Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: Histological changes and histological injury scores in lung tissues of all groups. (a) Sham group. (b) Intestinal ischemia/reperfusion (I/R) group. (c) Dexmedetomidine-treated I/R (DEX) group. (d) Dexmedetomidine- and AM630-treated I/R (DA) group. (e) AM630-treated I/R (AI) group. (f) Dexmedetomidine- and wortmannin-treated I/R (DW) group. (g) Wortmannin-treated I/R (WI) group. Red arrows indicate neutrophil infiltration (original magnification, ×200; scale bar = 50 μ m). (h) Histological injury scores of the lung. Data are expressed as mean ± SEM, n = 8 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus the I/R group, & P < 0.05 versus the DEX group.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: The effects of dexmedetomidine treatment on wet/dry ( W / D ) weight ratio and myeloperoxidase (MPO) activity in lung tissues. (a) W / D weight ratio. (b) MPO activity. Data are expressed as mean ± SEM, n = 8 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus the I/R group, & P < 0.05 versus the DEX group. I/R: intestinal ischemia/reperfusion; DEX: dexmedetomidine-treated I/R; DA: dexmedetomidine- and AM630-treated I/R; AI: AM630-treated I/R; DW: dexmedetomidine- and wortmannin-treated I/R; WI: wortmannin-treated I/R.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques: Activity Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: The effects of dexmedetomidine treatment on superoxide dismutase (SOD) activity and malondialdehyde (MDA) level in lung tissues. (a) SOD activity. (b) MDA level. Data are expressed as mean ± SEM, n = 8 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus I/R group, & P < 0.05 versus DEX group. I/R: intestinal ischemia/reperfusion; DEX: dexmedetomidine-treated I/R; DA: dexmedetomidine- and AM630-treated I/R; AI: AM630-treated I/R; DW: dexmedetomidine- and wortmannin-treated I/R; WI, wortmannin-treated I/R.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques: Activity Assay

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: The effects of dexmedetomidine treatment on the levels of tumor necrosis factor alpha (TNF- α ) and interleukin 6 (IL-6) in the lung tissues of all groups. (a) TNF- α level. (b) IL-6 level. Data are expressed as mean ± SEM, n = 8 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus the I/R group, & P < 0.05 versus the DEX group. I/R: intestinal ischemia/reperfusion; DEX: dexmedetomidine-treated I/R; DA: dexmedetomidine- and AM630-treated I/R; AI: AM630-treated I/R; DW: dexmedetomidine- and wortmannin-treated I/R; WI: wortmannin-treated I/R.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques:

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: Immunohistochemical analysis of macrophage infiltration in the lung tissues of all groups. Macrophage infiltration was investigated by detecting F4/80 expression with immunohistochemistry. Dexmedetomidine treatment reduced macrophage infiltration in the lung tissues following I/R. (a) Sham group. (b) Intestinal ischemia/reperfusion (I/R) group. (c) Dexmedetomidine-treated I/R (DEX) group. (d) Dexmedetomidine- and AM630-treated I/R (DA) group. (e) AM630-treated I/R (AI) group. (f) Dexmedetomidine- and wortmannin-treated I/R (DW) group. (g) Wortmannin-treated I/R (WI) group (original magnification, ×400). (h) Integrated optical density (IOD) of F4/80 expression in all groups. Data are expressed as mean ± SEM, n = 6 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus the I/R group, & P < 0.05 versus the DEX group.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques: Immunohistochemical staining, Expressing, Immunohistochemistry

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: Immunohistochemical analysis of the expression of p-Akt in all groups. (a) Sham group. (b) Intestinal ischemia/reperfusion (I/R) group. (c) Dexmedetomidine-treated I/R (DEX) group. (d) Dexmedetomidine- and AM630-treated I/R (DA) group. (e) AM630-treated I/R (AI) group. (f) Dexmedetomidine- and wortmannin-treated I/R (DW) group. (g) Wortmannin-treated I/R (WI) group (original magnification, ×400). (h) Integrated optical density (IOD) of p-Akt expression in all groups. Data are expressed as mean ± SEM, n = 6 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus the I/R group, & P < 0.05 versus the DEX group.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques: Immunohistochemical staining, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: The effects of dexmedetomidine treatment on the mRNA expressions of caspase-3, Bax, Bcl-2, and Akt in all groups. (a) Caspase-3 mRNA expression. (b) Bax mRNA expression. (c) Bcl-2 mRNA expression. (d) Akt mRNA expression. Data are expressed as mean ± SEM, n = 6 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus the I/R group, & P < 0.05 versus the DEX group. I/R: intestinal ischemia/reperfusion; DEX: dexmedetomidine-treated I/R; DA: dexmedetomidine- and AM630-treated I/R; AI: AM630-treated I/R; DW: dexmedetomidine- and wortmannin-treated I/R; WI: wortmannin-treated I/R.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques: Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: The effects of dexmedetomidine treatment on the protein expressions of cleaved caspase-3, Bax, Bcl-2, and p-Akt in all groups. (a) The expressions of cleaved caspase-3, Bax, Bcl-2, and p-Akt in lung tissues were detected using Western blot analysis. (b) Cleaved caspase-3 expression. (c) Bax expression. (d) Bcl-2 expression. (e) Bcl-2/Bax ratio. (f) p-Akt expression. Data are expressed as mean ± SEM, n = 6 rats per group. ∗ P < 0.05 versus the Sham group, # P < 0.05 versus the I/R group, & P < 0.05 versus the DEX group. I/R: intestinal ischemia/reperfusion; DEX: dexmedetomidine-treated I/R; DA: dexmedetomidine- and AM630-treated I/R; AI: AM630-treated I/R; DW: dexmedetomidine- and wortmannin-treated I/R; WI: wortmannin-treated I/R.

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques: Western Blot, Expressing

Journal: Oxidative Medicine and Cellular Longevity

Article Title: Dexmedetomidine Ameliorates Lung Injury Induced by Intestinal Ischemia/Reperfusion by Upregulating Cannabinoid Receptor 2, Followed by the Activation of the Phosphatidylinositol 3-Kinase/Akt Pathway

doi: 10.1155/2020/6120194

Figure Lengend Snippet: Schematic depicting model of potential molecular mechanism of protection of intestinal I/R-induced lung injury by dexmedetomidine. Dexmedetomidine may confer a protective role against intestinal I/R-induced lung injury by upregulating the CB 2 receptor expression or activating the PI3K/Akt pathway. Treatment with the CB 2 receptor antagonist, AM630, or the PI3K inhibitor, wortmannin, reverses the protective role of DEX by increasing the levels of apoptosis, inflammation, and oxidative stress (as marked by solid arrows). In addition, there may be a potential interaction between CB 2 receptor and the PI3K/Akt pathway. CB2 receptor upregulated by dexmedetomidine may also play a protective role by activating Akt (or other pathways), because treatment with AM630 reduces Akt activation (as shown by dotted lines).

Article Snippet: The CB 2 R antagonist AM630 was obtained from Tocris (Baldwin, MO, USA).

Techniques: Expressing, Activation Assay

Journal: Physiological Reports

Article Title: The endocannabinoid systemʼs involvement in motor development relies on cannabinoid receptors, TRP channels, and Sonic Hedgehog signaling

doi: 10.14814/phy2.15565

Figure Lengend Snippet: Reductions to swimming performance caused by singular MAGL or dual FAAH/MAGL inhibition are still observed when blocking CB2R. Escape swimming in response to an acute touch stimulus was assessed in zebrafish embryos at 2 days post‐fertilization (dpf). The effect of ~24‐h drug treatment on embryonic escape swimming distance (cm) and mean velocity (cm/s) was measured, and mean ranks of treatment groups were compared against each other. In each respective treatment group, the horizontal line within its boxplot represents the median value which is surrounded by the interquartile range, while the error bars represent the maximum and minimum values. (A, B) The effects of the vehicle control ( n = 35), 5 μM JZL 184 ( n = 36), 1 μM AM 630 ( n = 52), and 5 μM JZL 184 in the presence of 1 μM AM 630 ( n = 36) on escape swimming was compared. (C, D) The effects of the vehicle control ( n = 35), 2 μM JZL 195 ( n = 36), 1 μM AM 630 ( n = 52), and 2 μM JZL 195 in the presence of 1 μM AM 630 ( n = 28) on escape swimming was compared. (E) Representative tracings of the swimming paths are shown for vehicle, 1 μM AM 630, 5 μM JZL 184, 5 μM JZL 184 + 1 μM AM 630, 2 μM JZL 195 and 2 μM JZL 195 + 1 μM AM 630. N = 4 experiments with 7–13 animals per experiment for each treatment. Columns which share the same letter(s) of the alphabet are not statistically different from one another (Kruskal–Wallis, followed by Dunn's multiple comparisons test—where statistical significance was determined as p < 0.05).

Article Snippet: Along with these compounds, animals were co‐treated with the CB1R antagonist AM 251 (Selleck Chemicals), the CB2R antagonist AM630 (Adooq Bioscience), the TRPV1/TRPA1/TRPM8 inhibitor AMG 9090 (Alomone Labs), or the SMO agonist Purmorphamine (Sigma‐Aldrich).

Techniques: Inhibition, Blocking Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Activation of cannabinoid receptor 2 attenuates Angiotensin II-induced atrial fibrillation via a potential NOX/CaMKII mechanism

doi: 10.3389/fcvm.2022.968014

Figure Lengend Snippet: Under Angiotensin II stimulation, the RAAS was activated, and blood pressure (BP) rose. Ang II (2,000 ng/kg/min) was subcutaneously infused into the mice for 21 days. In addition, solvent (0.1 ml), AM1241 (20 mg/kg), and AM630 (3 mg/kg) were administered intraperitoneally every day to the animals. Before the treatment, there were no significant differences in blood pressure (BP) among the five groups. After 21 days of different treatments, BP increased significantly in all groups except the control group (A) . The content of Angiotensin II (B) , aldosterone (ALD) (C) , malondialdehyde (MDA) (D) and glutathione (GSH) (E), and the enzyme activity of superoxide dismutase (SOD) (F) were determined by serological examination. The data represent the mean ± SEM. * P < 0.05 vs. control group. & p < 0.05 vs. before treatment. # P < 0.05 vs. Ang II + AM1241 group.

Article Snippet: The CB2R agonist AM1241 (S1544, Selleck Chemicals, Houston, TX, USA) and CB2R antagonist AM630 (HY-15421, MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in solvents containing DMSO, Tween-20 and PBS (1:1:8).

Techniques: Solvent, Control, Activity Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Activation of cannabinoid receptor 2 attenuates Angiotensin II-induced atrial fibrillation via a potential NOX/CaMKII mechanism

doi: 10.3389/fcvm.2022.968014

Figure Lengend Snippet: Administration of AM1241 reduced the inducibility of atrial fibrillation (AF) in Angiotensin II-infused mice. (A) Simultaneous recordings of surface electrocardiogram (ECG) after 21-day infusion of Ang II (2,000 ng/kg/min). Solid underlines indicate the burst pacing period, and dashed underlines highlight the AF period. The above two ECGs recorded representative ECG waves of AF and SR after a burst of electrical stimuli. The bottom recorded ECG waves of AF followed by spontaneous conversion to SR. Bar = 300 ms. (B) Inducibility of AF. (C) Duration of AF. (D) Electrophysiological mapping images of spontaneous conduction in the LA. The bars from red to blue indicate the total time from the first to the last measurement within one heartbeat. (E) Mean conduction velocity. (F) Absolute inhomogeneity of left atrial electrical conduction. (G) Representative M-mode echocardiography of the LA. (H) Quantification of LAD. The data represent the mean ± SEM. * P < 0.05 vs. control group. # P < 0.05 vs. Ang II + AM1241 group.

Article Snippet: The CB2R agonist AM1241 (S1544, Selleck Chemicals, Houston, TX, USA) and CB2R antagonist AM630 (HY-15421, MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in solvents containing DMSO, Tween-20 and PBS (1:1:8).

Techniques: Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Activation of cannabinoid receptor 2 attenuates Angiotensin II-induced atrial fibrillation via a potential NOX/CaMKII mechanism

doi: 10.3389/fcvm.2022.968014

Figure Lengend Snippet: Administration of AM1241 attenuated oxidative stress in the atrial tissue of Angiotensin II-infused mice by activating cannabinoid receptor 2 (CB2R). (A) Representative western blot image and relative expression of CB2R. (B) Representative picture of reactive oxygen species (ROS) (red) by dihydroethidium staining. Bar = 100 μm. (C) Quantitative analysis of reactive oxygen species (ROS) mean intensity. (D) Representative western blot image and relative expression of NOX2 and NOX4. (E) Representative western blot image and relative expression of ox-CaMKII and CaMKII. Lane 1: control group; Lane 2: Ang II group; Lane 3: Ang II + Veh group; Lane 4: Ang II + AM1241 group; Lane 5: Ang II + AM630 group. The data represent the mean ± SEM. * P < 0.05 vs. control group. # P < 0.05 vs. Ang II + AM1241 group. @ P < 0.05 vs. Ang II group. $ P < 0.05 vs. Ang II + Veh group.

Article Snippet: The CB2R agonist AM1241 (S1544, Selleck Chemicals, Houston, TX, USA) and CB2R antagonist AM630 (HY-15421, MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in solvents containing DMSO, Tween-20 and PBS (1:1:8).

Techniques: Western Blot, Expressing, Staining, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Activation of cannabinoid receptor 2 attenuates Angiotensin II-induced atrial fibrillation via a potential NOX/CaMKII mechanism

doi: 10.3389/fcvm.2022.968014

Figure Lengend Snippet: Administration of AM1241 attenuated mitochondrial damage in Angiotensin II-infused mice. (A) Representative transmission electron microscopic images of atrial samples. Bar = 1 μm. (B) Fraction of swollen mitochondria. (C) Length ratio of the mitochondrial cristae membrane and outer membrane, a parameter of mitochondrial cristae density. For each analyzed mitochondrion, the total length of the cristae membrane was obtained by adding the lengths of all the cristae together and multiplying by 2. The ratio was determined by dividing the total length of the cristae membrane by the mitochondrion perimeter. (D) Representative western blot image and relative expression of p-Drp1 and Drp1. Lane 1: control group; Lane 2: Ang II group; Lane 3: Ang II + Veh group; Lane 4: Ang II + AM1241 group; Lane 5: Ang II + AM630 group. The data represent the mean ± SEM. * P < 0.05 vs. control group. # P < 0.05 vs. Ang II + AM1241 group.

Article Snippet: The CB2R agonist AM1241 (S1544, Selleck Chemicals, Houston, TX, USA) and CB2R antagonist AM630 (HY-15421, MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in solvents containing DMSO, Tween-20 and PBS (1:1:8).

Techniques: Transmission Assay, Membrane, Western Blot, Expressing, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Activation of cannabinoid receptor 2 attenuates Angiotensin II-induced atrial fibrillation via a potential NOX/CaMKII mechanism

doi: 10.3389/fcvm.2022.968014

Figure Lengend Snippet: Administration of AM1241 attenuated atrial fibrosis in Ang II-infused mice. (A) Representative images of Masson’s trichrome staining. Bar = 50 μm. (B) Relative myocardial fibrosis. (C) Representative images of H&E staining. Bar = 50 μm. (D) Representative western blot image and relative expression of TGF-β, MMP9, collagen I and collagen III. Lane 1: control group; Lane 2: Ang II group; Lane 3: Ang II + Veh group; Lane 4: Ang II + AM1241 group; Lane 5: Ang II + AM630 group. The data represent the mean ± SEM. * P < 0.05 vs. control group. # P < 0.05 vs. Ang II + AM1241 group.

Article Snippet: The CB2R agonist AM1241 (S1544, Selleck Chemicals, Houston, TX, USA) and CB2R antagonist AM630 (HY-15421, MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in solvents containing DMSO, Tween-20 and PBS (1:1:8).

Techniques: Staining, Western Blot, Expressing, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Activation of cannabinoid receptor 2 attenuates Angiotensin II-induced atrial fibrillation via a potential NOX/CaMKII mechanism

doi: 10.3389/fcvm.2022.968014

Figure Lengend Snippet: Proposed model describing the role of cannabinoid receptor 2 (CB2R) activation in the treatment of atrial fibrillation (AF).

Article Snippet: The CB2R agonist AM1241 (S1544, Selleck Chemicals, Houston, TX, USA) and CB2R antagonist AM630 (HY-15421, MedChemExpress, Monmouth Junction, NJ, USA) were dissolved in solvents containing DMSO, Tween-20 and PBS (1:1:8).

Techniques: Activation Assay

Journal: Frontiers in Immunology

Article Title: Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury

doi: 10.3389/fimmu.2022.993168

Figure Lengend Snippet: Expression patterns of CB2R in cord tissues post-SCI. (A) Representative protein band of CB2R on day 3 and weeks 1, 2, 4, and 6 post-SCI. (B) Quantitative analysis showing that CB2R expression was significantly increased on day 3 and weeks 1 and 2 post-SCI. (C) IF staining showing that CB2R (red) in cords of mice within 6W post-SCI. (D) IF staining of CB2R (red) with GFAP, Neun, or IBA-1 (green) in cords of sham mice. (E) IF staining of CB2R (red) with GFAP, Neun, or IBA-1 (green) in SCI mice on day 7 post-SCI. Scale bar = 100 μm. * p < 0.05.

Article Snippet: At 80% confluence, the microglia and HEK-293T cells were pretreated for 24 h with the CB2R activator JWH-133 (10 nM; B7941; APExBIO Technology LLC, Houston, TX, USA) or antagonist AM630 (40 nM; A3168; APExBIO Technology LLC) dissolved in 0.1% dimethyl sulfoxide (KGT5131; Nanjing KeyGen Biotech.

Techniques: Expressing, Staining

Journal: Frontiers in Immunology

Article Title: Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury

doi: 10.3389/fimmu.2022.993168

Figure Lengend Snippet: CB2R regulates inflammation-mediated polarization of microglia. (A) IF staining of iNOS (red) and ARG-1 (green) in BV2 microglia after LPS insult for 24 h with CB2R activation by JWH-133 (J) or inactivation by AM630 (A) . Scale bar = 50 μm. (B) Quantitative analysis indicating that CB2R significantly altered expression of the M1 marker iNOS during inflammation. (C) Quantitative analysis indicating that CB2R significantly altered expression of the M2 marker ARG-1 after LPS stimulation. (D) Flow cytometry results showing the relative proportion of M1 microglia (red) after LPS insult with and without CB2R activation or inactivation. (E) Quantitative analysis of M1 microglia. (F) Flow cytometry results showing the relative proportion of M2 microglia (red) after LPS insult with and without CB2R activation or inactivation. (G) Quantitative analysis of M2 microglia. ELISA results of IL-1β (H) , IL-6 (I) , and IL-10 (J) in the control, LPS, LPS+J (JWH-133), and LPS+A (AM630) groups. * p < 0.05. n.s means no significance.

Article Snippet: At 80% confluence, the microglia and HEK-293T cells were pretreated for 24 h with the CB2R activator JWH-133 (10 nM; B7941; APExBIO Technology LLC, Houston, TX, USA) or antagonist AM630 (40 nM; A3168; APExBIO Technology LLC) dissolved in 0.1% dimethyl sulfoxide (KGT5131; Nanjing KeyGen Biotech.

Techniques: Staining, Activation Assay, Expressing, Marker, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury

doi: 10.3389/fimmu.2022.993168

Figure Lengend Snippet: CB2R promoted ubiquitination and autophagy-induced degradation of NLRP3 via the AMPK/ULK signaling pathway. (A) IF staining of CB2R (red) and NLRP3 (green) after LPS stimuli for 24 h with CB2R activation by JWH-133 (J) or inactivation by AM630 (A) . Scale bar = 50 μm. (B) Quantitative analysis of CB2R. (C) Quantitative analysis of NLRP3. (D) Representative protein bands of p-AMPK, AMPK, p-ULK1, ULK1, LC3A, and LC3B in each group after 24 h of LPS insult. (E) Quantitative analysis of the p-AMPK/AMPK ratio. (F) Quantitative analysis of the p-ULK1/ULK1 ratio. (G) Quantitative analysis of the LC3B/LC3A ratio. (H) Representative protein bands of p-p65, p65, NLRP3, and p62 after 24 h of LPS stimulation. (I) Quantitative analysis of the p-p65/p65 ratio. (J) Quantitative analysis of NLRP3. (K) Quantitative analysis of p62. (L) IF staining of CB2R (red) and NLRP3 (green) after LPS stimulation for 24 h with CB2R activation by JWH-133 or CB2R inactivation by AM630. Scale bar = 50 μm. (M) Representative protein bands of NLRP3 and p62 in BV2 microglia after IP assay using an Ab against NLRP3. (N) Representative protein bands of ubiquitin linkage-specific K48 bound to NLRP3. * p < 0.05.

Article Snippet: At 80% confluence, the microglia and HEK-293T cells were pretreated for 24 h with the CB2R activator JWH-133 (10 nM; B7941; APExBIO Technology LLC, Houston, TX, USA) or antagonist AM630 (40 nM; A3168; APExBIO Technology LLC) dissolved in 0.1% dimethyl sulfoxide (KGT5131; Nanjing KeyGen Biotech.

Techniques: Staining, Activation Assay

Journal: Frontiers in Immunology

Article Title: Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury

doi: 10.3389/fimmu.2022.993168

Figure Lengend Snippet: CB2R alleviated inflammation by promoting NLRP3-autophagosome formation in injured cords post-SCI. On day 3 post-SCI with CB2R activation by JWH-133 or inactivation by AM630, (A) IF staining of iNOS (red) and IBA-1 (green); (B) IF staining of ARG-1 (red) and IBA-1 (green). Scale bar = 100 μm. (C) Quantitative analysis of the number of iNOS + microglia. (D) Quantitative analysis of the number of ARG-1 + microglia. (E) IF staining of NLRP3 (red) and LC3B (green) on day 3 post-SCI. Scale bar = 100 μm. * p < 0.05.

Article Snippet: At 80% confluence, the microglia and HEK-293T cells were pretreated for 24 h with the CB2R activator JWH-133 (10 nM; B7941; APExBIO Technology LLC, Houston, TX, USA) or antagonist AM630 (40 nM; A3168; APExBIO Technology LLC) dissolved in 0.1% dimethyl sulfoxide (KGT5131; Nanjing KeyGen Biotech.

Techniques: Activation Assay, Staining

Journal: Frontiers in Immunology

Article Title: Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury

doi: 10.3389/fimmu.2022.993168

Figure Lengend Snippet: CB2R ameliorated secondary neuronal apoptosis and demyelination post-SCI. (A) LFB staining of spinal cords on day 7 post-SCI. Scale bar = 200 μm. (B) Quantitative analysis of LFB-labeled area. (C) IF staining of NF200 or myelin basic protein (green) on day 7 post-SCI. Scale bar = 500 μm. (D) Quantitative analysis of NF200. (E) Quantitative analysis of myelin basic protein. (F) TUNEL staining of spinal cords on day 7 post-SCI. Scale bar = 100 μm. (G) Quantitative analysis of TUNEL-positive cells. (H) IF staining of Neun (green) and Annexin V (red) on day 7 post-SCI. Scale bar = 100 μm. (I) Quantitative analysis of Annexin V-positive neurons. (J) Nissl staining of spinal cords on day 7 post-SCI. Scale bar = 200 μm. (K) Quantitative analysis of the number of neurons. * p < 0.05. n.s means no significance.

Article Snippet: At 80% confluence, the microglia and HEK-293T cells were pretreated for 24 h with the CB2R activator JWH-133 (10 nM; B7941; APExBIO Technology LLC, Houston, TX, USA) or antagonist AM630 (40 nM; A3168; APExBIO Technology LLC) dissolved in 0.1% dimethyl sulfoxide (KGT5131; Nanjing KeyGen Biotech.

Techniques: Staining, Labeling, TUNEL Assay

Journal: Frontiers in Immunology

Article Title: Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury

doi: 10.3389/fimmu.2022.993168

Figure Lengend Snippet: CB2R mitigated expansion of neuronal damage and formation of glial scarring post-SCI. (A) IF staining of GFAP (green) and IBA-1 (red) on day 7 post-SCI. Scale bar = 500 μm. Quantitative analysis of the area of injured foci inside the white dotted line (B) , microglia-occupied area, (C) and astrocyte-occupied area (D) on day 7 post-SCI. (E) IF staining of GFAP (green) and IBA-1 (red) on day 28 post-SCI. Scale bar = 500 μm. Quantitative analysis of the area of injured foci inside the white dotted line (F) , microglia-occupied area, (G) and astrocyte-occupied area (H) on day 28 post-SCI. * p < 0.05.

Article Snippet: At 80% confluence, the microglia and HEK-293T cells were pretreated for 24 h with the CB2R activator JWH-133 (10 nM; B7941; APExBIO Technology LLC, Houston, TX, USA) or antagonist AM630 (40 nM; A3168; APExBIO Technology LLC) dissolved in 0.1% dimethyl sulfoxide (KGT5131; Nanjing KeyGen Biotech.

Techniques: Staining

Journal: Frontiers in Immunology

Article Title: Cannabinoid receptor-2 attenuates neuroinflammation by promoting autophagy-mediated degradation of the NLRP3 inflammasome post spinal cord injury

doi: 10.3389/fimmu.2022.993168

Figure Lengend Snippet: CB2R attenuated histological and functional damage post-SCI. (A) Representative images of H&E staining of spinal cords on day 3 post-SCI (injured cords in black frames). Scale bar (i) = 600 μm (ii) = 100 μm. (B) Quantitative analysis of defected area by H&E staining. (C) H&E staining of spinal cords on day 3 post-SCI (injured cords in black frames). Scale bar: (i) = 600 μm, (ii) = 100 μm. (D) Quantitative analysis of defected area by H&E staining. (E) Mouse footprint assay on day 28 post-SCI. Quantitative analysis of stride length (F) and width (G) of the hindlimbs of mice post-SCI. (H) Representative images of a mouse swimming on day 28 post-SCI. Statistical analysis of the Louisville Swim Scale score (I) and Basso Mouse Scale (J) over a 28-day period. * p < 0.05. n.s means no significance.

Article Snippet: At 80% confluence, the microglia and HEK-293T cells were pretreated for 24 h with the CB2R activator JWH-133 (10 nM; B7941; APExBIO Technology LLC, Houston, TX, USA) or antagonist AM630 (40 nM; A3168; APExBIO Technology LLC) dissolved in 0.1% dimethyl sulfoxide (KGT5131; Nanjing KeyGen Biotech.

Techniques: Functional Assay, Staining

Journal: Physiological Reports

Article Title: The endocannabinoid systemʼs involvement in motor development relies on cannabinoid receptors, TRP channels, and Sonic Hedgehog signaling

doi: 10.14814/phy2.15565

Figure Lengend Snippet: Escape swimming deficits caused by singular MAGL or dual FAAH/MAGL inhibition is rescued by blocking CB1R. Escape swimming in response to an acute touch stimulus was assessed in zebrafish embryos at 2 days post‐fertilization (dpf). The effect of ~24‐h drug treatment on embryonic escape swimming distance (cm) and mean velocity (cm/s) was measured, and mean ranks of treatment groups were compared against each other. In each respective treatment group, the horizontal line within its boxplot represents the median value which is surrounded by the interquartile range, while the error bars represent the maximum and minimum values. (A, B) The effects of 5 μM URB 597 ( n = 38) on escape swimming are compared relative to the vehicle control ( n = 34), where ns = not statistically significant (Mann–Whitney test—where statistical significance was determined as p < 0.05). (C, D) The effects of the vehicle control ( n = 34), 5 μM JZL 184 ( n = 30), 10 nM AM 251 ( n = 33), and 5 μM JZL 184 in the presence of 10 nM AM 251 ( n = 43) on escape swimming was compared. (E, F) The effects the vehicle control ( n = 34), 2 μM JZL 195 ( n = 39), 10 nM AM 251 ( n = 33), and 2 μM JZL 195 in the presence of 10 nM AM 251 ( n = 34) on escape swimming was compared. N = 4 experiments with 8–11 animals per experiment for each treatment. (G) Representative tracings of the swimming paths are shown for vehicle, 10 nM AM 251, 5 μM JZL 184, 5 μM JZL 184 + 10 nM AM 251, 2 μM JZL 195, and 2 μM JZL 195 + 10 nM AM 251. For (C–F), columns which share the same letter(s) of the alphabet are not statistically different from one another (Kruskal–Wallis, followed by Dunn's multiple comparisons test—where statistical significance was determined as p < 0.05).

Article Snippet: Along with these compounds, animals were co‐treated with the CB1R antagonist AM 251 (Selleck Chemicals), the CB2R antagonist AM630 (Adooq Bioscience), the TRPV1/TRPA1/TRPM8 inhibitor AMG 9090 (Alomone Labs), or the SMO agonist Purmorphamine (Sigma‐Aldrich).

Techniques: Inhibition, Blocking Assay, Control, MANN-WHITNEY