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Objective:To evaluate the role of cannabinoid receptor 2 (CB2R) in macrophage pyroptosis induced by lipopolysaccharide (LPS) in mice.Methods:Macrophage line RAW264.7 cells of mice were routinely cultured and divided into 3 groups ( n=18 each) using a random number table method: control group (group C), LPS group and LPS plus CB2R agonist HU308 group (group HU308). Group C received no mediation, LPS at the final concentration of 1 μg/ml was added in the other groups.After incubation for 15 min, HU308 with the final concentration of 10 μmol/L was added in group LPS+ HU308.All groups were then incubated for 6 h. The expression of nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3 (NLRP3), caspase-1, caspase-11 and gasdermin D (GSDMD) mRNA was measured by real-time polymerase chain reaction, the expression of NLRP3, caspase-1, caspase-11, GSDMD and C-terminal domain of human GSDMD (GSDMD-C) was detected by Western blot, and the concentrations of interleukin-18 (IL-18) and IL-1β in the supernatant were determined by enzyme-linked immunosorbent assay.GSDMD-C/GSDMD ratio was calculated. Results:Compared with group C, the expression of NLRP3, caspase-1, caspase-11, GSDMD and GSDMD-C was significantly up-regulated, GSDMD-C/GSDMD ratio was increased, and the concentrations of IL-18 and IL-1β in the supernatant were increased in group LPS ( P<0.05). Compared with group LPS, the expression of NLRP3, caspase-1, caspase-11, GSDMD and GSDMD-C was significantly down-regulated, GSDMD-C/GSDMD ratio was decreased, and the concentrations of IL-18 and IL-1β were decreased in group HU308 ( P<0.05). Conclusion:CB2R is involved in macrophage pyroptosis induced by LPS in mice.
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Objective To investigate the role of activated cannabinoid receptor 2 (CB2R) in lipopolysaccharide (LPS)-induced secretion of RAW264.7 macrophage inflammatory cytokines and its possible mechanism. Methods Macrophages were seeded in 6-well plates (2 mL/well) at the density of 1×105 cells/mL and randomly divided into four groups (n=6 each group): control group (group C), LPS group (group LPS), LPS plus CB2R agonist HU308 group (group LPS+HU308), and LPS plus HU308 plus 3-Methyladenine group (group LPS+HU308+3-MA). LPS with the final concentration of 1 μg/mL were added in group LPS, group LPS+HU308 and group LPS+HU308+3-MA. After incubation for 15 min, 3-MA with a final concentration of 10 mmol/L was added into group LPS+HU308+3-MA . HU308 with the final concentration of 10 μmol/L was added in group LPS+HU308 and group LPS+HU308+3-MA at 15 min after 3-MA intervention, and the cells were then incubated for 24 h. The concentrations of TNF-α, IL-18 and IL-1β in supernatant serum of each group were determined by ELISA. The expressions of ICAM-1 and NLRP3 mRNA were detected by RT-PCR. The expressions of LC3 and Beclin1 were detected by Western blot, and the ratio of LC3-Ⅱ/LC3-Ⅰ was calculated. LSD-t test was used for sample pairwise comparison, and one way ANOVA for inter-group comparison. A P<0.05 was considered statistically significant. Results Compared with group C, the concentrations of TNF-α [(228.86±10.20) pg/mL vs (140.05±5.54) pg/mL], IL-1β [(363.62±8.14) pg/mL vs (244.82±9.11) pg/mL], and IL-18 [(293.28±13.57) pg/mL vs (202.84±9.54) pg/mL] in supernatant serum were increased (all P<0.05), the expressions of ICAM-1 [(5.88±0.32) vs (1.00±0.03)] and NLRP3 [(8.07±0.93) vs (1.01±0.05)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.50±0.03) vs (0.40±0.06)] and Beclin1 [(0.51±0.04) vs (0.16±0.03)] were up-regulated in group LPS (all P<0.05). Compared with group LPS, the concentrations of TNF-α [(165.44±7.07) pg/mL], IL-1β [(272.09±3.35) pg/mL] and IL-18 [(220.41±6.01) pg/mL] in supernatant serum were significantly decreased (all P<0.05), the expressions of ICAM-1 [(3.21±0.35)] and NLRP3 [(1.54±0.30)] mRNA were decreased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.71±0.03)] and Beclin1 [(0.71±0.02)] were up-regulated in group LPS+HU308 (all P<0.05). Compared with group LPS+HU308, the concentrations of TNF-α [(197.06±5.59) pg/mL], IL-1β [(318.98±11.54) pg/mL] and IL-18 [(243.33±8.71) pg/mL] in supernatant serum were significantly increased (all P<0.05), the expressions of ICAM-1 [(4.04±0.21)] and NLRP3 [(5.87±0.77)] mRNA were increased (all P<0.05), the expressions of LC3-Ⅱ/LC3-Ⅰ ratio [(0.44±0.08)] and Beclin1 [(0.32±0.03)] were down-regulated in group LPS+HU308+3-MA (all P<0.05). Conclusions Activation of cannabinoid receptor 2 can alleviate LPS-induced the secretion of RAW264.7 macrophage inflammatory cytokines, and its mechanism may be related to enhanced autophagy.
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Objective To evaluate the role of M3 receptors in penehyclidine hydrochloride ( PHC)-induced reduction of lipopolysaccharide ( LPS)-induced injury to human pulmonary microvascular endotheli-al cells ( PMVECs) . Methods Human PMVECs transfected with M3 shRNA were seeded in 6-well plates (2 ml∕hole) or in culture flasks (4 ml∕flask) at the density of 1×105 cells∕ml and divided into 5 groups ( n=5 each) using a random number table method: control group ( group C) , LPS group, PHC plus LPS group ( group P+LPS) , LPS plus M3 shRNA transfection group ( group LPS+shRNA) , and PHC plus LPS plus M3 shRNA transfection group ( group P+LPS+shRNA) . Group C received no mediation, and LPS was added at the final concentration of 0. 1 μg∕ml in the other groups. PHC 2 μg∕ml was added at 1 h before adding LPS in P+LPS and P+LPS+shRNA groups. The cells were transfected with plasmid containing 2. 5 nmol∕L M3 receptor shRNA in LPS+shRNA group and P+LPS+shRNA group. Contents of filamentous actin ( F-actin) in endothelial cells were measured by flow cytometry at 1 h after adding LPS. The expression of myosin light chain kinase ( MLCK) and VE-cadherin protein was examined by immunofluorescence. The ex-pression of nuclear factor kappa B ( NF-κB) p65 and IκB was detected by Western blot. Contents of tumor necrosis factor-alpha ( TNF-α) and interleukin-6 ( IL-6) were determined by enzyme-linked immunosorbent assay. The M3 receptor mRNA transcription was detected by real-time polymerase chain reaction at 10, 30 and 60 min after adding LPS. Results Compared with group C, F-actin content was significantly de-creased, the expression of VE-cadherin and IκB was down-regulated, the contents of TNF-αand IL-6 were increased, and the expression of MLCK and NF-κB p65 was up-regulated in LPS and P+LPS groups ( P<0. 05) . Compared with group C, the expression of M3 receptor mRNA was significantly up-regulated in group LPS ( P<0. 05) , and no significant change was found in group P+LPS ( P>0. 05) . Compared with group LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB was up-reg-ulated, the contents of TNF-αand IL-6 were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+LPS and group LPS+shRNA ( P<0. 05) . Compared with group P+LPS, F-actin content was significantly increased, the expression of VE-cadherin and IκB protein was up-regulated, TNF-α and IL-6 contents were decreased, and the expression of MLCK, NF-κB p65 and M3 receptor mRNA was down-regulated in group P+LPS+shRNA ( P<0. 05) . Conclusion PHC re-duces LPS-induced injury to human PMVECs through interfering with M3 receptors and inhibiting NF-κB-mediated inflammatory responses.
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Objective To evaluate the effect of activating cannabinoid receptor 2 (CB2R) on sepsis-induced acute lung injury and the role of autophagy in mice.Methods Twenty-four SPF male C57BL/6 mice,aged 8-10 weeks,weighing 20-25 g,were divided into 4 groups (n=6 each) using a random number table method:sham operation group (group Sham),sepsis group (group Sep),sepsis plus CB2R agonist HU308 group (group Sep+HU308) and sepsis plus HU308 plus autophagy inhibitor 3-methyladenine group (group Sep+HU308+3-MA).Sepsis was induced by cecal ligation and puncture in anesthetized mice.HU308 2.5 mg/kg was intraperitoneally injected at 15 min after surgery in Sep+HU308 and Sep+ HU308+3-MA groups,and 15 min later 3-MA 10 mg/kg was intraperitoneally injected in group Sep+ HU308+3-MA.Lung tissues were obtained at 12 h after surgery and stained with haematoxylin and eosin for examination of the pathological changes which were scored and for determination of the expression of tumornecrosis factor-alpha (TNF-α),interleukin-18 (IL-18) and IL-1β mRNA (by real-time polymerase chain reaction),expression of autophagy-related protein 5 (Atg5) (by immuno-histochemistry),and expression of microtubule-associated protein 1 light chain 3 (LC3),Beclin-1 and p62 (by Western blot).The ratio of LC3Ⅱ to LC3Ⅰ (LC3Ⅱ/LC3Ⅰ ratio) was calculated.Results Compared with group Sham,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,and LC3 Ⅱ/LC3 Ⅰ ratio and lung injury score were increased in the other three groups,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep and group Sep+HU308,and the expression of p62 was significantly up-regulated in group Sep+HU308+3-MA (P＜0.05).Compared with group Sep,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly down-regulated,the expression of Atg5 was up-regulated,and lung injury score was decreased in group Sep+ HU308 and group Sep+ HU308 + 3-MA,LC3Ⅱ/LC3Ⅰ ratio was increased,the expression of Beclin-1 was up-regulated,and the expression of p62 was down-regulated in group Sep+HU308,and the expression of Beclin-1 was down-regulated,and the expression of p62 was up-regulated in group Sep + HU308 + 3-MA (P ＜ 0.05).Compared with group Sep+ HU308,the expression of TNF-α,IL-18 and IL-1β mRNA was significantly up-regulated,the expression of Atg5 and Beclin-1 was down-regulated,LC3Ⅱ/LC3Ⅰ ratio was decreased,the expression of p62 was up-regulated,and lung injury scores were increased in group Sep+HU308+3-MA (P＜0.05).Conclusion Activating CB2R can alleviate acute lung injury in septic mice,and the mechanism may be partially related to enhancing autophagy and reducing inflammatory responses.
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Objective To evaluate the role of M3 receptor in penehyclidine hydrochloride(PHC)-induced reduction of increased permeability of human pulmonary microvascular endothelial cells (PMVECs) caused by endotoxin and the relationship with mitogen-activated protein kinase (MAPK) signaling pathway.Methods Human PMVECs were seeded in 6-well plates (2 ml/hole) or in culture flasks (4 ml/flask) at the density of 1 × 105 cells/ml and randomly divided into 6 groups (n=5 each):control group (group C),M3 receptor shRNA transfection group (group shRNA),lipopolysaccharide (LPS) group,penehyclidine plus LPS group (group P+LPS),LPS plus M3 receptor shRNA transfection group (group LPS+shRNA) and PHC plus LPS plus M3 shRNA transfection group (group P+LPS+shRNA).The cells were transfected with shRNA plasmid containing 2.5 nmol/L M3 receptors in shRNA,LPS+shRNA and P+LPS+shRNA groups.LPS at the final concentration of 0.1 μg/ml was added at 24 h of incubation and then cells were incubated for 1 h in LPS and LPS+shRNA groups.PHC at the final concentration of 2 μg/ml was added at 24 h of incubation,cells were incubated for 1 h,then LPS at the final concentration of 0.1 μg/ml was added,and cells were incubated for another l h in P+LPS and P+LPS+shRNA groups.The permeability of PMVECs was measured using Transwell assay.The expression of phosphorylated p38 MAPK (p-p38 MAPK)and phosphorylated extracellular signal-regulated protein kinase 1/2 (p-ERK1/2) was detected by Western blot,the expression of heat shock protein 27 (HSP27) using immunofluorescent staining,and the expression of M3receptor mRNA by real-time polymerase chain reaction.Results Compared with group C,M3 receptor mRNA expression was significantly down-regulated in group shRNA,and the permeability of cells was significantly increased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was up-regulated in group LPS (P＜0.05).The permeability of cells was significantly decreased,and the expression of p-p38 MAPK,p-ERK1/2,HSP27 and M3 receptor mRNA was down-regulated in P+ LPS,LPS+shRNA and P+LPS+shRNA groups as compared with group LPS,and in group P+LPS+shRNA as compared with group LPS+shRNA (P＜0.05).Conclusion The mechanism by which PHC reduces endotoxin-caused increased permeability of human PMVECs is related to inhibiting activation of MAPK signaling pathway after down-regulating M3 receptor.