Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 3. Ferroptosis-inducing and immunostimulatory capabilities of the biomimetic hydrogel. (a) Schematic diagram for the tumor cell/immune cell co- incubation system in transwell plates. The tumor cells (B16F10, 4T1) or immune cells were inoculated in the bottom chamber of the 24-well transwell culture plate, while the hydrogel soaking solution was placed in the upper chamber. (b) Changes of GPX4 activity in B16F10 cells after different treatments. (I) Control, (II) Gel, (III) RSL3, (IV) Gel@RSL3 (n = 4). (c) Flow cytometric analysis on the lipid ROS levels in B16F10 cells after different treatments. (d) CLSM imaging of lipid ROS generation in B16F10 cells after different treatments. Higher green fluorescence intensity indicates greater lipid ROS production. (e) Quantitative fluorescence analysis of lipid ROS levels in panel D (n = 4). (f) Flow cytometric analysis on the hydrogel-mediated ferroptosis levels of B16F10 cells after different treatments. (g) ATP levels in the supernatants of cell culture after different treatments. (I) Control, (II) Gel, (III) RSL3, (IV) Gel@RSL3 (n = 4). (h) CLSM imaging of CRT expression in B16F10 cells after different treatments. Stronger red fluorescence indicates higher expression levels. (i) Quantitative fluorescence analysis of CRT expression levels in panel H (n = 4). (j) CLSM imaging of cellular HMGB1 abundance after different treatments. Lower red fluorescence indicates greater HMGB1 release into the extracellular compartment. (k) Quantitative fluorescence analysis of HMGB1 release in panel J (n = 4). (l) Flow cytometric analysis on the treatment-induced maturation of BMDCs. (m) Flow cytometric analysis on the activation status of macrophages by monitoring the CD80 expression levels. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: Incubation, Activity Assay, Control, Imaging, Cell Culture, Expressing, Activation Assay

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 4. Gel@RSL3 + GM-CSF + aPD-L1 activates immune response in vitro. (a-d) Flow cytometric analysis of the activation status of DCs (CD11c + /MHC II + ), M1 macrophages (F4/80 + /CD80 + ) and T cells (CD8 + /CD3 + and CD8a + /IFN- 𝛾+ ) in the co-incubation system of splenic immune cells and B16F10 cells after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3 and (V) Gel@RSL3 + GM-CSF (n = 4). (e) Secretion levels of immunostimulatory cytokines including IFN- 𝛾, TNF- 𝛼and antitumor effector molecule GzmB in the supernatant from the co-culture system after different treatments (n = 4). (f) PD-L1 expression in tumor cells with after the hydrogel-mediated ferroptosis-immunotherapy. Group set-up for panel e-f: (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3 and (V) Gel@RSL3 + GM- CSF). (g-h) Flow cytometric analysis of the expression levels of effector T cell marker CD4 + /CD8 + and CD8a + /IFN- 𝛾+ in T cells co-incubated with B16F10 cells after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD-L1. (i) Secretion levels of immunostimulatory cytokines including IFN- 𝛾, TNF- 𝛼and antitumor effector molecule GzmB in the supernatant from the co-culture system after different treatments (n = 4). (j) Evaluation on the GSH levels in B16F10 cells after different treatments (n = 4). (k) Western blot analysis of the expression level of CRT, HMGB1 and SLC7A11 in different groups. (l) Flow cytometric analysis on the lipid ROS levels in B16F10 cells after different treatments. (m) MDA levels in B16F10 cells after different treatments (n = 4). Group set-up for panel I-M: (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD- L1). (n) Flow cytometric analysis on the death rate of B16F10 cells after different treatments, including (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF + aPD-L1. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01, ∗ ∗ ∗ indicates significance at P < 0.001, ∗ ∗ ∗ ∗ indicates significance at P < 0.0001.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: In Vitro, Activation Assay, Incubation, Control, Co-Culture Assay, Expressing, Marker, Western Blot

Journal: Fundamental Research

Article Title: Engineered In-Situ-Forming Biomimetic Hydrogel with Self-Regulated Immunostimulatory Capacity Promotes Postoperative Tumor Treatment

doi: 10.1016/j.fmre.2023.02.029

Figure Lengend Snippet: Fig. 5. Antitumor effect of biomimetic hydrogel in vivo. (a) Schematic illustration of the treatment scheme of the B16F10-luc tumor-bearing mice (n = 7). (b) Treatment procedures on the tumor-bearing mice. (c) In vivo bioluminescence images of B16F10-luc tumor-bearing mice throughout the treatment period with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. (d) Tumor size changes during the incubation period after different treatments. (e) Survival analysis of mice after different treatments. (f) Body weight changes after treatment with different samples. (g) Evaluation on the GPX4 activity in tumor tissues after different treatments (n = 4). (h) Western blot analysis on the expression of CRT, HMGB1 and SLC7A11 in B16F10-luc tumors after treatment with (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. (i) MDA levels in tumor tissue after different treatments (n = 4). (j) H&E and TUNEL staining of tumor tissue samples after treatment (I) Control, (II) Gel, (III) Gel@GM-CSF, (IV) Gel@RSL3, (V) Gel@RSL3 + GM-CSF and (VI) Gel@RSL3 + GM-CSF-aPD-L1. ∗ indicates significance at P < 0.05, ∗ ∗ indicates significance at P < 0.01, ∗ ∗ ∗ indicates significance at P < 0.001.

Article Snippet: RT, HMGB1, and SLC7A11 were supplied by Proteintech (Wuhan, hina).

Techniques: In Vivo, Control, Incubation, Activity Assay, Western Blot, Expressing, TUNEL Assay, Staining

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 2. Upregulation of HMGB1, TLR4, pIKBα and GFAP in astrocytes and brain microvessel endothelial cells in MCAO rats. (A, B and D) Compared with the sham group, the expression of HMGB1, TLR4, pIKBα and GFAP was potentiated in the cerebral cortex tissues of MCAO rats. The scale bars represent 60 and 30 μm in (A) and (B) respectively. (C and E) Consistently, the HMGB1 (red) in astrocytes in MCAO group translocated from nucleus (Blue color represents DAPI) to the cytoplasm (Green color represents GFAP). The expression of TLR4 (yellow) and pIKBα (yellow) in brain microvessel endothelial cells (Green color represents VIII factor) in MCAO group was potentiated respectively. The expression of GFAP (green) in astrocytes (Blue color represents DAPI) in MCAO group was also increased. The scale bars represent 10 μm. (D and E) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 6. OGD activated astrocytes and induced HMGB1 release into the extracellular space. (A and C) OGD potentiated GFAP expression in astrocytes. GFAP was labelled with HRP (yellow) and nucleus was stained with DAPI (blue). (B (a and b), C, D, E and F) OGD increased HMGB1 expression (red) in astrocytes and induced HMGB1 release from nucleus to the cytoplasm. GFAP was labelled with FITC (green) and nucleus was stained with DAPI (blue). (B (c and d)) Expression of HMGB1 was not observed in rBMECs in both sham and OGD groups. VIII factor was labelled with FITC (green) and nucleus was stained with DAPI (blue). (G) OGD increased HMGB1 content in the medium. (C, D (b), E (b), F (b) and G (b)) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group. Scale bars represent 20 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: Expressing, Staining

Journal: European journal of pharmacology

Article Title: HMGB1 promoted P-glycoprotein at the blood-brain barrier in MCAO rats via TLR4/NF-κB signaling pathway.

doi: 10.1016/j.ejphar.2020.173189

Figure Lengend Snippet: Fig. 8. HMGB1 contributed to upregulation of P-gp via TLR4/NF-kB pathway. (A) The relationship among HMGB1 and changes of TLR4, TIRAP, NF-kB and P-gp in rBMECs after OGD was investigated using related positive agents. (B) Changes of TLR4 (green) and TIRAP (red) after OGD and treatment with positive agents in rBMECs. (C) Each value represents the mean ± S.D. (n = 10) and was measured as described in “Materials and Methods”. Note: *P < 0.05 vs. sham group; #P < 0.05 vs. OGD group. The scale bars represent 10 μm.

Article Snippet: Brain slices were treated with antibodies against HMGB1 (rabbit polyclonal IgG antibody; 1:100), TLR4 (rabbit polyclonal IgG antibody; 1:100), pIKBα (rabbit polyclonal IgG antibody; 1:100) and glial fibrillary acidic protein (GFAP) (rabbit polyclonal IgG antibody; 1:100) at 4 °C and reacted with avidin-biotin-peroxidase complex and DAB (Boster Bio-engineering, Wuhan, China).

Techniques: