AIM To investigate the role of tumor necrosisfactor(TNF)in lung injury during acutenecrotizing pancreatitis(ANP),and thetherapeutic effect of'Tong Xia'purgativemethod in minimizing the severity of lung injury....AIM To investigate the role of tumor necrosisfactor(TNF)in lung injury during acutenecrotizing pancreatitis(ANP),and thetherapeutic effect of'Tong Xia'purgativemethod in minimizing the severity of lung injury.METHODS Fourteen canines were randomlydivided into 3 groups:the'Tong Xia'treatmentgroup(n = 5)using Dachengqitang;salinecontrol group(n = 5),and the sham operationgroup(n = 4).TNF activity in serum and inbronchoalveolar lavage fluid(BALF),the serumendotoxin levels were measured,and theseverity of lung injury evaluated.RESULTS Elevation of TNF activity was moreprominent in BALF than in serum.TNF activity inserum at 6 and 12 hours and in BALI:wassignificantly decreased in the'Tong Xia'treatment group than in the saline control one(q=21.11,q=12.07,q=9.03,respectively,P【0.01)and the lung injury was significantlyalleviated at 12 hours as compared with that inthe saline group,manifested as amelioration otthe lung wet/ dry weight ratio,decrease inprotein concentration and neutrophils count inBALF,and improvement of pulmonaryinflammatory changes.A positive correlationwas demonstrated between serum TNF activity and endotoxin level.CONCLUSION Hypersecretion of TNF is shownto be one of the major causes of lung injuryduring ANP;'Tong Xia'purgative method couldalleviate the degree of lung injury mediated byTNF.展开更多
The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibi...The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.展开更多
基金the"8th 5-year Plan"of National Administration Bureau of Traditional Chinese Medicine and Pharmacy,No.H09301
文摘AIM To investigate the role of tumor necrosisfactor(TNF)in lung injury during acutenecrotizing pancreatitis(ANP),and thetherapeutic effect of'Tong Xia'purgativemethod in minimizing the severity of lung injury.METHODS Fourteen canines were randomlydivided into 3 groups:the'Tong Xia'treatmentgroup(n = 5)using Dachengqitang;salinecontrol group(n = 5),and the sham operationgroup(n = 4).TNF activity in serum and inbronchoalveolar lavage fluid(BALF),the serumendotoxin levels were measured,and theseverity of lung injury evaluated.RESULTS Elevation of TNF activity was moreprominent in BALF than in serum.TNF activity inserum at 6 and 12 hours and in BALI:wassignificantly decreased in the'Tong Xia'treatment group than in the saline control one(q=21.11,q=12.07,q=9.03,respectively,P【0.01)and the lung injury was significantlyalleviated at 12 hours as compared with that inthe saline group,manifested as amelioration otthe lung wet/ dry weight ratio,decrease inprotein concentration and neutrophils count inBALF,and improvement of pulmonaryinflammatory changes.A positive correlationwas demonstrated between serum TNF activity and endotoxin level.CONCLUSION Hypersecretion of TNF is shownto be one of the major causes of lung injuryduring ANP;'Tong Xia'purgative method couldalleviate the degree of lung injury mediated byTNF.
文摘The light chain of inter-α inhibitor, also known as bikunin or urinary trypsin inhibitor, is composed of two tandemly arranged Kunitz-type protease inhibitor domains. The second domain of bikunin has factor Xa inhibitory activity which previously was enhanced by mutating two amino acids, glutamine 19 and tyrosine 46 to lysine and aspartate, respectively. In this study, we tried to potentiate its inhibitory activity against leukocyte elastase. A molecular docking model of the second domain of bikunin with leukocyte elastase revealed that P5 arginine 11 was a candidate residue for a third substitution. We generated six triple point mutants using site-directed mutagenesis, compared their leukocyte elastase-inhibitory activities, and selected the most potent variant with arginine 11 substituted to serine. The IC50 values for factor XIa, factor Xa, and leukocyte elastase were 182, 302, and 273 nM, respectively. Moreover, this triple point mutant prolonged the activated partial thromboplastin time and moderately reduced leukocyte elastase-induced endothelial injury. Additionally, favorable conformations created by these mutations were speculated using the structure of the Kunitz protease inhibitor domain of protease nexin 2 complexed with factor XIa as a reference. We discovered a novel triple point mutant of the second domain of bikunin that has potent inhibitory activities against factor XIa, factor Xa, and leukocyte elastase. This variant exhibited anticoagulant activity in plasma and suppressed endothelial cell injury.
