Roles Of HO-1 In NO Donor-SNP Attenuating Acute Lung Injury Induced By Lipopolysaccharide In Rat

Objective To investigate the effect of NO donor-SNP on the expressions of HO-1 and iNOS in lung in lipopolysaccharide-induced acute lung injury in rat and its protective mechanism.Methods Intratracheal instillation of lipopolysaccharide (LPS) in rat was established as a model of acute lung injury. Thirty-two Wastar rats of both sexes, weighting 190-210g, were randomized into four groups (n=8): the sham-operation group (S group), the LPS intratracheal instillation group (LPS group), the inducer of HO-1 (hemin) pretreatment group (HM group), and the SNP intratracheal administration group (SNP group). Prior to experimentation, animals were fasted overnight but had free access to water. On the day of the experiment, rats were weighed, anesthetized by an intraperitoneal injection with 7% chloral hydrate (5ml·kg^-1). LPS was dissolved in 0.9% saline. An anterior midline incision was made to expose the trachea, and a 24-gauge needle attached to a 1-ml syringe containing 300μl of either 750μg·kg^-1 body weight of LPS in LPS group or drug vehicle (0.9% saline) in S group was inserted into the trachea. The fluid was injected slowly. In HM group, animals were injected intraperitoneally with hemin at a dose of 40μmol/kg body wt 12 h before LPS instillation. In SNP group, an equivalent volume (300μl) of LPS (750μg·kg^-1) and SNP (25μg·kg^-1) was instilled intratracheally at the same time. Blood sample was drawn from the carotid before (baseline) and 8h after LPS or vehicle instillation for artery gas analysis. 8h after LPS instillation, rats were killed by carotid exsanguination. The upper and middle lobes of right lung were harvested for the detections of wet weight to dry weight ratio (W/D) and malondialdehyde (MDA) content respectively. The left lung lobe was used for bronchoalveolar lavage. The bronchoalveolar lavage fluid (BALF) was centrifuged at 3,000 rpm for 10 minutes at 4℃ and stored at -70 ℃ for measurement of protein content. The lower of right lung was fixed in 10% formalin and paraffin embedded. The protein expressions of HO-1 and iNOS were observed by using immunohistochemical technique and assessed through HIPAS-2000 image analysis system semi-quantitatively. Arterial oxygen tension (PaO₂), Lung W/D, BALF protein content, MDA content and lung histopathological alteration were detected respectively.Results (1) 8h after LPS instillation, some of the inflammatory cells accumulating in lung and alveolar epithelium stained for HO-1. The expression of HO-1was significantly induced in LPS group compared with S group (PO.01). In HM and SNP groups, the expression was higher than that in LPS group and S group respectively (P<0.01, P<0.05), and there was intense staining of more inflammatory cells and alveolar epithelium for HO-1. (2) iNOS was barely detectable in the alveolar region in S group. LPS group showed intense staining of iNOS in macrophages, some inflammatory cells and alveolar epithelium. Compared with S group, the expression of iNOS was significantly increased in LPS group (P<0.01). In these cells of HM and SNP groups, there was light staining for iNOS and the induction of iNOS was markedly attenuated compared with LPS group (P<0.05, /><0.01), but the expression was still higher than that in S group (PO.01). (3) W/D was increased of 53% over S group (P<0.01). In HM and SNP groups it was decreased by 15% and 20% respectively over LPS group (P<0.01, P<0.05), but it was still higher than that in S group (P<0.01). (4) In LPS group, the BALF protein content was increased to 3-fold over S group (P<0.01), Compared with LPS group, it was decreased by 53% and 63% in HM group and SNP group (P<0.01, PO.01). There was no difference among HM group, SNP group and S group (P>0.05). (5) MDA content in LPS group was increased to 3.1-fold over S group (P<0.0I). In HM group, it was decreased by 38% over LPS group (PO.01), but it was still higher than that in S group (P<0.01). In SNP group, it was deceased by 53% (PO.01), and there was no difference compared with S group (P>0.05). (6) Compared with S group and the baseline of PaO2 in LPS group, the carotid PaO2 was decreased by 21% and 20% respectively at 8h after LPS injury (PO.01, PO.01), which was reversed by the pretreatment with hemin or administration with SNP (P<0.05, P<0.05). (7) Morphology revealed evidences for lung edema, hemorrhage, numerous inflammatory cells sequestration and compensatory emphysema in lung after 8 h of LPS intratracheal instillation. Under light microscope, the damage of lung in HM and SNP group was mild compared with that in LPS group.Conclusions The results suggest that the protective effect of HO-1 induction, at least in part, via preventing the activation of iNOS/NO system, may be involved in the pathophysiological process of LPS-induced acute lung injury. The NO donor-SNP ameliorates LPS-induced ALI, which may be related to the over induction of HO-1 and inhibition of iNOS.