Effect Of Crtc1 On Acute Lung Injury Induced By Sepsis

Objective Acute lung injury（ALI）is a refractory respiratory disease with high morbidity and mortality.The pathogenesis of ALI is still unclear.Crtcl is a member of the cAMP-regulated transcriptional coactivators（CRTC）family and is involved in the regulation of biological processes,such as cell proliferation and apoptosis.Studies have reported that Crtcl plays an important role in some diseases.This study was aimed to investigate the role of Crtcl in the progression of ALI,and to explore the relationship between the mechanism and the PI3K/Akt signaling pathway.Methods（1）In this experiment,mice received cecal ligation and puncture（CLP）surgery to establish acute lung injury（ALI）model.Experimental animals were grouped as followed:Crtc1+/++Sham,Crtcl+/++CLP,Crtc1-/-+Sham,Crtc1-/-+CLP.The mice were sacrificed after 24h.Bronchoalveolar lavage fluid（BALF）and lung tissues were collected.The secretion of inflammatory factors in BALF was detected by ELISA.Evan’s blue assay was used to assess the changes in pulmonary microvascular permeability.The pathological damage of lung tissues in mice was analyzed using HE staining.qRT-PCR was used to analyze the expression of inflammatory factors（IL-6,IL-1β and TNF-α）and chemokines（CXCL1）.TUNEL staining was used to evaluate the apoptosis rate.Western blot was applied to detect the protein expression of apoptosis-related makers and the activation of PI3K/Akt signaling pathway.（2）In this experiment,lipopolysaccharides（LPS）was used to stimulate RAW264.7 cells and an in vitro ALI model was established.RAW264.7 cells were transfected with siRNA or plasmid to knock down or overexpress Crtcl.The experiment was divided into the two parts.The first part was divided into the following four groups:EGFP,EGFP+LPS,CRTC1,CRTC1+LPS.The second part was divided into the following four groups:sineg,sineg+LPS,sicrtc1,sicrtc1+LPS.The cells were collected after 6h of LPS stimulation.The mRNA expression of Crtcl,inflammatory factors（IL-6,IL1β and TNF-α）and chemokines（CXCL1）in RAW264.7s were evaluated by qRT-PCR.The rate of apoptotic cells was assessed by TUNEL staining.The protein expression of apoptosis-related makers and the activation of PI3K/Akt signaling pathway were detected by Western blot.（3）PI3K/Akt inhibitor Triciribine was given to inhibit Akt activation,while Crtcl was knocked down by siRNA.Cells were collected at 6h after LPS stimulation.qRTPCR was used to analyze the expression of inflammatory factors and chemokines.TUNEL staining was used to evaluate the apoptosis rate.Western blot was applied to detect the protein expression of apoptosis-related makers and the activation of PI3K/Akt signaling pathway.Results（1）Animal experiment results:Compared with Crtcl+/++Sham group and Crtc1-/-+Sham group,CLP caused higher histopathological scores,greater accumulation of Evans blue in mice lung tissue,enhanced the levels of IL-6 and TNFα in BALF,and elevated the mRNA levels of inflammatory factors and chemokines,increased the number of TUNEL-positive cells,up-regulated the expression of proapoptotic proteins Bax and Cleaved-caspase3,down-regulated the expression of antiapoptotic protein Bcl2,and activated the PI3K/Akt signaling pathway（P<0.05）.Crtc1 KO mice exposed to CLP demonstrated obvious alleviation of the degree of lung damage,decline of histopathological scores（P<0.05）,decrease of accumulation of Evans blue in mice lung tissue（P<0.05）,distinct reduction on the mRNA expression of IL-1β and TNF-α（P<0.05）,up-regulation of the protein expression of Bax and Cleaved-caspase3（P<0.05）,down-regulation of the protein expression of Bcl2 and pAkt（P<0.05）,while compared with Crtcl+/++CLP group（P<0.05）.（2）Cell experiment results:Compared with EGFP group and siNeg group,LPS increased the mRNAlevels of inflammatory mediators IL-6,TNF-α,IL-1β and CXCL1（P<0.05）,raised the number of TUNEL-positive cells（P<0.05）,increased the protein expressions of Bax and Cleaved-caspase3（P<0.05）,decreased the protein expression of Bcl2（P<0.05）,and increased the expression of p-Akt protein（P<0.05）.Treatment with Crtcl plasmid significantly increased the LPS-induced TUNEL-positive cells（P<0.05）,enhanced the expression of Cleaved-capase3 and Bax induced by LPS and increased the inhibitory effect of LPS on Bcl-2 expression（P<0.05）,down-regulated the expression of p-Akt（P<0.05）,while the results of the Crtcl knockdown group showed the opposite trend.（3）Akt inhibitor Triciribine reduced the effect of Crtcl silencing on the expression of inflammatory factors.Compared with siCrtcl+LPS+DMSO group,the expression of mRNA of inflammatory factors IL-6 and IL-1β in si crtcl+LPS+triciribine group were increased significantly（P<0.05）,the number of apoptosis was increased（P<0.05）,the expression of Bax and Cleaved-caspase3 protein were increased（P<0.05）,and the expression of Bcl2 protein was decreased（P<0.05）.Conclusion Crtcl deficiency provided a protective effect on lung injury caused by sepsis,with the protective effects attributed to the activation of the PI3K/Akt pathway.