A Study On The Role Of CCN3 And Its Mechanism In LPS-Induced Acute Lung Injury

BackgroundAcute lung injury(ALI)/Acute respriatary distress syndrom(ARDS)is a common clinically life-threatening syndrome,which is mainly caused by internal or external lung diseases,such as severe lung infection,sepsis,aspiration,trauma and so on.The damagement of epithelial and capillary endothelial basement membrane,caused the diffuse pulmonary interstitial and alveolar edema,clinically mainly manifested as refractory hypoxemia and respiratory failure.According to the statistics,the occurrence of ARDS in the community was((1-5)/10000),but the mortality rate was as high as 30%-40%,which was a serious threat to human life and health.Its pathogenesis is extremely complicated,and it has not been clearly clarified.Diffuse alveolar injury(DAD)is the classical histopathological features of ARDS.Type ?alveolar epithelial cells(AEC ?),which cover the surface of the alveoli,as the main component of the respiratory membranes,have a barrier protection function.Therefore,to explore the possible mechanisms that affect the survival or apoptosis of AEC ? cells will help improving the prognosis of patients with ALI/ARDS,which is the basis for future research.Nephroblastoma overexpressed(NOV),also known as CCN3,belongs to a member of the CCN protein family.Its name derived from the the abbreviation of the first letter of the first three members Cyr61/CCN,CTGF/CCN2 and NOV/CCN3.CCN3 was first isolated from the neonatal chick Wilms tumor tissue infected with myeloblastosis-associatedvirus-1(MAV)by Joliot and other scholars in 1992.Later,they were found in the heart,liver,kidney,nervous system,lung and other tissues.They combined with the receptor on the cell surface,such as integrin,heparan sulfate proteoglycans,protein kinases,hormones,etc to participate in and regulate cell adhesion,migration,proliferation,apoptosis and differentiation.Thereby they play important roles in embryonic development,tumorigenesis and development,tissue repair,and wound healing.CCN3 has 50%homology similarity with CCN1 and CCN2 in structure.It is regarded as an emerging family in fine regulation of inflammatory response.It has been confirmed that the CCN protein family is involved in the occurrence and development of many acute and chronic inflammatory diseases,such as glomerulonephritis,rheumatoid arthritis,osteoarthritis,inflammatory bowel disease,nervous system and lung tissue diseases such as chronic obstructive pulmonary disease diseases,mechanical ventilation-related pulmonary infection(VAP),etc.However,little is known about the role of CCN3 in ALI/ARDS.At present,there is no effective serum molecular markers to diagnose ALI and predict the severity of the disease accurately.In a prospective cohort clinical study on ALI/ARDS,it was found that the expressions of more than a dozen of proteins including NOV/CCN3,IL-6,IGFBP-4,etc,were increased by gene chip technology combining with clinical informatics during ALI,especially in severe ARDS patients.And the extent of the increasements were closely related to the mortality of the disease.It is proposed that NOV/CCN3,IL-6,IGFBP-4,etc.are important biological markers to judge the severity of ARDS patients.Based on this discovery,it is prompted that highly expressed of CCN3 may be related to the acute inflammatory response of ALI.LPS,as the main component of bacterial endotoxins in the wall of gram-negative bacteria,is the main pathogenic factor that causes sepsis and ALI.A series of animal models of endotoxin-induced ALI have been established,such as intravenous injection,intratracheal injection and intraperitoneal injection.Intratracheal injection of LPS is the most common model of direct lung injury model among them.In vitro study it has been confirmed that NF-?B and TGF-? signaling pathways are important hubs in regulating the inflammation,apoptosis and fibrosis of ALI.Therefore,whether CCN3 is involved in the inflammation and apoptosis process of LPS-induced ALI,or regulated by NF-?B or TGF-?-mediated signaling pathways remain to be further explored.It has important clinical and social significance for the prevention and treatment of ALI/ARDS.Objective1.To study the expression of CCN3 in lung tissue of ALI animal model.2.To observe the expression of CCN3 in A549 cells,and to explore the role of CCN3 in inflammation and apoptosis on A549 cells.3.To explore the possible signal pathways of CCN3 involved in ALI,and provide new theoretical support for clinically targeted treatment of ALI.Method1.In vivo animal experiment1)It was divided into PBS group and LPS group randomly,0.5 mg/kg LPS was instilled directly into the trachea of C57BL/6 wild-type mice for 24 hours to construct a model of ALI in vivo.2)To stain the supernatant of the alveolar lavage fluid(BALF)in each group of mice by Wright-Gimsa to calculate the amount of the percentage of neutrophils;then BCA method was used to detect the total protein concentration in the BALF fluid.3)To detect the expression levels of inflammatory factors such as TNF-?,IL-1? and IL-6 in BALF and blood by ELISA method.4)To observe the morphological changes of lung injury by Hematoxylin and eosin(H&E)staining with the paraffin-embedded sections of mouse lung tissue.Then evaluate the effectiveness of the mouse ALI model by lung injury pathlogical score.5)To detect the expression of CCN3 protein in lung tissues by using immunofluorescence staining and Western blot.2.In vitro cell experiment1)Human alveolar type ? epithelial cell line(A549 cell line)was incubated with LPS with different concentrations(0 ?g/mL,0.01 ?g/mL,0.1 ?g/mL,1 ?g/mL)for 12 h.Then incubate the A549 cell line for 0 h,3 h,6 h,12 h,and 24 h at optimal concentration of LPS,to establish the vitro cell model of ALI.The expression of CCN3 protein was detected by Western blot method,and the optimal concentration and optimal time of LPS were screened for follow-up research.2)Transfect CCN3-siRNA to A549 cells by transient transfection technique,then detect the transfection effect of CCN3 by qPCR and Western blot method.3)They were divided into four groups:NC-siRNA group,NC-siRNA+LPS group,CCN3-siRNA group and CCN3-siRNA+LPS group.ELISA method was used to detect the expression levels of inflammatory factors(such as IL-1?,TNF-?,and TGF-?1)after silencing of CCN3 expression.4)Flow cytometry was used to detect the apoptosis rates of A549 cells,and the expression of apoptosis-related proteins Bcl-2 and Caspase-3 were detected by Western blot after silencing of CCN3 expression.5)To construct a CCN3 overexpression vector,they were divided into three groups:A549 cell group(blank control group),pLVX-Puro group(blank plasmid transfection group);and pLVX-Puro-CCN3 group(CCN3 overexpression plasmid).6)ELISA method was used to detect the expression levels of IL-1? and TNF-?.qPCR method and Western blot method were used to detect the efficiency of transfection.7)Flow cytometry was used to detect the rate of apoptosis,and Western blot was used to detect the expression of apoptosis-related proteins Bcl-2 and Bax.8)They were divided into four groups:NC-siRNA group,NC-siRNA+LPS group,CCN3-siRNA group and CCN3-siRNA+LPS group.9)Western blot method was used to detect the expression of TGF-?1,TGF-?R ? and p-Smad2/3 protein.10)Laser confocal was used to detect the changes of NF-?B fluorescence intensity,Western blot method was used to detect the levels of NF-?B p65 protein in the cytoplasm and nucleus.Result1.The total number of cells and total protein in BALF in LPS group were significantly increased,and the expressions of inflammatory factors IL-6,TNF-? and IL-1? in BALF and serum were also significantly up-regulated.2.H&E staining showed that the alveolar integrities in LPS group were totally destroyed,a large number of inflammatory cells were infiltrated.The pathological lung injury score in LPS group was also significantly higher than the PBS group.3.Immunofluorescence showed that the fluorescence intensity of CCN3 in LPS group was greatly increased.Western blot showed that the expression of CCN3 protein in LPS group was significantly increased.4.Western blot showed that when different concentrations(0 ?g/mL,0.01 ?g/mL,0.1?g/mL,1 ?g/mL)of LPS were applied to A549 cells for 12 h,the expression of CCN3 was increased significantly according with the increasement of LPS concentration.Then incubating A549 cells with 0.1 ?g/mL LPS for 0 h,3 h,6 h,12 h,and 24 h,the expression of CCN3 was also increased with the time,reached a peak at 12 h,and then decreased rapidly.5.The results of qPCR and Western blot showed that the expression of CCN3mRNA and CCN3 protein in the CCN3-siRNA transfection group was significantly reduced.6.ELISA results showed that after LPS stimulation,the expression of IL-1??TGF-?1 and TNF-? were increased significantly,while the expression of IL-1? and TGF-?1 in the CCN3-siRNA transfection group were decreased significantly.7.Flow cytometry showed that the apoptosis rate was increased significantly after LPS stimulation,while in the CCN3-siRNA transfection group was significantly reduced;Western blot results showed that the level of Bcl-2 protein in CCN3-siRNA transfection group was significantly higher than that of the control group,while the expression of Caspase-3 was downregulated than the control group.8.The results of qPCR and Western blot showed that the expression of CCN3 mRNA and CCN3 protein in the pLVX-Puro-CCN3 transfection group was significantly up-regulated.9.The ELISA method showed that the expression levels of IL-1? and TNF-? in the pLVX-Puro-CCN3 transfection group were significantly higher than those in the blank transfection group.10.Flow cytometry results showed that compared with the A549 blank group and the pLVX-Puro group,the apoptosis rate of the pLVX-Puro-CCN3 group was significantly increased;Western blot showed that the expression of the pro-apoptotic protein Bax in the pLVX-Puro-CCN3 group was significantly increased,while the expression of anti-apoptotic protein Bcl-2 was decreased significantly.11.Laser confocal showed that the fluorescence intensity of NF-?B nuclear translocation was increased significantly after LPS stimulation,while it was significantly lower in the CCN3-siRNA transfection group than that of the control group;Western blot results also showed that the expression of NF-?B p65 in the nucleus was siginificantly decreased,while the expression in the cytoplasm was greatly increased after CCN3-siRNA transfection.12.The results of Western blot showed that compared with the NC-siRNA group,the expressions of TGF-?1,TGF-?R ? and p-Smad2/3 were significantly increased after LPS stimulation;while the expression of TGF-?1,TGF-?R ? and p-Smad2/3 were significant declined after CCN3-siRNA intervention.Conclusion:CCN3 participates in ALI/ARDS disease process via promoting the secretion of inflammatory factors and the apoptosis of AEC ? cells through the TGF-?/p-Smad or NF-?B signaling pathways.