| Objective To explore the effects of TLR4/My D88/NF-?B signal pathway of spinal microglia on the sepsis-induced neuromuscular dysfunction and heterogenization of nicotinic acetylcholine receptor(nAChR);to explore whether the ulinastatin(ULI)could inhibit the TLR4/My D88/NF-?B signal pathway to attenuate the sepsis-induced neuromuscular dysfunction and heterogenization of nAChR;to compare the magnitudes of inward currents between the two nAChRs(?7-nAChR and ?-nAChR)under the sepsis status.MethodsSection ?Male Sprague Dawley(SD)rats,aged 2~3 months,weight 220~240 g,were purchased and randomly divided into Sham group(Sham,n = 6)and Sepsis group(Sepsis,N = 60).According to the different times after model established,the sepsis group then was divided into 6 subgroups(Sepsis-2h,Sepsis-4h,Sepsis-6h,Sepsis-12 h,Sepsis-24 h,and Sepsis-48 h,n=10).The sepsis model was established by cecal ligation and puncture(CLP),and the sham model was made by sham operations(laparotomy and suture).At the different time point after operations,the behavioral and organizational change,the level of IL-6 and TNF-? were used to evaluate the degree of sepsis;the CMAP,MCV and latency time were used to evaluate the muscular function;the spinal water content was used to evaluate the edema of spinal cord;the distribution and expression of TLR4 and Iba-1were detected by immunofluorescence staining;the protein expression level of the TLR4 and its downstream effectors(My D88 and NF-?B),the neuregulin-1(NRG-1),and the ?-and ?7-nAChR was measured using western blotting.Section ?Male Sprague Dawley(SD)rats,aged 2~3 months,weight 220~240 g,were purchased and randomly divided into Sham group(Sham,n = 6)and Sepsis group(Sepsis,N = 50).The sepsis group then was divided into 5 subgroups(0.02 mg/kg TAK-242,1 000 U/kg ULI,3 000 U/kg ULI,5 000 U/kg ULI,and 0.9 % N.S,n=10).At 60 min before model establishment,5 reagents(5 ?l)were intrathecally injected into the segments between lumbar 3 and 4.At 24 h after model established,each group was tested by the same methods in the section I.Section ?Male Kunming mice(KM),aged 2~3 months,weight 50~60 g,were purchased and randomly divided into Control group(Control),Sham group(Sham)and Sepsis group(Sepsis).The musculi flexor pollicis brevis and tibialis anterior muscle were harvested at 24 h after model established.The musculi flexor pollicis brevis were incubated for 3 h with Dulbecco's modified Eagle's medium(DMEM)containing 10 % fetal calf serum,100 ?g/ml penicillin-streptomycin,and 0.2 % collagenase I A in a shaking bath at 37 ?.A conventional patch clamp technique with a whole cell configuration was used to record the inner currents of nAChR with the stimulus of ACh and different blockers.The protein expression of the ?-and ?7-nAChR in tibialis anterior muscle was measured using western blotting.ResultsSection ?(1)Compared to the Sham group,the rats in the sepsis groups that survived exhibited pronounced symptoms of acute sepsis,including motionlessness,drowsiness,trembling,a hunched appearance,and exudation around the eyes and urethra.The peritoneal cavity contained a large amount of bloody and suppurate secreta after postmortem laparotomy,and feces covered the distended and gangrenous cecum.Throughout the observation period,the obvious decrease in spontaneous activity and the appearance of sepsis distinguished the septic rats from the sham rats.(2)Compared to the Sham group,the motality rate and cytokines of Sepsis groups was significantly higher after model established(P < 0.05);(3)Compared to the Sham group,the neuromuscular function of Sepsis groups was significantly decreased after model established(P < 0.05);(4)Compared to the Sham group,the cytokines and water content of spinal cord of Sepsis groups were significantly increased after model established(P < 0.05);(5)Compared to the Sham group,the distribution of TLR4/Iba-1in spinal cord of Sepsis groups was significantly increased after model established(P < 0.05);(6)Compared to the Sham group,the protein expression of TLR4,My D88,p-I?B-? and NF-?B in spinal cord and the protein expression of ?-nAChR and ?7-nAChR in skeletal muscle of Sepsis groups were significantly increased(P < 0.05),however,the expression of I?B-? in spinal cord and the neuregulin-1 in skeletal muscle of Sepsis groups were significantly decreased(P < 0.05).Section ?(1)Compared to the Sham group and the 0.9 % N.S group,the rats in the 5 000 U/kg ULI group showed the slight symptoms of sepsis,which were included of the behavioral and organizational change,the mortality rate,the cytokines(P < 0.05);(2)Compared to the Sham group and the 0.9 % N.S group,the neuromuscular function of 5 000 U/kg ULI group was significantly ibcreased after sepsis model established(P < 0.05);(3)Compared to the Sham group and the 0.9 % N.S group,the cytokines and water content of spinal cord of 5 000 U/kg ULI group were significantly decreased after sepsis model established(P < 0.05);(5)Compared to the Sham group and the 0.9 % N.S group,the distribution of TLR4/Iba-1in spinal cord of 5 000 U/kg ULI group was significantly decreased after sepsis model established(P < 0.05);(6)Compared to the Sham group and the 0.9 % N.S group,the protein expression of TLR4,My D88,p-I?B-? and NF-?B in spinal cord and the protein expression of ?-nAChR and ?7-nAChR in skeletal muscle of 5 000 U/kg ULI group were significantly decreased(P < 0.05),however,the expression of I?B-? in spinal cord and the neuregulin-1 in skeletal muscle were significantly increased(P < 0.05).Section ?(1)Compared to the Control group and the Sham group,the protein expressions of ?-nAChR and ?7-nAChR in Sepsis group were significantly increased(P < 0.05);(2)The EC50 of ACh to nAChR was 84.00 ± 0.016 ?mol/L;(3)Compared to the Sham group,the inner currents of Sepsis group was significantly decreased(1133.48 ± 475.12 vs 563.68 ± 138.26,P < 0.05);(4)The EC50 of waglerin-1 to ?-nAChR was 0.60 ± 0.02 ?mol/L;the EC50 of MLA to ?7-nAChR was 0.30 ± 0.34 ?mol/L;the EC50 of ?-BTX to nAChR was 0.65 ± 0.01 ?mol/L;(5)In the Sepsis group,compared to the inner currents of single-used ACh,the inner currents of ACh + waglerin-1 were significantly decreased(548.96 ± 163.65 vs.135.25 ± 39.85,P < 0.05);compared to the inner currents of ACh + waglerin-1,the currents were futher decreased when injected the ACh + waglerin-1 + MLA(135.25 ± 39.85 vs.86.21 ± 28.78,P < 0.05);however,compared to the inner currents of ACh + waglerin-1 + MLA,the ?-BTX could block all the currents(86.21 ± 28.78 vs.12.32 ± 3.25,P < 0.05).Conclusions(1)The activation of the TLR4/My D88/NF-?B signal pathway of spinal microglia in sepsis is respond to the spinal inflammation,neuromuscular dysfunction and heterogenization of nAChR;(2)The intrathecal injection of 5000 U/kg ulinastatin inhibited the TLR4/My D88/NF-?B signal pathway of spinal microglia in sepsis to alleviate the sepsis-induced spinal inflammation,neuromuscular dysfunction and heterogenization of nAChR;(3)In sepsis,the inner currents of nAChR were mainly generated by ?-nAChR,a small part of currents were generated by ?7-nAChR and ?-nAChR,of which ?-nAChR was mainly generated. |
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