| Background The acute respiratory distress syndrome (ARDS) is a common clinical mani-festation of acute lung injury (ALI) with a high mortality rat,with an age-adjusted incidence of 86.2/100,000 person-years, with a mortality of 40%, and with significant morbidity among the survivors.Remains one of the major challenges adult intensive care units. ALI/ARDS can lead to sepsis, trauma, and severe pneumonia, among which sepsis and in particular pneumonia are leading causes of acute lung injury.The presence of infiltrating leukocytes is the hallmark of pulmonary inflammation associated with acute lung injury. Activation of endothelial and infiltrating inflammatory cells results in the production of injurious cytokines and chemokines, and disruption of the alveolar epithelial and capillary barriers contributes to the accumulation of protein-rich fluid in the alveoli,decreasing lung compliance.The molecular mechanisms underlying the inflammatory by pattern recognition receptors, followed by the coordinated expression of proinflammatory cytokines (TNF-αand IL-1β).Increased nuclear translocation of the transcription factor nuclear factor(NF-κB) contributes to the pathogenesis of acute lung injury. NF-κB is transcriptional regulatory proteins that modulate the expressions of immunoregulatory genes related to critical organ injury. NF-κB activation in lung tissue is associated with cytokine and chemokine production,lung neutrophilia,epithelial permeability,and lipid peroxidation with increased expression of NF-κB–regulated proin?ammatory genes, including tumor necrosis factor-α(TNF-α) and (IL-1β).Intraalveolar and intravascular fibrin deposition is frequently found in the setting of ALI or ARDS. Fibrin deposits enhance inflammatory response by increasing vascular permeability,activating endothelial cells to produce proinflammatory cytokines and other mediators, inducing the accumulation of activated neutrophils, and modulating immunoregulatory responses in the lung.An improved understanding of the pathogenesis of ALI/ARDS, searching for effective and specific therapies is still urgently needed,ideally with the conventional pharmaceutical drugs.Tetramethylpyrazine (TMP) is an alkaloid contained in ligustrazine which has been used in traditional Chinese medicine for centuries.It widely used to treat cardiovascular and cerebrovascular diseases ,occasionally used to treat various causes of acute and chronic respiratory failure.TMP has also been reported to possess a diverse array of pharmacological functions in the modulation of arterial resistance, cerebral blood ?ow, platelet function, microcirculation, and capillary permeability. Moreover, Ligustrazine was reported to possess a very broad spectrum of pharmacological actions, such as antioxidant, anti-in?ammatory, anti-fibrosis, antitumor and immunomodulative effects.Therefore, the present investigation was designed to evaluate its effect on ALI/ARDS, we have chosen the OA-induced(Oleic acid- induced) ALI in rats as a model,implying that the use of TMP may be one of the useful novel options for ALI/ARDS management.Objective :⑴To research whether tetramethylpyrazine has the effect for ALI/ARDS rats;⑵To research the change of inflammatory cytokines TNF-αand IL-lβin the BALF of ALI/ARDS rats;⑶To research the change of NF-κB in the lung of ALI/ARDS rats;⑷To research the effect of tetramethylpyrazine on TNF-α, IL-lβ, NF-κB protein expression of ALI / ARDS rat;⑸To find a new drug candidates for the treatment of ALI/ARDS and the rich connotation of Chinese medicine.Methods :A total of 60 Male SD (Sprague-Dawley) rats were randomly divided into control,OA-treated,OA+saline treated and OA+ TMP treated groups. Acute lung injury was induced by injection of OA (dose, 0.15 ml/kg) via intravenous vein.The TMP treatment was administered after 30min the OA injection at a dose of 100 mg/kg.The dose of TMP was chosen according to previous reports and our pilot study.The rats in OA+saline treated received an equal volume of saline. At each of designated time points (4, 8and 12 h after OA challenge), five rats from each group were killed.To investigate the animal model reactionp,partial pressure of oxygen,lung wet/dry weight ratio, collection and analysis of bronchoalveolar lavage fluid, histopathological examination,immunohistochemistry, measurement of NF-κBp65 by western blot.Results 1. Symptoms and partial pressure of oxygenThe spirit of control rats was good and the respiratory rate (RR) was 40 to 50 times/min.After OA injection, the RR up to 135~160 times / min, listlessness,decreased activity.They were significantly reduced after administration of TMP compared with the ALI/ARDS rats(P<0.001).The partial pressure of oxygen of OA group was lower than C group (P<0.05).The levels of partial pressure of oxygen was improved after TMP administration (P<0.05).2. TMP reduced morphologic damage in lungs exposed to OAThe lungs of mice exposed to OA showed marked inflammatory alterations characterized by athickening of the alveolo capillary membrane, the presence of alveolar hemorrhage, and massive extravagation of both mono and polymorphonuclear leukocytes into the alveolar spaces when compared with those of mice.In contrast, histological damage was less pronounced in TMP-treated mice by dose dependent manner. The amount of both erythrocytes and leukocytes in the alveolar spaces was clearly reduced in TMP -treated mice. According to the above data, we decided to use the 100mg/kg of TMP on follow up examination.3. Wet/dry weight ratio and protein content in BALFCompared with the normal groups, respiratory rate increased significantly in OA groups (P<0.001);The indicators all decreased in OA+TMP treated groups.A hallmark of ARDS is the development of high permeability edema, characterized by high protein content in the edema fluid.Such abnormalities were noted in the lungs of mice exposed to OA. Respectively, the concentration of protein in the BALF was significantly lower in OA-treated mice that received TMP,the most obvious time point was 12h(P<0.05).3. Effect of TMP on the expression of TNF-αand IL-lβin BALFA substantial increase in TNF-αand IL-lβproduction was found in BALF collected from rats at 4h,8h,12h and peaked at 4 hours after OA administration(p<0.001).The levels of these pro-in?ammatory cytokines were significantly lower in OA-treated mice treated with TMP and peaked at 12 hours after OA administration(p<0.05).4. Immunofluorescence for the activations of NF-κBp65Immunofluorescence for NF-κBp65 show positive staining mainly localized along the alveolar nuclei from rats. At 4 h after OA administration,the intensity of the positive staining for NF-κBp65 was significantly more than control group 8h and 12h group(p<0.05).It reduced after TMP administration 4h,8h,12h and peaked at 12 hours after TMP administration in the lung (p<0.05).5. Effects of Tetramethylpyrazine on the activations NF-κBp65The rats in OA and OA+slaine group expressed higher NF-κBp65 than the normal control rats (p<0.05) at 4h,8h,12h and peaked at 4 hours after OA administration. Tetramethylpyrazine significantly suppressed NF-κBp65 protein at 4h,8h,12h and peaked at 12 hours after TMP administration (p<0.05).Conclusion :(1) Tetramethylpyrazine can improve pulmonary edema, hemorrhage and lung morphological changes of ALI / ARDS rats;(2) Tetramethylpyrazine can improve respiratory distress and it may be have potential therapeutic value in treatment of ALI/ARDS;(3)Tetramethylpyrazine decreased inflammatory TNF-α, IL-lβreleaset in ALI / ARDS after the injection of drugs;(4) Tetramethylpyrazine decreased NF-κB p65 protein express after the injection of drugs and the protective effect may be by inhibiting NF-κB activation in ALI / ARDS rats;...
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