Study On Protective Effects Of Penehyclidine Hydrochloride On The Lungs Against Acute Injury And Its Mechanisms Of Action

Acute lung injury(ALI) is a cause of acute respiratory failure that develops in patients of all ages from a variety of clinical disorders,including sepsis(pulmonary and nonpulmonary),pneumonia(bacterial,viral,and fungal),aspiration of gastric and oropharyngeal contents,major trauma,and several other clinical disorders including severe acute pancreatitis,drug overdose,and blood products.ALI is characterized by neutrophil accumulation in the lungs,loss of epithelial integrity,widening of the alveolar-arterial O₂ gradient,and the development of interstitial pulmonary edema and remains a significant cause of morbidity and mortality in critically ill patients.Penehyclidine hydrochloride(PHCD) a new anticholinerigic drug,which invented by the institute of Pharmacology and Toxicology,Academy of Military Medical Sciences in the People's Republic of China,is different from anisodamine with long duration and slight effection on M₂ receptor.It is noteworthy that PHCD can decrease significantly the nasal mucosa capillary permeability.Thus,PHCD is potentially beneficial for treatment of ALI.In this study we aimed to investigate whether PHCD has in vivo protective effect on ALI induced by LPS and to explore the mechanisms for the inhibitory effects of PHCD on ALI.The principal contents were divided into some sections as follows: 1.Effects of Penehyclidine hydrochloride on ALI induced by LPSPHCD(0.03,0.1,0.3mg/kg,ig) significantly increased the partial pressure of oxygen and decreased partial pressure of carbondioxide of arterial blood.Furthermore, PHCD markedly reduced lung water content,wet-to-dry weight ratio of lung,the lung permeability index and the bronchoalveolar lavage protein concentration.Lung histopathological and electron microscopy examination showed PHCD could also ameliorate lung injury and alveolar type cell injury induced by LPS.All data showed that PHCD had significant protective effects in ALl induced by LPS.2.Effects of PHCD on the neutrophil accumulation and production of oxygen free radical in lung induced by LPSPHCD(0.03,0.1,0.3mg/kg,ig) significnatly reduced MDA level in lung and serum and increased SOD level in lung and serum in LPS-induced ALI rats.PHCD also decreased activities of MPO in lung and activities of LDH in BALF.PHCD also significantly decreased the apoptosis of lung cell with method of TUNEL.All this showed that PHCD attenuates LPS-induced acute lung responses through inhibition massive accumulation of neutrophils in the lungs and the production of reactive oxygen species and apotosis of lung cell.3.Effect of PHCD on the apoptosis of activated PMNFlow cytometry was used to analyze the apoptosis rate of PMN obtained from blood.The protein expressions of Mcl-1 and bax of PMN were measured by Western-blot.Fas/FasL mRNA expression of PMN was analyzed by semi-quantitative RT-PCR.The result showed that the apoptosis rate(16.05±0.93%) of PMN with PHCD(0.3mg/mg) treatment was significantly decreased,when compared with that (1.85%±0.31%) in the model group(P＜0.05).Moreover the expression of Mcl-1/bax was down-regulated and Fas/FasL mRNA expression was up-regulated after PHCD(0.1, 0.3mg/mg) treatment respectivily.But,pretreatment with PHCD(0.03mg/mg) could not decrease the apoptosis rate of PMN.4.Effects of PHCD on the expression of NF-κB p65 and NF-κB activation,expression of proinflammatory cytokines and NOS in lung tissueNF-κB is an essential transcription factor that regulates the gene expression of various cytokines,chemokines,growth factors,and cell-adhesion molecules.The expression of NF-κB p65 and NF-κB activation in lung measurd by Immunohistochemical staining and EMSA.The mRNA and levels of TNF-αand IL-1βin lung tissue were measured by semi-quantitative RT-PCR and ELISA methods respectively.The NOS mRNA and protein expressions were measured by semi-quantitative RT-PCR and Western-blot methods respectively and the levels of NO in lung and serum were also measured.The results showed that PHCD could significantly inhibite the LPS-induced the induction of NF-κB activation and reduced TNF-αand IL-1βexpression.Furthermore, PHCD also substantially reduced the LPS-induced NOS mRNA and protein expression in lung tissue,dcreased activity of NOS and levels of NO in lung and serum.These results suggest that PHCD attenuates LPS-induced acute lung responses through inhibition of LPS-induced NF-κB DNA binding activity,down- regulation gene expression of IL-1βand TNF-a cytokines in lung tissue.In addition,the level of NO might contribute to the lung injury by LPS.NF-κB activation appears to be an important mechanism mediating LPS-induced TNF-αand IL-1βproduction,NO production,which resulting neutrophil recruitment associated with acute lung inflammation and injury.In summary.,PHCD could possess obvious protective effect on ALI induced by LPS in our experiment without does-dependently in vivo.Its mechanisms of protective effect might be reletive to(1)antioxidation,(2) increase apoptosis rate of PMN, mechanisms of which might be associated with its down-regulation protein expression rato of Mcl-1/bax and up-regulation Fas/FasL mRNA expression,(3) inhibition NF-κB activativity,which might mediate LPS-induced TNF-αand IL-1βproduction,NO production.