Mechanisms Of ADMA/DDAH Pathway In Acute Lung Injury Induced By Cerebral Ischemia-reperfusion In Rat

BackgroundCerebral ischemia-reperfusion injury (CIRI) often causes acute lung injury (ALI) and infection, which are primary causes of death among complications of ischemic cerebrovascular disease. Though lung infection occurs mainly one week after acute cerebral ischemia, pulmonary edema has happened2hours later. At present, the mechanisms of ALI induced by CIRI is still unclear. In recent years, it is a hot topic to find out the underlying mechanisms, which are the theoretical basis to look for methods for prevention ischemia-reperfusion from leading to ALI. Pulmonary microvascular endothelial cells (PMVEC) constitute pulmonary vascular lining barriers which regulate the exchange, such as liquid and micromolecular substances, between blood and organizational gap. Vascular endothelial barriers dysfunction is the main pathological link of acute lung injury, which is closely related to microvascular endothelial cells and regulation of nitric oxide (NO). Reported as an crucial risk factor for endothelial dysfunction recently, NO effects vascular permeability. NO is produced by Nitric oxide synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA). The increase of ADMA leads to the decrease of NO. ADMA is synthesized when arginine residues in proteins are methylated by the action of protein arginine methyltransferases (PRMT) with methyl from S-adenosylme-thionine(SAM). However, ADMA is degraded into citrullin and dimethylamine by dimethylargininedimethy laminohydrolase (DDAH), and excreted by the kidney. In ADMA/DDAH pathway, ADMA concentration is regulated by PRMT and DDAH. PMVEC injury played an important role in ALI. And protein kinase C a (PKCa) and myosin light chain (MLCK) are two critical factors for PMVEC injury. PKCa is related to rescheduling of scaffolding proteins in microvascular endothelial cells. Activitied PKCa will phosphorylate its downstream effect proteins, such as MLCK. And activitied MLCK phosphorylates myosin light, which regulates muscle ball-actin interactions and then leads to the contraction of endothelial cells. This contributes to paracellular gap formation and vascular hyperpermeability. Reports showed that ADMA metabolism are closely related to pulmonary microvascular endothelial barriers function.ObjectiveTo research Mechanisms of ADMA/DDAH pathway in acute lung injury induced by cerebral ischemia-reperfusion in rat to provide theoretical basis for prevention CIRJ from leading to ALI.MethodsThis report includes4parts:Part1Ischemia-reperfusion (I/R) rat model was created in accordance with modified Zea Longa method. Sprague-Dawley rats were divided into sham(S) operated group and I/R group. We evaluated the model that CIRI leads to ALl via various methods including observation of pathological changes of the lung tissue dyed with HE, detection of the lung weight ratio that the wet compares to the dry (W/D), assay myeloperoxidase activity and detection of MDA by ELISA, and count of white blood cells in lung lavage fluid.Part2Rats were divided into normal group, sham(S) operated group, I/R group, ADMA group (I/R model treated with ADMA) and DDAH group (I/R model treated with DDAH). After reperfusion for24hours, content of NO in lung tissue was detected by the method of Nitric acid reductase and NO in lung lavage fluid was detected by chemical colorimetry. ADMA concentration in lung lavage fluid was Analyzed by High Performance Liquid Chromatography (HPLC). Expression of PKCa and MLCK protein and mRNA in lung tissue was detected by western blot and reverse transcriptase PCR. Part3We created the ALl model induced by CIRI in vitro. After subculture for three generation, PMVEC of normal rats were treated with serum of normal rat, or sham operated rats, or I/R rats or with PKCa inhibitor (A-3hydrochloride). To evaluate the model in vitro, growth of PMVEC was bserved by microscope, and PKCa, MLCK, MDA and ADMA in PMVEC were detected by ELISA. Part4PMVEC were treated with equal serum, or0.5mL ADMA(20g/L)or0.5mL DDAH(20g/L), respectively. Serum includes inactivated normal rat serum, activated normal rat serum, inactivated I/R rat serum and activated I/R rat serum. Morphology of PMVEC was observed by microscope. PMVEC vitality and proliferation were detected by MTT. NO in medium was detected by chemical colorimetry. Expression of PKCa and MLCK protein and mRNA were detected by western blot and reverse transcriptase PCR, and PKCa and MLCK protein were detected by immunocytochemistry.ResultsPart1Except mild lung inflammation such as capillary expansion and congestion of some field, there are no significant pathological changes in sham groups. However, in I/R groups, there are diffuse congestion and bleeding, scattered red blood cells and inflammatory cells within alveolar space, and congestion, incrassation and exudation in alveolar walls. Compared with sham groups, MPO activity, MDA content and W/D ratio were increased markedly in I/R groups (ρ<0.05) and white blood cells in lung lavage fluid were increased dramaticlly in I/R groups (P<0.01).Part2Compared with S group and DDAH group, ADMA in BALF and pulmonary vein blood (into lung)was increased markedly in I/R group and ADMA group (ρ<0.05). However, ADMA in pulmonary artery blood (out lung)has no significant change. Expression of PKCa and MLCK protein and mRNA were increased in I/R group and ADMA group. There are no significant changes in ADMA of plasma and expression of PKCa and MLCK protein and mRNA in lung tissue between S group and DDAH group, as well as ADMA group compared with I/R. Compared with S group, NOS activity and NO content in lung tissue were decreased markedly in I/R group (ρ<0.05or0.01); NOS activity and NO content in lung tissue were increased in ADMA group, but there are no significant differences. Compared with I/R group, NOS activity and NO content in lung tissue were decreased in ADMA group.Part3MDA content in PMVEC was increased in the group treated with I/R rat serum (ρ<0.05). There are no significant differences in MDA content of PMVEC in the other3groups. Expression of PKCa and MLCK protien have a consistent change. AMDA concentration in PMVEC increases significantly in the group treated with PKC inhibitor (ρ<0.05). However, compared to the model, AMDA concentration in PMVEC has no significant difference in the group treated with PKC inhibitor.Part4Vitality and proliferation in PMVEC were decreased significantly and expression of PKCa and MLCK protein in PMVEC were increased markedly in the group treated with inactivated I/R rat serum and the group treated with ADMA (ρ<0.05). However, compared with the group treated with ADMA, there has no significant difference in PKCa and MLCK protein in PMVEC in the group treated with inactivated I/R rat serum.ConclusionsADMA/DDAH pathway plays an important role in the mechanisms of ALI induced by CIRI. ADMA may lead to PMVEC injury through regulating expression of PKCa and MLCK.