The Effect Of Anesthetics On The Expression Of CD14 And TLR4 In Lung Tissue In Acute Lung Injury Rat Induced By Lipopolysaccharide

Acute lung injury(ALI) and acute respiratory distress syndrome(ARDS) are major causes of acute respiratory failure,which increases the risk of morbidity and mortality in critically ill patients.Intratracheal instillation of lipopolysaccharides(LPS) in the rat is a well characterized model of acute lung injury,and closely resembles in many important aspects the clinical presentation of ALI/ARDSBy triggering a physical association between CD14 and TLR4,LPS is a principal initiator of host antimicrobial innate immune responses.Inflammatory response to endotoxins is largely mediated through CD14 and TLR4.The activation of CD14 and TLR4 leads to proinflammatory cascade through NF-κB,which induces expression of inflammatory mediators {tumor necrosis factor(TNF)-α,etc}.Elevated CD 14 is reported to be associated with increased infection and greater mortality in critically ill patients.The level of expression of TLR4 is closely associated with the extent of acute pulmonary response to inhaled endotoxin.Anaesthetics are administered to sedate intubated,mechanically ventilated ALI and ARDS patients.These anaesthetics may modulate the immune system and influence the clinical course and outcome of patients.Previous reports have shown that anaesthetics affect inflammatory mediation in ALI.However,the exact mechanism of anaesthetic-induced effects on ALI has yet to be clarified.The lung represents a site for the invasion of various bacteria or bacterial products. Along with alveolar macrophages,pulmonary epithelial cells are the first cells to be challenged by pathogenic microorganisms.Rat ATII cells synthesize and secrete surfactant,control the volume and composition of fluid in the alveolar space,and proliferate and differentiate into alveolar typeâ… epithelial cells after lung injury to maintain the integrity of the alveolar lining.Recently,there have been publications reporting the involvement of ATâ…¡in modulating the development of inflammatory reactions within the alveolus.ATâ…¡secrete chemokines in response to inflammatory stimuli in vitro,suggesting a potential physiologic role in acute inflammatory lung injury.Little is known about the interaction of generally used intravenous anesthetic propofol with ATâ…¡.As a means of investigating how anaesthetics affect the inflammatory system,we designed a study to examine the effects of propofol,midazolam,and remifentanil on the mRNA and protein expression of both CD14 and TLR4 in rats with LPS-induced ALI and to investigate whether LPS induced inflammation in ATâ…¡is through CD14 and TLR4 and the effect of different dosage of propofol on the inflammation.Materials1.Animals:Adult male Wistar rats weighting 250-350g for in vivo experiment and weighting 180-250 g for in vitro experiment,were provided by the Experimental Animal Center of China Medical University2.Major reagents:LPS,propofol,midazolam,remifentanil,Takara reverse transcript kit,rabbit anti-rat CD-14 primary antibody,goat anti-rat TLR4 primary antibody,mouse anti-ratβ-actin primary antibody,goat anti-rabbit secondary antibody,rabbit anti-goat secondary antibody,TNF-αELISA kit,SYBR PrimeScriptTM real-timePCR kit.3.Major instruments:Respiratory machine,micro-infusion pump,photo analysis system electrophoresis apparatus,semi-dry blotter,spectrophotometer,bechtop,CO₂ Incubators,Olympus BX61 photo analysis system,ABI 7500 Real-Time PCR system.Methods1.Animal model of the acute lung injuryAnaesthesia was induced with 50 mg kg^-1 pentobarbital sodium administered intraperitoneally and maintained with pentobarbital sodium and pipecuronium bromide via left jugular vein.Tracheal cannulation(14 gauge) was performed after tracheostomy.The right carotid artery was catheterized for continuous arterial blood pressure measurements and blood sampling.The animals were ventilated using Servo 900C ventilator.LPS 5mg/kg was instilled intratracheally to induce acute lung injury.2.In vivo experimentAccording to the intravenous anaesthetics they received,the rats were randomly assigned to 5 groups with 8 in each group:Control group:saline IT and salineâ…³LPS group:LPS IT and salineâ…³Propofol group:LPS IT and propofolâ…³Midazolam group:LPS IT and midazolamâ…³Remifentanil group:LPS IT and remifentanilâ…³This intravenous infusion continued to the end of five-hour protocol.At 0 h,and at 1,3 and 5 h,MAP and HR were recorded and arterial blood was sampled for blood gas analysis.After the protocol,CD14 and TLR4 gene expression and protein expression in lung tissues were evaluated using real time PCR and western blot,respectively.TNF-αin bronchoalveolar lavage fluid(BALF) was detected using ELISA.3.Primary culture of typeâ…¡alveolar epithelial cellATâ…¡were isolated from male SPF degree Wistar rats(180-250g),following the protocol according to the method of Richard et al¹².Briefly,after intraperitoneal sodium pentobarbital and heparin(400U.kg^-1),the thoracic cavity was opened and the inferior vena cava was cut.The lungs were perfused with sterile saline.After removal from the thoracic cavity,they were washed 6 times with normal saline and digested enzymatically with typsin.The lungs were then minced,and the resulting suspension was filtered through 150μm and 30μm mesh once.The cell mixture was purified by centrifugation on a discontinuous Percoll gradient.The interface was collected and after rewashing the cells were seeded onto 6-well culture dishes in DMEM at 37℃in the humidified atmosphere of 95%air/5%CO2 and cultured for 18-20 hours.4.In vivo experimentThe cultured ATâ…¡cells from the same rat were randomly assigned to one of the following five groups:Group C:ATâ…¡cells was cultured in the absence of propofol and LPS; Group LPS:treated with 1μg.^-1 ml LPS;Group P1:treated with 1μg^-1 LPS and 25μM propofol;Group P2:treated with 1μg^-1 LPS and 50μM propofol;Group P3:treated with 1μg^-1 LPS and 100μM propofol.ATâ…¡cells in untreated control group and propofol groups were cultured for 3 h. CD14 and TLR4 mRNA were detected using real-time PCR.Western blot were used to detect CD14 and TLR4 protein expression.CD14 and TLR4 expression on the ATâ…¡cells were imaged using immunofluorescence.TNF-α.production were determined using ELISA kit.5.Data analysis and statistics:The data were normally distributed as determined by the Levene's test.ANOVA followed by a post hoc Tukey's test was used for multiple-group comparisons.Values are expressed as means±SD.The significance level was set at P<0.05.SPSS 13.0 software was used for all statistical work.Results1.Rat model of acute lung injuryThis study regards PaO₂/FiO₂≤300mmHg as the successful setup of ALI rat model. After LPS 5mg/kg IT,LPS group,C,D and E all satisfied this standard.PaO₂ values at 3 h and 5 h were lower than at 0 h.HE stain showed edema,hemorrhage and thickness of alveolar wall as well as obvious inflammatory ceils infiltration into the septum.2.Results of in vivo experimentCD 14 mRNA expression in LPS group,propofol group,midazolam group and remifentanil group were significantly increased compared with control group.TLR4 mRNA expression in LPS group and remifentanil group were significantly increased compared with control group.TLR4 mRNA expression in propofol group and midazolam group were lower than in midazolam group.TLR4 mRNA in propofol group was lower than in LPS group.CD14 protein expression in LPS,propofol,midazolam and remifentanil groups were significantly higher than in control group and there were no statistical difference among LPS,propofol,midazolam and remifentanil groups.TLR4 protein expression in LPS,midazolam and remifentanil groups were higher than in control group.TLR4 protein expression in propofol and midazolam groups were lower than in remifentanil group and TLR4 protein expression in propofol group was lower than in LPS group.Propofol and midazolam group showed milder lung injury compared with remifentanil group in HE stain.Compared with LPS group,TNF-αin BALF was decreased in propofol group and elevated in remifentanil group.3.Primary culture of ATâ…¡cellsThe cell mix,obtained by the discontinuous Percoll gradient centrifugation method,contained 85±5%of ATâ…¡which were stained blue with AKP histochemical staining.Transmission electron microscopy showed typical lamella bodies in ATâ…¡.The cell viability in each group,determined by trypan blue dye exclusion test ranged from 90 to 95%.4.Results of in vitro experimentLPS stimulation resulted in an increased CD 14 and TLR4 expression and increased TNF-αproduction in ATâ…¡cells.Propofol,at concentrations≥50μM, significantly and dose-dependently decreased CD14 and TLR4 mRNA expression as well as their protein expression in ATâ…¡cells.This was accompanied by decreases in TNF-αproduction.Conclusion1.Both the mRNA and protein expressions of CD14 and TLR4 can be increased by LPS stimulation in the lung tissue in vivo and the ATâ…¡cells in vitro,so as with TNF-αproduction.2.Propofol can downregulate both the mRNA and protein expressions of CD14 and TLR4 in the lung tissue in vivo and the ATâ…¡cells in vitro,and can decrease TNF-αproduction.3.Compared with remifentanil,propofol and midazolam can relieve acute lung injury.4.The study showed the collocation of CD14 and TLR4 expression.