CYP2J2 Gene Overexpression Protects Against Pulmonary Hypertension With Lung Ischemia-reperfusion Injury In Rats Via Anti-inflammation

ObjectivesTo investigate the protective effect and possible mechanism of cytochrome P450 epoxygenase(CYP)2J2 overexpression on pulmonary hypertension(PAH)with lung ischemia-reperfusion injury(LIRI)in rats.Methods(1)Animal grouping30 Sprague-Dawley(SD)male rats(8 weeks old,body weight of 250-300g)were randomly assigned into six groups(n=5 in each group): Control group,PAH group,lung ischemia-reperfusion group(IR),PAH+IR group,PAH+IR and r AAV-GFP gene transfection group(PAH+IR+GFP)and PAH+IR and r AAV-CYP2J2 gene transfection group(PAH+IR+ CYP2J2).(2)Animal model of PAHRats received one intraperitoneal injection of monocrotaline(MCT,60 mg/kg of body weight).(3)CYP2J2 gene deliveryRecombinant adeno-associated viral(r AAV)vectors containing green fluorescent protein(GFP)or CYP2J2 were prepared.Rats received one intravenous injection of r AAV-GFP or r AAV-CYP2J2(1*10^7PFU/kg of body weight)24hours after MCT administration.(4)Animal model of LIRIA non-invasive arterial clamp was used to clamp the left hilum to cause lung ischemia and the clamp was released after 1 hour of sustained clamping to restore blood flow for 2 hours.(5)Animal model of PAH with LIRILung ischemia-reperfusion was performed 4 weeks after MCT administration to establish model of PAH with LIRI.(6)Mean pulmonary arterial pressure(mPAP)and right ventricular systolic pressure(RVSP)were measured by right cardiac catheterization immediately after modeling.The rats were sacrificed after the measurement and the lung and heart tissues and blood were collected for further analysis.(7)Right ventricular hypertrophy index(RVHI)was measured in each group.(8)Changes in lung tissues were observed by visual inspection and light microscopy.(9)Lung wet-to-dry weight ratio(W/D)was measured.(10)Determination of IL-1β,IL-6,IL-10 and TNF-α in rat serum by ELISA.(11)Determination of CYP2J2,PPARγ,NF-κBp65 and IκBα in lung tissues by Western blot.Results(1)CYP2J2 gene transfection significantly increased CYP2J2 protein content in rat lung tissue.(2)Effects of CYP2J2 overexpression on mPAP and RVSPmPAP and RVSP in PAH+IR group were significantly higher than those in PAH and Control groups(P<0.05).mPAP and RVSP in PAH+IR+2J2 group were significantly lower than those in PAH+IR group(P<0.05).(3)Effects of CYP2J2 overexpression on RVHIRVHI in PAH+IR and PAH group were significantly higher than that of Control group.However,there was no significant statistical difference between PAH+IR group and PAH group(P>0.05).RVHI in PAH+IR+2J2 group was significantly lower than that of PAH+IR group(P<0.05).(4)Effects of CYP2J2 overexpression on lung histopathologyThe appearance of lung in the Control and PAH groups was generally normal.The congestion and edema were obvious in IR group,which was aggravated in the PAH+IR group.CYP2J2 overexpression significantly alleviated the congestion and edema.Lung tissues in Control group under light microscopy showed normal architecture.Endothelial proliferation and thickening of pulmonary arteries were observed in each group with MCT administration.Inflammatory cell infiltration and edema were obvious in IR group,which were aggravated in the PAH+IR group.CYP2J2 overexpression significantly alleviated the inflammatory cell infiltration and edema.(5)Effects of CYP2J2 overexpression on lung W/DW/D in PAH+IR and PAH+IR+2J2 groups was significantly higher than that of Control group(P<0.05),but W/D in PAH+IR+2J2 group was significantly lower than that in PAH+IR group(P<0.05).(6)Effects of CYP2J2 overexpression on inflammatory cytokines and inflammation-related proteinCompared with Control group,the serum levels of IL-1β,IL-6 and TNF-αwere significantly increased in PAH+IR,PAH and IR groups(P<0.05).CYP2J2 overexpression significantly inhibited the increase of the above inflammatory cytokines and increased the levels of anti-inflammatory factor IL-10(P<0.05).Besides,PPARγ and IκBα in PAH+IR group was significantly decreased compared with Control group(P<0.05),while NF-κB p65 in PAH+IR group was significantly increased(P<0.05),which were significantly inhibited by CYP2J2 overexpression(P<0.05).Conclusions(1)PAH aggravated LIRI,and LIRI further increased pulmonary artery pressure.(2)CYP2J2 gene overexpression significantly protects against PAH with LIRI.(3)CYP2J2 can inhibit the inflammatory response of PAH with LIRI,which may be mediated by the activation of PPARγ.CYP2J2 gene overexpression can protect against PAH with LIRI via anti-inflammatory,which may be mediated by the activation of PPARγ/NF-κB pathway.