| Objectives Acupuncture can treat acute lung injury caused by endotoxin,but the specific mechanism is still unclear.Our previous studies found that increased HO-1 expression could promote mitochondrial fusion and inhibit mitochondrial fission in endotoxin acute lung injury,and its endogenous organ protection effect might be related to mitochondrial fusion/ fission.Electroacupuncture can play a protective role in acute lung injury by up-regulating the expression of HO-1.These results have drawn our attention to the correlation between acupuncture,HO-1 and mitochondrial dynamics.In view of this,this study intends to clarify the effect of electroacupuncture on mitochondrial fusion/fission in lung tissues of endotoxin-induced acute lung injury.To investigate whether electroacupuncture can improve mitochondrial function and reduce lung injury by up-regulating the dynamic balance of mitochondrial fusion/ fission in lung tissues of endotoxin-induced acute lung injury through HO-1.Methods To clarify the effect of electroacupuncture on mitochondrial fusion/fission in lung tissues of endotoxin-induced acute lung injury.To investigate whether electroacupuncture can improve mitochondrial function and reduce lung injury by up-regulating the dynamic balance of mitochondrial fusion/fission in the lung tissue of endotoxin-induced acute lung injury by HO-1.This study was divided into experiment 1 and experiment 2.Experiment 1: We selected 40 New Zealand white rabbits and divided them into 4 groups by random number table: normal control group(group C),acute lung injury group(group M),non-acupoint electroacupuncture + acute lung injury group(group SEAM)and acupoint electroacupuncture + acute lung injury group(group EAM).LPS 5 mg/kg(dissolved in 0.9% normal saline 2 ml)was intravenously injected into the ear margin of the rabbits in M group,group SEAM and group EAM to prepare the model of endotoxin-induced acute lung injury.Before intravenous injection of LPS for 4,3,2,1 d and 30 min,the group EAM selected zusanli and feishu points,and the group SEAM selected the non-acupoint site to establish the electric acupuncture model.After the injection of LPS 6 h animals were killed by exsanguination under anesthesia for lung tissue,then observe the pathology results and calculate the lung injury score,to detect W/D,electron microscope mitochondria ultrastructure changes,detection of active oxygen(Reactive oxygen species,ROS)generation,adenosine triphosphate(adenosine triphosphate,ATP)content,respiratory control ratio(RCR)and mitochondrial membrane potential(MMP)to understand the state of mitochondrial function,the expressions of Drp1,Mfn1,Mfn2,OPA1 mRNA and protein were determined by real-time PCR and Western blot.The changes of mitochondrial fusion/fission were understood from the expression of mitochondrial fusion/fission markers.Experiment 2: To investigate whether electroacupuncture can regulate the dynamic changes of fusion/fission of HO-1 by regulating mitochondrial dynamics-related proteins in lung tissue,we selected 70 healthy and clean male New Zealand white rabbits.Divided into 7 groups by random number table in the normal control group(group C),LPS to establish bacterial endotoxin(group LPS),non-acupoint electric acupuncture plus LPS to establish a bacterial endotoxin ALI model group(group LPS + non-acupoint EA),establishes a model of endotoxin ALI electroacupuncture acupoints plus LPS group + EA group(group LPS + EA),electroacupuncture acupoints + endotoxin ALI model + HO-1 blockers(group LPS + EA + ZnPP),electroacupuncture acupoints + HO-1 inducer + endotoxin ALI model group(group LPS + EA + hemin),acupoint electroacupuncture stimulation + endotoxin ALI model + HO-1 inducer + HO-1 blocker group(group LPS+EA+hemin+ZnPP).Group LPS,group LPS+non-acupoint EA,group LPS+EA,group LPS+EA+ZnPP,group LPS+EA+hemin,group LPS+EA+hemin+ZnPP were intravenous injected with LPS 5 mg/kg(dissolved in 0.9% normal saline 2 ml)through the ear margin to prepare the endotoxin-induced acute lung injury model.The group LPS+EA,the group LPS+EA+ZnPP,the group LPS+EA+hemin,the group LPS+EA+hemin+ZnPP were selected for electroacupuncture stimulation 4,3,2,1 d and 30 min before the intravenous injection of LPS.Group LPS+non-acupoint EA was given non-acupoint electrical stimulation,and the stimulation parameters were the same as group LPS+EA.Group LPS+EA+hemin,group LPS+EA+hemin+ZnPP were intravenously injected with 100 mg/kg HO-1 inducer hemin(dissolved in 0.1 mol/l sodium hydroxide)1h before LPS,Group LPS+EA+ZnPP and group LPS+EA+hemin+ZnPP were intravenously injected with 10 mol/kg HO-1 inhibitor ZnPP(dissolved in 1ml sodium bicarbonate)1h before LPS.After the injection of LPS 6 h animals were killed by exsanguination under anesthesia for lung tissue,then observe the pathology results and calculate the lung injury score,to detect W/D,electron microscope mitochondria ultrastructure changes,detection of ROS generation,ATP content,RCR and MMP to understand the state of mitochondrial function,the expressions of Drp1,Mfn1,Mfn2,OPA1,HO-1 mRNA and protein were determined by real-time PCR and western blot.The changes of mitochondrial fusion/fission were understood from the expression of mitochondrial fusion/fission markers.ResultsExperiment 1: Compared with group C,lung injury scores and W/D of lung tissues in group M,group SEAM and group EAM were all increased,ATP content was decreased,ROS content was increased,MMP and RCR were decreased,mRNA and protein expressions of Mfn1,Mfn2 and OPA1 were down-regulated,and Drp1 mRNA and protein expressions were up-regulated(P<0.05).Compared with group M,group EAM of lung injury score of lung tissue and W/D are reduced(P < 0.05),there was no significant difference of lung injury score and W/D statistical significance in group SEAM(P > 0.05),ATP content in lung tissue,ROS generation is reduced,MMPS and RCR were higher,the lung tissue Mfn1,Mfn2 and mRNA and protein expression of OPA1 raising,mRNA and protein expression of Drp1 were decreased in group SEAM and EAM(P < 0.05);Compared with group SEAM,lung injury score and W/D in group EAM and group EAM were decreased,ATP content was increased,ROS content was decreased,MMP and RCR were increased,mRNA and protein expressions of Mfn1,Mfn2 and OPA1 were up-regulated,mRNA and protein expressions of Drp1 were down-regulated in group EAM and group EAM(P<0.05).Experiment 2: Compared with group C,lung injury score and W/D were elevated,ATP content was reduced,the production of ROS was increased,MMP and RCR were decreased,mitochondrial fusion markers Mfn1,Mfn2,OPA1 mRNA and protein expression significantly were reduced,and mitochondria marker Drp1 mRNA and protein expression levels,the expression levels of HO-1 mRNA and protein were increased in group LPS,group LPS + non-acupoint EA,group LPS + EA,group LPS + EA + ZnPP,group LPS + EA + hemin and group LPS + EA + hemin + ZnPP(P < 0.05).Compared with group LPS and group LPS + EA lung injury score and W/D lung tissue were lower(P < 0.05),there was no significant difference of lung injury score and W/D statistical significance in group LPS + non-acupoint EA(P > 0.05),group LPS + non-acupoint EA and group LPS + EA ATP content increased,the generation of ROS was reduced,MMPS and RCR all rise,mitochondria fusion markers Mfn1,Mfn2,OPA1 mRNA and protein expression significantly increases,The expression levels of mitochondrial division marker Drp1 mRNA and protein were decreased,while the expression levels of HO-1 mRNA and protein were increased(P < 0.05).Compared with group non-acupoint LPS + EA,group LPS + EA lung injury score of lung tissue and W/D were reduced,the ATP content increased,the production of ROS reduced,MMP and RCR increased,mitochondria fusion markers Mfn1,Mfn2,OPA1 rise,and mitochondria Drp1 mRNA and protein expression level decreased,HO-1 mRNA and protein expression levels increased(P < 0.05);Compared with LPS + EA group,group LPS + EA + hemin lung injury score and W/D were reduced,the ATP content increased,the generation of ROS reduced,MMPand RCR all rised,mitochondria fusion markers Mfn1,Mfn2,OPA1 had a significant rise in mRNA and protein expression,and mitochondria markers Drp1 mRNA and protein expression level decreased,HO-1 mRNA and protein expression levels increased(P < 0.05),lung injury score,W/D were higher,the ATP content is lower,The expression levels of mitochondrial fusion markers Mfn1,Mfn2,OPA1 mRNA and protein were significantly decreased,while the expression levels of mitochondrial fission marker Drp1 mRNA and protein were increased,and the expression levels of HO-1 mRNA and protein were decreased in group LPS + EA + ZnPP(P < 0.05).Compared with group LPS + EA + hemin,lung tissue lung injury score,W/D were elevated,ATP content is reduced,the production of ROS,MMPS and RCR decreased,mitochondrial fusion markers Mfn1,Mfn2,OPA1 mRNA and protein expression significantly were reduced,and mitochondria marker Drp1 mRNA and protein expression levels increased,HO-1 mRNA and protein expression levels decreased in group LPS + EA + ZnPP,group LPS + EA + hemin + ZnPP(P < 0.05);Compared with group LPS + EA + ZnPP,lung tissue lung injury score,W/D are reduced,the ATP content increased,the production of ROS reduced,MMP and RCR rise,mitochondria fusion markers Mfn1,Mfn2,OPA1 mRNA and protein expression significantly increased,and mitochondria Drp1 mRNA and protein expression levels decreased,HO-1 mRNA and protein expression levels increased in group LPS + EA + hemin + ZnPP(P < 0.05).Conclusion 1.Electroacupuncture can maintain the balance of mitochondrial fusion/ fission in lung tissues of endotoxin ALI.2.The mechanism is related to the up-regulation of HO-1 by acupuncture. |
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