| Objective:LY294002,a PI3 K inhibitor,was used to inhibit PI3 K / Akt signaling pathway and infected rat lung tissue by mi R21-5p overexpression of adeno-associated virus vector.It was to verify that mi R21-5p of hyperoxia-induced acute lung injury(HALI)can target PTEN,thus activating PI3K/Akt signaling pathway to regulate AEC-? apoptosis and play a protective role in lung injury.Methods:Sixty male SD rats(SPF 200-250g)were random Ly divided into 5 groups(n=12): the HALI group(fed for three weeks + established hyperoxia injury model),the LY+HALI group(fed for three weeks + injected PI3K/AKT pathway inhibitor LY294002 of 0.3mg/kg via tail vein + established hyperoxia injury model),the LY+mi R21+HALI group(infected mi R21-5p over-expression virus vector by the trachea and fed for 3 weeks + injected PI3K/AKT pathway inhibitor LY294002 of 0.3mg/kg via tail vein + established hyperoxia injury model),the mi R21+HALI group(infected mi R21-5p overexpression virus vector by trachea and fed for 3 weeks + established hyperoxia injury model)and DMSO+HALI group(fed for three weeks + injected 0.5% DMSO via tail vein + established hyperoxia injury model).At 0h,24 h,48h and 60 h,the following lung injury related indicators were tested: 1)the carotid artery blood was collected to test oxygenation index(OI)and respiratory index(RI);2)the intact upper left lung was taken to measure Wet/Dry(W/D)weight ratio;3)the lower lobe of left lung was taken for HE staining and pathological score of lung injury was standardized by international standard;4)real-time quantitative PCR(RT q PCR)was used to test the relative expression of mi R21-5p in AEC-II of each experimental group;5)the relative expression levels of PI3 K,PTEN and Akt / p-AKT in each experimental group were tested by western blot;6)apoptosis of AEC-II was tested by flow cytometry(FCM)using Annexin V-FITC/PI double staining.Results:1.Oxygenation index(OI)and respiratory index(RI):The OI of rats in each group decreased gradually with time,and the RI increased gradually with time(P<0.05).At 24 h,48 h and 60 h,the OI was significantly lower and RI was significantly higher in LY++HALI group than those in HALI group(P<0.05);the OI was significantly higher and RI was significantly lower in mi R21+HALI group than those in the other four groups(P<0.05).2.Wet/Dry(W/D)weight ratio: The W/D weight ratio of each group increased gradually with time points(P<0.05).At 48 h and 60 h,the W/D weight ratio of LY+HALI group was significantly higher than those of HALI group(P<0.05)and the W/D weight ratio of mi R21 +HALI group was significantly lower than that of other four groups(P<0.05).3.HE staining: At 0h,2-3 neutrophils were occasionally detected in lung interstitial;at 24 h,neutrophils were seen in lung interstitial of all groups,neutrophils increased most significantly and thickened lung interstitial was observed in LY+HALI group;at 48 h,a large number of neutrophils were found in alveoli and lung interstitial in each group,and protein fragments and hyaline membrane were found in LY+HALI group;at 60 h,except the mi R21+HALI group,protein fragments and hyaline membrane were found in alveoli of the other four groups,and alveolar interstitial were significantly thickened,especially in LY+HALI group.4.The standardized pathological score of lung injury: The pathological score of each group gradually increased with time points.There was no significant change in the pathological score of each group at 0h(P>0.05)and at 24 h,48h and 60 h,the pathological score of the LY +HALI group was significantly higher than those of the other groups(P<0.05).The mi R21+HALI group had significantly lower pathological scores at 24 h,48h and 60 h than the other 4 groups(P<0.05).5.Detection of AEC-? apoptosis by flow cytometry: The apoptosis rate of AEC-II increased gradually with the increase of time;at 0h,there was no significant change in apoptosis rate(P>0.05);at 24 h,48h and 60 h,the apoptosis rate of AEC-II in LY+HALI group were significantly higher than those in HALI group(P<0.05),and the apoptosis rate in mi R21+HALI group were significantly lower than those in other four groups(P<0.05).6.RT-q PCR detection of mi R21-5p levels: At 24 h,48h and 60 h,the HALI group,LY+HALI group and DMSO+HALI group were significantly lower than those of the 0h(P<0.05);In LY+mi R21+HALI and mi R21+HALI groups,there were no significant changes in mi R21-5p at 0h,24 h,48h and 60h(P>0.05).7.Western blot detection of related protein expression results: at 24 h and 48 h,p-AKT was significantly increased in mi R21+HALI group(P<0.05),and p-AKT was decreased significantly in LY+HALI group(P<0.05);at 60 h,p-AKT was not found in the other groups except for the mi R21+HALI group in which p-AKT was increased significantly(P<0.05);at 0h,24 h,48h and 60 h,PTEN in mi R21+HALI group and LY+mi R21+HALI group were significantly decreased(P<0.05)and PI3 K in LY+HALI group and LY+mi R21+HALI group were significantly decreased(P<0.05).Conclusions: In HALI animal models,mi R21-5p can regulate AEC-II apoptosis by targeting PTEN to activate the PI3K/AKT signaling pathway and reduce the occurrence of HALI to play a lung protective effect. |
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