| Objective: To investigate the mechanism of sepsis-induced neuromuscular dysfunction and heterogenization of nicotinic acetylcholine receptors(n ACh Rs)in skeletal muscle;to verify neural agrin can reduce the heterogenization of n ACh R to alleviate neuromuscular dysfunction through agrin/Mu SK singaling pathway.Methods:Male healthy Sprague-Dawley(SD)rats weighing 250-280 g,2-3months old,were obtained.Rats were randomly divided into 3 groups(n=12): control group,sham group and sepsis group(CLP group).The rats of CLP group suffered cecal ligation and puncture(CLP)operation to establish the model of sepsis,while the rats of sham group were only exposed their abdominal cavity without CLP.The measurements were performed and the tissues of tibialis anterior muscles and blood were harvested at 24 h after the operation.The cytokine levels of IL-6 and TNF-α were detected by Enzyme-linked immunosorbent assay(ELISA).The neuromuscular function were measured by compound muscle action potentials(CMAP)by calculating the amplitude,duration,latency of CMAP and motor nerve conduction velocity.The expression and distribution of γ-n ACh R and α7-n ACh R were detected by immunofluorescence staining.Quantitative real-time PCR(q RT-PCR)was used to detect the gene transcription level of γ-subunit and α7-subunit.The protein expression levels of agrin,Mu SK,rapsyn,γ-n ACh R,α7-n ACh R and ε-n ACh R were measured by western blot.Twenty-four male healthy SD rats weighed 250-280 g were purchased.All of the animals went CLP surgery and were randomly divided into 2 groups: group CLP + S and group CLP + A.After the operation,rats in group CLP + A were intramuscularly injected with 10 ug agrin(100 ug/ml)at bilateral tibialis anterior muscles,while rats in the other group were injected with the same amount of saline at the same place.The measurements were performed and the tissues were harvested as the same methods in section 1 at 24 h after the operation.Results:Section 1(1)Compared to the control group and sham group,the cytokine levels of IL-6 and TNF-α in CLP group increased significantly(P<0.05).(2)Compared to the control group and sham group,the neuromuscular function was impaired in the CLP group,which was shown as the increased duration,added latency,decreased amplitude and nerve conduction velocity(P<0.05).(3)Compared to the control group and sham group,the distribution expression of γ-n ACh R and α7-n ACh R increased in the CLP group.(4)Compared to the control group and sham group,the gene transcription level of Chrng and Chrna7,which were the gene of γ-subunit and α7-subunit,increased significantly in the CLP group(P<0.05).(5)Compared to the control group and sham group,the expression ofγ-n ACh R 、 α7-n ACh R and ε-n ACh R increased,the expression of agrin decreased,the expression of Mu SK and rapsyn increased in the CLP group(P<0.05).Section 2(1)There was no significant difference in the plasma level of cytokines among the two groups(P>0.05).(2)Compared to the CLP + S group,the neuromuscular function was ameliorated in the CLP + A group,which was shown as the decreased duration,reduced latency,increased amplitude and nerve conduction velocity(P<0.05).(3)Compared to the CLP + S group,the distribution expression of γ-n ACh R and α7-n ACh R decreased in the CLP + A group.(4)Compared to the CLP + S group,the gene transcription level of Chrng and Chrna7 decreased significantly in the CLP + A group(P<0.05).(5)Compared to the CLP + S group,the protein expression of γ-n ACh R 、 α7-n ACh R and ε-n ACh R decreased,the expression of agrin increased,the expression of Mu SK and rapsyn decreased in the CLP + A group(P<0.05).Conclusions(1)The abnormal expression of agrin/Mu SK signaling pathway may be associated with the heterogenization of n ACh Rs and neuromuscular dysfunction caused by sepsis.(2)Exogenous neural agrin can inhibited the protein expression and gene transcription of n ACh Rs and ameliorate the neuromuscular function through the related pathway. |
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