The Effect Of BIGU Theory Based Every-other-day Fasting On NF-?B Signal Pathway In Rats After Spinal Cord Injury

Objective: 1.Under the guidance of "meridian theory" and "treatment targeting spleen",taking every-other-day fasting(EODF)and spinal cord injury(SCI)as its carrier,to study BIGU concept in the application of syndrome of Du damaged,in order to provide experimental evidence for expanding its scope of application.2.To establish a rat model of SCI with medical aneurysm clip,take EODF as intervention,to observe the effect of long tern EODF on pathological changes and functional recovery of rat model with clip-compression injury of spinal cord,and detect the expression and status of inflammatory cytokine expression and NF-?B signal pathway in acute stage of SCI,in order to provide an experimental basis for future study of its mechanisms in SCI recovery.Methods: 1.Animal origin and model preparation: Sprague-Dawley(SD)adult rats(experimental animal quality certificate number: SCXK CHUAN 2015-030)were purchased from Chengdu Dasuo Biotechnology Co.,Ltd.,female,clean grade,weighing 280-310 g.The acute spinal cord injury rat model in T10 was established by using medical cerebrovascular temporary aneurysm clip(70g).2.Experiment grouping design: The rats were randomized into the following four groups: Group A,spinal cord injury with EODF as inteervention;Group B,spinal cord injury group;Group C: sham operation with EODF;Group D: sham operation group.The sham operation group received only laminectomy.Rats in EODF group start fasting for 24 hours(only drinking water,the same below)immediately after operation,and receive conventional food for 24 hours,then fasting again for 24 hours,and so forth,until the end of the study.Rats in Non-EODF group had free access to food(ad libitum,AL)and water.3.Thiry-six SD rats were randomly assigned to group A,B,C and D,with nine rats in each group.This part of experiment was designed to observe the effects of EODF on the pathological changes and motor function recovery of SCI rats model with clamped spinal cord injury: observation period: 12 weeks.Food intake and body weight were observed.Motor functional recovery in rats was assessed by Basso-Beattie-Bresnahan score before and at 1 day before and1 day,1,2,4,6,8,10,12 weeks after operation.At 12 weeks after operation,hematoxylin-eosin staining and toluidine blue staining was performed for pathological observation.4.One hundred and eighty SD rats were randomly assigned to group A,B,C and D,with forty-five rats in each group.At 6 hours,12 hours,24 hours,3 days,7 days after operation,serum and spinal cord tissue samples were collected.Real-time PCR was used in order to detect the expression level of NF-?B p65 and I-?B? m RNA in spinal cord injury area;Western blot was used in order to detect the expression level of I-?B? protein in spinal cord injury area of rats;Electrophoretic mobility shift assay(EMSA)was used to detect the transcriptional activity of NF-?B p65 in spinal cord injury area of rats;Serum levels of TNF-? and IL-10 were measured through using enzyme-linked immunosorbent assay(ELISA).All of these tests were to observe the effects of EODF on NF-?B signal pathway and inflammatory cytokines in each group.Results: 1.The effect of EODF on motor function recovery and spinal cord tissue structure of rats with SCI ?There were no significant differences in daily food intake and body weight of rats among each group before operation.In 4 weeks after operation,rats in EODF groups comsumed more in feeding day than rats in AL groups(P<0.01).At 2 and 3 weeks after operation,food comsumption in group A in feeding day increased about 90% compared with group B(P<0.01,P=0.028),at 2-4 weeks after operation,food comsumption in group C in feeding day increased about 75% compared with group D(P<0.01).However,despite the fact that the EODF group had some compensation for the zero intake during fasting on the day of eating,the overall food intake was reduced(about 10%)compared with the AL group.As for body weight,body weight of rats in group A,B and C declined for about 1-3 weeks and gradually increase later,then body weight of rats in group B and C returned to preoperative level at 8 week and 3 week after operation,respectively.Body weight of rats in group A still not recovered to preoperative level at 12 week after operation.?The BBB score of rats in group A and B reached lowest after operation,and gradually increased with time.1 day after operation,rats in group A and B after rats showed typical signs of paraplegia,there was no visible hind limb movement,BBB scores were significantly lower than the sham operation group: group C and D,the difference was statistically significant(F = 5272.076,P = 0.000).At 8,10,12 weeks after operation,rats in group A had improved hind limb activity,with frequent motion with claw palm bearing the weight,hind limbs coordinated action,the BBB scores of rats in group A were higher than that in group B,with significant differences between the groups(P<0.05).?HE staining: In group A,vacuoles cloud be seen in the white matter of spinal cord,spinal nerve fiber axon atrophy and myelin swelling could be seen in spinal cord.In group B,much of vacuoles cloud be seen in the white matter of spinal cord,significant spinal nerve fiber axon atrophy and myelin swelling could be seen,fibroblast proliferation could be seen,and there exists visible inflammatory cell infiltration.In group C and D,there is normal histological structure and no obvious pathological changes.The results of HE staining showed that the rats in group A had milder degree of pathological changes compared with group B,EODF exerts certain protective effect on spinal cord injury rats.?Toluidine blue staining: in group A,serious visible butterfly gray area damage,and structure in this area is not clear,focal necrotic shedding cloud be seen,a significant reduction in nerve cells,connective tissue hyperplasia with inflammatory cell infiltration,the structure of residual neuronal cell is complete,nissl body cloud clearly be seen in cytoplasm with equitable coloring and amber shape.In group B,serious visible butterfly gray area damage,and structure in this area is not clear,arge area necrotic shedding cloud be seen,a significant reduction in nerve cells,connective tissue hyperplasia with inflammatory cell infiltration,the strcture of parts of residual neuronal cell and nissl body is not clear,the structure of parts of nerve fiber is not clear in white area.In group C and D,complete butterfly gray area,clear structure,abundant nerve cells,nissl body cloud clearly be seen in cytoplasm with equitable coloring and amber shape,clear structure and well arrangement of nerve fiber in white area.The results of toluidine blue staining showed that EODF exerts certain protective effect on spinal cord injury rats.2.The effect of EODF on NF-?B signal pathway and inflammatory cytokines of rats in acute stage of SCI ?Results comparison of the model group and sham operation group: in the AL groups,group B(with SCI)is the model group,group D(sham operation group)is the sham operation group.Compared with the sham group,the expression of NF-?B p65 m RNA had the tendency of increasing in model group after SCI;at 6h,12 h and 3d after operation,the expression of I-?B? m RNA was increased significantly(P<0.05);at 6h,12 h after operation,the expression of I-?B? protein was decreased significantly(P<0.05),and increased at 3d(P<0.05);at 6h,12 h after operation,NF-?B transcription activity was significantly enhanced(P<0.05);at 12 h after operation,the level of serum TNF-? increased significantly(P<0.05),and showed a trend of first increasing and then decreasing,and return to sham group level at 7d(P<0.05);the level of serum IL-10 were significantly increased in model group at each time point after operation(P<0.05).The above results showed that compared with sham group,the NF-?B signal pathway was activated in the model group in acute phase after SCI,and the secretion of proinflammatory and anti-inflammatory cytokines were started,these results were consistent with the results of other SCI animal studies.? Results comparison of the experimental group and model group: in SCI operation groups,group A(SCI+EODF)is the experimental group,group B(SCI)is the model group.Compared with the model group,the expression of NF-?B p65 m RNA was increased significantly at 12 h,1d after operation in experimental group(P<0.01,P<0.05);there was no significant difference in the expression level of I-?B? m RNA between the two groups;6h after operation,the expression of I-?B? protein was decreased significantly(P<0.05);NF-?B transcription activity had the tendency of increasing in experimental group after SCI,at 3d after operation,the activity was significantly enhanced(P<0.01);at 1d after operation,the level of serum TNF-? decreased significantly(P<0.05);there was no significant difference in the level of serum IL-10 between the two groups;The above results showed that the NF-?B signal pathway was activated in the experimental group in acute phase after SCI,and compared with model group,NF-?B transcription activity in the experimental group had the tendency of increasing,and has certain inhibitory effect on acute inflammatory reaction.?Results comparison of sham operation groups with different dietary intervention: in sham operation groups,group C received EODF,group B had free access to food and water.There was no significant difference in the expression level of NF-?B p65 m RNA and I-?B? m RNA between the two groups(P>0.05).Compared with the group D,the expression of I-?B? protein was decreased significantly at 3d after operation in group C(P<0.05),the NF-?B transcription activity was significantly decreased at 1d after operation in group C(P<0.05),the the level of serum TNF-? was significantly decreased at 1d and 7d after operation in group C(P<0.05).There was no significant difference in the level of serum IL-10 between the two groups(P>0.05).The above results showed that EODF could inhibit inflammatory reaction,and had the tendency of reducing NF-?B transcription activity,which means EODF may exert different effect on NF-?B signal pathway in intact and injured spinal cord.Conclusions: 1.BIGU concept shows the beneficial effect in diseases and syndromes in syndrome of Du damaged.2.EODF has certain neuroprotective effec on the rats with spinal cord clipping injury,and further verify the effectiveness of this treatment in experiment models of SCI.3.Long-term EODF can promote the hind limb motor recovery in rats with spinal cord clip-compression injury.Compared with the AL group,there was significant difference starting at 8 week after operation.4.Long-term EODF can alleviate the degree of pathological damage after spinal cordinjury,indicating EODF exerts certain neuroprotective effect on SCI rats with clipcompression injury.5.In acute stage of SCI,short-term EODF might has certain inhibitory effect on acute inflammatory reaction,but might further degree and time course of NF-?B signal pathway activation,which may be correlated with its molecular mechanism in neuroprotective effect.