The Role Of Autophagy In The Regulation Of Cannabinoid Receptor 2 In Sepsis-induced Acute Lung Injury

Objective To investigate the role of autophagy in the regulation of cannabinoid receptor 2 in sepsis-induced acute lung injury.Methods(1)Twenty-four SPF male C57BL/6 mice,aged 8-10 weeks,weighing20-25 g,were divided into 4 groups(n=6 each)using a random number table method:sham operation group(group Sham),sepsis group(group CLP),sepsis plus cannabinoid 2 receptor agonist HU308 group(group CLP+HU308)and sepsis plus cannabinoid 2 receptor agonist HU308 plus 3-Methyladenine group(group CLP+HU308+3-MA).Sepsis model was induced by cecum ligation and perforation.HU308 2.5mg/kg was intraperitoneally injected at 15min after surgery in CLP+HU308 and CLP+HU308+3-MA groups,and 15min later 3-MA 10 mg/kg was intraperitoneally injected in group CLP+HU308+3-MA.Lung tissues were obtained at 12h after surgery and stained with haematoxylin and eosin for examination of the pathological changes which were scored and for determination of the expression of CB2R,TNF-α,IL-18 and IL-1βmRNA by RT-PCR,expression of autophagy-related protein 5(Atg5)by immuno-histochemistry,and expression of microtubule-associated protein 1 light chain 3(LC3),Beclin1 and p62 by Western blot.The ratio of LC3Ⅱto LC3Ⅰ(LC3Ⅱ/Ⅰ)was calculated.(2)Macrophages were seeded in 6-well plates(2ml/well)at the density of 1×10~5 cells/ml and divided into 4 groups(n=6 each)using a random number table method: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 1ug/ml were added in LPS,LPS+HU308 and LPS+HU308+3-MA groups.After incubation for15min,3-MA with a final concentration of 10mM was added into the LPS+HU308+3-MA group.15min later HU308 with the final concentration of 10uM was added in LPS+HU308 and LPS+HU308+3-MA groups,and the cells were then incubated for 24h.The concentrations of TNF-α,IL-18 and IL-1βin supernatant serum of each group were determined by ELISA.The expressions of CB2R and Atg5mRNA were detected by RT-PCR.The expressions of LC3,Beclin1 and p62 were detected by Western blot,then the ratio of LC3Ⅱto LC3Ⅰ(LC3Ⅱ/Ⅰ)was calculated.The expression of NLRP3 was detected by RT-PCR and immunofluorescent staining.Results(1)Compared with group Sham,the expression of CB2R mRNA was elevated(P<0.05),lung injury score and the expressions of TNF-α,IL-18 and IL-1βmRNA were significantly increased(P<0.05),the expressions of Atg5,LC3Ⅱ/Ⅰratio and Beclin1 were up-regulated(P<0.05),and the expression of p62 was down-regulated in group CLP(P<0.05).After activating CB2R with HU308,there was no significant difference in the expression of CB2R mRNA(P>0.05),lung injury score and the expressions of TNF-α,IL-18 and IL-1βmRNA were significantly decreased(P<0.05),the expressions of Atg5,LC3Ⅱ/Ⅰratio and Beclin1 were up-regulated(P<0.05),and the expression of p62 was down-regulated(P<0.05).There was no significant difference in the expression of CB2R mRNA after autophagy inhibition by 3-MA(P>0.05),lung injury score and the expressions of TNF-α,IL-18 and IL-1βmRNA were significantly increased(P<0.05),the expressions of Atg5,LC3Ⅱ/Ⅰratio and Beclin1 were down-regulated(P<0.05),and the expression of p62 was up-regulated(P<0.05).(2)Compared with group C,the expression of CB2R mRNA was elevated(P<0.05),the concentrations of TNF-α,IL-18 and IL-1βin supernatant serum were significantly increased(P<0.05),the expressions of Atg5 mRNA,LC3Ⅱ/Ⅰratio and Beclin1 were up-regulated(P<0.05),the expression of p62 was down-regulated(P<0.05),and the expression of NLRP3 was elevated.After activating CB2R with HU308,there was no significant difference in the expression of CB2R mRNA(P>0.05),the concentrations of TNF-α,IL-18 and IL-1βin supernatant serum were significantly decreased(P<0.05),the expressions of Atg5 mRNA,LC3Ⅱ/Ⅰratio and Beclin1 were up-regulated(P<0.05),the expression of p62 was down-regulated(P<0.05),and the expression of NLRP3 was decreased(P<0.05).There was no significant difference in the expression of CB2R mRNA after autophagy inhibition by 3-MA(P>0.05),the concentrations of TNF-α,IL-18 and IL-1βin supernatant serum were significantly increased(P<0.05),the expressions of Atg5 mRNA,LC3Ⅱ/Ⅰratio and Beclin1 were down-regulated(P<0.05),the expression of p62 was up-regulated(P<0.05),and the expression of NLRP3 was elevated(P<0.05).Conclusion Activating CB2R can alleviate acute lung injury in sepsis and the mechanism may be related to enhancing autophagy and reducing inflammatory responses.