Role Of Cystine/Glutamate Transporter In Rabbits With Ventilator-induced Lung Injury

Objective:In order to investigate the pathophysiological significance of cystine/glutamate transporter (Xc") system in the development process of ventilator-induced lung injury (VILI), the New Zealand White Rabbit model of mechanical ventilation (MV) was developed.and the effect of L-HCA, an inhibitor of Xc", on VILI were observed. The results of this project may therefore contribute to the further pathogenic research of VILI and and be a new target for the clinical prevention and treatment of VILI.Methods:1. New Zealand white rabbits treated with MV were randomly divided into four groups:low tidal volume (LV), high tidal volume(HV), HV+L-Homocysteic acid (L-HCA), and LV+L-HCA.2. Ventilation strategies of low tidal volume: VT=8mL/kg, E:I=2:1, f=35times/min, FiO2=0.21, PEEP=3cmH2O, t=4h. Ventilation Strategies of high tidal volume:n=35mL/kg, E:I=2:1, f=8times/min, FiO2=0.21, PEEP=0, t=4h.3.The rabbits were sacrificed4hours after the start of MV and the lungs were collected from the experimental animals for determination of the following indicators:(1) The change of lung appearance and the ratio of wet-to-dry weight (W/D) in lung;(2) The lung tissue morphology by conventional HE staining;(3) The contents of malondialdehyde (MDA), the activity of myeloperox idase (MPO) and the contents of total glutathione (T-GSH) in lung tissue by colorimetry assay.(4) The mRNA expression level of tumor necrosis factor alpha (TNF-a) and interleukin1beta (IL-1β) in the lung tissue by Real-time PCR;(5) The contents of TNF-a and IL-1β in the lung tissue by ELISA;(6) The mRNA expression level of xCT and4F2heavy chain(4F2hc) in the lung tissue by Real-time PCR.Results:1. The effect of L-HCA on the changes of appearance and morphology of the lung tissue:The lungs in HV group showed an obvious edema, a significant increase in size, and obvious point or plaque of bleeding were observed in the lung lobe,, while all the symptoms mentioned above were improved by the treatment with L-HCA. The morphological results showed that the mechanical ventilation with high tidal volume had induced an obvious inflammatory cell infiltration, and had significantly reduced alveolar number and increased septal thickness, indicating significantly altered lung structure in the HV group.The pathological changes of the lung in HV group were, however, reduced at different level after the treatment with L-HCA.2.The effect of L-HCA on W/D ratio in rabbits:Compared with LV group, the lung W/D ratio in HV group was significantly higher (P<0.01); the treatment with L-HCA could reverse the HV induced increase in lung W/D ratio.3.The effect of L-HCA on the content of MDA in lung tissue: Compared with LV group, the content of MDA was dramatically increased (P<0.01) in the HV group; The content of MDA response to HV was, however, obviously inhibited by the treatment with L-HCA (P<0.01).4.The effect of L-HCA on the activity of MPO in lung tissue: Compared with LV group, the activity of MPO was significantly increased(P<0.05)in the HV group, the activity of MPO response to HV was, however, significantly attenuated by the treatment with L-HCA (P <0.01).5.The effect of L-HCA on the content of T-GSH in lung tissue: Compared with LV group, the content of T-GSH was significantly increased (P<0.01) in the HV group; While the treatment with L-HCA could obviously inhibit the increase in the content of T-GSH(P<0.05).6.The effect of L-HCA on the expression level of TNF-a, IL-1β and IL-8mRNA in lung tissue:Compared with LV group, the mRNA expression of TNF-a (P<0.05),IL-1β (P<0.01) and IL-8(P<0.05) were significantly increased in the HV group; compared with HV group, the expression of IL-1β mRNA was significantly decreased after the treatment with L-HCA (P<0.01), however, there was no significant difference in the expression of TNF-a and IL-8(P>0.05).7.The effect of L-HCA on the contents of TNF-a and IL-1β in lung tissue:Compared with LV group, the contents of TNF-a (P<0.05) and IL-1β (P<0.05) were significantly increased in the HV group; there was no significant difference between HV+L-HCA group and HV group in the content of TNF-a (P>0.05); However, compared with HV group, the content of IL-1β was significantly decreased after the treatment with L-HCA (P<0.01).8.The effect of L-HCA on the expression level of xCT mRNA in lung tissue:Compared with LV group, the expression level of xCT mRNA was obviously increased in the HV group (P<0.01), While the treatment with L-HCA could significantly reduce the increase in xCT mRNA expression (P<0.01).9.The effect of L-HCA on the expression level of4F2hc mRNA in lung tissue:Compared with LV group, the expression level of4F2hc mRNA was increased in the HV group, but there is no significant difference in statistics (P>0.05) Treatment with L-HCA could reduce the increase in4F2hc mRNA expression, but there was also no significant difference in statistics (P>0.05)Conclusions:1. This study has for the first time found the positive expression of Xc mRNA in the lung tissue in normal rabbits, especially the4F2hc, a subunit of Xc-showed a very high mRNA expression in the lung tissue.2. We pointed out for the first time that VILI may up-regulate the expression of xCT mRNA, which contributes to the genesis and development of VILI. so that L-HCA has a protective effect on VILI.3. L-HCA, an inhibitor of Xc-, can inhibit the VILI induced increase in the expression of xCT mRNA.