| Objective: The animal model of clamped C5 spinal cord injury was established and intervened by every-other-day fasting.By detecting the expression of apoptosis and necroptosis-related proteins in spinal cord tissue,the effect of every-other-day fasting on the cell death of acute spinal cord injury was preliminarily explored,which provided experimental basis for the mechanism of every-other-day fasting on promoting the recovery of spinal cord injury.Methods: The model of spinal cord injury in rats was established by aneurysm clamp with unilateral C5 spinal cord for 30 seconds.Four weeks before injury,240 rats were randomly divided into pre-AL group(180 rats)and pre-EODF group(60 rats).Rats in pre-AL group were given free access to food and water.Rats in pre-EODF group were given EODF intervention.Four weeks later,rats in pre-AL group were randomly divided into Sham group,AL group and post-EODF group(60 rats in each group).Sham group and AL group took food and water freely after injury,post-EODF group began EODF intervention immediately after injury,and pre-EODF group continued EODF intervention after injury.EODF means that fasting and free feeding alternate every 24 hours,but there is no restriction on drinking water.The injured spinal cord tissues of each group were collected at 5 hours,12 hours,24 hours,3 days,and 7 days after injury.The expression of protein Bcl-2,Bax and Caspase-3 in spinal cord was detected by immunohistochemistry,apoptosis was detected by TUNEL,and the expression of protein TNF-?,RIPK1 and RIPK3 in spinal cord was detected by immunoblotting.The activity of MPO in the spinal cord was detected by myeloperoxidase kit.Result: 1.Diet and weight of rats(1)Rat diet: 1-4 weeks before injury,the pre-EODF rats had more food intake on the feeding day than the pre-AL rats in the same day,but overall,the average daily food intake of the pre-EODF rats was lower.In the pre-AL group,the total food intake of the pre-EODF group was 81% of pre-AL rats within 4 weeks.After injury,the total food intake of pre-EODF and post-EODF was lower than that of AL group,and the total food intake was 79% and 63% of AL rats respectively.(2)Rat weight: Four weeks before injury,there was no significant difference in body weight between pre-AL group and pre-EODF group(P>0.05).After one week of EODF intervention,the body weight of pre-EODF group was significantly lower than that of preAL group(P<0.05).After the injury,the body weight of the four groups all decreased,and then gradually increased.2.Effect of EODF on apoptosis of spinal cord tissue(1)Immunohistochemical detection of Bcl-2,Bax and Caspase-3 showed that there was no significant positive expression of Bcl-2,Bax and Caspase-3 in Sham group at all time points after injury,and the other three groups had different degrees of positive expression.On the 3rd day after injury,the expression of Bcl-2 protein in pre-EODF group was significantly higher than that in AL group(P<0.05).The expression of Bax in pre-EODF group was significantly lower than that in AL group at all time points after injury(P<0.05),and was lower than that in post-EODF group at 12 hours after injury(P<0.05),while the expression of Bax in post-EODF group was also lower than that in AL group at 24 hours,3 days and 7 days(P<0.05).The expression of Caspase-3 in pre-EODF group was significantly lower than that in AL group at 6h,12 h,24h and 7d after injury(P<0.05),and significantly lower than that in post-EODF group at 6h and 24 h after injury(P<0.05),while the expression of Caspase-3 in post-EODF group was also lower than that in AL group at 12 h and 7d after injury(P<0.05).At the same time,compared with AL group,pre-EODF and post-EODF increased the ratio of Bcl-2: Bax by 16% and 12%,respectively.(2)TUNEL assay showed that there were few apoptotic positive cells of Sham group at each time after injury.The apoptotic rate of pre-EODF group was significantly lower than that of AL group at 6h,12 h,24h and 3d after injury(P<0.05),and the apoptotic rate of preEODF group was significantly lower than that of post-EODF group at 6h,12 h and 24 h after injury(P<0.05),but there was no significant difference in apoptotic between post-EODF group and AL group at all time points after injury(P>0.05).3.Effect of EODF on necropoptosis of spinal cord tissue(1)Western blotting detection of TNF-?,RIPK1 and RIPK3 showed that the protein expression of TNF-?,RIPK1 and RIPK3 in the Sham group was at a low level at each time point after injury.The expression of TNF-a in pre-EODF group was significantly lower than that in AL group at all time points after injury(P<0.05),and significantly lower than that in post-EODF group at 3 days after injury(P<0.05),while the expression of TNF-a in postEODF group was significantly lower than that in AL group at 24 hours and 3 days after injury(P<0.05).The expression of RIPK1 in pre-EODF group was significantly lower than that in AL group at 6,12 and 24 hours after injury(P<0.05),while post-EODF group was significantly lower than that in AL group only at 24 hours after injury(P<0.05).The expression of RIPK3 in pre-EODF group was significantly lower than that in AL group at 6h,12 h,3d and 7d after injury(P<0.05),and significantly lower than that in post-EODF group at 7d after injury(P<0.05),while the expression of RIPK3 in post-EODF group was also significantly lower than that in AL group at 6h and 7d after injury(P<0.05).(2)MPO results showed that MPO activity in AL group was significantly higher than that in Sham group from 12 hours to 7 days after injury(P<0.05),pre-EODF group was significantly lower than that in AL group from 12 hours to 7 days after injury(P<0.05),from 24 hours to 7 days after injury,MPO activity in post-EODF group was also significantly lower than that in AL group(P<0.05).Conclusion: 1.EODF can reduce the production of pro-apoptotic factors Bax and Caspase-3,promote the expression of anti-apoptotic factor Bcl-2,and preventive EODF intervention 4 weeks before spinal cord injury inhibits apoptosis after spinal cord injury.2.EODF can inhibit the activation of necrotizing apoptotic pathway and decrease the activity of MPO by inhibiting the expression of TNF-?,RIPK1 and RIPK3 proteins,which may be one of the mechanisms of its anti-inflammatory effect.3.Compared with EODF intervention after spinal cord injury,preventive EODF intervention 4 weeks before spinal cord injury showed better neuroprotective effect. |
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