Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 1. Serum and urine HMGB1 levels upon diagnosis of sepsis. (A) Serum and (B) urine levels of HMGB1 were measured by ELISA in the different patient groups prior to the initiation of treatment. Data are presented as the mean ± standard deviation. #P<0.05 vs. Control. HMGB1, high mobility group box protein 1; CRRT, continuous renal replacement treatment; HP, hemoperfusion.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Biomarker Discovery, Enzyme-linked Immunosorbent Assay, Standard Deviation, Control

Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 2. Changes in serum and urine HMGB1 levels over 24 h. (A) Serum and (B) urine levels of HMGB1 in the sepsis (no apheresis intervention) group at different time points. Data are presented as the mean ± standard deviation. #P<0.05 vs. 0‑h value. HMGB1, high mobility group box protein 1.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Standard Deviation

Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 3. Association of serum HMGB1 level with urine HMGB1 level and APACHE II score. Correlations were identified between (A) urine and serum HMGB1 levels, and (B) APACHE II score and serum HMGB1 level in the sepsis (no apheresis intervention) group. HMGB1, high mobility group box protein 1; APACHE II, Acute Physiology and Chronic Health Evaluation II.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques:

Journal: Experimental and therapeutic medicine

Article Title: Blood purification treatment initiated at the time of sepsis diagnosis effectively attenuates serum HMGB1 upregulation and improves patient prognosis.

doi: 10.3892/etm.2017.4854

Figure Lengend Snippet: Figure 4. Changes in serum and urine HMGB1 levels in the different patient groups over 24 h. (A) Serum and (B) urine levels of HMGB1 were measured by ELISA at different time points after the initiation of treatments. Data are presented as the mean ± standard deviation. #P<0.05 vs. 0‑h value; *P<0.05 vs. sepsis group; ^P<0.05 vs. 12‑h value. HMGB1, high mobility group box protein 1; CRRT, continuous renal replacement treatment; HP, hemoperfusion.

Article Snippet: Serum and urine HMGB1 levels were measured using a Human HMGB1 ELISA kit (E-EL-H1554c, Elabscience Biotechnology Co., Ltd., Wuhan, China) according to the manufacturer's protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Standard Deviation

Journal: Journal of Cellular Physiology

Article Title: Akt‐independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS‐CoV2 infection

doi: 10.1002/jcp.30343

Figure Lengend Snippet: TCBN suppresses HMGB1‐induced ACE2 expression in A549 cells. (a) and (b) Western blot images and bar graphs of band densitometry analysis of ACE2 expression induced by various doses of HMGB1 treatment in A549 cells. (c)–(e) Western blot images and bar graphs of band densitometry analysis of 10 nM HMGB1‐induced ACE2 expression and Akt phosphorylation, and the effect of TCBN in A549 cells, respectively. Data are shown as mean ± SEM . ACE2, angiotensin‐converting enzyme 2; SEM , standard error of mean; TCBN, triciribine

Article Snippet: HMGB1 (Cat No. 1690‐HMB‐050) was obtained from R&D Systems®, and d ‐Glucose (Cat No. D16‐500) was obtained from Thermo Fisher Scientific.

Techniques: Expressing, Western Blot, Phospho-proteomics

Journal: Journal of Cellular Physiology

Article Title: Akt‐independent effects of triciribine on ACE2 expression in human lung epithelial cells: Potential benefits in restricting SARS‐CoV2 infection

doi: 10.1002/jcp.30343

Figure Lengend Snippet: Schematic representation of the potential effects of HMGB1, hyperglycemia, and TCBN on ACE2 expression and SARS‐CoV2 binding to lung epithelial cells. (a) HMGB1 or high glucose‐induced AGE (advanced glycation end‐products) promotes the expression of ACE2 receptors, thereby increasing the risk of SARS‐CoV‐2 interaction with the lung epithelial cell surface ACE2 leading to their internalization and infection. (b) Whereas Akt inhibitor MK‐2206 does not affect the expression of ACE2 receptors, treatment with TCBN blunts HMGB1, and hyperglycemia‐induced ACE2 expression in lung epithelial cells potentially in an Akt‐independent but adenosine‐receptor dependent mechanism. ACE2, angiotensin‐converting enzyme 2; SARS‐CoV‐2, severe acute respiratory syndrome coronavirus 2; TCBN, triciribine

Article Snippet: HMGB1 (Cat No. 1690‐HMB‐050) was obtained from R&D Systems®, and d ‐Glucose (Cat No. D16‐500) was obtained from Thermo Fisher Scientific.

Techniques: Expressing, Binding Assay, Infection

Journal: OncoImmunology

Article Title: Carbon ion radiotherapy triggers immunogenic cell death and sensitizes melanoma to anti-PD-1 therapy in mice

doi: 10.1080/2162402x.2022.2057892

Figure Lengend Snippet: Figure 1. CIRT-induced immunogenic cell death in vitro. Human osteosarcoma U2OS cells were irradiated by X-ray and carbon ion radiotherapy (CIRT), and biomarkers of immunogenic cell death were evaluated at 6 h and 24 h after irradiation. Mitoxantrone (MTX, 1 µM) was used as a prototype immunogenic cell death inducer Flow cytometry, immunofluorescence, and immunoblot assays showed that CIRT induced CALR exposure and elevated the phosphorylation level of eIF2α, enhanced ATP release by quinacrine staining and ELISA, augmented HMGB1 exodus from the nucleus, and significantly up-regulated the expression of type I interferon at the mRNA level. Representative images and quantification are shown (mean ± SD of triplicate assessments, Student’s t test, **p < .01, ***p < .001).

Article Snippet: The HMGB1 (#E-EL-M0676c) and IFN-γ (#E-EL-M0048c) ELISA kits were purchased from Elabscience (Wuhan, China); the ENLITEN ATP assay kits (#ff2000) was purchased from Promega (Madison, WI, USA).

Techniques: In Vitro, Irradiation, Flow Cytometry, Immunofluorescence, Western Blot, Phospho-proteomics, Staining, Enzyme-linked Immunosorbent Assay, Expressing

Journal: OncoImmunology

Article Title: Carbon ion radiotherapy triggers immunogenic cell death and sensitizes melanoma to anti-PD-1 therapy in mice

doi: 10.1080/2162402x.2022.2057892

Figure Lengend Snippet: Figure 2. CIRT-induced immunogenic cell death in vivo. 5 × 105 B16 and S91 cells were irradiated with 5 GyE of carbon ion beams or treated with 2 μM MTX for 24 h respectively, then subcutaneously inoculated in C57BL/6 mice. 2 weeks after, rechallenged with 5 × 104 B16 and S91 cells and the tumor growth was documented regularly. C57BL/6 mice bearing subcutaneous B16 or S91 melanoma were locally irradiated with X-rays (XR, 5 Gy) or carbon ions (CIRT, 5 GyE) at the tumor site. Mitoxantrone (MTX, 2.0 mg/Kg)-treated animals were used as positive control. Eight days after irradiation, CALR exposure, eIF2α phosphorylation, and HMGB1 exodus were examined by immunofluorescence. The secretion of HMGB1, ATP, and IFN-γ in the serum was determined by ELISA; and infiltration of IFN-γ expressing cell in the tumor bed was assessed by immunohistocheistry. Representative images and quantification are shown (mean ± SD of triplicate assessments, Student’s t test, ***p < .001).

Article Snippet: The HMGB1 (#E-EL-M0676c) and IFN-γ (#E-EL-M0048c) ELISA kits were purchased from Elabscience (Wuhan, China); the ENLITEN ATP assay kits (#ff2000) was purchased from Promega (Madison, WI, USA).

Techniques: In Vivo, Irradiation, Positive Control, Phospho-proteomics, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Expressing

Journal: OncoImmunology

Article Title: Carbon ion radiotherapy triggers immunogenic cell death and sensitizes melanoma to anti-PD-1 therapy in mice

doi: 10.1080/2162402x.2022.2057892

Figure Lengend Snippet: Figure 3. Combination therapy with CIRT and anti-PD-1 stimulates immunogenic cell death. C57BL/6 mice bearing subcutaneous B16 and S91 melanoma (Model) were injected intraperitoneally with anti-PD-1 (α-PD-1) or locally irradiated with 5 GyE of carbon ions (CIRT) at tumor sites, or received treatment with 5 Gy CIRT plus anti-PD-1 (CIRT+α-PD-1). Eight days after irradiation, we examined CALR exposure, eIF2α phosphorylation, and HMGB1 exodus by immunofluorescence; secretion of IFN-γ in serum by ELISA; and IFN-γ infiltration in the tumor by immunohistochemical assay. Representative images and quantification are shown (mean ± SD of triplicate assessments, Student’s t test, ***p < .001).

Article Snippet: The HMGB1 (#E-EL-M0676c) and IFN-γ (#E-EL-M0048c) ELISA kits were purchased from Elabscience (Wuhan, China); the ENLITEN ATP assay kits (#ff2000) was purchased from Promega (Madison, WI, USA).

Techniques: Injection, Irradiation, Phospho-proteomics, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Immunohistochemical staining

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The expression of HMGB1, TLR4, and RAGE proteins and mRNA in the spinal dorsal horn. (a) Diagram of the timeline of this experiment. (b-d) Images showing HMGB1 expression in the spinal dorsal horn of naïve rats (b) and rats receiving intrathecal (i.t.) injection of saline (c) and i.t. injection of morphine (d). (e-g) Images showing TLR4 expression in the spinal dorsal horn of naïve rats (e) and rats receiving i.t. injection of saline (f) and i.t. injection of morphine (g). (gh-j) The images showing RAGE expressed in spinal dorsal horn in rats of naïve rats (h) and rats receiving i.t. injection of saline (i) and i.t. injection of morphine (j). (k) Repeated intrathecal (i.t.) injections of morphine led to a significant increase in the expression of HMGB1, TLR4, and RAGE proteins in the spinal dorsal horn. * P < 0.05, ** P < 0.01 vs. control group (i.t. injection of saline daily for 6 days). (l) Repeated i.t. injections of morphine led to a significant increase in the expression of HMGB1, TLR4, and RAGE mRNA in the spinal dorsal horn. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. saline group (i.t. injection of saline daily for 6 days). (m) Repeated subcutaneous (s.c.) injections of morphine caused increased expression of HMGB1 mRNA in the spinal dorsal horn of mice. * P < 0.05; ** P < 0.01 vs. control (s.c. saline daily for 9 days). Data are presented as the mean ± SEM and were analyzed with one-way ANOVA. Images in b-j, scale bar = 200 μm. Sa: saline

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Injection, Saline, Control

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The cell types that express HMGB1, TLR4, and RAGE in the rats spinal dorsal horn following repeated intrathecal (i.t.) injections of morphine. (a-c) Representative images showing that HMGB1 colocalized with the spinal neuronal marker NeuN (a), the astrocytic marker GFAP (b), and the microglial marker OX42 (c). (d-f) Representative images showing TLR4 colocalization with NeuN (d), GFAP (e), and OX42 (f). (g-i) Representative images showing RAGE colocalization with NeuN (g) and GFAP (h) but not with OX42 (i). Images in a-i, scale bar = 50 μm.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Marker

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: Effects of morphine exposure on the expression and release of HMGB1 in primary cultured spinal neurons. (A) Diagram of the timeline of this experiment. (b-g) The cultured neuron purity was calculated by labeling neurons with the neuronal marker MAP2 and glial cells with GFAP (an astrocytic marker) and OX42 (a microglial marker). The images were merged in (d) and (g). Scale bar = 50 μm. (h-j) Representative images showing the colocalization of the neuronal marker NeuN (h) with the TLR4 protein (i) in cultured cells (j). (k-m) Images showing the neuronal marker NeuN (k) colocalized with TLR4 mRNA (i) in cultured cells (m). Images in h-m, scale bar = 50 μm. (n, o) Morphine exposure promoted the expression of HMGB1 in primary cultured spinal neurons in a dose- (neurons were cultured with different concentrations of morphine for 12 h) (n) and time- (neurons were cultured in medium containing 20 μM morphine for different amounts of time) (o) dependent manner. * P < 0.05; ** P < 0.0; *** P < 0.001 vs. control group. (p, q) Morphine challenge resulted in the increased release of HMGB1 from cultured neurons in a dose- (p) and time- (q) dependent manner. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. control group. Data are presented as the mean ± SEM and were analyzed with two-way ANOVA.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Cell Culture, Labeling, Marker, Control

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The role of the chronic i.t. morphine exposure-induced upregulation of HMGB1 in the spinal dorsal horn in the development of analgesic tolerance and hyperalgesia. (a) Diagram of the timeline of this experiment. (b) Repeated i.t. injections of morphine led to a significant reduction in morphine’s maximal possible analgesic effect (% MPAE). # P < 0.05; ## P < 0.01 vs. day one. Data are presented as the mean ± SEM and were analyzed by two-way ANOVA. Intrathecal coadministration of morphine (Mor) plus glycyrrhizin (GL), an inhibitor of HMGB1, dose-dependently prevented the decrease in the MPAE. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle (Veh) group (one-way ANOVA). (c, d) Chronic i.t. morphine exposure resulted in decreased paw withdrawal threshold (PWT) (c) and paw withdrawal latency (PWL) (d) in the left hind paw. ## P < 0.01 vs. baseline. Repeated intrathecal coinjections of morphine plus GL prevented the reduction in PWT and PWL after morphine withdrawal. * P < 0.05; ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT and PWL were not changed by repeated i.t. injections of GL alone. (e) Repeated i.t. injections of morphine plus vehicle (Veh: transfection regent) or morphine plus scramble (sc) RNA resulted in significant reductions in the MPAE at day 5 and day 7. ## P < 0.01 vs. day one (two-way ANOVA). This effect was clearly prevented by the coadministration of morphine with HMGB1 siRNA intrathecally. ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle or morphine plus HMGB1 scRNA (one-way ANOVA). (f, g) Repeated i.t. injections of morphine plus vehicle or morphine plus HMGB1 scRNA led to significant decreases in PWT (f) and PWL (g) in the left hind paw, but these reductions were prevented by i.t. coinjections of morphine plus HMGB1 siRNA. # P < 0.05, ## P < 0.01 vs. baseline; * P < 0.05, ** P < 0.01 vs. morphine plus vehicle or morphine plus HMGB1 scRNA (Student’s t-test). The basal tail-flick response, PWT, and PWL were not changed by repeated i.t. injections of HMGB1 siRNA or transfection regent alone. (h, i) Repeated i.t. coinjections of morphine plus vehicle or morphine plus HMGB1 scRNA led to significantly increased expression of the HMGB1 protein (h) and HMGB1 mRNA (i) in the spinal dorsal horn. These effects were inhibited by i.t. coadministration of morphine with HMGB1 siRNA. * P < 0.05, ** P < 0.01 vs. vehicle group; ### P < 0.001 vs. morphine plus vehicle or morphine plus scramble RNA (one-way ANOVA).

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Tail Flick Test, Transfection, Expressing

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The effects of repeated i.t. administration of glycyrrhizin (GL) and an HMGB1 neutralizing antibody on established morphine tolerance and hyperalgesia. (a) Diagram of the timeline of this experiment. (b-d) Repeated i.t. coinjections of morphine plus vehicle (saline) resulted in a significant decrease in the MPAE on day 7, day 9 and day 11. ### P < 0.001 vs. day one (two-way ANOVA) (b). This reduction was reversed by the i.t. coadministration of morphine with GL started at day 7. ** P < 0.01, *** P < 0.001 vs. morphine plus vehicle group (Student’s t-test). The morphine withdrawal-induced reductions in PWT (c) and PWL (d) in the left hind paw were also partially reversed in the morphine plus GL group. # P < 0.05, ### P < 0.001 vs. baseline. * P < 0.01, ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT, and PWL were not changed by repeated i.t. injections of GL or saline alone. (e) The reduction in the MPAE induced by repeated i.t. injections of morphine were also partially reversed by the i.t. coinjection of morphine with an HMGB1 neutralizing antibody starting at day 7. * P < 0.05, ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). (f, g) Coadministration of morphine with an HMGB1 neutralizing antibody alleviated morphine withdrawal-induced mechanical allodynia (f) and thermal hyperalgesia (g). * P < 0.05 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT, and PWL were not changed by repeated i.t. injections of control IgG or ACSF alone. ACSF: artificial cerebrospinal fluid; Mor: morphine; Veh: vehicle.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Saline, Tail Flick Test, Control

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The role of spinal HMGB1 in morphine tolerance and morphine withdrawal-induced hyperalgesia in female rats. (a) Repeated intrathecal (i.t.) injections of morphine led to a significant increase in the expression of HMGB1, TLR4, and RAGE proteins in the spinal dorsal horn. * P < 0.05, ** P < 0.01 vs. control group (i.t. injection of saline daily for 6 days). (b) Intrathecal coadministration of morphine (Mor) plus glycyrrhizin (GL), an inhibitor of HMGB1, prevented the decrease in the MPAE. * P < 0.05; ** P < 0.01; *** P < 0.001 vs. morphine plus vehicle (Veh) group (one-way ANOVA). (c, d) Repeated intrathecal coinjections of morphine plus GL prevented the reduction in PWT and PWL after morphine withdrawal. * P < 0.05; ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). (e) The reduction in the MPAE induced by repeated i.t. injections of morphine were also partially reversed by the i.t. coinjection of morphine with an HMGB1 neutralizing antibody starting at day 7. * P < 0.05, ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). (f, g) Coadministration of morphine with an HMGB1 neutralizing antibody alleviated morphine withdrawal-induced mechanical allodynia (f) and thermal hyperalgesia (g). * P < 0.05 vs. morphine plus vehicle group (Student’s t-test). (h, i) All rats, male (h) and female (i) showed equivalent time in the pairing chambers prior to conditioning day. After rats received vehicle, clonidine, and GL i.t. injection, the times that spent in chambers between vehicle-, clonidine-, and GL-paired group were no reached statistical difference at testing day in male(h) and female rats (i).

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Control, Injection, Saline

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: The signaling pathway involved in the morphine-mediated increase in the expression and release of HMGB1 in primary cultured spinal neurons. (a) Diagram of the timeline of this experiment. (b-e) The morphine plus vehicle-induced increased expression of HMGB1 in cultured spinal neurons was not blocked by the treatment of morphine plus CTOP (b) but was blocked by morphine plus TAK-242 (c), morphine plus naloxone (d), and morphine plus TLR4 siRNA (e). The concentration of morphine in the media was 20 μM, and the neurons were cultured for 12 hours. (f-i) The treatment of morphine plus CTOP in cultured spinal neurons did not reduce the morphine-stimulated increase in the release of HMGB1 (f). However, morphine plus TAK-242 (g), morphine plus naloxone (h), and morphine plus TLR4 siRNA (i) treatments resulted in significant reductions in the release of HMGB1 from cultured neurons. Compared to the normal control neurons (in which nothing was added to the media), the neurons treated with vehicle alone did not exhibit a change in the levels of HMGB1 released. * P < 0.05, ** P < 0.01 vs. control (Vehicle). # P < 0.05, ## P < 0.01 vs. morphine plus vehicle (Veh: b, c, d, f, g, and h: normal saline containing 10% DMSO; e and i: transfection regent). Data are presented as the mean ± SEM and were analyzed by one-way ANOVA. (j-m) Images showing the results of the cell death assay for cultured spinal neurons. (j) and (k) are the normal cultured neurons under different magnifications. (l) and (m) are the neurons treated with morphine and costained with Hoechst/propidium iodide (PI). Images in j scale bar = 100 μm; k-m scale bar = 50 μm.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Expressing, Cell Culture, Concentration Assay, Control, Saline, Transfection

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: Effects of TLR4/NF-κB signaling activation on the expression of HMGB1 in the spinal cord and the development of morphine tolerance and hyperalgesia. (a) Repeated i.t. coinjections of morphine with TAK-242 inhibited the phosphorylation of NF-κB p65 and reduced HMGB1 expression. (b) Repeated i.t. coinjections of morphine with PDTC reduced HMGB1 expression. Data shown in (a) and (b) are presented as the mean ± SEM and were analyzed by one-way ANOVA. * P < 0.05, ** P < 0.01 vs. control (i.t. saline containing 10% DMSO); # P < 0.05, ## P < 0.01 vs. morphine plus vehicle group. (c) Repeated i.t. coinjections of morphine plus TAK-242 dose-dependently prevented the decrease in morphine’s MPAE. ### P < 0.001 vs. day one (two-way ANOVA); * P < 0.05, ** P < 0.01, *** P < 0.001 vs. morphine plus vehicle group (one-way ANOVA). Repeated i.t. injections of saline or TAK-242 alone did not change the basal MPAE value. (d, e) Chronic i.t. morphine exposure resulted in decreased paw withdrawal threshold (PWT) (d) and paw withdrawal latency (PWL) (e) in the left hind paw. ## P < 0.01 vs. baseline. Repeated i.t. coinjections of morphine plus TAK-242 prevented the reduction in PWT and PWL after morphine withdrawal. * P < 0.05; ** P < 0.01 vs. morphine plus vehicle group (Student’s t-test). The basal tail-flick response, PWT and PWL were not changed by repeated i.t. injections of TAK-242 alone.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Control, Saline, Tail Flick Test

Journal: Neurotherapeutics

Article Title: Chronic morphine-mediated upregulation of high mobility group box 1 in the spinal cord contributes to analgesic tolerance and hyperalgesia in rats

doi: 10.1007/s13311-019-00800-w

Figure Lengend Snippet: Signaling pathway of spinal HMGB1-mediated morphine tolerance and hyperalgesia. (a) Compared to morphine plus vehicle treatment, intrathecal administration of morphine with HMGB1 siRNA inhibited the increase in NF-κB p-p65 levels and the decrease in cytoplasmic IκB-α levels. However, p-p38 and p-JNK levels were not changed by this treatment. Data are presented as the mean ± SEM and were analyzed by one-way ANOVA. * P < 0.05, ** P < 0.01 vs. vehicle (Veh: transfection regent); # P < 0.05, ## P < 0.01 vs. morphine plus vehicle or morphine plus scramble (sc) RNA group. (b, c) A single i.t. injection of recombinant HMGB1 resulted in the increased expression of p-p65, TNF-α and IL-1β in the dorsal horn at 3 hours, which persisted to 6 hours after the injection (b). These effects were partially blocked by the coinjection of HMGB1 with TAK-242 (c). * P < 0.05, ** P < 0.01 vs. control (i.t. ACSF); # P < 0.05 vs. HMGB1 plus vehicle (Veh: ACSF). (d) A bolus i.t. coinjection of morphine with HMGB1 reduced the analgesic efficacy of morphine. * P < 0.05 vs. morphine plus ACSF group. (e-f) Bolus i.t. injection of HMGB1 led to a reduction in the paw withdrawal threshold (e) and paw withdrawal latency (f) in the left hind paw, which occurred at 3 hours and persisted to 6 hours after the injection. * P < 0.05 vs. baseline; # P < 0.05 vs. ACSF group. ACSF: artificial cerebrospinal fluid. Data of (d), (e), and (f) were analyzed by Student’s t-test.

Article Snippet: The HMGB1 small interfering RNA (siRNA) target rat hmgb1 was purchased from OriGene (OriGene Technologies, Rockville, SR506455).

Techniques: Transfection, Injection, Recombinant, Expressing, Control

Journal: Biomolecules & Therapeutics

Article Title: Curzerene Ameliorates Depression-Like Behaviors and Cognitive Impairment by Modulating the Gut Microbiota and HMGB1/RAGE/TLR4 Pathway

doi: 10.4062/biomolther.2025.120

Figure Lengend Snippet: Curzerene reduce the mRNA expression of IL-1β (A), TNF-α (B), IL-6 (C), NF-κB p65 (D), IKK (E), IκB (F), TLR4 (G), RAGE (H) and HMGB1 (I) in the hippocampus of LPS-treated mice. Data were showed as means ± SD (n=3). # p <0.05, ## p <0.01 and ### p <0.001 vs the control group; * p <0.05, ** p <0.01 and *** p <0.001 vs the model group.

Article Snippet: Next, the membrane was blocked with 7.5% no-fat milk for 1.5 h at 25°C and then incubated with primary antibodies against HMGB1 (#3935, CST), NF-κB p65 (#8242, CST), RAGE (ab30381, Abcam), IKK (ab124957), IκB (ab32518), TLR4 (19811-1-AP, Proteintech), β-actin (20536-1-AP, Proteintech) and GAPDH (10494-1-AP, Proteintech) in a refrigerator at 4°C overnight.

Techniques: Expressing, Control

Journal: Biomolecules & Therapeutics

Article Title: Curzerene Ameliorates Depression-Like Behaviors and Cognitive Impairment by Modulating the Gut Microbiota and HMGB1/RAGE/TLR4 Pathway

doi: 10.4062/biomolther.2025.120

Figure Lengend Snippet: Effects of curzerene on neuroinflammatory response and HMGB1/RAGE/TLR4 signaling pathways activation in LPS-induced mice. (A) Western blots of HMGB1, RAGE, TLR4, IKK and IκB in the hippocampus. (B-F) Relative expression of HMGB1, RAGE, TLR4, IKK and IκB protein. (G) Western blots of nuclear HMGB1 and NF-κB in the hippocampus. (H-I) Relative expression of nuclear HMGB1 and NF-κB protein. Data were showed as means ± SD (n=3). # p <0.05, ### p <0.001 vs the control group; * p <0.05, ** p <0.01, *** p <0.001 vs the model group.

Article Snippet: Next, the membrane was blocked with 7.5% no-fat milk for 1.5 h at 25°C and then incubated with primary antibodies against HMGB1 (#3935, CST), NF-κB p65 (#8242, CST), RAGE (ab30381, Abcam), IKK (ab124957), IκB (ab32518), TLR4 (19811-1-AP, Proteintech), β-actin (20536-1-AP, Proteintech) and GAPDH (10494-1-AP, Proteintech) in a refrigerator at 4°C overnight.

Techniques: Protein-Protein interactions, Activation Assay, Western Blot, Expressing, Control

Journal: Biomolecules & Therapeutics

Article Title: Curzerene Ameliorates Depression-Like Behaviors and Cognitive Impairment by Modulating the Gut Microbiota and HMGB1/RAGE/TLR4 Pathway

doi: 10.4062/biomolther.2025.120

Figure Lengend Snippet: Curzerene promotes HMGB1 nuclear translocation. (A, B) Immunofluorescence of nuclear HMGB1 in the hippocampus. Data were showed as means ± SD (n=3). # p <0.05 vs the control group; * p <0.05 vs the model group.

Article Snippet: Next, the membrane was blocked with 7.5% no-fat milk for 1.5 h at 25°C and then incubated with primary antibodies against HMGB1 (#3935, CST), NF-κB p65 (#8242, CST), RAGE (ab30381, Abcam), IKK (ab124957), IκB (ab32518), TLR4 (19811-1-AP, Proteintech), β-actin (20536-1-AP, Proteintech) and GAPDH (10494-1-AP, Proteintech) in a refrigerator at 4°C overnight.

Techniques: Translocation Assay, Immunofluorescence, Control

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Role of mTOR in the Development of Asthma in Mice With Cigarette Smoke-Induced Cellular Senescence

doi: 10.1093/gerona/glab303

Figure Lengend Snippet: Effects of rapamycin on the CSE-induced changes of inflammatory and senescence-associated markers in lung and glutathione in serum. ( A ) Experimental protocol (5–6 mice in each group). ( B ) Inflammatory cells in bronchoalveolar lavage fluid. ( C ) SA-β-gal activity in lung homogenate. Y axis represents relative fluorescence unit (RFU). ( D ) A plot of p21+ population in lung cells. ( E ) The frequency of p21+ population in lung cells. ( F ) Total glutathione, oxidized glutathione and reduced glutathione levels, and reduced/oxidized glutathione ratios in serum were measured, and the detection time was 2 minutes. TNF-α, Pro-MMP9, IL-6, IL-1β, S100A8/9, and HMGB1 levels in bronchoalveolar lavage fluid were measured. # p < .05, ## p < .01 compared to control (PBS) group; * p < .05, ** p < .01 between 2 groups. PBS = phosphate-buffered saline; SA-β-gal = senescence-associated beta-galactosidase; CSE = cigarette smoke extract.

Article Snippet: Tumor necrosis factor (TNF)-α (BioLegend, CA), Pro-MMP9 (R&D Systems, Abingdon, UK), interleukin(IL)-6 (BioLegend), IL-1β (BioLegend), S100A8/9 (R&D Systems), and HMGB1 (CUSABIO, Houston, TX) levels in bronchoalveolar lavage (BAL) fluid and Der p-specific immunoglobulin E (IgE) level in serum were measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions.

Techniques: Activity Assay, Fluorescence, Control, Saline

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Role of mTOR in the Development of Asthma in Mice With Cigarette Smoke-Induced Cellular Senescence

doi: 10.1093/gerona/glab303

Figure Lengend Snippet: Murine asthma model with CSE-induced cellular senescence and effects of rapamycin on this model. ( A ) Experimental protocol (4–5 mice in each group). ( B ) SA-β-gal activity in lung homogenate. Y axis represents relative fluorescence unit (RFU). ( C ) Airway hyperresponsiveness. Y axis represents cmH 2 O.s/mL. ( D ) Inflammatory cells in bronchoalveolar lavage fluid. ( E ) Serum Der p -specific IgE. Y axis represents OD (optimal density). ( F ) Gating plot of MHCII+ CD86+ population in CD11c+ cells from lung-draining lymph node. ( G ) The frequency of MHCII+ CD86+ population with CD11c+ in total cells from lung-draining lymph node. ( H ) Lung tissue (H&E stain, ×200). ( I ) The frequencies of IL-5+, IL-13+, IL-17+, and IFN-γ+ populations in CD4+ cells. ( J ) Gating plot of S100A8/9+ HMGB1+ population in CD45− Epcam+ cells. ( K ) The frequency of S100A8/9+ HMGB1+ population in CD45− Epcam+ cells. # p < .05, ## p < .01 compared to control (PBS) group; * p < .05, ** p < .01 between 2 groups. Der p = Dermatophagoides pteronyssinus ; Meth con = methacholine concentration; Macro = macrophage; Neu = neutrophil; Eos = eosinophil; Lym = lymphocyte; Rapa = rapamycin; CSE = cigarette smoke extract.

Article Snippet: Tumor necrosis factor (TNF)-α (BioLegend, CA), Pro-MMP9 (R&D Systems, Abingdon, UK), interleukin(IL)-6 (BioLegend), IL-1β (BioLegend), S100A8/9 (R&D Systems), and HMGB1 (CUSABIO, Houston, TX) levels in bronchoalveolar lavage (BAL) fluid and Der p-specific immunoglobulin E (IgE) level in serum were measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions.

Techniques: Activity Assay, Fluorescence, Staining, Control, Concentration Assay

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Role of mTOR in the Development of Asthma in Mice With Cigarette Smoke-Induced Cellular Senescence

doi: 10.1093/gerona/glab303

Figure Lengend Snippet: Effects of rapamycin on the CSE-induced changes of S100A8/9+ or HMGB1+ populations in various lung cells. ( A ) Gating plot and the frequency of S100A8/9+ population in CD45− EpCAM+ cells. ( B ) Gating plot and the frequency of S100A8/9+ F4/80+ population in CD45+ cells. ( C ) Gating plot and the frequency of S100A8/9+ CD11c+ population in CD45+ cells. ( D ) Gating plot and the frequency of S100A8/9+ population in CD4+ cells. ( E ) Gating plot and the frequency of HMGB1+ population in CD45− EpCAM+ cells. ( F ) Gating plot and the frequency of HMGB1+ F4/80+ population in CD45+ cells. ( G ) Gating plot and the frequency of HMGB1+ CD11c+ population in CD45+ cells. ( H ) Gating plot and frequency of HMGB1+ population in CD4+ cells. # p < .05 compared to control (PBS) group; * p < .05, ** p < .01 between 2 groups. PBS = phosphate-buffered saline; CSE = cigarette smoke extract.

Article Snippet: Tumor necrosis factor (TNF)-α (BioLegend, CA), Pro-MMP9 (R&D Systems, Abingdon, UK), interleukin(IL)-6 (BioLegend), IL-1β (BioLegend), S100A8/9 (R&D Systems), and HMGB1 (CUSABIO, Houston, TX) levels in bronchoalveolar lavage (BAL) fluid and Der p-specific immunoglobulin E (IgE) level in serum were measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions.

Techniques: Control, Saline

Journal: The Journals of Gerontology Series A: Biological Sciences and Medical Sciences

Article Title: Role of mTOR in the Development of Asthma in Mice With Cigarette Smoke-Induced Cellular Senescence

doi: 10.1093/gerona/glab303

Figure Lengend Snippet: Effects of rapamycin on CSE-stimulated MLE-12 cells and effects of S100A9 or HMGB1 overexpressing MLE-12 cells treated by low-dose Dermatophagoides pteronyssinus allergen on dendritic cell activation. ( A ) Plot (upper) and histogram (lower) of DCF+ population in CSE-stimulated MLE-12 cells (with or without rapamycin). ( B ) The frequency of DCF+ population. Y axis of graph represents % of DCF+ cells. ( C ) Gating plot of SA-β-gal+ population in total cells from CSE-stimulated MLE-12 cells (with or without rapamycin). ( D ) The frequency of SA-β-gal+ population. ( E ) Gating plot of p-mTOR+ population in S100A8/9+ HMGB1+ cells in singlets from CSE-stimulated MLE-12 cells (with or without rapamycin). ( F ) The frequency of p-mTOR+ population in S100A8/9+HMGB1+ cells. ( G ) Experimental procedure. ( H ) Gating plot of MHCII+ CD11c+ population in BMDC after coculture with S100A9 or HMGB1 overexpressing MLE-12 cells. ( I ) The frequency of MHCII+ CD11c+ cells in BMDCs after coculture with S100A9 or HMGB1 overexpressing MLE-12 cells. # p < .05, ## p < .01 compared to media; * p < .05, ** p < .01 between 2 groups. DCF = dichlorofluorescein; CSE = cigarette smoke extract.

Article Snippet: Tumor necrosis factor (TNF)-α (BioLegend, CA), Pro-MMP9 (R&D Systems, Abingdon, UK), interleukin(IL)-6 (BioLegend), IL-1β (BioLegend), S100A8/9 (R&D Systems), and HMGB1 (CUSABIO, Houston, TX) levels in bronchoalveolar lavage (BAL) fluid and Der p-specific immunoglobulin E (IgE) level in serum were measured using enzyme-linked immunosorbent assay according to the manufacturer’s instructions.

Techniques: Activation Assay