Effects Of Pentraxin 3 On Ventilator-induced Lung Injury

VIABILITY OF A549 CELLS AND EXPRESSION OF PTX3 IN A549 CELLS INDUCED CYCLIC STRETCHBackground and Objective Amount of data has confirmed ventilator- induced lung injury (VILI) is relevant with mechanical stretch. One of the underlying mechanisms of ventilator- induced lung injury (VILI) is that mechanical stretch leads to trigger the expression of stretch-sensitive genes and the release of pro-inflammatory mediators in lung cells in response to mechanical stretch. The stretch-sensitive genes initiate a train of biological reactions to cause a series of. biological injury. Long pentraxin PTX3 is a newly discovered acute phase protein produced at the sites of infection and inflammation as a marker of inflammation. It plays an important part in inflammation cascade reaction and also participates in the inflammation reaction leaded cyclic stretch and has stretch-response-element in its promoter. As a result, we guess PTX3 is possibly one of the stretch-sensitive genes. This experiment is to study the effect of mechanical stretch on viability of the cells in human alveolar epithelial cells(A549) and expression of PTX3 in A549.Methods Cells were subjected to cyclic stretch and relaxation by using the Flexercell Strain Unit FX-4000 Tension plus (Flexcell International) which is a computer-driven, vacuum-operated, stress-providing instrument. The vacuum-operated pump suck the special flexible cell culture, forming the cell elongating, thus the cell growing on the film accept tension, to analogue with stimulus of mechanical ventilation to pulmonary alveolar II type epithelium. The cell stress size is in proportion to the stretch extent of the culture .Computer controlled the duration, amplitude, and frequency of the applied stretch.The cells grown on collagenⅠBioFlex plates were divided into 3 groups. Grpup A and group B were exposed to square cyclic stretch at 0.3Hz using the Flexercell system with 6% and 20% elongation of cells. Loading time is 1 h , 2 hs , 4 hs and 6 hs For group C, We used a"Flexstop,"which is a rubber stopper inserted into the underside of the BioFlex culture plate wells of the control nonstretched cells to prevent the vacuum-induced flexing of the BioFlex growth surface.The apoptosis and viability of the A549 cells were detected by flow cytometry and laser confocal microscopy following staining by Annexin V/PI, transmission electron microscope and fluorescence microscope after staining by Hoechst33258.The secretion of TNF-αby A549 cells was detected by ELISA. Following treatments, conditioned media and cells were collected to measure the levels of PTX3 protein by Western blotting. of Real-time RT-PCR was performed to measure PTX3 mRNA extracted from stretched cells.Results Compared with group C, the percent of apoptosis cells in group B significant increased after stretching for more than 1 hour(p<0.01), but in group A it didn't(p>0.05), which was caused by apoptosis in later period and early phase. But not notable difference of the percent of cell death was not found between group A and group B (p> 0.05).The percent of apoptosis A549 cells of group B time-dependently increased and at the same time so did the secretion of TNF-αCompared with group C, expression of PTX3 mRNA and protein of group B cells time-dependently increased. PTX3 mRNA and protein of group B significantly increased by 6.25 and 2.58 times after stretching for 2 hs(p< 0.01).The expression of group A cells didn't change.Conclusion These results suggest that 20%mechanical stretch not only induces apoptosis of alveolar epithelial cells and secretion of TNF-α, but also induces expression of PTX3,which may play an important role during ventilator-induced lung injury.PartⅡINHIBITION OF EXPRESSION OF PTX3 IN A549 CELLS BY RNA INTERFERENCEBackground and Objective RNA interference (RNAi) has been used to mediate sequence-specific gene silencing and has become a powerful and widely used tool for the analysis of gene function and gene therapy .That basal principle is to through minor 21-23 nucleotide fragments to identify complementary mRNA , urge complementary mRNA to degradate and make the relevance gene keep silence after transcription and the cell show phaenotype of definite gene deletion thereby.At present, methods of preparation of siRNA consist of the extracorporeal chemosynthesis , extracorporeal transcribing , expression vector combining in vitro and so on . Among them, extracorporeal chemosynthesis has the convenient simple characteristic. Our study is to screen for chemosynthesized siRNAs that inhibit the expression of PTX3 in A549 cells.Methods Three PTX3 siRNAs, one GAPDH siRNA as positive control ,one random siRNA as negative control were synthesized and labeled with FAM siRNA were for measurement of transfection effect by confocal microscopy and flow cytometry. SiRNAs were transfected into A549 cells by LipofectamineTM 2000. The expression of PTX3 was analyzed by Real-time PCR and Western blot.Results The transfection effect of LipofectamineTM 2000 for A549 was 89.18%±3.26%.Compared with blank control, siRNAs targeted PTX3 were identified to be able to respectively down-regulate the PTX3 expression (P<0.01). SiRNA which was most effective specifical for PTX3 restrained PTX3 mRNA to 3.41% , PTX3 protein to 4.94%.In negative control group, PTX3 mRNA and protein expressing had not notable difference with blank control(P>0.05).Conclusion LipofectamineTM 2000 can transfect siRNA into A549 cells with highly efficiency. RNAi technique can significantly inhibit expression of PTX3 in A549 cells.PartⅢEFFECT OF INHIBITION OF PTX3 EXPRESSION IN A549 CELLS ON STRETCH-RESPONSEObjective To study the effect of inhibition of PTX3 expression in A549 cells. Methods The A549 cells cultured in vitro were divided into 5 groups,i.e., normal control group, stretched group, RNAi group, RNAi and stretched group, static control group. Cells grown on collagenⅠBioFlex plates were exposed to square cyclic stretch at 0.3Hz using the Flexercell system with 20% elongation of cells for 4 hours in stretched group. The cells of RNAi group were transfected with chemosynthetic PTX3 specific siRNA by RNAi technique. In RNAi and stretched group, cells were were transfected with chemosynthetic PTX3 specific siRNA by RNAi technique. before stretch. Cells were unstretched using"Flexstop"as static control group. Real-time PCR of PTX3 mRNA extracted from cells was performed .The conditioned media were collected and the levels of PTX3 protein were measured by Western blotting. Apoptosis of the cells following treatments were routinely monitored.Results Compared with static control group,cyclic stretch led to increased PTX3 gene expression and protein in lung epithelial cells. PTX3 mRNA of strech group increased by 7.397±0.276 times(P<0.01), PTX3 protein of stretch group and static control group were 0.647±0.019 vs1.590±0.060(P<0.01);Compared with stretch group, PTX3 mRNA and protein expression notably suppressed in RNAi and stretched group. Compared with static control group,the percentage of apoptotic cells significantly increased in stretch group; Compared with stretch group, the percentage of viable cells in RNAi and stretched group significantly increased( 85.377%±1.395% vs 93.727%±0.492%,p<0.01).Conclusion Stretch can induce apoptosis of A549 cells and excessive inflammatory reaction through up-regulation of PTX3 which may be important mediator and play an important role during ventilator-induced lung injury.PartⅣEXPRESSION OF PTX3 IN LUNG OF RAT WITH VENTILATOR-INDUCED LUNG INJURYObjective As a life support for critically ill-patients, mechanical ventilation is often indispensable. However, mechanical ventilation can induce and/or worsen acute lung injury. Ventilator-induced lung injury has been considered as one of the most important contributing factors to the high mortality in ARDS. This study aims to investigate the expression of PTX3 in lung tissue of rat with ventilator-induced lung injury.Methods Forty-eight Sprague-Dawley rats weighing 300～350g were randomly divided into the following experimental groups (16 rats in each group): group C spontaneously breathed; group PV were ventilated with protective tidal volume (VT= 6 ml/kg, PEEP 3～5cmH2O, RR=70); group HV were ventilated with high tidal volume (VT= 20 ml/kg, PEEP 0 cmH2O, RR=20). The expression of PTX3mRNA in lung tissue was measured by real-time polymerase chain reaction (real-time PCR); PTX3 protein in lung tissues was measured by Western Blot and immune histochemistry; Lung pathological changes were examined with optical microscopy; Total protein, wet/dry ratios (W/D),MPO activity of the lung tissue or lavage fluid were measured with corresponding methods.Results Compared with group C, in group HV PaO2 was significantly decreased(P < 0.05)and LPI,W/D and MPO significantly increased (P < 0.05). Compared with group C and group PV, TNF-αlevel significantly increased in group HV. But there is not obvious difference between group C and PV(P > 0.05). Lung cell apoptosis can be seen in group HV detected by TUNEL. The expression of PTX3 in group HV were significantly higher than group C, Compared to group C , PTX3 mRNA and protein expressing of group B significantly increase by 3.493 times and 3.799 times(P < 0.01).But there was no significantly difference between group C and group PV(P>0.05) Conclusion High volume ventilation induces significant VILI and remarkably increases the expression of PTX3 in lung tissue, which probably plays an important role in VILI.