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Objective:To explore the correlation between cannabinoid 2 receptor (CB2R) and pyroptosis-related indicators in mice with septic lung injury.Methods:Mice were randomly (ramdon number) divided into four groups ( n=6 per group): sham operation group (sham), mild sepsis group (ALIMi), moderate sepsis group (ALIMo) and severe sepsis group (ALIS). The model of septic lung injury was established by cecal ligation and puncture. The wet-dry weight ratio of lung tissues and lung injury scores were measured 12 hours after operation. The expression of CB2R protein was measured by western blot, and the expression of mRNA of CB2R, NLRP3, caspase-1/11, GSDMD were detected by RT-PCR. Meanwhile ELISA was used to measure the level of inflammatory factor IL-6 and TNF-α. SPSS 22.0 software was used for data analysis. Multiple comparison was analyzed by one-way analysis of variance (one-way ANOVA) and comparison between two groups was performed by LSD test or Games-Howell test. Then, the correlation between the expression of CB2R mRNA and the level of inflammatory cytokines as well as the expression of the pyroptosis-related indicators mRNA was analyzed by pearson correlation analysis, respectively. Results:The statistical value F was obtained by one-way ANOVA and comparison between two groups was performed. Compared to sham group, all above indicators increased with the aggravation of inflammation in the sepsis groups ( P<0.05). Compared to ALIMi group, the concentrations of IL-6 [(277.31±41.07) vs.(140.09±27.56), P<0.05] and TNF-α [(501.09±73.91) vs. (261.36±40.73), P<0.05] in lung tissue homogenate increased in ALIMo group. And the level of CB2mRNA [(2.99±0.28) vs. (2.02±0.19), P<0.05], the expression of CB2 protein [(0.44±0.08) vs.(0.23±0.05), P<0.05] and the level of NLRP3 [(2.53±0.26) vs.(1.61±0.15), P<0.05], caspase-1 [(6.02±0.35) vs.(3.60±0.38), P<0.05], caspase-11 [(11.43±0.83) vs.(6.30±0.65), P<0.05] and GSDMD [(10.46±0.62) vs. (5.67±0.54), P<0.05] mRNA also increased. Compared to ALIMo group, the concentrations of IL-6 [(475.90±67.65) vs. (277.31±41.07), P<0.05] and TNF-α [(713.93±58.85) vs. (501.09±73.91), P<0.05] in lung tissue homogenate increased in ALIS group. And the level of CB2mRNA [(4.00±0.19) vs.(2.99±0.28), P<0.05], the expression of CB2 protein [(0.61±0.05) vs.(0.44±0.08), P<0.05] and the level of NLRP3 [(4.75±0.40) vs.(2.53±0.26), P<0.05], caspase-1 [(8.76±0.72) vs.(6.02±0.35), P<0.05], caspase-11 [(16.31±1.13) vs.(11.43±0.83), P<0.05] and GSDMD [(16.46±1.22) vs. (10.46±0.62), P<0.05] mRNA also increased. Furthermore, correlation analysis showed that there was a highly positive correlation between the expression of CB2R mRNA and the expression of mRNA of NLRP3, caspase-1/11, and GSDMD respectively ( r>0.9, P<0.01). Conclusion:The correlation between the aggravation of inflammation, the indicators of pyroptosis and CB2R mRNA was highly positive in different degrees of septic lung injury. Consequently, CB2R may play a role in the regulatory process of inflammation.
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Objective:To evaluate the role of histone deacetylase 6 (HDAC6) in the maintanence of neuropathic pain (NP) and the relationship with myeloid differentiation factor 88 (MyD88)/nuclear factor kappa B (NF-κB) signaling pathway in the rats.Methods:Twenty-four clean-grade healthy male Sprague-Dawley rats, aged 6-8 weeks, weighing 200-260 g, were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), sham operation group (group S), NP group and NP plus HDAC6 inhibitor ACY-1215 group (group NP+ ACY). The rat model of NP was established by ligating the L 5 spinal nerve in anesthetized rats.The L 5 spinal nerve was only exposed without ligation in group S. In NP+ ACY group, ACY-1215 25 mg/kg was intraperitoneally injected daily for 21 days after the end of model establishing.The equal volume of solvent was intraperitoneally injected in S and NP groups, and group C was reared normally.The mechanical paw withdrawal threshold (MWT) was measured on 3 days before establishing the model (T 0), the day before establishing the model (T 1) and 1, 3, 7, 10, 14 and 21 days after establishing the model (T 2-7). The rats were sacrificed after measurement of MWT on day 21 after ligation, and the spinal dorsal horn tissues of L 4-6 were removed for determination of the expression of MyD88, NF-κB and phosphorylated NF-κB (p-NF-κB) (by Western blot) and expression of interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α) mRNA (by real-time polymerase chain reaction). Results:Compared with C and S groups, the MWT was significantly decreased at T 2-7, and the expression of MyD88, NF-κB, p-NF-κB, TNF-α mRNA and IL-1β mRNA was up-regulated in NP and NP+ ACY groups ( P<0.05). Compared with group NP, the MWT was significantly increased at T 5-7, and the expression of MyD88, NF-κB, p-NF-κB, TNF-α mRNA and IL-1β mRNA was down-regulated in group SNL+ ACY ( P<0.05). Conclusion:HDAC6 activation is involved in the maintanence of NP, which is related to activating MyD88/NF-κB signaling pathway in the rats.
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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 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 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.
