| Background:Intensive care unit-acquired muscle weakness(ICUAW)is a common disease among patients in ICU.Immobilization is an important independent risk factor for ICUAW,and neuromuscular dysfunction(NMD)is one of the main manifestations.NMD increases the risk of lung infection and aggravates respiratory failure,which may lead to prolonged mechanical ventilation and hospital stay.NMD not only affects muscle function in the acute phase of the disease,but also lasts for several months or even more than one year after discharged from the hospital,seriously affecting the quality of life of patients.Electroacupuncture(EA)plays an important role in neuromuscular injury and repair in clinical practice.At the same time,a large number of studies have confirmed that EA plays a protective role in the treatment of skeletal muscle atrophy.Our previous study found that heterogeneity and remodeling of nicotinic acetylcholine receptor(nAChR)in skeletal muscle is an important cause of NMD in sepsis,and neuregulin-1(NRG-1)plays an important protective role in it.However,the effect of NRG-1/ErbB mediated heterogeneity and remodeling of nAChR in NMD caused by immobilization has not been reported.The role of NRG-1/ErbB regulated by EA in NMD caused by immobilization has not been investigated.This study will provide new therapeutic ideas and targets for NMD caused by immobilization.ObjectiveIn present study,we investigate whether nAChR heterogeneity and remodeling occurs in rat skeletal muscle under immobilization condition,and to explore the relationship between nAChR heterogeneity and remodeling,NMD and NRG-1 at different time points of immobilization.Subsequently,we manipulate the changes of NRG-1/ErbB to explore whether immobilization leads to NMD through nAChR heterogeneity and remodeling.We hope these could provide new therapeutic target and strategies for NMD caused by immobilization.Finally,we aim to observe whether NRG-1/ErbB-mediated heterogeneity and remodeling of nAChR plays a role in the improvement of NMD by EA,and to determine whether EA plays a protective role in NMD through NRG-1/ErbB signaling pathway.MethodsPart Ⅰ:Healthy adult male Sprague-Dawley(SD)rats aged 2 to 3 months and weighing 220-250g were selected and unilateral hindlimb immobilization model was established.Rats were randomly divided into the two groups:sham operation group(Sham)and the immobilization group(Immobilization).According to the time point,each group was divided into four subgroups named Day 1,Day 3,Day 7,and Day 14.Muscle fiber size and Cross·sectional area(CSA)were measured according to H&E staining.Then the weight of rats and wet weights of tibialis anterior muscles were quantified.To investigate the neuromuscular function of tibialis anterior muscle,amplitude,duration,latency time and nerve conduction velocity(NCV)of compound muscle action potential(CMAP)were detected.The skeletal muscle samples were harvested at different immobilization time point,the c-nAChR,γ-nAChR,a7-nAChR and NRG-1 level were detected by Western blot.Meanwhile,the nAChRs expression in skeletal muscle tissue was also estimated by immunofluorescent assay.Part Ⅱ:Experiment 1:In order to investigate whether the exogenous O addition of human recombinant neuregulim-1(rhNRG-1)could alleviate NMD in immobilization.After establishing the rat immobilization model,different doses of rhNRG-1(0,1,10,20,50ug/kg/d)were administered lntraperitoneally daily.After 14 days,behavioral,morphological analysis of tibialis anterior muscle and CMAP of the tibialis anterior muscle was estimated to choose the optimum dose of rhNRG-1 to improve the neuromuscular function of the immobilization rats.Experiment 2:To elucidate whether NMD regulated by NRG-1/ErbB signaling pathway,rhNRG-1 and(or)ErbB inhibitor PD-158780 were administered intraperitoneally in immobilization rats.Immobilization rats were randomly divided into 4 groups:Immobilization[rhNRG-1(-)PD-158780(-)],Immobilization+rhNRG-1[rhNRG-1(+)PD-158780(-)1 Immobilization+PD-158780[rhNRG-1(-)PD-158780(+)],Immobilization+rhNRG-1+PD-158780[rhNRG-1(+)PD-158780(+)].CMAP of tibialis anterior muscle was quantified to verify the neuromuscular function after 14 days.To test whether NRG-1/ErbB signaling pathway was involved in the process of NMD following immobilization,not only the expression of ErbB2,ErbB3,ErbB4,Erkl/2 and their phosphorylation protein,but also GABPα、GABPp in skeletal muscle were estimated by Western-blot.The ε-nAChR,γ-nAChR,andα7-nAChR protein expression was estimated by Western blot and immunofluorescent assay.Part Ⅲ:Experiment 1:To investigate the influence of EA on NMD,Immobilization models were established in healthy adult male Sprague-Dawley rats aged 2 to 3 months,rats were randomly divided into four groups:normal group(Control),sham operation group(Sham),immobilization(Immobilization),immobilization+electroacupuncture(EA).Experiment 2:To demonstrate the influence of non-acupoint electroacupuncture on heterogeneity and remodeling of nAChR and NMD.Then,Rats were randomly divided into three groups:immobilization(Immobilization),immobilization+electroacupuncture(EA)and immobilization+non-acupoimts electroacupuncture(NEA).After establishing immobilization model,the rats in EA group received electroacupuncture treatment at the acupoints of the "Zusanli(ST36)","Huantiao(GB30)" with a certain frequency and intensity.A set of non-acupoint located on the ulna side of the metacarpus served as controls in the NEA group to vefify the effect of the acupoint.Muscle fiber size and CSA were measured according to H&E staining.Then the weight of rats and wet weights of tibialis anterior muscles were quantified to verify degree of muscle atrophy.Subsequently,Principal component analysis(PCA)and Hierarchical cluster analysis(HCA)were used to estimated neuromuscular function according to the CMAP data.Then the NRG-1/ErbB pathway protein expression(ErbB2,ErbB3,ErbB4,Erkl/2,GABPa,nAChR and GABPβ)were estimated by Western blot or lmmunofluorescent assay to verify the connection of NRG-1/ErbB pathway and heterogeneity and remodeling of nAChR.Experiment 3:In order to further elucidate whether NRG-1/ErbB pathway participated in regulation of EA in skeletal muscle NMD caused by immobilization,NRG-1/ErbB signaling pathway was inhibited through intraperitoneal injection ErbB inhibitor PD-158780.Immobilized SD rats were randomly divided into four groups,Immobilization[EA(-)PD-158780(-)],Immobilization+EA[EA(+)PD-158780(-)],Immobilization+PD-158780[EA(-)PD-158780(+)],and Immobilization+EA+PD-158780[EA(+)PD-158780(+)].Subsequently,the skeletal muscle of immobilization 14days’injury was harvested and evaluated.Then,CMAP of tibialis anterior muscle was quantified to verify the neuromuscular function.To test whether NRG-1/ErbB signaling pathway was involved in the process of EA triggered skeletal muscle protection following immobilization,not only the expression of ErbB2,ErbB3,ErbB4,Erkl/2 and their phosphorylation protein,but also GABPα、GABPβ in skeletal muscle were estimated by Western-blot.Theγ-nAChR,ε-nAChR and α7-nAChR protein expression was estimated by Western blot and immunofluorescent assay.ResultsPart Ⅰ(1)Firstly,rearing number and distance gradually decreased in Immobilization group,while the distance and rearing number gradually increased in the Sham group with a time dependent manner(P<0.05).Compared with those in the Sham group,the rearing number and distance decreased on Day 1,3,7,and 14 in the immobilization group(P<0.05).(2)According to H&E staining,we found that tibialis anterior muscle atrophied caused by immobilization,as the immobilization time prolonged,the muscle cells atrophied and the muscle fiber gap widened.There was no significant difference in muscle cell CSA and muscle fiber size at each time point in the Sham group(P>0.05).There was a significant difference in CSA and muscle fiber size at each time point in the Immobilization group(P<0.05).The CSA and muscle fiber size of muscle cells in Day 3,Day 7 and Day 14 groups were significantly different from those in Sham group(P<0.05).With the immobilization time prolonged,the muscle atrophy became most severe in Day 14 group.(3)There was no significant difference of rats weight between immobilization and sham group at each time point(P>0.05).The ratio of wet weights of tibialis anterior muscles/weight of rats and weights of tibialis anterior muscles gradually decreased on Day 3,7,and 14 in the immobilization group compared with those in the Sham group(P<0.05).(4)After the immobilization,we found that the nerve conduction velocity(NCV),latency time of CMAP decreased,and the duration and amplitude of CMAP increased,each index of CMAP was significantly different in immobilization group at different time point(P<0.05).Compared with Sham group,NCV,latency time,duration and amplitude of CMAP,there was a significant difference in immobilization group(P<0.05).(5)Immunofluorescence staining showed that all three nAChRs were distributed on the skeletal muscle cell membrane.Compared with the Sham group,the expression levels of α7-nAChR and γ-nAChR in the tibialis anterior musclo membrane of the rats in Day 1,Day 3,Day 7 and Day 14 were gradually increased in immobilization group;while the expression of normal receptor ε-nAChR was gradually decreased(P<0.05).The expression of α7-nAChR and γ-nAChR was the highest and the expression of ε-nAChR was the lowest at 14 days after immobilization.Western-blot results indicated that the expression changes of α7-nAChR,γ-nAChR andε-nAChR proteins were consistent with the results of immunofluorescence.(6)Compared with the Sham group,the expression of neuregulim-1 protein in the tibialis anterior muscle was significantly decreased after immobilization in the Day 14 group(P<0.05).Part Ⅱ(1)The results of behavior and H&E stanning of anterior tibialis muscle suggested that exogenous administration of rhNRG-1 can increase the horizontal movement distance and rearing number of immobilized rats,and ameliorate the atrophy of anterior tibialis muscle.(2)First,we found with the increase of the exogenous dose of rhNRG-1in immobilization rats,the better recovery of each index of CMAP in rats,indicating the better recovery of neuromuscular function.There was no significant difference in neuromuscular function at the dose of 20 μg/kg and 50 μg/kg When rhNRG-1 was administered intraperitoneally daily(P>0.05).We concluded that intraperitoneal administration of 2Oμg/kg/d rhNRG-1 is the optimal dose for the treatment of neuromuscular dysfunction caused by immobilization in rats.(3)Then,compared with Immobilization group[rhNRG-1(-)PD-158780(-)],Immobilization+rhNRG-1 group[rhNRG-1(+)PD-158780(-)]showed increased expression of ε-nAChR on the skeletal muscle membrane,α7-nAChR and γ-nAChR expression were decreased(P<0.05).However,Immobilization+PD-158780 group[rhNRG-1(-)PD-158780(+)],Immobilization+EA+PD-158780 group[rhNRG-1(+)PD-158780(+)]exhibited the expression levels of α7-nAChR andγ-nAChR increased,but the expression of ε-nAChR decreased(P<0.05),which was in accordance with the trend of estern-blot results.(4)Then we found from Western blot that accompanied with the exogenous administration of rhNRG-1,the phosphorylation levels of ErbB2,ErbB3,ErbB4,Erkl/2 were all significantly increased,as well as the expression level of GABPa,GABPB(P<0.05).Then we observed that the ratios of P-ErbB2/ErbB2,P-ErbB3/ErbB3,P-ErbB4/ErbB4,and P-Erk1/2/Erkl/2,GABPα,GABPβ were significantly decreased with the treatment of PD-158780(p<0.05).Part Ⅲ(1)Compared with the Immobilization group,the CSA and muscle fiber size of the anterior muscle of the EA group increased(P<0.05).CSA and muscle fiber size in the Control group exhibited no significant difference with the Sham group(P>0.05).Also,muscle fiber size and CSA in the Immobilization group exhibited no significant difference with the NEA group(P>0.05).(2)After the administration of EA,compared with the Immobilization group,the anterior tibialis anterior muscle weight and the ratio of tibialis anterior muscle weight/weight of rats were significantly increased in the EA group(P<0.05).The anterior tibialis anterior muscle weight and the ratio of tibialis anterior muscle weight/weight of rats in NEA group revealed no significant difference compared with Immobilization group(P>0.05).There was no significant difference between the control group and the Sham group(P>0.05).(3)After 14 days of immobilization,amplitude,NCV and twitch tension of CMAP decreased significantly compared with Sham group(P<0.05).However,with EA administration,immobilization-induced reduction in amplitude,NCV and twitch tension were significantly improved in the EA group,EA decreased the duration of CMAP compared with Immobilization group(P<0.05).There was no significant difference between the NEA group and Immobilization group(P>0.05).Amplitude,duration,NCV and twitch tension of CMAP in Control group exhibited no significant difference compared with Sham group(P>0.05).(4)Based on the results of CMAP data,we performed PCA and HCA analysis,the normal and pathological conditions of neuromuscular function in rats could be separated by CMAP data.The results of PCA and HCA indicated that there was no significant separation in neuromuscular function between Control group and Sham group.Neuromuscular function of rats in EA group was distinguished from Immobilization group with the EA treatment indicating EA alleviated immobilization-induced neuromuscular dysfunction.(5)Immunofluorescence staining showed that the expression ofα7-nAChR and γ-nAChR in skeletal muscle membrane of Immobilization group was significantly increased compared with Sham group,while the expression of normal receptor s-nAChR was decreased(P<0.05).With EA administration,the expression levels of αa7-nAChR and γ-nAChR decreased in the skeletal muscle membrane while the expression of normal receptor s-nAChR was increased compared with immobilization group(P<0.05).Also,NEA group revealed no significant difference with the Immobilization group(p>0.05).The results of western-blot showed the same trend with the immunofluorescence of three nAChRs.(6)Then we found from Western blot that accompanied with the rapid induction of NRG-1 expression,the ErbB2,ErbB3,ErbB4,phosphorylation levels of Erkl/2,and expression level of GABPα,GABPβ were all significantly decreased after 14 days immobilization(P<0.05).Then we observed that the ratios of p-Erkl/2/Erkl/2,ErbB2,ErbB3,ErbB4,GABPa,GABPβ and NRG-1 expression increased with EA treatment(P<0.05).(7)In further research,we found Immobilization+EA group[EA(+)PD-158780(-)]showed the increased amplitude,NCV,and the latency time,duration were decreased compared with Immobi lization group[EA(-)PD-158780(-)](P<0.05),while Immobilization+PD-158780 group[EA(-)PD-158780(+)],and Immobilization+EA+PD-158780 group[EA(+)PD-158780(+)]exhibited the latency time,duration were increased,but the amplitude,NCV were decreased compared with Immobilization group[EA(-)PD-158780(-)](P<0.05)(8)Immunofluorescence showed that compared with the Immobilization group[EA(-)PD-158780(-)],the expression of ε-nAChR on the skeletal muscle membrane was increased,ε7-nAChR and γ-nAChR expression level decreased in Immobilization+EA group[EA(+)PD-158780(-)](P<0.05),while Immobilization+PD-158780 group[EA(-)PD-158780(+)],Immobilization+EA+PD-158780 group[EA(+)PD-158780(+)]showed the expression levels of α7-nAChR and γ-nAChR increased,but the expression of s-nAChR decreased(P<0.05),which was consistent with the trend of western-blot results(9)Compared with the Immobilization group[EA(-)PD-158780(-)],The protein expression of NRG-1,phosphorylation levels of ErbB2,ErbB3,ErbB4,and Erkl/2,and expression level of GABPa,GABPB were all increased in Immobilization+EA group[EA(+)PD-158780(-)](P<0.05).However,the up-regulation of the phosphorylation levels of ErbB2,ErbB3,ErbB4,and Erkl/2,and expression level of GABPα,GABPβ were abolished with the treatment of PD-158780Conclusion(1)Immobilization-induced muscle atrophy,as well as the NMD,which was associated with the reduction of NRG-1 and heterogeneity and remodeling of nAChR(2)Heterogeneity and remodeling of nAChR regulated by NRG-1/ErbB signaling Pathway leads to NMD.Exogenous administration of rhNRG-1 could ameliorate muscle atrophy,inhibit the heterogeneity and remodeling of nAChR,and attenuate NMD caused by immobilization through NRG-1/ErbB signaling pathway,which provided a new target for the treatment of NMD caused by immobilization(3)EA would enhance NRG-1 signaling pathway,and inhibit heterogeneity and remodeling of nAChR through NRG-1/ErbB signaling pathway,resulting in suppressed immobilization-induced muscle loss and alleviated neuromuscular dysfunction. |
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