Ulinastatin Attenuates Paraquat-induced Myocardial Injury In Rabbits

PART I:Protective effect of ulinastatin in myocardial injury by acute paraquat poisoningObjective:To investigate the effect of ulinastatin (UTI), trypsin inhibitor from human urine, on myocardial injury induced by acute paraquat poisoning in rabbits.Methods:Twenty-four Japan white rabbits were divided into four groups through random number table. A normal control group, a paraquat poisoning model group with37mg/kg paraquat intraperitoneally, two UTI intervention groups with25kU-kg-1·d-1and50kU-kg-1·d-1UTI for9days respectively, began at l weeks pre-poisoning. Rabbits were sacrificed24h after the last UTI administration. Left ventricle of hearts were harvest. Tissue hydroxyproline(HYP) content was determined by alkaline hydrolysis; Matrix metalloproteinase-2(MMP-2) and tissue inhibitor of metalloproteinase-2(TIMP-2) mRNA levels were obtained respectively through reverse transcription PCR (RT-PCR); expression levels of MMP-2in tissue of left ventricle were quantified through immunohistochemistry.Results:Compared with normal myocardium, acute paraquat poisoning elevated HYP (μg/mg) content significantly(3.85±0.36to2.52±0.29, P<0.05); through RT-PCR and immunohistochemistry, both transcription and expression levels of MMP-2(all P<0.05) were much higher than normal concentrations (2.07±0.57to1.00±0.35,2.24±0.82to1.40±0.62, all P＜0.05); TIMP-2, the major cellular inhibitor of MMP-2, appeared downregulated in transcriptional level though there was no significant difference (0.78±0.24to1.00±0.17, P>0.05); and disorganized cadiocytes were abtained. UTI attenuated these changes. Compare with paraquat poisoning model,low and high UTI administration depressed tissue HYP contents (μg/mg)(3.40±0.48,3.12±0.43to3.85±0.36, P>0.05and P<0.05, respectively); transcriptional levels and immunohistochemical scores of MMP-2in left ventricle with low and high dose UTI intervention reduced (mRNA:1.86±0.44,1.58±0.46to2.07±0.57, P>0.05and P<0.05; immunohistochemical score:1.93±0.86,1.75±0.67to2.24±0.82, all P<0.05); TIMP-2mRNA level was increased slightly, though there was no significant difference (0.82±0.35,0.94±0.33to0.78±0.24, all P>0.05); improvements of disorderd myocardium were observed.Conclusion:UTI significantly attenuated myocardial injury induced by acute paraquat poisoning, its mechanisms might be related to reduce expression level of MMP-2. PART Ⅱ:Ulinastatin attenuates paraquat-induced myocardial oxidative stress injuryObjective:To investigate the effect of Ulinastatin (UTI), trypsin inhibitor from human urine, on myocardial oxidative stress injury induced by paraquat (PQ).Methods:Thirty Japan white rabbits were radomly allocated to a sham control group, a PQ intoxication model group and three UTI groups. PQ(30mg/kg) was administered to the PQ intoxication model group through intreperitoneal injections, UTI with15000,30000,50000U/kg was administered intravenously to three UTI groups respectively for7days after PQ intoxication. After euthanasia with10%chloral hydrate, left ventricle was collected. Myocardial geometric change was assessed using HE staining. Hydroxyproline,a measure of collagen content, was measured by alkaline hydrolysis method, myocardial oxidative stress evaluated by4-hydroxy-2-nonenal (4-HNE) using immunohistochemistry, transcription levels of transforming growth factor-β1(TGF-β1), adiponectin, peroxisome proliferator-activated receptor a (PPAR-a), AMP-activated protein kinase-β1(AMPK-β1), uncoupling protein1(UCP-1) by reverse transcription PCR.Results:Myocardial stracture was disarranged, HYP increased, scores of4-HNE augmented in PQ intoxication model animals compared with the sham control animals (all p<0.05). PQ with30mg/kg increased mRNA expression of TGF-β1and decreased the transcription levels of PPAR-a and AMPK-β1(all p<0.05).UTI treatment reduced the increase of HYP concentration (all p<0.05) as well as intensities of4-HNE (all p<0.01) in PQ model animals, the mRNA expressions of TGF-β1in myocardium with UTI30000U/kg and50000U/kg were significantly reduced (t=11.024, p<0.01; t=14.697, p＜0.01), UTI mitigated myocardial oxidative stress induced by PQ. The mRNA expressions of PPAR-a together with UCP-1were activated with30000U/kg (vs sham control, t=18.708, p<0.01; T=180,p<0.01), which was much more prominent in animals with50000U/kg. Significantly, the mRNA expression levels of PPAR-a and UCP-1positively correlated with the dose of UTI in the PQ intoxicated rabbits (rs=0.948, p<0.01; rs=0.802,p<0.01).Conclusion:The present study suggests that UTI protects against PQ-induced myocardial injury, its mechanisms might be related to reduction of TGF-β1、activation of PPAR-a and UCP-1.