The Role Of NRG-1/ErbB/Erk Signaling Pathway In The Compensatory Increase Of AChR Transcription In Skeletal Muscle Of Myasthenia Gravis

Myasthenia Gravis(MG)is an autoimmune disease of the Neuromuscular Junction(NMJ).Its pathogenesis depends on the type of autoantibody.Most of the myasthenia gravis patients were caused by anti-acetylcholine receptor antibody(anti-AChR Ab).The pathological mechanism of AChR MG is that AChR levels in the postsynaptic membrane are blocked or damaged by anti-AChR ab,and then the NMJ transmission efficiency of the NMJ is reduced.Our previous study found that the transcription level of AChR-ε subunit in the gastrocnemius muscle tissue of experimental autoimmune myasthenia gravis(EAMG)rats was significantly increased.Other studies have shown that after the protein expression of AChR in the postsynaptic membrane is lower than 40%,the skeletal muscle AChR subunit m RNA transcription and protein expression levels will be significantly increased,and the AChR transcription level is negatively correlated with the severity of MG.Taken together,we hypothesize that skeletal muscle is not only an attacking target in the pathophysiological process of MG.When the AChR protein expression level drops below the certain threshold,it will initiate a self-compensatory mechanism through transcription,translation and secretion of some unknown bioactive factors and then activate some of their signaling pathways to promote the transcription of AChR subunits.However,it is still unclear which mechanism triggers the compensatory increase in AChR subunit transcription in response to the pathological process of AChR reduction in MG.The Neuregulin-1(NRG-1)/ErbB/Erk signaling pathway is involved in the regulation of nervous system development,including the promotion of synaptic AChR subunit transcription and clustering of AChR on NMJ postsynaptic membrane.However,so far,the role of NRG-1/ErbB in MG have not been studied in detail.Therefore,in this study,we will explore the mechanism of the NRG-1/ErbB/Erk signaling pathway to AChR expression in MG.At the same time,we will evaluate the therapy value of exogenous NRG-1 to EAMG rats.This study will provide new therapeutic strategies and potential targets for MG.Part Ⅰ: The expression of NRG-1/ErbB/Erk1/2 signaling protein in skeletal muscle of EAMG ratsObjective To establish the experimental autoimmune myasthenia gravis rat model(EAMG)and explore the changes in the NRG-1/ErbB/Erk signaling pathway in the skeletal muscle tissue of model rats.Methods Twenty-two Lewis rats were divided into 3 groups: the Control group(CTL)(n=6),the immune adjuvant(IA)group(n=6)and the EAMG group(n=10).Then,the body weight,grip strength,Lennon clinical symptom score and anti-AChRα R97-116 Ig G antibody level of the rats in each group were determined.The changes of AChR-ε subunit on the surface of the skeletal muscle of EAMG rats were detected by immunofluorescence.The changes in the expression levels of NRG-1,p-ErbB-2/ErbB-2,p-Erk/Erk,p-ErbB-4/ErbB-4,AChR-ε expression levels in the gastrocnemius tissue were analyzed by real-time PCR(RT-PCR)and western blot(WB)methods,respectively.Results 1.6 EAMG rats of 10 rats were successfully established by evaluating muscle strength,clinical scores and anti-AChRα R97-116 Ig G levels.The success rate of EAMG was 60%.Compared with the CTL group and the IA group,the rats’ body weight and muscle strength in the EAMG group were significantly decreased(p < 0.05).In contrast,the Lennon clinical score(p < 0.05)and anti-AChRα R97-116 Ig G were significantly increased(p < 0.01).The immunofluorescence results showed that the density of AChR-ε subunit in the gastrocnemius muscle of rats in the EAMG group decreased and the fluorescence intensity was weakened.Compared to the CTL and IA groups,the AChR-ε subunit protein in the gastrocnemius muscle tissue of the EAMG group was significantly decreased(P<0.01),while the AChR-ε subunit m RNA was significantly increased(P<0.01);the expression levels of the NRG-1 protein and m RNA in the gastrocnemius muscle tissue of the EAMG group also increased significantly increased(P<0.01).The phosphorylation of ErbB-2,ErbB-4 and Erk1/2(p-ErbB-2/ErbB-2、p-ErbB-4/ErbB-4 and p-Erk/Erk)in the gastrocnemius muscle tissue of the EAMG group increased significantly as compared to the CTL and IA groups(P<0.01).Conclusion 1.Active subcutaneous immunization of Lewis rats with the AChRα R97-116 peptide can establish a stable and reliable EAMG rat model.2.EAMG rat muscle may have a compensatory effect in response to AChR reduction.The NRG-1/ErbB/Erk signaling pathway may be involved in the compensatory process of muscles responding to AChR reduction.Part Ⅱ: Neuregulin-1 enhances the protection of C2C12 myotube to the AChR damage of sreum from AChR-GMG patients through ErbB/Erk signaling pathwayObjective To observe the changes in the NRG-1/ErbB/Erk pathway in C2C12 myotubes with reduced Ach R protein expression after being incubated with serum from AChR antibody positive generalized myasthenia gravis(AChRGMG)patients.At the same time,to investigate whether exogenous rhNRG-1β1could protect against reducing AChR in myotubes by serum from AChR-GMG patients.Methods The first part of the experiment:The C212 myotube cell model was incubated with serum from AChR-GMG at various time points after Agrininduced AChR clustering.The changes of m RNA and protein expression of NRG-1,p-ErbB-2/ErbB-2,p-Erk/Erk and AChR-ε at each time point were detected by RT-PCR and WB.The second part of the experiment: C2C12 myotube cell model was divided into 6 groups: CTL group,NRG group,MG serum group,NRG+MG Serum,PD98059(a Mek/Erk inhibitor)+NRG+MG serum and PD158780(an ErbB inhibitor)+NRG+MG serum group after Agrin-induced AChR clustering.After different interventions,RT-PCR and WB were used to detect the changes in m RNA and protein expression of NRG-1,p-ErbB-2/ErbB-2,p-Erk/Erk,AChR-εin each group.The number of AChR clusters on the surface of myotubes in each group was observed by immunofluorescence.Results The first part of the experiment: After incubation with the serum of AChR-GMG patients,the protein level of the AChR-ε subunit first gradually decreased,reached the lowest point at 300 min,and then slowly recovered,however,the changing trend of the AChR-ε transcription is opposite,rising gradually at first,at a high transcription level in the range of 60-420 min,and then gradually decreasing.The changing trend of NRG-1,p-ErbB-2/ErbB-2 and pErk/Erk expression level are similar to that of the AChR-ε subunit transcription level.The protein expression level of the NRG-1 was negatively correlated with the level of AChR-ε protein(r=-0.953,P<0.001)and positively correlated with the transcription level of AChR-ε(r =0.852,P<0.001).The second part of the experimental results: After different interventions,compared to the CTL group,the relative protein expression levels of AChR-ε in other experimental groups were: NRG group(1.34±0.03),MG serum group(0.70±0.03),NRG group +MG serum group(0.93±0.03),PD98059+NRG+MG serum group(0.67±0.03),PD158780+NRG+MG serum group(0.69±0.01).There was a significant difference in AChR-ε protein expression levels between the six experiments(F=399.50,P<0.0001).Further multiple comparison results showed that the AChR-ε protein level in the NRG+MG Serum group was significantly higher than that in the MG Serum group(p<0.001).Compared to the CTL group,the relative transcript levels of AChR-ε in other experimental groups were: NRG group(4.90±0.68),MG serum group(4.18±0.68),NRG+MG serum group(6.22±1.01),PD98059+NRG+MG serum group(1.09±0.32)and PD158780+NRG+MG serum group(1.25±0.25).There was a significant difference in AChR-ε transcript levels between the six experiments(F=44.63,P<0.0001).Further multiple comparison results showed that the AChR-ε transcript level in the NRG+MG Serum group was significantly higher than that in the MG Serum group(p<0.05).The results of AChR immunofluorescent staining indicated that the fluorescence intensity and number of AChR clusters in NRG+MG Serum group were more obvious than those in MG Serum group.Conclusion 1.When myotube cells responding to the AChR decline caused by AChR-GMG patients’ serum,myotube cells themselves have a compensatory mechanism.These mechanisms include increasing AChR-εtranscription level,NRG-1 transcription and translation levels in myotubes and phosphorylation level of the ErbB/Erk1/2 signaling pathway.2.Exogenous rhNRG-1β1 can promote the transcription and protein expression of AChR-ε and AChR clustering.3.Exogenous NRG-1β1 increases AChR-ε protein and transcription level by activating the ErbB/Erk1/2 signaling pathway and may provide new therapeutic strategies for MG.Part III The therapeutic effect and mechanism of rhNRG-1β1 in EAMG ratsObjective To understand the effect of exogenous rhNRG-1β1 on EAMG rats.By inhibiting the NRG-1/ErbB/Erk signaling pathway with inhibitors,to explore whether exogenous NRG-1β1 exhibit its effects on EAMG through NRG-1/ErbB/Erk pathway in order to provide new therapeutic targets for MG.Methods After EAMG rat models were established,NRG-1β1 and(or)ErbB receptor inhibitor PD158780 and Me K/Erk inhibitor PD98059 were injected intraperitoneally.To evaluate the therapeutic effect of exogenous NRG-1 and clarify its mechanism of action.The experimental rats were randomly divided into5 groups: IA group,EAMG group,EAMG + NRG-1 group [NRG-1β1(+)],EAMG + NRG-1β1 + PD98059 group [ NRG-1β1(+)+ PD98059(+)],EAMG +NRG-1β1 + PD158780 group [ NRG-1β1(+)+ PD158780(+)].The rats in each experimental group received different intervention on the 28 th day after the first immunization.Rats’ body weight,muscle strength,and Lennon clinical scores were evaluated every two days during the experimental period.At the end of the experiment(day 62),the serum level of anti-AChRα R97-116 Ig G of rats in each experimental group was detected by the ELISA method.WB,RT-PCR,and immunofluorescence methods were used to detect the changes of the AChR-εsubunit on skeletal muscle;WB was used to detect the phosphorylation levels of the ErbB-2 and ErbB-4 receptors as well as the phosphorylation levels of the downstream signaling molecules Erk1/2.Results 1.Exogenous NRG-1β1 decreased the Lennon score and increased muscle strength and body weight in EAMG rats.At the same time,ErbB inhibitor PD158780 and Mek/Erk inhibitor PD98059 could aggravate these effects of NRG-1β1(p<0.05).2.Administration of exogenous NRG-1β1 could reduce the serum level of anti-AChRα R97-116 Ig G of EAMG rats(p<0.0001).The use of the ErbB inhibitor PD158780 but not Mek/Erk inhibitor PD98059 could reverse the antibody reduction effect of rhNRG-1β1(p<0.0001).3.Compared to the EAMG group,the AChR-ε transcription and protein expressions in the gastrocnemius muscle(P<0.01)and the expression of AChR-ε on the muscle cell membrane increased in the EAMG + NRG-1β1 group.Both ErbB inhibitor PD158780 and Mek/Erk inhibitor PD98059 attenuated the effect of rhNRG-1β1on the transcription and expression of AChR-ε(P<0.01),and even further aggravated the reduction of AChR-ε transcription and expression levels in EAMG rats.Conclusion 1.Exogenous NRG-1β1 reduces the serum level of antiAChRα R97-116 antibody of EAMG rats,indicating that NRG-1β1 has a role in regulating immunity.2.Administration of exogenous NRG-1β1 through the ErbB/Erk signaling pathway promotes the transcription and expression of AChR-ε,relieves the clinical symptoms of EAMG rats,and increases body weight and muscle strength of EAMG rats.These results provide new therapeutic targets and strategies for MG.