| Purpose:This study will explore the regulation and mechanism of autophagy on nerve cells after spinal cord injury,reveal the intervention effect of low-frequency electrical stimulation combined with acupuncture at jiaji point on autophagy of nerve cells after spinal cord injury,and provide a theoretical basis for the repair of urinary retention after spinal cord injury.Materials and methods: 1.50 female SPF grade SD rats were randomly divided into 5 groups with 10 rats in each group.The blank group(K): does not do any processing;Sham operation group(J): exposed only the spinal dural membrane and did not give blows;Model group(M): Allen's strike was administered without any treatment;Low frequency group(D): Allen's strike was performed to treat the model of urinary retention caused by spinal cord injury 24 hours after the strike.The treatment sites were divided into two groups,with a total of 4 electrodes: the first group had 2 electrodes.The negative electrode was placed on the bladder area above the pubis of the rats,and the positive electrode was placed on the third sacral vertebra of the rats.In the second group,two electrodes were placed at the clipping points in the upper and lower segments of the spinal cord injury,with the positive stage at the top and the negative electrode at the bottom.The output waveform was triangular wave,and the low-frequency electrical stimulation(50 Hz)was conducted.The maximum current intensity was no more than 50 m A,with moderate muscle contraction as the degree.Low-frequency + acupuncture group(DZ): undertake Allen 's first strike,strike caused by spinal cord injury model of urinary retention after 24 h treatment,first in the low frequency electrical stimulation(50 Hz)treatment,10 min after the filiform needle(0.35 x 25 mm)needle stab into the T6 clip ridge hole 2 mm,T9,T11,20 min retaining needle,needle don't give fill diarrhea during technique,not electricity,1 / d,7 d for intervention.Urodynamics recorded the maximum bladder volume before and after treatment and calculated the difference.Autophagy in spinal cord was observed by transmission electron microscopy.The expression of LC3 B and P62 proteins in the spinal cord was detected by immunofluorescence method.The expression of NGF and Trk A proteins in the spinal cord was detected by immunohistochemistry.Western Blot was used to detect the expression of PI3 K,p-akt,AKT,p-mtor,m TOR,LC3 B and P62 proteins in spinal cord tissues,and to observe the interaction and mechanism of autophagy in urinary retention after spinal cord injury.The positive cell expression of LC3 B and P62 in modified Allen's model of urinary retention after spinal cord injury was observed.The expression changes of p-AKT and p-m TOR in the PI3K/AKT signaling pathway were detected by Western Blot to determine the autophagy related signaling pathway.2.With improved Allen 's caused urinary retention after spinal cord injury rat model,transmission electron microscopy(sem)method is used to directly observe autophagy,immunofluorescence and Western Blot method to detect LC3 B,P62 protein expression of indirect measurement autophagy flow,low frequency electric stimulation combined acupuncture clip ridge point on urinary retention in rats after spinal cord injury PI3K/AKT signaling pathway,the influence of discuss the regulation of cell autophagy and the mechanism of action of repair of spinal cord injury.Results: 1.Maximum bladder volume The maximum bladder volume of the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group were 1.014±0.107,0.927±0.036,4.110±0.165,4.114±0.150 and 4.284±0.201,respectively.Compared with the blank group,the maximum bladder volume of the sham group was not abnormal(n=6,P>0.05).Compared with the sham group,the maximum bladder volume in the model group increased significantly(n=6,P<0.01).Compared with the model group,there was no difference in the maximum bladder volume between the lowfrequency plus acupuncture group and the low-frequency group(n=6,P>0.05).Compared with the low-frequency + acupuncture group,there was no significant difference in the maximum bladder volume of the low-frequency group(n=6,P>0.05).After treatment,themaximum bladder volume of blank group,sham operation group,model group,lowfrequency + acupuncture group and low-frequency group were 1.037±0.116,0.937±0.026,4.023±0.173,2.503±0.160 and 3.497±0.277,respectively.Compared with the blank group,the maximum bladder volume of the sham group was not abnormal(n=6,P>0.05).Compared with the sham group,the maximum bladder volume in the model group increased significantly(n=6,P<0.01).Compared with the model group,the maximum bladder volume of both the low-frequency plus acupuncture group and the low-frequency group decreased significantly(n=6,P<0.01).Compared with the low-frequency + acupuncture group,the maximum bladder volume in the low-frequency group increased significantly(n=6,P<0.01).Before and after treatment,the difference values of bladder volume in the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group(d)were-0.023±0.036,-0.010±0.013,0.087±0.052,1.611±0.212 and 0.786±0.135,respectively.Compared with the blank group,there was no abnormal bladder volume difference(d)in the sham group(n=6,P>0.05).Compared with the sham group,the difference value of bladder volume(d)in the model group increased significantly(n=6,P<0.01).Compared with the model group,the difference value of bladder volume(d)between the low-frequency plus acupuncture group and the low-frequency group increased significantly(n=6,P<0.01).Compared with the low-frequency + acupuncture group,the bladder volume difference(d)of the low-frequency group was significantly reduced(n=6,P<0.01).2.Filling bladder pressure and bladder compliance In the blank group and the sham group,the nuclear structure was complete,the nuclear membrane was clear,and the mitochondrial morphology was basically normal.Compared with the sham group,the nuclear membrane of the model group was less clear,and the mitochondria were vacuolated and accompanied by autophagy.Compared with the model group,there was no difference in nuclear membrane morphology in the low-frequency + acupuncture group,mitochondria cavitation but no obvious autophagy,and no difference in nuclear membrane morphology in the low-frequency group,but vacuolated mitochondria and autophagy were clearly observed.Morphological changes of spinal cord.3.Observation of NGF expression in spinal cord tissue by immunohistochemical method The main part of NGF expression is in the cytoplasm and nucleus,which is brownish yellow or brown.The grayscale values of NGF in blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group were 621.375±54.255,614.780±43.754,473.651±66.564,597.441±36.070,545.574±39.440,respectively.Compared with the blank group,there was no abnormal NGF expression in the sham group(n=6,P>0.05).Compared with the sham group,the expression of NGF in the model group decreased significantly(n=6,P<0.01).Compared with the model group,the expression of NGF in the low-frequency + acupuncture group was significantly up-regulated(n=6,P<0.01),and the expression of NGF in the low-frequency group was significantly upregulated(n=6,P<0.05).Compared with the low-frequency + acupuncture group,the expression of NGF in the low-frequency group decreased(n=6,P<0.05).4.Observation of Tr KA expression in spinal cord tissue by immunohistochemistry Tr KA is mainly expressed in cytoplasm and nucleus,which is brownish yellow or brown.The Tr KA gray values of the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group were 701.770±26.727,698.174±24.618,461.207±85.214,630.975±66.938 and 576.011±63.233,respectively.Compared with the blank group,no abnormal Tr KA expression was found in the sham group(n=6,P>0.05).Compared with the sham group,Tr KA expression in the model group decreased significantly(n=6,P<0.01).Compared with the model group,Tr KA expression in the lowfrequency + acupuncture group was significantly up-regulated(n=6,P<0.01),and Tr KA expression in the low-frequency group was up-regulated(n=6,P<0.05).Compared with the low-frequency + acupuncture group,there was no difference in Tr KA expression in the lowfrequency group(n=6,P>0.05).5.The expression of PI3 K protein in spinal cord tissues was detected by Western Blot The contents of PI3 K protein in blank group,sham operation group,model group,lowfrequency + acupuncture group and low frequency group were 1.073±0.024,1.068±0.034,0.487±0.025,0.994±0.013 and 0.701±0.015,respectively.Compared with the blank group,there was no abnormal expression of PI3 K protein in the sham group(n=6,P>0.05).Compared with the sham group,the expression of PI3 K protein in the model group decreased significantly(n=6,P<0.01).Compared with the model group,the expression of PI3 K protein in the low-frequency + acupuncture group and the low-frequency group was significantly up-regulated(n=6,P<0.01).Compared with the low-frequency + acupuncture group,the expression of PI3 K protein in the low-frequency group decreased significantly(n=6,P<0.01).6.P-AKT content in spinal cord tissues was determined by Western Blot The p-akt/AKT ratios in the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group were 0.753±0.036,0.782±0.034,0.643±0.028,0.788±0.019,and 0.700±0.015,respectively.Compared with the blank group,there was no difference in the phosphorylation level in the sham group(n=6,P>0.05).Compared with the sham group,the phosphorylation level of the model group decreased significantly(n=6,P<0.01).Compared with the model group,the phosphorylation levels of the lowfrequency + acupuncture group and the low-frequency group were significantly increased(n=6,P<0.01).Compared with the low-frequency + acupuncture group,the phosphorylation level of the low-frequency group decreased significantly(n=6,P<0.01).7.P-m TOR content in the spinal cord was determined by Western Blot The p-mtor /m TOR ratios were 1.099±0.066,1.121±0.081,0.776±0.035,1.044±0.040 and 1.029±0.031 in the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group,respectively.Compared with the blank group,there was no difference in the phosphorylation level in the sham group(n=6,P>0.05).Compared with the sham group,the phosphorylation level of the model group decreased significantly(n=6,P<0.01).Compared with the model group,the phosphorylation levels of the lowfrequency + acupuncture group and the low-frequency group were significantly increased(n=6,P<0.01).Compared with the low-frequency + acupuncture group,there was no difference in the phosphorylation level in the low-frequency group(n=6,P>0.05).8.Western Blot was used to detect the expression of LC3 B protein in spinal cord tissue The contents of LC3 B protein in the blank group,sham operation group,model group,lowfrequency + acupuncture group and low-frequency group were 0.500±0.009,0.493±0.013,1.509±0.020,0.611±0.017 and 1.099±0.009,respectively.Compared with the blank group,LC3 B protein expression was not abnormal in the sham group(n=6,P>0.05).Compared with the sham group,LC3 B protein expression in the model group was significantly upregulated(n=6,P<0.01).Compared with the model group,LC3 B protein expression was significantly decreased in the low-frequency plus acupuncture group and the low-frequency group(n=6,P<0.01).Compared with the low-frequency + acupuncture group,LC3 B protein expression in the low-frequency group was significantly up-regulated(n=6,P<0.01).9.The expression of P62 protein in spinal cord tissues was detected by Western Blot The P62 protein content in the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group were 1.473±0.010,1.472±0.013,1.033±0.008,1.347±0.008,1.093±0.023,respectively.Compared with the blank group,there was no abnormal P62 protein expression in the sham operation group(n=6,P>0.05).Compared with the sham group,the expression of P62 protein in the model group decreased significantly(n=6,P<0.01).Compared with the model group,P62 protein expression in the low-frequency + acupuncture group and the low-frequency group was significantly upregulated(n=6,P<0.01).Compared with the low-frequency + acupuncture group,the expression of P62 protein in the low-frequency group decreased significantly(n=6,P<0.01).10.LC3 B expression in spinal cord was detected by immunofluorescence assay The average optical density of LC3 B in the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group was 0.608±0.069,0.622±0.284,0.969±0.039,0.673±0.043 and 0.776±0.053,respectively.Compared with the blank group,LC3 B expression was not abnormal in the sham group(n=6,P>0.05).Compared with the sham group,LC3 B expression in the model group was up-regulated (n=6,P<0.05).Compared with the model group,LC3 B expression in both the low-frequency plus acupuncture group and the low-frequency group decreased significantly(n=6,P<0.01).Compared with the low-frequency + acupuncture group,LC3 B expression in the lowfrequency group was significantly up-regulated(n=6,P<0.01).11.The expression of P62 in the spinal cord was detected by immunofluorescence method The average optical density of P62 in the blank group,sham operation group,model group,low-frequency + acupuncture group and low-frequency group were 0.627±0.045,0.608±0.039,0.362±0.068,0.597±0.061 and 0.506±0.061,respectively.Compared with the blank group,there was no abnormal P62 expression in the sham group(n=6,P>0.05).Compared with the sham group,P62 expression in the model group decreased significantly(n=6,P<0.01).Compared with the model group,P62 expression in the low-frequency + acupuncture group and the low-frequency group was significantly up-regulated(n=6,P<0.01).Compared with the low-frequency + acupuncture group,the expression of P62 in the low-frequency group decreased(n=6,P<0.05).Conclusion: 1.Low frequency electrical stimulation can be used to treat urinary retention in rat spinal cord injury by inhibiting autophagy,and low frequency electrical stimulation combined with acupuncture at Jiaji point is better.2.Urinary retention in spinal cord injury is associated with increased autophagy levels in neuronal cells.3.The proliferation of spinal cord neuronal cell NGF and its receptors in rats with urinary retention Tr KA spinal cord injury by treatment,and activation of downstream PI3K/AKT/m TOR pathways mediate autophagy,promote phosphorylation levels of this signaling pathway and inhibit autophagy. |
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