PCR. Female ICR mice were immunized with the recombinant SjHMGB1 pro-tein and infected with 45±2 cercariae of S. japonicum after three immunizations. Forty-two days post-infection,the worms and eggs of S. japonicum were recovered from the portal vein and liver tissue,respectively. The worm and egg reduction rates were calculat-ed respectively. Results A 530 bp of specific DNA fragment was amplified from mRNA of S. japonicum by RT-PCR,which was the open reading frame(ORF)encoding SjHMGB1protein confirmed by DNA sequencing analysis. The recombinant expression plasmid SjHMGB1-pET28a was constructed by cloning the ORF of SjHMGB1 into a expression vector pET28a(+). The bacterium transformants containing the recombinant plasmid expressed a soluble recombinant protein about 28 kDa after induced by IPTG, and the recombinant SjHMGB1 protein was purified by nickel chelating affinity chromatography. The gel retard experiment showed that the recombinant SjHMGB1 protein could bind to both supercoiled DNA and linear DNA,and the recombinant protein immu-nized mice produced high titers of antiserum IgG. Western bloting indicated that the recombinant SjHMGB1 protein was recognized specifically by the S. japonicum-infected mice serum. Above results showed that the recombinant SjHMGB1 protein possessed both functional activity and immunogenicity as the natural protein. RT-PCR and Western blot results showed that SjHMGB1 was abun-dantly expressed in the adult and egg stages whereas barely detectable in the cercaria stage. The immune protection experiment showed that the recombinant SjHMGB1 induced mice to produce high titers of specific antibody IgG but failed to conduct an effec-tive immune protection against S. japonicum. Conclusion The gene encoding HMGB1 from S. japonicum and the soluble recombi-nant SjHMGB1 protein with natural functional activity are obtained,and the recombinant SjHMGB1 has a high immunogenicity but is not able to induce an effective immune protection against S. japonicum.

Hypoxia Activates Toll-like Receptor 4 Signaling in Primary Mouse Hepatocytes Through the Receptor Clustering within Lipid Rafts

PURPOSE: Transient hypoxia is an initial event that accentuates ischemia/reperfusion (I/R) injury in the liver. Hepatic ischemia/reperfusion (I/R) injury is largely related to innate immunity via Toll-like receptor 4 (TLR4) signaling. However, the mechanism by which hypoxia could lead to activate TLR4 signaling remains unclear. Therefore, the aim of this experimental study investigates how TLR4 signalling is activated by hypoxia. METHODS: Hepatocytes were isolated from male wild-type (C57BL/6) mice (8~12 weeks old) by an in situ collagenase (Type IV, Sigma-Aldrich) perfusion technique. In this study, using primary mouse hepatocytes in culture to 1% oxygen, detection of TLR4 translocation to the lipid rafts on the cell membrane by immunofluorescence staining and immunoblotting was saught. RESULTS: Hypoxia caused TLR4/MD2 and beta2-Integrin (CD11b/CD18) translocation to lipid rafts associated with CD14 in hepatocytes. The cholesterol sequestering agent, Nystatin and Filipin prevented hypoxia-induced TLR4/MD2 translocation to lipid rafts. Consistent with a role for oxidative stress in this effect, in vitro H2O2 treatment of hepatocytes similarly caused TLR4/MD2 translocation to lipid rafts. In addition, translocation of hypoxia-induced TLR4 complex was inhibited by N-acetylcysteine (NAC) demonstrating that the activation of TLR4 signaling is dependent on ROS. Further, the cholesterol sequestering agent, nystatin, prevented hypoxia-induced high mobility group box 1 (HMGB1) release in hepatocytes. CONCLUSION: These results suggest that ROS dependent TLR4 signaling is achieved following receptor translocation to the lipid raft in hepatocytes. We hypothesized that this mechanism is required for the release of HMGB1, an early mediator of injury and inflammation in hepatic I/R injury.

Mechanism and Interference therapy of HMGB1 after Ischemia/Reperfusion Injury / 国际外科学杂志

After operations such as organ transplantation or cardiopulmonary bypass complicated with is-chemia/reperfusion injury,activated inflammatory cells can express and secret HMGB1,and cooperate with other inflammation factor to induce tissue damage.The mechanism is HMGB1 actives such receptors as TLRs,RAGE,TM,and NF-κB transcription factor,P38MAPK pathway,induce releasing of HMGB1 itself and other inflammation factors.Different with sepsis,HMGB1 emerges much earlier,lasting longer.Inter-ference therapy of HMGB1 could effectively decrease secretion of HMGB1 after ischemia/reperfusion injury.