| Part1.Correlation between microvesicles and ventilator-induced lung injury in mice.ObjectiveTo investigate the role of microvesicles(MVs)in ventilator-Induced lung injury(VILI).MethodThirty-six adult male C57BL/6 mice were randomly divided into three groups(each n=12):spontaneous breathing group(group A),normal tidal volume(VT)group(group B,VT was 7 mL/kg)and high VT group(group C,VT was 20 mL/kg).All mice underwent trachea intubation under intraperitoneal anesthesia(ketamine:100 mg/kg).Group A were kept to have spontaneous breathing while mice in group B and group C received respectively mechanical ventilation(ventilator parameters:PEEP=0cmH2O,RR=80 beats/min).After 4hours,the mice were sacrificed by lethal dose of anesthetic agent,and the blood serum,bronchoalveolar lavage fluid(BALF)and lung tissues were harvested.MVs in each group was further extracted from BALF by multi-step differential centrifugation.BALF was centrifuged to remove larger particles(400 g,5 min at 4°C).The pellet was discarded,and the supernatant was centrifuged to remove cells(2000 g,10 min at 4°C).The supernatant was then centrifuged successively at high speed(50000 g,120 min at 4°C)to pellet microvesicles.The microvesicle pellets were washed twice in PBS,pooled and centrifuged(50000 g,120 min at 4°C)to purify the microvesicles.The lung wet/dry weight(W/D)ratio was measured.Light microscopy and electron microscopy were performed to respectively observe the pathological changes in lung tissues and the ultrastructural changes in MVs.The inflammatory cells and total protein in BALF were respectively measured by hemocytometer and bicinchoninic acid methods(BCA).The levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)in serum and BALF were measured by enzyme linked immunosorbent assay(ELISA).The expression of Flotillin-2(microvesicle marker)in lung tissue and MVs was determined by Western Blot,and the expression of NLRP3,COX IV,IL-1βand TNF-αin MVs was detected.ResultsLight microscopy showed that no obvious pathological changes in lungs were found in group A and group B whereas group C illustrates a marked increase in alveolar rupture and fusion,alveolar wall thickening and inflammatory cell infiltration in the lungs.Compared with group A and B,the wet/dry ratios in lung tissues,the total cell counts and the levels of total proteins as well as IL-1βand TNF-αin BALF showed a significant increase in group C[W/D ratio:5.08±0.36 vs.4.19±0.30,4.29±0.44;total cell count in BALF(×10~4):112.4±21.3 vs.26.4±8.6,33.4±11.6;total proteins in BALF(g/L):1.157±0.161 vs.0.482±0.193,0.544±0.215;IL-1βin BALF(pg/mL):130.25±28.58 vs.74.88±20.32,84.25±11.96;TNF-αin BALF(pg/mL):517.700±40.72 vs.404.31±31.84,421.62±48.04,all P<0.05];and the protein expressions of Flotillin-2 in lung tissues also showed a significant increase in group C[Flotillin-2(R value):1.607±0.107 vs.1.131±0.107,1.233±0.113,all P<0.05],but there was no significant difference among the parameters mentioned above between group A and B(all P>0.05).The ultrastructure of MVs were shown by transmission electron microscope(TEM)to observe the differences in the size and morphology of MVs after mechanical ventilation.The results demonstrated that MVs appeared round or ellipse,and the structure of MVs in group C significantly increased compared with group A and group B.Further,The overexpression of IL-1βand TNF-αin MVs of group C was higher than that of group A and B[IL-1βin MVs(pg/mL):1225.29±26.38 vs.974.50±51.52,1010.70±37.40;TNF-αin MVs(pg/mL):488.46±13.75 vs.410.25±28.02,411.35±10.02,all P<0.05].And there was no or low expression of NLRP3 and COX IV in MVs of the three groups.ConclusionThe lung injury induced by mechanical ventilation in C57BL/6 mice may be closely related to the microvesicles carrying IL-1βand TNF-α.Part2.Inflammatory effect of microvesicles in ventilator-induced lung injury in mice.ObjectiveTo investigate the inflammatory effect of microvesicles(MVs)on lung tissue of mice.MethodMulti-step differential centrifugation was used to extract and purify MVs from groups A,B and C(part1).The supernatant obtained from the final centrifugation step of group C was retained as a control to exclude the potential influence of soluble factors during the production of MVs.Thirty-six adult male C57BL/6 mice were randomly divided into three groups(each n=12):control group(Control group),test A group(Test A),and test C group(Test C).All mice underwent trachea intubation under intraperitoneal anesthesia(ketamine:100 mg/kg).The mice in control group received 30μL of the supernatant obtained from the final centrifugation step of group C,while the mice in Test A and Test C groups received 30μL of MVs suspension from group A and C,respectively.After 4 hours,the mice were sacrificed by lethal dose of anesthetic agent,and the blood serum,bronchoalveolar lavage fluid(BALF)and lung tissues were harvested.The lung wet/dry weight(W/D)ratio was measured.Light microscopy were performed to observe the pathological changes in lung tissues.The inflammatory cells and total protein in BALF were respectively measured by hemocytometer and BCA.The levels of interleukin-6(IL-6)in serum and BALF were measured by ELISA.ResultsLight microscopy showed that compared with the Control group,the lung tissue of Test A and Test C groups showed a obvious increase in scattered bleeding points,alveolar septum thickening and inflammatory cell infiltration;And compared with the Test A group,the pathological changes of lung tissue in the Test C group were more notable.The lung W/D ratios,inflammatory cell counts,and the levels of total proteins and IL-6 in the BALF of Test A and Test C groups were significantly higher than those in the Control group:[W/D ratio:4.78±0.31,5.56±0.42 vs.4.33±0.21;total cell count in BALF(×10~4):75.3±37.9,142.6±62.4 vs.20.4±8.9;total proteins in BALF(g/L):0.396±0.039,0.531±0.090 vs.0.257±0.044;IL-6 in BALF(pg/mL):10.1±1.1,14.5±2.9 vs.5.8±1.2,all P<0.05];In addition,these lung inflammation indicators of mice in Test C group were significantly higher than those in Test A group.ConclusionMicrovesiclespackagingIL-1βandTNF-αenhancelung inflammation.Part3.The role of cofilin in the formation of microvesicles in VILI mice.ObjectiveIt is well-known that cofilin plays a key regulatory role in actin dynamics,and actin filaments can produce protrusions of tissue cells by dynamic polymerization and depolymerization.Therefore,the study is aimed to investigated the effect of cofilin in the formation of microvesicles(MVs)in VILI mice.MethodForty-eight adult male C57BL/6 mice were randomly divided into four groups(each n=12):control group(PBS),p-cofilin mAb group(diluted 1:50),high-Vt group(20 mg/kg,PBS),and p-cofilin mAb(diluted 1:50)+high-Vt group(20 mg/kg).All mice underwent trachea intubation under intraperitoneal anesthesia(ketamine:100 mg/kg).To begin with,the mice in p-cofilin mAb group and p-cofilin mAb+high-Vt group were administered intratracheally with30μL p-cofilin mAb(1:50),and the control and high-Vt mice were offered with PBS.After 1 h,high-Vt group and p-cofilin mAb+high-Vt group were combined with sedation,muscle relaxation(xylazine hydrochloride,10 mg/kg)and mechanical ventilation with Vt of 20 mL/kg(ventilator parameters:PEEP=0cmH2O,RR=80 beats/min).4 hours later,the mice were sacrificed by lethal dose of anesthetic agent,and the blood serum,BALF and lung tissues were harvested.The lung W/D ratio was measured.Light microscopy were performed to observe the pathological changes in lung tissues.The inflammatory cells and total protein in BALF were respectively measured by hemocytometer and BCA methods.The levels of IL-6 in serum and BALF were measured by ELISA,and the expression of p-cofilin,cofilin and Flotillin-2 in lung tissue were evaluated by western blot analysis.ResultsCompared with the control and p-cofilin mAb mice,the high-Vt group had significantly higher expression of p-cofilin and Flotillin-2 in the lung tissue;compared with the high-Vt group,the expression of p-cofilin and Flotillin-2 in the lung tissue of p-cofilin mAb+high-Vt group of were significantly decreased(P<0.05),indicating that when the antibody blocked the phosphorylation of cofilin caused by mechanical ventilation,the corresponding MVs production caused by mechanical ventilation was also significantly reduced.Light microscopy showed that compared with the the control and p-cofilin mAb mice,the lung tissue of high-Vt and p-cofilin mAb+high-Vt mice showed a significant increase in scattered bleeding points,alveolar septum thickening and inflammatory cell infiltration,while compared with the high-Vt group,the p-cofilin mAb+high-Vt group showed a significant decrease in these pathological changes of lung tissue.At the same time,these lung inflammation indexes(pulmonary W/D ratio,inflammatory cell count,total protein and IL-6)were significantly higher in the high-Vt and p-cofilin mAb+high-Vt groups than in Control and p-cofilin,while the lung inflammation in the p-cofilin mAb+high-Vt group was significantly lower than that in the high-Vt group(P<0.05).ConclusionBlocking the phosphorylation of cofilin can inhibit the formation of microvesicles in the lung,thereby reducing lung injury. |
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