| Background and ObjectiveAcute lung injury(ALI)has become one of the leading causes of death in intensive care units,and its more severe form is Acute respiratory distresssyndrome(ARDS).Acute respiratory distress syndrome was the first time defined by the United States in 1967-European consensus meeting,although approximately 150,000 people are diagnosed with ARDS in the United States each year,ARDS patients account for 10% of the intensive care unit worldwide,and each year more than 3 million cases.The mortality rate of ALI is still as high as 30-40%,and ARDS mortality is still 40% ~ 50%,but there is no cure for the effective treatment,and standard treatment plan.Because of its rapid progress,high motality and lack of effective treatment,it is particularly important to find the treatment drugs of ALI/ARDS and explore its mechanism.Lidocaine as clinical commonly used local anesthetics,its anti-inflammatory effects have been widely recognized.The research on the mechanism of anti-inflammatory,found that lidocaine can reduce cytokine-induced damage to endothelial and vascular smooth muscle cells by adenosine triphosphate salt sensitive potassium channels;Block Potassium and sodium ion channels to inhibit the activation of NF-κ B;Decrease the production of pro-inflammatory cytokines induced by microglia extracellular ATPby inhibiting the increase of intracellular calcium and Activation of P38 MAPK,or reduce inflammation by down regulating TLR4(Toll like receptor 4,TLR4)signaling pathway.In vivo and in vitro experiments,there have proved lidocaine can inhibit the expression of inflammatory markers,such as interleukin 1 beta(IL-1 beta),interleukin 6(IL-6),tumor necrosis factor alpha(TNF alpha),interleukin-18(IL-18),the High mobility of protein B1(High mobility group box 1,HMGB1)etc.And relieving acute lung injury.on the animal model,found that lidocaine can not only reduce the expression of inflammatory cytokines,also reduce alveolar artery blood oxygen partial pressure difference,the lung tissue neutrophils sticking and thus reduce lung injury degree;In clinical studies,it is also found that lidocaine can reduce the chemotaxis and metastasis of neutrophils,reduce the proliferation of T cells and the release of inflammatory factors.The P2X7 receptor is an adenosine triphosphate(ATP)gated ion channel,belonging to the P2 family,P2X7 receptors can not be only through smaller cations such as sodium,potassium,and calcium,but also larger cations such as N-methyl-D-glucosamine and nanoscale dyes.Numerous studies have shown that the damaged tissue or cell damage can elevate extracellular ATP concentration to activate P2X7 receptors and its downstream NLRP3 / Caspase 1 pathway,further more,induce the IL-1 beta release and mature,at last,start the inflammatory cascade reaction.Research of the pathway is more popular in recent years,and found it plays a crucial role in some known inflammation,such as ischemia reperfusion injury,myocardial infarction,acute liver damage,and acute lung injury.P2X7 receptors inhibitor BBG(brilliant blue G)through the noncompetitive manner downgrade its downstream pathways NLRP3/Caspase 1 to inhibition of inflammation,proving the important role of P2X7 receptors ininflammation chain.According to previous studies,lidocaine anti-inflammatory effects can be found the related mechanism is numerous,lidocaine and P2X7 receptors are involved in a variety of inflammatory reaction process,and all related to ion channels.But the research about the effect of lidocaine on P2X7R/NLRP3/Caspase 1 pathway and whether through the pathway inhibit inflammation is few,So we guess whether lidocaine by inhibiting ATP-gated ion channel P2X7 receptor downstream pathway NLRP3 / Caspase 1 to reduce the degree of acute lung injury,its protective effect is consistent with the classical P2X7 receptor inhibitor BBG.Based on the previous studies,we established an ALI model in rats by lipopolysaccharide(LPS)to explore the protective effect of lidocaine on ALI and the possible mechanism from lung tissue pathology,inflammatory factor expression level,P2X7R/NLRP3/Caspase 1 expression levels,Hope to bring new idea to the treatment of ALI/ARDS.Methods40 SPF male Sprague Dawley(SD)rats weighing about 180 g ~220g were purchased from the Experimental Animal Center of Guangdong Province and were raised in the animal house of the Central Laboratory in Guangzhou First People’s Hospital.Day and night alternate 12 h,free feeding and drinking water,all of them adapt to 1 ~ 2 weeks before acute lung injury model established.40 male SD rats were randomly divided into normal saline control group(NS group),lipopolysaccharide group(LPS group),5 mg/kg lidocaine treatment group(5LD group),10 mg/kg lidocaine treatment group(10 LD group)and 20 mg/kg lidocaine treatment group(20 LD group).NS group was received the same amount of vehicle as the rest of the four groups which were intraperitoneally treated with 5 mg/kg LPS to set up ALI model,and the lidocaine treated groups were intraperitoneally administered lidocaine at 10 min before the LPS injection,1 h and 3 h after LPS injection.Twenty-four hours after LPS administration,the rats were sacrificed,lung tissue specimens were collected,hematoxylin-eosin(HE)staining to observe the lung tissue pathology,measuring lung wet/dry weight ratio(W/D),real-time fluorescent quantitative PCR(RT-PCR and QPCR)technology to detect in specimens P2X7 R mRNA,NLRP3 mRNA,Western blotting to detect P2X7 R content,NLRP3 content and caspase-1 content,enzyme-linked immunosorbent method(ELISA)to detect interleukin 1 beta(IL – 1β)and high mobility group box-1 protein(HMGB1)content,colorimetry to detect myeloperoxidase(MPO)activity.Statistical analysis was performed using SigmaPlot 13.0 software.Measured data with normal distribution were expressed as mean±standard deviation(x±s).Comparison between groups was performed using One-Way ANOVA Dunnet-t test,And drawing with Graphpad prism5 software.Results1.Histopathological observation of hematoxylin-eosin staining: Lidocaine alleviates pathological damage of lung tissueThe lung tissue HE staining was observed under a microscope,the NS group had complete lung structure,Mainly manifested in no alveolar septum dilation,no inflammatory exudation,and clear alveolar cavity;LPS group showed significantly impaired lung structure compared with NS group,Mainly manifested in alveolar telangiectasia,alveolar erythrocyte exudation,alveolar septa edema,massive inflammatory cell infiltration,and hemorrhage;20LD group compared with LPS group,alveolar structure is basically complete,alveolar septa edema,inflammatory cell infiltration,and hemorrhage are significantly reduced compared with LPS group The degree of lung injury,alveolar septal edema,inflammatory cell infiltration,and hemorrhage in the 5LD group and the 10 LD group were all lower than those in the LPS group,but not as significantly as in the 20 LD group.The above results showed that LPS can induce lung pathological injury,and lidocaine can reduce LPS induced pathological lung injury in a dose-dependent manner.2.Lidocaine reduce the degree of pulmonary edema,reduce the W / D ratio Compared with the NS group.W/D values in the LPS group were significantly higher(P<0.001),and pulmonary edema was severe.The 5LD group showed that the W/D value was decreased,indicating that the degree of pulmonary edema was reduced but the difference was not statistically significant(P>0.05)compared with LPS group;W/D values of 10 LD group and 20 LD group were significantly lower than those of LPS group(P<0.01),indicating that the degree of pulmonary edema was significantly reduced in 10 LD group and 20 LD group.The above results showed that LPS caused lung tissue edema,the W/D ratio of lung tissue increased,and lidocaine can reduced the degree of lung tissue edema and the W/D value induced by LPS in a dose-dependent manner.3.Lidocaine dose-dependently reduced P2X7 R protein expression in lung tissueThe expression of P2X7 R protein was significantly increased in LPS group compared with NS group(P<0.001).The expression of P2X7 R protein in 5LD group was lower than that in LPS group,but the difference was not statistically significant(P>0.05).The 10 LD group showed P2X7 R protein expression was decreased compared with LPS group(P<0.05).The expression of P2X7 R protein in the 20 LD group was significantly lower than that in the LPS group(P<0.001).The above results showed that LPS up-regulated the expression of P2X7 R protein.5mg/kg lidocaine had little effect on P2X7 R protein expression.10mg/kg lidocaine and 20mg/kg lidocaine can reduce the expression of P2X7 R protein in a dose-dependent manner.4.Lidocaine reduced the expression level of NLRP3 in a dose-dependent mannerCompared with NS group,the expression of NLRP3 protein in LPS group was all significantly increased(P<0.001).Compared with LPS group,the expression of NLRP3 protein in 5LD group was decreased,but the difference was not statistically significant(P>0.05),the expression of NLRP3 protein was decreased in 10 LD group(P<0.01),and the expression of NLRP3 protein was significantly decreased in the 20 LD group(P<0.001).The above results showed that LPS up-regulates NLRP3 protein expression.5mg/kg lidocaine has little effect on NLRP3 protein expression.10mg/kg lidocaine and 20mg/kg lidocaine can reduce NLRP3 protein expression in a dose-dependent manner.5.Lidocaine reduced the expression level of Caspase-1 in the lung tissue in a dose-dependent mannerIn the control group,The Caspase-1 protein had a small number of expressions.Compared with the NS group,the expression of Caspase-1 protein was significantly increased in the LPS group(P<0.01).Compared with LPS group,the expression of Caspase-1 protein decreased in 5LD group,but the difference was not statistically significant(P > 0.05),the expression of Caspase-1 protein was decreased in the 10 LD group,and the degree of reduction was more relative to the 5LD group,but the difference was also not statistically significant(P > 0.05);the expression of Caspase-1 protein was significantly decreased in the 10 LD group(P <0.05).The above results indicated that LPS up-regulated Caspase-1 protein expression.5mg/kg lidocaine and 10mg/kg lidocaine had negligible effect on Caspase-1 protein expression.20mg/kg lidocaine could significantly reduce the expression of caspase-1 protein.6.Lidocaine reduced P2X7 R mRNA content and NLRP3 mRNA content in lung tissue in a dose-dependent mannerP2X7R mRNA content and NLRP3 mRNA content were slightly expressed in NS group.Compared with NS group,P2X7 R mRNA content and NLRP3 mRNA content were significantly increased in LPS group(P<0.05).P2X7 R mRNA content and NLRP3 mRNA content were decreased in 5LD group compared with LPS group,but the difference was not statistically significant(P>0.05).Compared with the LPS group,the content of P2X7 R mRNA was significantly decreased(P<0.05),and the NLRP3 mRNA content was significantly decreased(P<0.01)in the 10 LD group;P2X7R mRNA content was significantly decreased(P<0.05),and NLRP3 mRNA content was significantly reduced(P<0.001)in the 20 LD group.These results indicated that LPS up-regulates P2X7 R mRNA and NLRP3 mRNA expression,5mg/kg lidocaine has little effect on P2X7 R mRNA and NLRP3 mRNA expression,while 10mg/kg and 20mg/kg lidocaine can significantly reduce P2X7 R mRNA and NLRP3 mRNA content as a dose-dependent manner.7.Lidocaine dose-dependently reduced the expression of IL-1β and HMGB1 in lung tissueCompared with NS group,IL-1β and HMGB1 levels in LPS group were significantly increased(P<0.001).Compared with LPS group,IL-1β and HMGB1 expression levels in 5LD group were significantly lower(P<0.05),10 LD group was remarkably lower,the expression of IL-1β was significantly decreased(P<0.001),and the expression of HMGB1 was significantly decreased(P<0.01).The expression of IL-1β was significantly lower in the 20 LD group(P<0.001),and the expression of HMGB1 was significantly decreased(P<0.01).The above results showed that LPS induced the release of pro-inflammatory cytokines IL-1β and HMGB1,while lidocaine dose-dependently reduced the expression of IL-1β and HMGB1.8.Lidocaine reduces the accumulation of neutrophils in lung tissue and reduces the MPO content in lung tissue in a dose-dependent mannerCompared with NS group,the MPO activity level in LPS group was significantly higher(P<0.001);the activity level of MPO in 5LD group was lower than that in LPS group(P<0.05);the activity level of MPO was significantly lower in 10 LD group compared with LPS group(P< 0.01);20LD group compared with LPS group,MPO activity levels decreased significantly(P <0.001).The above results indicated that LPS induced increased neutrophil infiltration and increased MPO activity.Lidocaine reduced neutrophil infiltration and MPO activity in a dose-dependent manner.ConclusionLidocaine can reduce the release of inflammatory cytokines induced by lipopolysaccharide in a dose-dependent manner and improve the degree of acute lung injury in rats.The mechanism may be related to the inhibition of the P2X7R/NLRP3/Caspase-1 axis and thus the release of inflammatory factors. |
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