The Therapeutic Effects And Mechanisms Of Naja Naja Atra Venom On Acute Lung Injury And Pulmonary Fibrosis

Part1The protective effects of NNAV on LPS-induced acute lunginjury in miceObjective: To determine possible therapeutic effects of NNAV on lipopolysaccharide(LPS)-induced acute lung injury.Methods：KM mice were randomly divided into control, model and Naja naja atravenom (NNAV,30,90,270μg/kg) groups. LPS (5mg/kg,0.1ml/10g) wasintraperitoneally injected into all the mice except those in control group, and the first doseof oral Naja naja atra venom (30、90、270μg/kg/day) was given4days before the LPSadministration. All mice were sacrificed6h after LPS to determine the pulmonary index,TNF-α and IL-1β levels in serum. The effects of NNAV on the expression of IκB-α andNF-κB in lung tissue of mice were assessed with Western blot analysis andimmunohistochemisty. HE staining was used to histopathological examination of the lungtissue.Results：Compared with control group, the LPS increased pulmonary index andserum levels of TNF-α and IL-1β (P<0.05, P<0.01). NNAV administrated micesignificantly decreased pulmonary index and the levels of TNF-α and IL-1β (P<0.05,P<0.01) compared with model group. Histological examination of LPS-treated miceshowed the alveolar septa were significantly thickened, accompanied by the infiltration ofinflammatory cells and damage of alveolus structure. NNAV robustly reduced suchdamages in lung tissues. The nuclear translocation of NF-κB was markedly increased andthe expression of IκB-α was significantly decreased in the lung tissue. However, NNAVinhibited NF-κB nuclear translocation and up-regulated the levels of IκB-α. Conclusion: NNAV has a protective effect on LPS-induced acute lung injury, via amechanism thought inhibiting the activation of NF-κB, decrease synthesis and release ofthe cytokines and inflammatory factors (TNF-a、IL-1β). Part2The protective effects of NNAV on BLM-and LPS-inducedpulmonary fibrosis in miceObjective: To determine possible therapeutic effects of NNAV on BLM-inducedpulmonary fibrosis and LPS-induced pulmonary fibrosis.Methods：90male KM mice were separated into two parts and each part wasrandomly divided into control, model and Naja naja atra venom (NNAV,30,90,270μg/kg)groups. The first dose of oral Naja naja atra venom (30、90、270μg/kg/day,0.2ml/10gbody weight) was given4days before bleomycin (BLM) or LPS. Model1: Except forcontrol group, model group were produced by intratracheal injection of BLM (5mg/kg) onthe fifth day and control group were administrated with the equal volume physiologicalsaline. Model2: LPS (5mg/kg,0.1ml/10g) was intraperitoneally injected into all the miceexcept those in control group on the fifth day and control group were administrated withthe equal volume physiological saline. Later on, LPS (5mg/kg,0.1ml/10g) wasintraperitoneally injected once a week to induce pulmonary fibrosis. The mice in eachNNAV treatment group were intragastric administrated corresponding dose of NNAV andcontrol and model group were intragastric administrated the equal volume physiologicalsaline. All the mice were sacrificed8weeks after BLM or LPS to determine the pulmonaryindex and HYP content in the lung. HE staining was used to histopathological examinationof the lung tissue.Results：Compared with control group, BLM and LPS model groups reduced bodyweight, significantly increased lung index and HYP content (P<0.05, P<0.01). Comparedwith respective model group, the oral administrated of NNAV mice increased body weight,decreased pulmonary index and HYP content (P<0.05, P<0.01). Histological examination of BLM-treated and LPS-treated mice showed the infiltration of inflammatory cells,increased collagen contents and thickened alveolar septa, while NNAV reduced thesepathologies. Part3The therapeutic effects of Naja naja atra venom onBLM-induced pulmonary fibrosis and its mechanisms of action in ratsObjectives: To determine possible therapeutic effects of NNAV onbleomycin-induced pulmonary fibrosis and its mechanism.Methods: Sprague-Dawley rats were randomly divided into control, model and Najanaja atra venom (NNAV,30,90,270μg/kg) groups. Bleomycin (5mg/kg) wasintratracheally instilled into all the rats except those in control group, and the first dose oforal Naja naja atra venom (30、90、270μg/kg/day) was given4days before the bleomycinadministration and continued until to the end of the study. Control and model group wereintragastric administrated the equal volume physiological saline. After8weeks all the micewere sacrificed. Lung weight was measured. Oxidative stress markers (MDA, SOD andGSH), cytokines (IL-1β, TNF-α), hydroxyproline (HYP), were detected with kits. TGF-β1,p-Smad2/3, Smad7, collagen I were detected with Western blot analysis.Hematoxylin-eosin (HE) and Masson staining were used to histopathological examination.Blood gas testing was used to evaluate pulmonary functions.Results：BLM induced increases in MDA, HYP, PCO2, lactic acid, IL-1β, TNF-α,TGF-β1, p-Smad2/3and Collagen I, while decreased SOD, GSH, blood pH, PO2, SO2andSmad7. NNAV treatment significantly inhibited BLM-induced these pathological changes.Histological examination of BLM-treated rats showed the infiltration of inflammatory cells,increased collagen contents and thickened alveolar septa. NNAV robustly reduced thesepathologies.Conclusion: This study indicates that NNAV could reduce lung damage, decrease collagen content and prevent bleomycin-induced pulmonary fibrosis. This protective effectmay be related to the inhibition of inflammation cytokines and the TGF-β1-mediatedtranscription of profibrotic genes.