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Chrysin is a natural flavonoid with anti-cancer effects. Despite its beneficial effects, little information is available regarding its immunogenic cell death (ICD) properties. In this work, we hypothesized that chrysin can potentiate radiotherapy(RT)-induced immunogenicity in melanoma cell line (B16-F10). We examined the effects of chrysin alone and in combination with radiation on ICD induction in B16-F10 cells. Cell viability was assessed using an MTT assay. Cell apoptosis and calreticulin (CRT) exposure were determined using flow cytometry. Western blotting and ELISA assay were employed to examine changes in protein expression. Combination therapy exhibited a synergistic effect, with an optimum combination index of 0.66. The synergistic anti-cancer effect correlated with increased cell apoptosis in cancer cells. Compared to the untreated control, chrysin alone and in combination with RT induced higher levels of DAMPs, such as CRT, HSP70, HMGB1, and ATP. The protein expression of p-STAT3/STAT3 and PD-L1 was reduced in B16-F10 cells exposed to chrysin alone and in combination with RT. Conditioned media from B16-F10 cells exposed to mono-and combination treatments elicited IL-12 secretion in dendritic cells (DCs), inducing a Th1 response. Our findings revealed that chrysin could induce ICD and intensify the RT-induced immunogenicity., (© 2024. The Author(s).)

Objective: To analyze and explore the effects of Huayu Jiedu Decoction on the malignant biological characteristics of multiple myeloma (MM) cells and its molecular mechanism, so as to provide experimental basis and theoretical basis for the alternative therapy of anti-MM in traditional Chinese medicine., Methods: Different concentrations of Huayu Jiedu Decoction were used to intervene myeloma U266 cells. The changes of cell proliferation activity were detected by CCK-8 assay, apoptosis was detected by Annexin V/PI double staining flow cytometry, and apoptosis and protein expression of related signaling pathways were detected by Western blot. Real-time quantitative PCR was used to detect mRNA expression changes of high mobility group protein B1 (HMGB1), CXC chemokine receptor 4 (CXCR4) and interleukin-6 (IL-6)., Results: Huayu Jiedu Decoction inhibited the proliferative activity of U266 cells and induced their apoptosis in a concentration and time dependent manner ( r =-0.713, r =-0.827). After treatment with Huayu Jiedu Decoction for 48 h, the expressions of anti-apoptotic protein Bcl-2 and survivin were down-regulated, while the expression of pro-apoptotic protein Bax was up-regulated, and the phosphorylation level of TLR4/NF-κB signaling pathway was inhibited. After intervention of Huayu Jiedu decoction, the expressions of HMGB1 and IL-6 mRNA were significantly decreased, while the expression of CXCR4 was not significantly decreased., Conclusion: Huayu Jiedu Decoction can inhibit the proliferative activity of U266 cells and induce programmed death. Its molecular mechanism may be related to regulating the expression of apoptotic proteins, inhibiting the activation of TLR4/NF-κB pathway and down-regulating the expression of HMGB1 and IL-6 mRNA.

Objective: To explore the effect of quercetin on renal inflammation and cell apoptosis in diabetic rats and its possible mechanisms., Methods: Twenty-four adult male SD rats were randomized equally into normal control group, high-glucose and high-fat feeding group, streptozotocin (STZ) -induced diabetic model group, and quercetin treatment (daily dose 100 mg/kg) group. Pathological changes of the renal tissues of the rats were observed with HE staining, serum inflammatory factor levels were determined with ELISA, and renal expression of NF‑κB was observed by immunohistochemistry. Fast blood glucose (FBG), serum levels of triglyceride (TG), BUN, and Scr, and 24-h urine protein content of the rats were measured, and renal expressions of HMGB1, RAGE, NF‑κB, Bax, Bcl-2, and caspase-3 were detected with Western blotting., Results: The diabetic rats showed significantly increased levels of FBG, TG, BUN, and Scr, renal hypertrophy index, 24-h urinary protein content, serum IL-1β, IL-6 and TNF-α levels and renal expressions HMGB1, RAGE, NF‑κB, Bax, and caspase-3 with decreased renal expression of Bcl-2. All these changes were significantly alleviated by quercetin treatment of the rats., Conclusion: Quercetin can ameliorate kidney injury in diabetic rats possibly by inhibiting the HMGB1/RAGE/NF-κB inflammatory signaling pathway to reduce renal inflammation and renal cell apoptosis.

Tanshinone IIA (Tan IIA), a neuroprotective natural compound extracted from Salvia miltiorrhiza, is used in stroke treatment. However, elucidating Tan IIA's neuroprotective mechanisms remains challenging due to limitations in assessing drug efficacy and biochemical parameters in clinical studies. This study investigated Tan IIA's impact on neuroinflammatory responses and its neuroprotective mechanisms using HMGB1- or TNF-α-stimulated BV2 microglia in a co-culture system with primary neuron cells. The results indicated that Tan IIA significantly reduced microglial activation induced by TNF-α or HMGB1. Concurrently, Tan IIA disrupted the interactions between HMGB1 and toll-like receptor 4 (TLR4), and between TNF-α and TNF receptor 1 (TNFR1), modulating the HMGB1/TLR4/nuclear factor-kappa B (NF-κB) and TNF-α/TNFR1/NF-κB signaling pathways and related protein expressions. Moreover, co-culture experiments showed that neuronal apoptosis induced by microglial activation was reversed by Tan IIA. In conclusion, Tan IIA provides neuroprotection by modulating signaling pathways in microglia, thus preventing neuronal apoptosis. This study offers new insights into therapeutic targets for ischemic stroke., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to dec., (Copyright © 2024. Published by Elsevier Ltd.)

Objectives: Chemoprevention can be a treatment for potentially malignant lesions (PMLs). We aimed to evaluate whether artemisinin (ART) and cisplatin (CSP) are associated with apoptosis and immunogenic cell death (ICD) in vitro, using oral leukoplakia (OL) and oral squamous cell carcinoma (OSCC) cell lines, and whether these compounds prevent OL progression in vivo., Methods: Normal keratinocytes (HaCat), Dysplastic oral cells (DOK), and oral squamous cell carcinoma (SCC-180) cell lines were treated with ART, CSP, and ART + CSP to analyze cytotoxicity, genotoxicity, cell migration, and increased expression of proteins related to apoptosis and ICD. Additionally, 41 mice were induced with OL using 4NQO, treated with ART and CSP, and their tongues were histologically analyzed., Results: In vitro, CSP and CSP + ART showed dose-dependent cytotoxicity and reduced SCC-180 migration. No treatment was genotoxic, and none induced expression of proteins related to apoptosis and ICD; CSP considerably reduced High-mobility group box-1 (HMGB-1) protein expression in SCC-180. In vivo, there was a delay in OL progression with ART and CSP treatment; however, by the 16th week, only CSP prevented progression to OSCC., Conclusion: Expression of proteins related to ICD and apoptosis did not increase with treatments, and CSP was shown to reduce immunogenic pathways in SCC-180, while reducing cell migration. ART did not prevent the malignant progression of OL in vivo; CSP did despite significant adverse effects., (© 2024. The Author(s).)

Acute lung injury (ALI) is a severe inflammatory disorder that has a high morbidity and mortality rate. Urolithin A (UA) is reported to have anti-inflammatory and anti-oxidative effects in ALI. However, its molecular mechanisms in ALI remain to be explored. Mice and BEAS-2B cells were administrated with lipopolysaccharide (LPS) to mimic the ALI model in vivo and in vitro. Hematoxylin-eosin (HE) staining was used to detect the pathological injury of lung tissues. The levels of proinflammatory cytokines in bronchoalveolar lavage fluid (BALF) and culture supernatant and the levels of oxidative stress markers in lung tissues were measured using ELISA. DCFH-DA probe was used to assess the reactive oxygen species (ROS) level. TUNEL staining and flow cytometry were performed to determine cell apoptosis. The key targets and pathways were confirmed by immunohistochemistry (IHC) and western blot. UA suppressed the pathologic damage, wet/dry weight ratio, and total protein and inflammatory cells in BALF. UA decreased neutrophil infiltration and proinflammatory cytokines production. UA reduced the level of malondialdehyde (MDA) and increased the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in pulmonary tissues. UA also inhibited cell apoptosis in lung tissues by decreasing Bax expression and increasing Bcl-2 expression. In addition, UA suppressed LPS-induced inflammatory factor production, ROS level, and cell apoptosis in BEAS-2B. Importantly, UA decreased the expression of HMGB1 in LPS-treated mice and BEAS-2B cells. HMGB1 overexpression greatly abrogated the inhibition of UA on inflammation, ROS, and cell apoptosis in LPS-administrated BEAS-2B. Furthermore, UA treatment suppressed the phosphorylated levels of p38, JNK, ERK, and p65 in LPS-administrated mice and BEAS-2B cells. UA alleviated lung inflammation, oxidative stress, and apoptosis in ALI by targeting HMGB1 to inactivate the MAPK/NF-κB signaling, suggesting the potential of UA to treat ALI., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: The newly discovered CircUBE2D2 has been shown to abnormally upregulate and promote cancer progression in a variety of cancers. The present study explored circUBE2D2 (hsa_circ_0005728) in Ovarian Cancer (OC) progression., Methods: CircUBE2D2, miR-885-5p, and HMGB1 were examined by RT-qPCR or WB. SKOV-3 cell functions (including cell viability, apoptosis, migration, and invasion) were validated using the CCK-8, flow cytometry, scratch assay, and transwell assay, respectively. The direct relationship between miR-885-5p and circUBE2D2 or HMGB1 was confirmed by a dual-luciferase reporter and RNA pull-down analysis. circUBE2D2's role in vivo tumor xenograft experiment was further probed., Results: OC tissue and cell lines had higher circUBE2D2 and HMGB1 and lower miR-885-5p. Mechanically, CircUBE2D2 shared a binding relation with miR-885-5p, while miR-885-5p can directly target HMGB1. Eliminating circUBE2D2 or miR-885-5p induction inhibited OC cell activities. However, these functions were relieved by down-regulating miR-885-5p or HMGB1 induction. Furthermore, circUBE2D2 knockout reduced tumor growth., Conclusion: CircUBE2D2 regulates the expression of HMGB1 by acting as a sponge of ceRNA as miR-885-5p, thereby promoting the control of OC cell proliferation and migration and inhibiting cell apoptosis. Targeting CircUBE2D2 could serve as a new potential treatment strategy for OC., Competing Interests: Conflicts of interest The authors declare no conflicts of interest., (Copyright © 2024 HCFMUSP. Published by Elsevier España, S.L.U. All rights reserved.)

Objective: Endothelial glycocalyx (EG) maintains vascular homeostasis and is destroyed after one-lung ventilation (OLV)-induced lung injury. Long noncoding RNAs (lncRNAs) are critically involved in various lung injuries. This study aimed to investigate the role and regulatory mechanism of KCNQ1 overlapping transcript 1 (KCNQ1OT1) in OLV-induced lung injury and LPS-induced type II alveolar epithelial cell (AECII) apoptosis., Methods: The rat OLV model was established, and the effects of KCNQ1OT1 on OLV-induced ALI in vivo were explored. Bax and Caspase-3 expression in rat lung tissues was measured by immunochemistry (IHC). AECIIs were isolated from rat lungs and treated with LPS or normal saline (control) for in vitro analysis. The expression of KCNQ1OT1, miR-129-5p, and HMGB1 was measured by quantitative real-time PCR (qRT-PCR) or Western blot (WB). Cell proliferation and apoptosis were examined by 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) and flow cytometry. The downstream targets of KCNQ1OT1 were predicted by bioinformatics, and the binding relationship between KCNQ1OT1 and miR-129-3p was verified by dual-luciferase reporter assays. The potential target of miR-129-5p was further explored on the Targetscan website and revealed to target HMGB1. Enzyme-linked immunosorbent assay (ELISA) or WB was adopted to determine the levels of IL-1β, TNF-α, MDA, SOD, heparanase (HPA), matrix metalloproteinase 9 (MMP9), heparan sulfate (HS) and syndecan-1 (SDC-1)., Results: KCNQ1OT1 and HMGB1 were up-regulated during OLV-induced lung injury, and their expression was positively correlated. KCNQ1OT1 knockdown reduced OLV-induced pulmonary edema and lung epithelial cell apoptosis, increased vascular permeability, reduced IL-1β, TNF-α, MDA, and SOD levels and glycocalyx markers by targeting miR-129-5p or upregulating HMGB1. Overexpressing KCNQ1OT1 promoted cell apoptosis, reduced cell proliferation, aggravated inflammation and oxidative stress, and up-regulated HMGB1, HPA and MMP9 in LPS-treated AECIIs, while the HMGB1 silencing showed the opposite effects. MiR-129-5p mimics partially eliminated the KCNQ1OT1-induced effects, while recombinant HMGB1 restored the effects of miR-129-5p overexpression on AECIIs. Additionally, KCNQ1OT1 was demonstrated to promote the activation of the p38 MAPK/Akt/ERK signaling pathways in AECIIs via HMGB1., Conclusion: KCNQ1OT1 knockdown alleviated AECII apoptosis and EG damage during OLV by targeting miR-129-5p/HMGB1 to inactivate the p38 MAPK/Akt/ERK signaling. The findings of our study might deepen our understanding of the molecular basis in OLV-induced lung injury and provide clues for the targeted disease management., (© 2024 Wiley Periodicals LLC.)

Neutrophils and T lymphocytes are closely related to occurrence of immunosuppression in sepsis. Studies have shown that neutrophil apoptosis decreases and T lymphocyte apoptosis increases in sepsis immunosuppression, but the specific mechanism involved remains unclear. In the present study, we found Toll-like Receptor 2 (TLR2) and programmed death-ligand 1 (PD-L1) were significantly activated in bone marrow neutrophils of wild-type mice after LPS treatment and that they were attenuated by treatment with C29, an inhibitor of TLR2. PD-L1 activation inhibits neutrophil apoptosis, whereas programmed death protein 1 (PD-1)activation promotes apoptosis of T lymphocytes, which leads to immunosuppression. Mechanistically, when sepsis occurs, pro-inflammatory factors and High mobility group box-1 protein (HMGB1) passively released from dead cells cause the up-regulation of PD-L1 through TLR2 on neutrophils. The binding of PD-L1 and PD-1 on T lymphocytes leads to increased apoptosis of T lymphocytes and immune dysfunction, eventually resulting in the occurrence of sepsis immunosuppression. In vivo experiments showed that the HMGB1 inhibitor glycyrrhizic acid (GA) and the TLR2 inhibitor C29 could inhibit the HMGB1/TLR2/PD-L1 pathway, and improving sepsis-induced lung injury. In summary, this study shows that HMGB1 regulates PD-L1 and PD-1 signaling pathways through TLR2, which leads to immunosuppression., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Intervertebral disc degeneration (IDD) is one of the most common disorders related to the spine. Inflammation, apoptosis and extracellular matrix (ECM) degradation contribute to disc degeneration in nucleus pulposus cells (NPCs). This study focused on the role and mechanism of the p38 inhibitor TAK-715 in intervertebral disc degeneration., Methods: NPCs were treated with IL-1β to mimic apoptosis, followed by the addition of TAK-715. It was determined that apoptosis, inflammatory mediators (COX-2), inflammatory cytokines (HMGB1), and ECM components (collagen II, MMP9, ADAMTS5, and MMP3) existed in NPCs. In addition, the p38MAPK signaling pathways were examined. The role of TAK-715 in vivo was determined by acupuncture-induced intervertebral disc degeneration. Following an intradiscal injection of TAK-715, MRI and a histopathological analysis were conducted to assess the degree of degeneration., Results: IL-1β-induced apoptosis was alleviated by TAK-715 in vitro, and antiapoptotic proteins were upregulated. Furthermore, TAK-715 blocked IL-1β-induced inflammatory mediator production (COX-2) and inflammatory cytokine production (HMGB1) and degraded the ECM (collagen II, MMP9, ADAMTS5, and MMP3). By inhibiting the phosphorylation of p38, TAK-715 exerted its effects. In a rat tail model, TAK-715 ameliorates puncture-induced disc degeneration based on MRI and histopathology evaluations., Conclusion: TAK-715 attenuated intervertebral disc degeneration in vitro and in vivo, suggesting that it might be an effective treatment for IDD., (© 2023. The Author(s).)

Dasatinib, a second-generation BCR-ABL inhibitor, is currently used as first-line treatment for patients with chronic myeloid leukemia. However, dasatinib treatment increases the risk of severe cutaneous toxicity, which limits its long-term safe use in clinic. The underlying mechanism for dasatinib-induced cutaneous toxicity has not been clarified. In this study, we tested the toxicity of dasatinib on human immortal keratinocyte line (HaCaT) and normal human epidermal keratinocytes (NHEK). We found that dasatinib directly caused cytotoxicity on keratinocytes, which could be the explanation of the clinical characteristic of pathology. Mechanistically, dasatinib impaired mitophagy by downregulating HMGB1 protein level in keratinocytes, which led to the accumulation of dysfunctional mitochondria. Mitochondria-derived ROS caused DNA damage and cell apoptosis. More importantly, we confirmed that overexpression of HMGB1 could reverse dasatinib-induced keratinocyte apoptosis, and preliminarily explored the intervention effect of saikosaponin A, which could increase HMGB1 expression, on cutaneous toxicity caused by dasatinib. Collectively, our study revealed that dasatinib induced keratinocyte apoptosis via inhibiting HMGB1-mediated mitophagy and saikosaponin A could be a viable strategy for prevention of dasatinib-induced cutaneous toxicity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objectives: The lung injury is often secondary to severe trauma. In the model of crush syndrome, there may be secondary lung injury. We hypothesize that high-mobility group box 1 (HMGB1), released from muscle tissue, mediates the apoptosis of alveolar epithelial cells (AEC) via HMGB1/Receptor of advanced glycation end-products (RAGE)/c-Jun N-terminal kinase (JNK) pathway. The study aimed to investigate how HMGB1 mediated the apoptosis of AEC in the rat model., Methods: Seventy-five SD male rats were randomly divided into five groups: CS, CS + vehicle, CS + Ethyl pyruvate (EP), CS + FPS-ZM1 group, and CS + SP600125 groups. When the rats CS model were completed after 24 h, the rats were sacrificed. We collected the serum and the whole lung tissues. Inflammatory cytokines were measured in serum samples. Western blot and RT-qPCR were used to quantify the protein and mRNA. Lastly, apoptotic cells were detected by TUNEL. We used SPSS 25.0 for statistical analyses., Results: Nine rats died during the experiments. Dead rats were excluded from further analysis. Compared to the CS group, levels of HMGB1 and inflammatory cytokines in serum were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups. Western blot and RT-qPCR analysis revealed a significant downregulation of HMGB1, RAGE, and phosphorylated-JNK in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups, compared with the CS groups, excluding total-JNK mRNA. Apoptosis of AEC was used TUNEL to assess. We found the TUNEL-positive cells were downregulated in CS + EP, CS + FPS-ZM1, and CS + SP600125 groups., Conclusion: The remote lung injury begins early after crush injuries. The HMGB1/RAGE/JNK signaling axis is an attractive target to abrogate the apoptosis of AEC after crush injuries., (© 2022. The Author(s).)

Background: Diabetic nephropathy (DN) is a severe microvascular complication of diabetes. Recently, many circular RNAs can exert crucial roles in DN progression. This study intended to explore the role and mechanism of circ_0000491 in DN., Methods: The DN mouse model was constructed by streptozotocin injection, and the DN cell model was established using high glucose (HG) treatment in mouse mesangial cells (SV40-MES13). The expression of circ_0000491 and microRNA-455-3p (miR-455-3p) was detected by quantitative real-time polymerase chain reaction. Cell apoptosis was evaluated by flow cytometry. The expression levels of high-mobility group box 1 (Hmgb1) protein, apoptosis-related proteins, and fibrosis-related proteins were examined by the Western blot assay. The release of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. The oxidative stress factors were analyzed by corresponding kits. The predicted interaction between miR-455-3p and circ_0000491 or Hmgb1 was verified by dual-luciferase reporter assay and RNA immunoprecipitation assay., Results: Circ_0000491 was overexpressed in the DN mouse model and HG-induced SV40-MES13 cells. Knockdown of circ_0000491 weakened HG-induced apoptosis, inflammation, oxidative stress, and fibrosis in SV40-MES13 cells. miR-455-3p was a direct target of circ_0000491, and miR-455-3p inhibition could reverse the role of circ_0000491 silencing in HG-induced SV40-MES13 cells. Moreover, Hmgb1 was a target gene of miR-455-3p, and miR-455-3p played a protective role against HG-induced cell injury by targeting Hmgb1. In addition, circ_0000491 regulated Hmgb1 expression by sponging miR-455-3p., Conclusion: Circ_0000491 knockdown inhibited HG-induced apoptosis, inflammation, oxidative stress, and fibrosis in SV40-MES13 cells by regulating miR-455-3p/Hmgb1 axis., (© 2021 S. Karger AG, Basel.)

Corilagin is the primary active component of the Euphorbia phyllanthus plant and has significant anti‑cancer properties. However, the biological effects and mechanisms of corilagin on acute myeloid leukemia (AML) have not been clarified. The Cell Counting Kit‑8 and Carboxyfluorescein Diacetate Succinimidyl Ester assay results showed that corilagin significantly inhibited proliferation of the AML cell line HL‑60 in a time‑ and dose‑dependent manner. Western blotting and flow cytometry analysis were performed to determine the levels of apoptosis in HL‑60 cells. The protein levels of cleaved caspase‑3 and Bak were upregulated, while Bcl‑xl was downregulated in cells treated with corilagin. The percentage of early‑ and late‑stage apoptotic cells increased following corilagin treatment in a dose‑dependent manner, indicating that the intrinsic mitochondrial apoptosis pathway was activated by corilagin. Simultaneously, western blotting and immunofluorescence results revealed that autophagy was suppressed; this was accompanied by a decrease in light chain 3‑II (LC3‑II) conversion and autophagosomes. MicroRNA (miRNA/miR) profile analysis showed that corilagin elevated the expression of the tumor suppressor miR‑451, while the mRNA and protein levels of high mobility group protein B1 (HMGB1), the target of miR‑451, decreased following exposure to corilagin. Knockdown of miR‑451 decreased the downregulation of HMGB1 caused by corilagin, indicating negative regulation of HMGB1 by miR‑451 during corilagin treatment. Furthermore, knockdown of miR‑451 also attenuated corilagin‑induced proliferation inhibition of HL‑60 cells, implying that miR‑451 was essential for the proliferation inhibitory effect of corilagin. In conclusion, these results indicated that corilagin induced apoptosis and inhibited autophagy in HL‑60 cells by regulating the miR‑451/HMGB1 axis, and corilagin may be a novel therapeutic drug for the treatment of AML.

Objective: To explore the therapeutic effects of quercetin on rats with encephalitis, especially on cell apoptosis, and the levels of HMGB-1 and TLR-4., Materials and Methods: 32 healthy rats were equally assigned into ZC group (healthy group), NY group (encephalitis group), DJ group (60 ml quercetin group), and GJ group (240 ml quercetin group) followed by analysis of cell apoptosis, brain tissue water content, neurons, HMGB-1, TLR-4 and other inflammatory factors., Results: The ZC group showed normal neuron volume and equitable staining; compared with ZC group, NY group showed neuron volume shrinkage and chromatin condensation; the neuron and color in DJ group were slightly better than NY group; the neuron volume in GJ group increased significantly and chromatin is distributed evenly. TLR-4, IL-4, IL-6, HMGB-1 in ZC and DJ group were significantly lower than those in NY group (p<0.05); IL-4, IL-6, HMGB-1 in GJ group significantly decreased compared with DJ group (p<0.05); MMP-9 enzyme activity in ZC and DJ group was significantly lower than NY group (p<0.05) with lower level in GJ group than DJ group (p<0.05). The water content was higher in brain tissue of NY group than ZC group (p<0.05) and lower in DJ group than NY group (p<0.05) with lower level in GJ group than DJ and NY group (all p<0.05). The hippocampal neurons and cortical neurons in ZC and DJ group were higher than those in NY group (p<0.05) and elevated in GJ group compared with DJ group and NY group (p<0.05)., Conclusions: Quercetin is effective in treating encephalitis rats possibly through inhibition of neuronal cell apoptosis and level of HMGB-1 and TLR-4.

Objectives: Secondary spinal cord injury (SCI), a reversible pathological change, involves neural inflammation and apoptosis. This study explored how microRNA (miR)-488, an inflammatory regulator as reported affected secondary SCI., Methods: In vivo, Wistar rats were clipped on the spinal cord for SCI induction. In vitro, PC-12 cells were treated with lipopolysaccharide (LPS) to induce cell injuries to mimic the environment during the secondary SCI. Cell viability and apoptosis were measured by CCK-8 assay and flow cytometry. The levels of inflammation-related factors (interleukin (IL)-6, IL-1β and tumor necrosis factor (TNF)-α) in the serum and PC-12 cells were determined by ELISA. The expressions of miR-488, high mobility group box 1 (HMGB1), B-cell lymphoma 2 (Bcl-2), Bcl-2-associated X protein (Bax), cleaved caspase-3, toll-like receptor 4 (TLR4), phosphorylated (p)-p65 and total-p65 in rat spinal cord or PC-12 cells were analyzed by quantitative reverse transcription PCR or western blot., Results: After SCI induction, rats exhibited low Basso-Beattie-Bresnahan scores, promoted the release of inflammation-related factors and downregulated miR-488. LPS treatment decreased cell viability, enhanced apoptosis and downregulated miR-488. Upregulating miR-488 neutralized LPS-induced releases of inflammation-related factors and expressions of Bax and cleaved caspase-3 and counteracted LPS-induced inhibition on Bcl-2 expression. MiR-488 directly targeted HMGB1 and miR-488 mimic decreased LPS-induced HMGB1 expression. Overexpressing HMGB1 counteracted miR-488 mimic-induced decreases in the expressions of TLR4 and p-p65 and the ratio of p-p65 to Total-p65 in LPS-treated PC-12 cells., Conclusion: MiR-488 inhibited neural inflammation and apoptosis in SCI via its binding with HMGB1-mediated restraint on the TLR4/NF-κB signaling pathway., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

The p53 tumor suppressor gene contributes to a series of life processes of cells. Previously, we have shown that T-17, a spirostanol saponin extracted from Tupistra chinensis induces cell cycle arrest, apoptosis and autophagy in gastric cancer cells. The p53 is essential in the cell cycle arrest induced by T-17, however, the effect of p53 on T-17-induced apoptosis and autophagy is still unclear. Here, our study shows that T-17 has no difference in the sensitivity of gastric cancer cells with different p53 status. By transfecting p53 siRNA into AGS cells (p53 wild type cells) or wild-type p53 into KATO-III cells (p53 deficiency cells), T-17 was found to induce apoptosis and autophagy in gastric cancer cells in a p53-independent manner. Pre-treatment with N-acetylcysteine (NAC, a ROS scavenger) demonstrated that reactive oxygen species (ROS) mediated T-17-induced p53-independent apoptosis. Besides, T-17 induces apoptosis and autophagy in gastric cancer cells by decreasing the expression of HMGB1, also in a p53-independent manner. But when we detected the inhibitory effect of T-17 on gastric cancer cell migration, it was found that p53 is essential. These experimental results showed that T-17 induced apoptosis and autophagy in gastric cancer cells in a p53-independent manner, but inhibited the migration of gastric cancer cells in a p53-dependent manner. Our research indicates that T-17 is a potential candidate for gastric cancer and provides support for better utilization of Tupistra chinensis., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Fluvastatin, a traditional fat-decreasing drug, is widely used for curing cardiovascular disease. Previous reports demonstrated that fluvastatin pretreatment protected against myocardial ischemia/reperfusion (I/R) by inhibiting TLR4 signaling pathway and/or reducing proinflammatory cytokines. However, whether fluvastatin has a cardioprotective effect against apoptosis and autophagy remains unknown. This study aims to evaluate whether the cardioprotective role of fluvastatin in I/R is mediated by high-mobility group box 1 (HMGB1)/toll-like receptor 4 (TLR4) pathway via anti-apoptotic and anti-autophagic functions. Sprague-Dawley rats were anesthetized, artificially ventilated and subjected to 30 min of coronary occlusion, followed by 4 h of reperfusion. The animals were randomized into four groups: (i) Sham operation; (ii) I/R; (iii) I/R + low-dosage fluvastatin (10 mg/kg); and (iv) I/R + high-dosage fluvastatin (20 mg/kg). After reperfusion, the hemodynamic parameters, myocardial infarct size, structural alteration of myocardium, apoptosis index, pro-inflammatory cytokine production, Beclin-1, Light chain 3 (LC3), HMGB1, TLR4 and Nuclear factor kappa B (NF-κB) protein levels were measured and recorded. It was found that fluvastatin preconditioning improved left ventricular dysfunction, reduced HMGB1/TLR4/NF-κB expressions, and inhibited cardiomyocyte apoptosis, autophagy, and inflammation reaction. Moreover, treatment with fluvastatin ameliorated myocardial injury by reducing infarct size, causing less damage to cardiac structure, downregulating autophagy-related protein expression and releasing pro-inflammation mediators. Our findings indicate that fluvastatin exerts beneficial effects on cardiac ischemic damage, which may be associated with its anti-autophagic and anti-apoptotic functions via inhibition of HMGB1/TLR4-related pathway during I/R injury.

High mobility group box-1 (HMGB1) participates actively in oxidative stress damage, and the latter relates closely to diabetes and diabetic complications including osteoporosis, though the underlying mechanisms are elusive. This study aimed to investigate the effect of high glucose on bone marrow stromal cells (BMSCs) apoptosis and the role of HMGB1 in this process. BMSCs were isolated from 2-week-old Sprague-Dawley rats and cultured in medium containing normal glucose (NG), high glucose (HG), high glucose + glycyrrhizin (HMGB1 inhibitor, HG+GL), and high glucose + glycyrrhizin + dorsomorphin (AMPK inhibitor, HG+GL+Dm), respectively. Cell apoptosis, expression of HMGB1, AMPK, apoptotic markers, and mitochondrial functions were detected. By these approaches, we demonstrated that HG treatment significantly upregulated the expression of HMGB1 in BMSCs, which could be attenuated by GL treatment. Inhibiting HMGB1 by GL improved AMPK activation, decreased mitochondrial ROS levels, increased mitochondrial membrane potential, normalized mitochondrial fission/fusion balance, and consequently reduced apoptosis of BMSCs under HG condition. The addition of AMPK inhibitor dorsomorphin hampered this protective effect. Taken together, our data show that inhibition of HMGB1 can be an effective approach to alleviate HG-induced BMSCs apoptosis by activation of AMPK pathway and relieving mitochondrial dysfunction.

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage-associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour-suppressive mechanism., Competing Interests: LJ, GN, GW, NR, GC, DS, SB, JM, LM, MM, AR, AD, MO No competing interests declared, (© 2021, Julian et al.)

Endotoxin lipopolysaccharide (LPS) is one of the primary causes of myocardial injury. Propofol confers protective effects against LPS‑induced myocardial damage; however, the biological functions and mechanisms underlying propofol are not completely understood. The present study aimed to investigate the effects of propofol on LPS‑induced myocardial injury. Primary neonatal rat cardiomyocytes were treated with LPS to establish a myocardial injury model. LDH release in the culture media was measured using a LDH assay kit. The interactions between NLR family pyrin domain containing 3 (NLRP3), apoptosis‑associated speck‑like protein containing A CARD (ASC) and pro‑caspase‑1 were determined using a co‑immunoprecipitation assay. Cell viability was measured using an MTT assay, and the levels of cell apoptosis were determined using flow cytometry, JC‑1 staining (mitochondrial membrane potential) and caspase‑3 activity assays. The mRNA expression levels of TNF‑α, IL‑6, IL‑1β and IL‑18, and the protein expression levels of NLRP3, ASC, pro‑caspase‑1, caspase‑1 p10, pro‑IL‑1β, IL‑1β, pro‑IL‑18, IL‑18, high mobility group box‑1 (HMGB1) and peroxisome proliferator‑activated receptor γ (PPARγ) were analyzed using reverse transcription‑quantitative PCR and western blotting analyses, respectively. ELISAs were performed to measure the production of inflammatory mediators, including TNF‑α, IL‑6, IL‑1β and IL‑18. The present results demonstrated that pretreatment with propofol significantly attenuated LPS‑induced neonatal rat cardiomyocyte injury in a concentration‑ and time‑dependent manner. Propofol pretreatment also significantly inhibited LPS‑induced cardiomyocyte inflammation and apoptosis. The results suggested that propofol pretreatment inactivated HMGB1‑dependent NLRP3 inflammasome signaling, which involved PPARγ activation. Therefore, the results indicated that propofol reduced endotoxin‑induced cardiomyocyte injury by inhibiting inflammation and apoptosis via the PPARγ/HMGB1/NLRP3 axis, suggesting that propofol may serve as a potential therapeutic agent for septic myocardial damage.

Anti-inflammatory therapy for early brain injury after subarachnoid hemorrhage is a promising treatment for improving the prognosis. HMGB1 is the initiator of early inflammation after subarachnoid hemorrhage. Oleanolic acid is a natural pentacyclic triterpenoid compound with strong anti-inflammatory activity. It can relieve early brain injury in subarachnoid hemorrhage rats, but its mechanism is not very clear. Here, we study the potential mechanism of Oleanolic acid in the treatment of subarachnoid hemorrhage. First, we demonstrated that oleanolic acid alleviated early brain injury after subarachnoid hemorrhage, including improvement of grading score, neurological score, brain edema and permeability of brain blood barrier. Then we found that oleanolic acid could inhibit the transfer of HMGB1 from nucleus to cytoplasm and reduce the level of serum HMGB1. Furthermore, we found that oleanolic acid decreased the acetylation level of HMGB1 by increasing SIRT1 expression rather than by inhibiting JAK/STAT3 pathway. SIRT1 inhibitor sirtinol eliminated all beneficial effects of oleanolic acid on subarachnoid hemorrhage, which indicated that oleanolic acid inhibited the acetylation of HMGB1 by up regulating SIRT1. In addition, oleanolic acid treatment also reduced the levels of TLR4 and apoptosis related factors and reduced neuronal apoptosis after subarachnoid hemorrhage. In summary, our findings suggest that oleanolic acid may activate SIRT1 by acting as an activator of SIRT1, thereby reducing the acetylation of HMGB1, thus playing an anti-inflammatory role to alleviate early brain injury after subarachnoid hemorrhage., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Long non-coding RNAs (lncRNAs) are bound up with the regulation of various diseases. Here, we probed into the effect of lncRNA colorectal neoplasia differentially expressed (CRNDE) on heart failure (HF). The pathological alterations and cell apoptosis of heart tissues were observed by hematoxylin-eosin and TUNEL staining. The viability or apoptosis of mouse myocardial cells HL-1 was tested by XTT or flow cytometry. The interaction between lncRNA CRNDE and poly-ADP-ribose polymerase 1 (PARP-1) was verified by RNA immunoprecipitation and RNA pull-down. The stability of the PARP-1 protein and the acetylation level of high mobility group box-1 (HMGB1) were determined by cycloheximide-chase and immunoprecipitation, respectively. LncRNA CRNDE expression was decreased in HF mice tissues and doxorubicin (Dox)-treated HL-1 cells, whereas PARP-1 and HMGB1 were increased. The overexpression of lncRNA CRNDE restrained HL-1 cell apoptosis induced by Dox. Moreover, the interaction between CRNDE and PARP-1 was corroborated, CRNDE negatively regulated PARP-1 expression, and the overexpression of CRNDE reduced PARP-1 protein stability. In HL-1 cells, PARP-1 positively regulated the acetylation level and cytoplasm translocation of HMGB1. CRNDE restrained Dox-induced apoptosis in mouse myocardial cells via the PARP-1/HMGB1 pathway.

The toxicity of doxorubicin (DOX) limits its clinical application. Nevertheless, at present, there is no effective drug to prevent DOX-induced cardiac injury. miR-204 is a newly discovered miRNA with many protective effects on cardiovascular diseases. However, little research has been done on the effects of miR-204 on DOX-induced cardiac injury. Our study is aimed at investigating the effect of miR-204 on DOX-induced myocardial injury. An adenoassociated virus system was used to achieve cardiac-specific overexpression of miR-204. Two weeks later, the mice were intraperitoneally injected with DOX (15 mg/kg) to induce cardiac injury. H9c2 myocardial cells were used to validate the role of miR-204 in vitro. Our study showed that miR-204 expression was decreased in DOX-treated hearts. miR-204 overexpression improved cardiac function and alleviated cardiac inflammation, apoptosis, and autophagy induced by DOX. In addition, our results showed that miR-204 prevented DOX-induced injury in cardiomyocytes by directly decreasing HMGB1 expression. Moreover, the overexpression of HMGB1 could offset the protective effects of miR-204 against DOX-induced cardiac injury. In summary, our study showed that miR-204 protected against DOX-induced cardiac injury via the inhibition of HMGB1, and increasing miR-204 expression may be a new treatment option for patients with DOX-induced cardiac injury., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Youyou Du et al.)

Background: Both apoptosis and necroptosis have been recognized to be involved in ischemia reperfusion-induced lung injury. We aimed to compare the efficacies of therapies targeting necroptosis and apoptosis and to determine if there is a synergistic effect between the two therapies in reducing lung ischemia reperfusion injury., Methods: Forty Sprague-Dawley rats were randomized into 5 groups: sham (SM) group, ischemia reperfusion (IR) group, necrostatin-1+ischemia reperfusion (NI) group, carbobenzoxy-Val-Ala-Asp-fluoromethylketone+ischemia reperfusion (ZI) group, and necrostatin-1+carbobenzoxy-Val-Ala-Asp-fluoromethylketone+ischemia reperfusion (NZ) group. The left lung hilum was exposed without being clamped in rats from the SM group, whereas the rats were subjected to lung ischemia reperfusion by clamping the left lung hilum for 1 hour, followed by reperfusion for 3 hours in the IR group. 1 mg/kg necrostatin-1 (Nec-1: a specific necroptosis inhibitor) and 3 mg/kg carbobenzoxy-Val-Ala-Asp-fluoromethylketone (z-VAD-fmk: a pan caspase inhibitor) were intraperitoneally administrated prior to ischemia in NI and ZI groups, respectively, and the rats received combined administration of Nec-1 and z-VAD-fmk in the NZ group. Upon reperfusion, expressions of receptor-interacting protein 1 (RIP1), receptor-interacting protein 3 (RIP3), and caspase-8 were measured, and the flow cytometry analysis was used to assess the cell death patterns in the lung tissue. Moreover, inflammatory marker levels in the bronchoalveolar lavage fluid and pulmonary edema were evaluated., Results: Both Nec-1 and z-VAD-fmk, either alone or in combination, significantly reduced morphological damage, inflammatory markers, and edema in lung tissues following reperfusion, and cotreatment of z-VAD-fmk with Nec-1 produced the optimal effect. The rats treated with Nec-1 had lower levels of inflammatory markers in the bronchoalveolar lavage fluid than those receiving z-VAD-fmk alone ( P < 0.05). Interestingly, the z-VAD-fmk administration upregulated RIP1 and RIP3 expressions in the lung tissue from the ZI group compared to those in the IR group ( P < 0.05). Reperfusion significantly increased the percentages of necrotic and apoptotic cells in lung tissue single-cell suspension, which could be decreased by Nec-1 and z-VAD-fmk, respectively ( P < 0.05)., Conclusions: Nec-1 synergizes the pan caspase inhibitor to attenuate lung ischemia reperfusion injury in rats. Our data support the potential use of Nec-1 in lung transplantation-related disorders., Competing Interests: The authors declared that there was no conflict of interest regarding the publication of this paper., (Copyright © 2020 Liangrong Wang et al.)

Allergic rhinitis (AR) is an allergic disease characterized by immunoglobulin E (IgE)-mediated type I hypersensitivity disorder. In the current study, we illuminated the potential roles of microRNA-141-3p (miR-141-3p) in lipopolysaccharide (LPS)-induced mucus production and the apoptosis in nasal epithelial cells (NECs). We demonstrated that miR-141-3p was significantly downregulated in nasal tissues from patients with AR and LPS-treated NECs. Upregulation of miR-141-3p decreased the level of mucin 5AC (MUC5AC) in LPS-treated NECs and induced NECs apoptosis. High Mobility Group Box 1 (HMGB1) was proved as a target of miR-141-3p and miR-141-3p negatively regulated its expression. In addition, we observed that HMGB1 was overexpressed in nasal mucosal tissues from patients with AR and LPS-treated NECs. Finally, we proved that miR-141-3p decreased the level of MUAC5AC in LPS-treated NECs through regulating HMGB1. In conclusion, miR-141-3p inhibited LPS-induced MUAC5AC production and the apoptosis of LPS-treated NECs by targeting HMGB1., (© 2020 The Authors. The Kaohsiung Journal of Medical Sciences published by John Wiley & Sons Australia on behalf of Kaohsiung Medical University.)

Objective: High-fat-diet- (HFD-) induced hepatic cell apoptosis is common in mice with nonalcoholic fatty liver disease (NAFLD). We aim to investigate the effect of Ginsenoside Rb1 (GRb1) on hepatocyte apoptosis., Methods: C57BL/6J mice with HFD were used to induce a liver-injured model with cell apoptosis. In addition, GRb1 was used to treat HFD-induced apoptosis in a liver with or without inhibitor of peroxisome proliferator-activated receptor γ (PPAR- γ )., Results: Compared with C57BL/6J mice with common chow, there are downregulated PPAR- γ but upregulated cell apoptosis in the liver of mice with HFD. Furthermore, GRb1 elevated the hepatic PPAR- γ level and suppressed hepatocytic apoptosis. However, GW9662 abolished the effects of GRb1 on apoptosis in the liver., Conclusions: GRb1 alleviated HFD-induced apoptosis of hepatocytes of mice via PPAR- γ ., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2020 Bing Song et al.)

Objective: Acute lung injury (ALI) is the most common organ damage in sepsis and sepsis-induced ALI is a clinically extremely dangerous disease. Therefore, it is essential to find an effective way to treat ALI. We hope to provide a new target for the treatment of clinical ALI by studying the effect of GDF11 on LPS-induced ALI., Materials and Methods: C57BL/6 male mice and lipopolysaccharide (LPS) were used to induce mouse ALI. Recombinant GDF11 protein was used to treat mice to detect the effect of GDF11 on mouse ALI. In addition, BEAS-2B cells were used to further validate the effects of GDF11 on inflammation and apoptosis of alveolar epithelial cells., Results: Recombinant GDF11 protein significantly reduced the expression of inflammatory factors and apoptosis-related pathways in mouse lung tissues. Overexpression of GDF11 in BEAS-2B cells also significantly attenuated the levels of inflammation and apoptosis in the cells. In addition, GDF11 can reduce the activity of TLR2/HMGB1/NF-κB signaling pathway, which is an important mechanism for GDF11 to play a role in lung protection., Conclusions: GDF11 can exert lung protection effects by inhibiting the TLR2/HMGB1/NF-κB signaling pathway and reduce the level of inflammation and apoptosis of the lung.

Antineoplastic drugs cause severe cytotoxicity for normal cells, especially hematopoietic stem cells (HSCs). However, bleomycin (BLM) is glycopeptide antibiotic that is effective on various cancers and has either low or no myelosuppression effects. The aim of the present study was to investigate the effect of BLM on 5-Azacitidine (5-AZA) induced cytotoxicity in bone marrow HSCs. 5-AZA reduced HSC cell viability in a time and dose-dependent manner with an IC 50 value of 16 μM. However, pretreatment of the cells with BLM for 4 h induced an antagonistic cytotoxicity with an increased IC 50 of 64 μM. 5-AZA decreased the colony formation ability of HSC cells in semi-solid agar culture and this effect was attenuated by BLM. 5-AZA significantly downregulated high mobility group Box1 (HMGB1) and Bcl-2 gene expression but upregulated Bax gene expression, while BLM impeded the action of 5-AZA. Pretreatment with BLM remarkably decreased HMGB1 release into culture media that was induced by 5-AZA. The cells were distribution at the sub/G1 phase. Annexin/PI staining of the cells, poly (ADP-ribose) polymerase (PARP) cleavage, and anion superoxide production indicated that BLM limited 5-AZA induced apoptotic cell death. In conclusion, BLM in combination with 5-AZA effectively reduces the adverse cytotoxic effects of 5-AZA on bone marrow hematopoietic stem cells, providing a new chemotherapeutic strategy., Competing Interests: Declaration of Competing Interest The authors declare that there is no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: Acute liver injury (ALI) is mainly characterized by the symptom of metabolic disorders, homeostasis unbalance, and loss of liver function. There are no effective treatment methods at present stage except the liver transplantation. Effective treatment for early ALI is of great significance for the treatment of liver injury thereof. Glycyrrhizin (GL) is a promising inhibitor of the high-mobility group box-1 gene (HMGB1) which is expressed much higher in an inflammatory injury. However, it is not clear whether GL improves ALI via the inhibition of HMGB1. The present study is to probe the function and mechanism of glycyrrhizin on acute liver injury., Materials and Methods: The expression of HMGB1 and inflammation in liver macrophages were analyzed. Lipopolysaccharide (LPS) was used in stimulating the macrophages to activate inflammatory response and recombined human HMGB1 was used to resist the function of GL to explore whether GL acted via the target of HMGB1. Then, LPS injection was utilized to induce ALI in mice, and then we evaluated GL treatment in ALI model., Results: The results showed that the expressions of HMGB1 and inflammatory factors were markedly increased in LPS-activated liver macrophages. GL inhibited the progress of macrophages inflammation by restraining HMGB1, and the administration of GL could reverse the effects of LPS-induced ALI in mice. Moreover, PI3K/mTOR pathway was significantly suppressed by GL application., Conclusions: These results suggest that GL prevents inflammation in liver macrophages via inhibition of HMGB1. GL restrains inflammation and cell apoptosis by inhibiting HMGB1 via PI3K/mTOR signaling pathway in ALI. GL may become a novel drug for the therapy of ALI in the future.

Knee osteoarthritis (KOA) is a common joint disease with a high incidence rate among middle‑aged and elderly individuals. However, the precise underlying pathological mechanisms and effective treatment of this disease remain to be determined. To explore the effect of high mobility group box 1 (HMGB1) on chondrocyte apoptosis and catabolism, the ATDC5 cell line was cultured as an in vitro model for cartilage research. Cultured cells were treated with recombinant HMGB1 at different concentrations. Hoechst staining and flow cytometry demonstrated that HMGB1 administration significantly induced apoptosis of ATDC5 cells, which was the same as the effect of interleukin‑1β treatment. HMGB1 also induced cartilage matrix degradation, as shown by Alcian blue staining. Moreover, HMGB1 markedly upregulated the expression levels of matrix metallopeptidases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS), while genetic silencing of HMGB1 significantly suppressed their expressions. The glycogen synthase kinase (GSK)‑3β/β‑catenin pathway was activated upon HMGB1 treatment. Pharmacological inhibitors or HMGB1 knockdown inactivated the GSK‑3β/β‑catenin pathway, inhibited the expression levels of downstream genes, including MMPs and ADAMTS, and attenuated the apoptosis of ATDC5 cells. Furthermore, the data demonstrated that HMGB1 promoted chondrocyte dysfunction via the regulation of estrogen sulfotransferase and Runt‑related transcription factor 2. Thus, the findings of the present study demonstrated that HMGB1 induces chondrocyte cell apoptosis via activation of GSK‑3β/β‑catenin and the subsequent expression of multiple targeted genes.

BACKGROUND Spinal cord injury (SCI) is a serious nervous system injury, causing extremely low quality of life and immensurable economic losses. However, there is few therapies that can effectively cure the injury. The goal of the present study was to explore the potential therapeutic effects of dihydrotanshinone I (DI) for SCI and the involving mechanism. MATERIAL AND METHODS A SCI rat model was structured to investigate the effects of DI on recovery of SCI. Tarlov's scale was employed to assess the neuronal function and histopathological examination was carried out by hematoxylin and eosin staining. In addition, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-1ß, inducible nitric oxide synthase (iNOS), total oxidant status (TOS) and total antioxidant status (TAS) levels were detected. Tunel assay and western blot analysis were performed to evaluate cell apoptosis. Furthermore, western blot assay was used to measure the protein expressions. RESULTS The results demonstrated that the treatment of DI alleviated the pathological damage induced by SCI and promoted the neuronal functional recovery. DI suppressed TNF-alpha, IL-1ß, IL-6, iNOS, and TOS levels while improved the TAS level. Moreover, increased cell apoptosis in SCI rats was inhibited by administration of DI. Most importantly, DI reserved the soaring of TLR4, MyD88, HMGB1, and NOX4 level after induction of SCI. Thus, the observation revealed that the HMGB1/TLR4/NOX4 pathway may be involved in the protective effects of DI on SCI. CONCLUSIONS In conclusion, the findings suggest that DI alleviates SCI by restraining secretion of inflammatory factors, and occurrence of oxidative stress and apoptosis in vivo. DI may be developed into an effective alternative therapy for SCI in clinic.

Chondrocytes in joints are responsible for the formation and remodeling of articular cartilage. The accumulation of advanced glycation end products (AGEs) in cartilage is detrimental to the survival of chondrocytes. Linagliptin is one of the most commonly used anti-diabetes agents, and recent work indicates that it exerts an anti-inflammatory effect in different cell types. In this study, we showed that Linagliptin had a protective role in AGEs-induced chondrocyte injury. The presence of Linagliptin ameliorated AGEs-induced reactive oxygen species (ROS) induction and reduced cellular protein carboxyl content. Linagliptin mitigated AGEs-induced mitochondrial membrane potential ( Δ Ψm) reduction and NAPDH oxidase subunit NOX-4 induction, indicating that Linagliptin is a potent anti-ROS agent in chondrocytes. Additionally, Linagliptin inhibited AGEs-induced production of high mobility group box chromosomal protein 1 (HMGB-1), and the expression of matrix metalloproteases (MMPs)-2 and -9. Flow cytometry experimentation showed that Linagliptin inhibited AGEs-induced apoptotic subpopulation. Moreover, Linagliptin inhibited the AGEs-induced increased ratio of Bax to Bcl-2, translocation of cytochrome C from mitochondria to the cytoplasm, and release of cleaved caspase-3. Collectively, our data indicate that the anti-diabetes drug Linagliptin has a new role in rescuing chondrocyte from insult by AGEs, and may, therefore, have the potential to treat joint disorders., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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

Acetaminophen (APAP)-induced acute liver failure (ALF) is a life-threatening disease with only a few treatment options available. Though extensive research has been conducted for more than 40 years, the underlying pathomechanisms are not completely understood. Here, we studied as to whether APAP-induced ALF can be prevented in mice by silencing the BH3-interacting domain death agonist (Bid) as a potential key player in APAP pathology. For silencing Bid expression in mice, siRNA Bid was formulated with the liver-specific siRNA delivery system DBTC and administered 48 h prior to APAP exposure. Mice which were pre-treated with HEPES (vehicle HEPES ) and siRNA Luci served as siRNA controls. Hepatic pathology was assessed by in vivo fluorescence microscopy, molecular biology, histology and laboratory analysis 6 h after APAP or PBS exposure. Application of siRNA Bid caused a significant decrease of mRNA and protein expression of Bid in APAP-exposed mice. Off-targets, such as cytochrome P450 2E1 and glutathione, which are known to be consumed under APAP intoxication, were comparably reduced in all APAP-exposed mice, underlining the specificity of Bid silencing. In APAP-exposed mice non-sterile inflammation with leukocyte infiltration and perfusion failure remained almost unaffected by Bid silencing. However, the Bid silencing reduced hepatocellular damage, evident by a remarkable decrease of DNA fragmented cells in APAP-exposed mice. In these mice, the expression of the pro-apoptotic protein Bax, which recently gained importance in the cell death pathway of regulated necrosis, was also significantly reduced, in line with a decrease in both, necrotic liver tissue and plasma transaminase activities. In addition, plasma levels of HMGB1, a marker of sterile inflammation, were significantly diminished. In conclusion, the liver-specific silencing of Bid expression did not protect APAP-exposed mice from microcirculatory dysfunction, but markedly protected the liver from necrotic cell death and in consequence from sterile inflammation. The study contributes to the understanding of the molecular mechanism of the APAP-induced pathogenic pathway by strengthening the importance of Bid and Bid silencing associated effects.

Background: Podocyte injury, characterized by podocyte hypertrophy, apoptosis, and epithelial-mesenchymal transition (EMT), is the major causative factor of diabetic nephropathy (DN). Autophagy dysfunction is regarded as the major risk factor for podocyte injury including EMT and apoptosis. High mobility group box 1 (HMGB1) is involved in the progression of DN through the induction of autophagy. However, the underlying mechanism remains unknown., Methods: Plasma HMGB1 concentrations were determined in DN patients using ELISA. Apoptosis of DN serum-treated podocytes was evaluated by flow cytometry. Podocyte autophagy flux was measured using immunofluorescence. Western blotting analysis was used to investigate HMGB1 expression, EMT, and autophagy-related signaling pathways., Results: Upregulation of HMGB1 was found in DN patients and DN serum-treated podocytes. Removal of HMGB1 inhibited DN serum-mediated podocyte apoptosis by inhibiting autophagy and activating AKT/mammalian target of rapamycin (mTOR) signaling. In addition, HMGB1 depletion repressed the progression of podocyte EMT by inhibiting transforming growth factor (TGF)-β/smad1 signaling in vitro and in vivo. The combination of HMGB1 short interference (si) RNA and the autophagy activator rapamycin protected against podocyte apoptosis and EMT progression by inhibiting the AKT/mTOR and TGF-β/smad signaling pathway, respectively., Conclusions: Although HMGB1 siRNA and rapamycin treatment had opposite effects on autophagy and AKT/mTOR signaling, there was no contradiction about the role of HMGB1 siRNA and rapamycin on AKT/mTOR pathway because autophagy and AKT/mTOR signaling play dual roles in intracellular biological processes. Based on the findings of this study, we may assume that HMGB1-initiated autophagy is harmful, whereas rapamycin is beneficial to podocyte survival. Possibly combined treatment with HMGB1 siRNA and rapamycin improved podocyte damage and EMT by regulating autophagy homeostasis., (© 2019 Ruijin Hospital, Shanghai Jiaotong University School of Medicine and John Wiley & Sons Australia, Ltd.)

High mobility group box-1 (HMGB1) is involved in various diseases and is associated with the resistance of many types of human cancers to chemotherapy; however, its role in cancer metastasis remains unexplored. This study examined the HMGB1 status of both highly and poorly metastatic cancer cells in response to genotoxic stress. The weakly and highly metastatic mouse melanoma cell lines (B16 vs . B16-F10), human melanoma cell lines (SK-MEL-28 vs . SK-MEL-24), colon cancer cell lines (DLD-1 vs . LS174T), and wild-type (WT) vs. HMGB1 knockout (KO) mouse embryonic fibroblasts (MEFs) were treated with doxorubicin (Dox) and camptothecin (CPT), and then cellular morphology, senescence-associated β-galactosidase staining, lactate dehydrogenase release, and caspase-3 activation were used to assess cell fate. To investigate the role of HMGB1 in p21 expression, HMGB1 and p21 expressions were examined by Western blotting, and the HMGB1-mediated p21 promoter luciferase assay was performed after small interfering RNA or overexpression of HMGB1 prior to Dox treatment. Although highly metastatic mouse melanoma B16-F10 cells preferred senescence, with persistent HMGB1 expression, poorly metastatic B16 cells entered apoptosis, with decreasing HMGB1 levels via cleavage under Dox treatment. Similarly, more metastatic human melanoma SK-MEL-24 and human colon cancer LS174T cells underwent senescence, whereas fewer metastatic melanoma SK-MEL-28 and DLD-1 cells exhibited apoptosis under Dox stimulation. In senescent B16-F10, SK-MEL-24, and LS174T cells treated with Dox, p21 levels were increased by persistent HMGB1 expression. Furthermore, HMGB1 depletion caused a senescence-apoptosis shift with p21 down-regulation in B16-F10 cells, and HMGB1 overexpression switched from apoptosis to senescence concomitantly with increased p21 expression in B16 cells after Dox treatment. The same effects were observed in both cell pairs of mouse melanoma and human colon cancer cells treated with CPT, another genotoxic stressor. Indeed, although WT MEF entered senescence accompanied by p21 increase, HMGB1 KO underwent apoptosis with p21 decrease by Dox treatment. In our cell model system, we demonstrated that highly metastatic cancer cells preferentially enter senescence, whereas apoptosis predominates in weakly metastatic cancer cells under genotoxic stress, which depends on the presence or absence of HMGB1, suggesting that the HMGB1-p21 axis is required for genotoxic stress-induced senescence. These findings suggest that HMGB1 modulation of cancers with different metastatic status could be a strategy for selectively enforcing tumor suppression.-Lee, J.-J., Park, I. H., Rhee, W. J., Kim, H. S., Shin, J.-S. HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.

Objective: High-Mobility Group Box 1 (HMGB1) promotes vascular injuries induced by limb Ischemia and Reperfusion (IR), but the molecular mechanisms are not well understood. This study aimed to investigate the role of Receptor for Advanced-Glycation End products (RAGE) in HMGB1-regulated inflammatory response and vascular injury in limb IR using the rat IR and cellular Hypoxia and Reoxygenation (HR) models., Methods: We analyzed the vascular structure and elastic fiber deposition in rat femoral arteries by histological staining. We determined gene expression in vascular tissues and cells by quantitative RT-PCR, Western blotting and immunofluorescence; analyzed the protein levels in rat serum or cell supernatant by ELISA; and assessed protein interaction by co-immunoprecipitation. We used CCK-8 for analyzing cell viability, and assessed apoptosis by Hoechst staining and flow cytometry., Results: RAGE inhibition by FPS-ZM1 significantly repressed rat vascular injury that was induced by limb IR treatment. HMGB1 and RAGE expression, P38, ERK1/2, P65 and IKBa phosphorylation, as well as HIF-1a, NLRP3, Caspase-1, TNF-a and IL-6 expression and P65 in nucleus, increased in femoral arteries of a rat IR model and HUVEC undergoing HR treatment, whereas all the factors except HMGB1 and RAGE were inhibited by FPS-ZM1 treatment. In addition, we found that HMGB1 binds with RAGE in HUVEC undergoing HR treatment, which was suppressed by FPS-ZM1. Finally, the apoptosis of HUVEC also increased by HR treatment, but repressed under FPS-ZM1 treatment., Conclusion: HMGB1 binds with RAGE to promote vascular inflammation and endothelial cell apoptosis, which mediates vascular injury during acute limb IR., (Copyright © 2019. Published by Elsevier Masson SAS.)

As an inhibitor of high mobility group protein B1 (HMGB1), ethyl pyruvate (EP) has been associated with various inflammatory diseases. Recent studies have investigated the relationship between EP and cancer. The present study aimed to determine the antitumor efficacy of EP in non‑small cell lung cancer (NSCLC) cells and elucidate the underlying mechanism. A549, H520 and PC‑9 cells were treated with EP in suitable concentrations. RT‑qPCR and western blot analysis were performed to evaluate HMGB1 and RAGE expression levels. MTT and colony formation assays assessed the effect of EP on cell growth. A Transwell assay was used to evaluate invasion and migration potential and flow cytometry was performed to analyze cell apoptosis. Bcl‑2 family proteins were identified by western blot analysis. The results demonstrated that an increased EP concentration effectively reduced HMGB1 and RAGE expression, thus inhibiting the HMGB1/RAGE axis. EP decreased level of PCNA and MMP‑9 and increased P53 levels. Bcl‑2 and Mcl‑1 were also decreased, whereas Bax expression was increased. Furthermore, a high concentration of EP (30 mmol/l) significantly inhibited NF‑κB and STAT3 activation. In summary, EP inhibited NSCLC cell growth, invasion and migration and induced apoptosis by suppressing the HMGB1/RAGE axis and the NF‑κB/STAT3 pathway, thus suggesting that EP may be a valuable therapeutic agent for NSCLC.

Hepatic ischemia reperfusion (IR) injury (IRI) occurs during liver transplantation, hepatectomy, and hemorrhagic shock. Oleanolic acid (OA) is a natural compound with antioxidant and anti-inflammatory activity that has been used to treat liver disorders in clinical practice for several years. Here, we investigated the effects and underlying mechanisms of OA in hepatic IRI. A 60-minute partial (70%) hepatic, warm, ischemic reperfusion model was established in BALB/c mice, and two doses (30 and 60 mg/kg) of OA were administered intragastrically for 7 consecutive days prior to hepatic IR. Orbital blood and liver specimens were collected at 2, 8, and 24 h after IR. The results showed that OA preconditioning significantly alleviated hepatic injury, as evidenced by decreased alanine aminotransferase and aspartate aminotransferase levels; improved histology, inhibition of JNK phosphorylation, and high mobility group box 1 (HMGB1); and tumor necrosis factor- α downregulation in hepatic IR mice. OA upregulated Bcl-2 and downregulated caspase-3, caspase-9, Bax, Beclin 1, and LC3, which play crucial roles in the regulation of apoptosis and autophagy. These findings highlighted the protective effects of OA against hepatic IRI mediated by the inhibition of apoptosis and autophagy and the release of HMGB1, which acted as a late inflammatory mediator in hepatic IRI.

Receptor-interacting protein kinase 3 (RIPK3) is a key regulator of programmed cell death and inflammation during viral infection or sterile tissue injury. Whether and how bacterial infection also activates RIPK3-dependent immune responses remains poorly understood. Here we show that bacterial lipids (lipid IVa or lipid A) form a complex with high mobility group box 1 (HMGB1), released by activated immune cells or damaged tissue during bacterial infection, and that this complex triggers RIPK3- and TIR domain-containing adapter-inducing IFN-β (TRIF)-dependent immune responses. We found that these responses lead to macrophage death, interleukin (IL)-1α release, and IL-1β maturation. In an air-pouch inflammatory infiltration model, genetic deletion of Ripk3 , Trif, or IL-1 receptor ( Il-1R ), or monoclonal antibody-mediated HMGB1 neutralization uniformly attenuated inflammatory responses induced by Gram-negative bacteria that release lipid IVa and lipid A. These findings uncover a previously unrecognized mechanism by which host factors and bacterial components work in concert to orchestrate immune responses., (© 2019 Meng et al.)

Autophagy, which works to remove stress and maintain cellular homeostasis, is usually considered a "pro-survival" signal. Contrarily, apoptosis is programmed "pro-death" machinery. Polychlorinated biphenyls (PCBs) are a group of ubiquitous industrial pollutants. Our previous studies illustrated that a PCB quinone metabolite, PCB29-pQ, elicited both autophagy and apoptosis. However, the signaling underlying the autophagy and apoptosis cross-talk has not been characterized. Here, we found that PCB29-pQ-induced autophagy mainly occurred at a lower concentration (5 μM), while apoptosis mostly arose at a higher concentration (15 μM) in HepG2 cells. Next, we demonstrated the elevation of intracellular calcium levels and calpain activity with PCB29-pQ treatment; however, the unaffected subcellular location of truncated ATG5 and Beclin1 suggested the irrelevance of calpain towards the autophagy-to-apoptosis signaling shift. HMGB1 and p53 both serve as transcription factors that play crucial roles in the regulation of PCB29-pQ-induced autophagy and apoptosis. PCB29-pQ not only enhanced the expression of HMGB1 and p53 but also promoted their binding and cytosolic translocation. Interestingly, HMGB1 rather than p53 plays a primary role in 5 μM of PCB29-pQ-induced autophagy in the nucleus; however, p53 promoted apoptosis to a great extent in the cytosol at the dose of 15 μM PCB29-pQ. Together, HMGB1 and p53 provided a subtle balance between autophagy and apoptosis, thus determining the fate of PCB29-pQ-treated cells., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Purpose: Aloin (ALO), a bioactive ingredient extracted from aloe vera, has anti-tumor effects. High Mobility Group Box 1 (HMGB1), a highly conserved nuclear DNA-binding protein, has been implicated in various cancer types. Highly expressed HMGB1 is closely associated with tumor cells apoptosis, proliferation and migration. We investigated the specific molecular mechanisms by which ALO-induced apoptosis by targeting HMGB1 in gastric cancer cells. Materials and methods: Human gastric cancer HGC-27 cells were treated with different doses of ALO (100, 200 and 400 µg/ml) for 24 h, after which DAPI staining was used to observe the nuclear morphology, Annexin V/PI double staining assay was used to determine the rate of apoptosis; Western blotting was used to detect the levels of PARP, pro-caspase3, HMGB1 and RAGE; nuclear translocation of HMGB1 was determined by conducting a nucleoplasm separation experiment. The Enzyme linked immunosorbent assay (ELISA) assay was used to detect release of HMGB1. The HGC-27 cells, transfected with HMGB1 shRNA plasmids, were stimulated with ALO for 24 h, after which a flow cytometry assay was used to detect the rate of apoptosis. HGC-27 cells were pre-treated with or without ALO and then stimulated with rhHMGB1, the phosphorylation of Akt, mTOR, P70S6K, S6, 4EBP1, ERK, P90RSK, cAMP regulatory element binding (CREB) were detected by Western blotting. Results: After different doses of ALO treatment, the nuclei showed morphological changes characteristic of apoptosis. Apoptotic rates were enhanced in a dose dependent manner. The level of cleaved PARP was enhanced and pro-caspase3, HMGB1 and RAGE levels were reduced, HMGB1 nuclear translocation and release were inhibited. The activation of rhHMGB1-induced Akt-mTOR-P70S6K and ERK-CREB signalling pathways was inhibited by ALO. Blocking these signalling pathways by special inhibitors and HMGB1 knockdown could enhance ALO-induced HGC-27 cell apoptosis. Conclusion: ALO- induced HGC-27 cell apoptosis by down-regulating expressions of HMGB1 and RAGE, inhibiting HMGB1 release and then suppressing rhHMGB1-induced activation of Akt-mTOR-P70S6K and ERK-P90RSK-CREB signalling pathways., Competing Interests: The authors report no conflicts of interest in this work.

Changes in the expression and subcellular localization of high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) molecule, have been implicated in tumorigenesis and tumor cell death in response to cancer therapy. Specifically, HMGB1 release has been shown to occur with a specific form of induced cell death known as necroptosis. In the present study, we examined the role of HMGB1 in the necroptosis of acute myeloid leukemia (AML) cells. In two AML cell lines and primary AML cells from two patients, etoposide induced necroptosis via cIAP1/2 degradation when caspase activity was inhibited by Z-VAD-fmk, but treatment with extracellular HMGB1 prevented this necroptosis. Interestingly, HMGB1 did not prevent the degradation of cIAP1/2, but rather activated the nuclear factor kappa B pathway. The results of the present study provide evidence that extracellular HMGB1 is not only an important DAMP molecule released by cells upon necrosis, but also a regulatory factor that prevents necroptosis in AML cells., (Copyright © 2019 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

The traditional view holds that apoptosis is non-immunogenic and does not induce an inflammatory response. However, recent studies have suggested that certain chemotherapeutic drugs that induce tumor cell apoptosis can induce immunogenic cell death (ICD) in cancer cells. This process is characterized by not only up-regulation of a series of signaling molecules in cancer cells, including expose of calreticulin (CRT), secretion of adenosine triphosphate (ATP) and release of high mobility group box 1 (HMGB1). In this review, we summarize recent progress in identifying and classifying ICD inducers; concepts and molecular mechanisms of ICD; and the impact and potential applications of ICD in clinical studies. We also discuss the contributions of ICD inducers in combination with other anticancer drugs in clinical applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Increased high mobility group box 1 (HMGB1) in dialysis effluence is associated with the presence of peritoneal dialysis-related peritonitis in patients and peritoneal dysfunction in acute peritonitis mice model, but it remains unclear whether HMGB1 is involved in peritoneal mesothelial cell injury and functions via molecular posttranslational modifications by acetylation in this process. Here we first showed correlation between HMGB1 acetylation level in dialysis effluence of patients and occurrence of Gram-negative peritonitis. The increased level of acetylated HMGB1 was similarly observed under the lipopolysaccharides (LPS) treatment in both human peritoneal mesothelial cell line (HMrSV5) and mice visceral peritoneum tissue. Overexpression of wild-type, but not hypoacetylation mutant of HMGB1, enhanced LPS-induced apoptosis in HMrSV5 cells, which was accompanied by elevated protein levels of BAX and cleaved-caspase 3 compared to the control. Pretreatment of HMrSV5 cell with JNK inhibitor attenuated LPS-induced HMGB1 acetylation. Consistently, primary peritoneal mesothelial cells from Jnk1 mice showed a lower protein contents of acetylated HMGB1, fewer apoptosis, and decreased protein expression of BAX and cleaved-caspase3 after LPS exposure, as compared to those from wild-type mice. In conclusion, our data demonstrated HMGB1 promotes LPS-induced peritoneal mesothelial cells apoptosis, which is associated with JNK1-mediated upregulation of HMGB1 acetylation. -/- mice showed a lower protein contents of acetylated HMGB1, fewer apoptosis, and decreased protein expression of BAX and cleaved-caspase3 after LPS exposure, as compared to those from wild-type mice. In conclusion, our data demonstrated HMGB1 promotes LPS-induced peritoneal mesothelial cells apoptosis, which is associated with JNK1-mediated upregulation of HMGB1 acetylation.

This study was design to investigate preventive function of Tongxinluo (TXL) capsule on micro vascular function and endothelial survival in rats model of intestine ischemia/reperfusion (I/R) injury. We randomly divided fifty male Sprague-Dawley rats into Sham group, I/R group, TXL0.4+I/R group, TXL0.8+I/R group, TXL1.6+I/R group (10 rats each). Rat intestine I/R injury was carried out using a model of acute superior mesenteric artery occlusion with 30 min ischemia followed by 60 min reperfusion. The distribution of endothelial apoptosis in intestine was determined by CD31+TUNEL immunofluorescent double staining analysis. VE-Cadherin, ANGPTL4, HMGB1 and NF-κB were determined by immunohistochemical analysis. I/R induced massively endothelial cell apoptosis, accompanied with reduced expression of adherens junction protein VE-Cadherin and up regulation of inflammatory mediator HMGB1 and NF-κB. TXL pretreatment groups (TXL0.4+I/R, TXL0.8+I/R and TXL1.6+I/R group) significantly attenuated endothelial cell apoptosis with a dose-dependent effect. TXL pretreatment could maintain the expression of VE-Cadherin and promote the expression of ANGPTL4 which help to maintain endothelial integrity. TXL pretreatment also exert great influence in inhibiting HMGB1 expression and NF-κB expression induced by I/R. It could be concluded from this study that micro vascular dysfunction and endothelial damage play a causal role in rat intestine I/R injury. TXL pretreatment could significantly prevent the I/R induced pathology of endothelial apoptosis, micro vascular integrity disruption and inflammatory reaction.

Necroptosis is an inflammatory form of cell death that depends on receptor-interacting serine-threonine kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) and displays the morphological characteristics of necrosis. To date, it is unclear to what extent necroptosis contributes to subarachnoid hemorrhage (SAH) induced brain injury. The present study aimed to investigate the RIPK3-mediated necroptosis and the effects of the RIPK3 selective inhibitor GSK'872 in early brain injury following SAH. After SAH, RIPK3 expression increased as early as 6 h and peaked at 72 h. Double immunofluorescence staining revealed that RIPK3 was mainly located in neurons. Most necrotic cells were neurons, which were further confirmed by TEM. Intracerebroventricular injection of GSK'872 (25 mM) could attenuate brain edema and improve neurological function following SAH and reduce the number of necrotic cells. In addition, GSK'872 could also decrease the protein levels of RIPK3 and MLKL, and cytoplasmic translocation and expression of HMGB1, an important pro-inflammatory protein. Taken together, the current study provides the new evidence that RIPK3-mediated necroptosis is involved in early brain injury and GSK'872 decreases the RIPK3-mediated necroptosis and subsequent cytoplasmic translocation and expression of HMGB1, as well as ameliorates brain edema and neurological deficits., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Necroptosis is a regulated form of necrosis that depends on receptor-interacting protein kinase (RIPK)3 and mixed lineage kinase domain-like (MLKL). While danger-associated molecular pattern (DAMP)s are involved in various pathological conditions and released from dead cells, the underlying mechanisms are not fully understood. Here we develop a fluorescence resonance energy transfer (FRET) biosensor, termed SMART (a sensor for MLKL activation by RIPK3 based on FRET). SMART is composed of a fragment of MLKL and monitors necroptosis, but not apoptosis or necrosis. Mechanistically, SMART monitors plasma membrane translocation of oligomerized MLKL, which is induced by RIPK3 or mutational activation. SMART in combination with imaging of the release of nuclear DAMPs and Live-Cell Imaging for Secretion activity (LCI-S) reveals two different modes of the release of High Mobility Group Box 1 from necroptotic cells. Thus, SMART and LCI-S uncover novel regulation of the release of DAMPs during necroptosis.

Ischemia-reperfusion (I/R) injury is a pathological process which magnifies with the ensuing inflammatory response and endures with the increase of oxidants especially during reperfusion. The present study was conducted to assess the possible modulatory effects of plumbagin, the active constituent extracted from the roots of traditional medicinal plant Plumbago zeylanica L., on the dire role of high mobility group box 1 (HMGB1) as well as the associated inflammation, oxidative stress and apoptotic cell death following hepatic I/R. Four groups of rats were included: sham-operated, sham-operated treated with plumbagin, I/R (30 min ischemia and 1 h reperfusion) and I/R treated with plumbagin. Pretreatment with plumbagin markedly improved hepatic function and structural integrity compared to the I/R group, as manifested by depressed plasma transaminases and lactate dehydrogenase (LDH) activities as well as alleviated tissue pathological lesions. Plumbagin prominently hampered HMGB1 expression and subsequently quelled inflammatory cascades, as nuclear factor κB (NF-κB), tumor necrosis factor-alpha (TNF-α) and myeloperoxidase (MPO) activity. It also interrupted reactive oxygen species (ROS)-HMGB1loop as evident by restored liver reduced glutathione (GSH), elevated glutathione peroxidase (GPx) activity, along with decreased liver lipid peroxidation. Simultaneously, plumbagin significantly ameliorated apoptosis by amending the mRNA expressions of both anti-apoptotic (Bcl-2) and pro-apoptotic (Bax). The present results revealed that plumbagin is endowed with hepatoprotective activity ascribed to its antioxidant, anti-inflammatory and anti-apoptotic properties which are partially mediated through dampening of HMGB1 expression., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)