| Objective:To investigate the effect of different concentrations of zinc diet on neurobehavioral,serum zinc concentration and seizure threshold of secondary strike in rats with developmental convulsions induced by lithium chloride-pilocarpine and its intervention mechanism.Methods:145 Sprague-Dawley(SD)rats of 27 days old(Postnatal days 27,P27)were randomly divided into two groups:control group(n=36)and modeling group(n=109).The control group was randomly divided into 3 subgroups:normal zinc diet group(Control group,n=12),normal low zinc diet group(ZD group,n=12),and normal high zinc diet group(ZS group,n=12).The remaining 109 rats were intraperitoneally injected with lithium chloride(127 mg/kg)in P27;24 hours later,rats were intraperitoneally injected with scopolamine hydrobromide(lmg/kg)to reduce peripheral cholinergic response;30min after intraperitoneal injection Pilocarpine 320mg/kg,observed behavioral changes in rats.Rats with successful modeling were randomly assigned to each subgroup of the convulsion group:convulsion zinc group(SE group),convulsion low zinc diet group(SE+ZD group),convulsion high zinc diet group(SE+ZS group).During the modeling period(P27,P28),each group was fed a regular zinc diet;after the convulsions,the Control group and the SE group continued to be fed with the zinc diet(zinc 44mg/Kg)for 28 days;the ZD group and the SE+ZD group were fed with the low-zinc diet(Zinc 2.7 mg/Kg)for 28 days;ZS group and SE+ZS group were fed with the high zinc diet(zinc 246 mg/Kg)for 28 days.Negative trend test,open field test,novel object recognition test,and passive avoidance test were performed on P42 and P56;blood was taken from the tail vein of P43 and P57;the body weight changes in rats were measured periodically.All rats were intraperitoneally injected with penicillin to construct two-hit seizure model and the convulsion threshold(time of first episode seizure)and survival rate were recorded within 1 h,brains were taken from each group of 4 surviving rats after perfusion,and the hippocampal mossy fiber germination was observed by Neo-Timm's staining;Western-blot was used to detect the expression of GPR39,Calbindin,KCC2 and MBP in cerebral cortex and hippocampus.Results:1.The effect on weight:(1)1-3 weeks after convulsions(during P35-P49 period),There was no significant difference between ZD group and Control group,ZS group and Control group,SE+ZD group and SE group,SE+ZS group and SE group;SE group compared with Control group,SE+ZD group compared with ZD group,SE+ZS group compared with ZS group,body weight decreased significantly(P<0.05).(2)After 4 weeks of convulsions(P56-P57),the body weight of the ZD group was significantly lower than that of the Control group(P<0.05);the ZS group was significantly increased compared with the Control group(P<0.05);the body weight of the SE group was significantly lower than that of the Control group,the body weight of the SE+ZD group was significantly lower than that of the SE group(P<0.05);the body weight of the SE+ZS group was significantly lower than that of the ZS group(P<0.05);the weight of the SE+ZS group was higher than that of the SE group,but there was no statistical difference.2.Negative reflection:(1)P42,there was no significant difference in reflex time between ZD group and Control group,ZS group and Control group,SE+ZD group and SE group,SE+ZS group and SE group.Compared with the Control group,the reflex time of the SE group was significantly prolonged(P<0.05).Compared with the ZD group,the reflex time of the SE+ZD was significantly prolonged(P<0.05).Compared with the ZS group,the reflex time of the SE+ZS was significantly prolonged(P<0.05).(2)P56,compared with the Control group,the reflex time of the ZD group was significantly prolonged(P<0.05).Compared with the Control group,the reflex time of the ZS group was significantly shortened(P<0.05).Compared with the Control group,the reflex time of the SE group was significantly prolonged(P<0.05).Compared with the ZD group,the reflex time of the SE+ZD group was significantly prolonged(P<0.05).Compared with the ZS group,the reflex time of the SE+ZS group was significantly prolonged(P<0.05).Compared with the SE group,the reflex time of the SE+ZD group was significantly prolonged(P<0.05).Compared with the SE group,the reflex time of the SE+ZS group was significantly shortened(P<0.05).3.Open field experiment:?Delay time:(1)P42,there was no significant difference in delay time between ZD group and Control group,ZS group and Control group,SE+ZD group and SE group,SE+ZS group and SE group.Compared with the Control group,the delay time of the SE group was significantly prolonged(P<0.05).Compared with the ZD group,the reflex time of the SE+ZD was significantly prolonged(P<0.05).Compared with the ZS group,the reflex time of the SE+ZS was significantly prolonged(P<0.05).(2)P56,compared with the Control group,the delay time of the ZD group was significantly prolonged(P<0.05).Compared with the Control group,the delay time of the ZS group was significantly shortened(P<0.05).Compared with the Control group,the delay time of the SE group was significantly prolonged(P<0.05).Compared with the ZD group,the delay time of the SE+ZD group was significantly prolonged(P<0.05).Compared with the ZS group,the delay time of the SE+ZS group was significantly prolonged(P<0.05).Compared with the SE group,the delay time of the SE+ZD group was significantly prolonged(P<0.05).Compared with the SE group,the delay time of the SE+ZS group was significantly shortened(P<0.05).?Opening score:(1)P42,there was no significant difference in opening score between ZD group and Control group,ZS group and Control group,SE+ZD group and SE group,SE+ZS group and SE group.Compared with the Control group,the opening score of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the opening score of the SE+ZD was significantly decreased(P<0.05).Compared with the ZS group,the opening score of the SE+ZS was significantly decreased(P<0.05).(2)P56,compared with the Control group,the opening score of the ZD group was significantly decreased(P<0.05).Compared with the Control group,the opening score of the ZS group was significantly increased(P<0.05).Compared with the Control group,the opening score of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the opening score of the SE+ZD group was significantly decreased(P<0.05).Compared with the ZS group,the opening score of the SE+ZS group was significantly decreased(P<0.05).Compared with the SE group,the opening score of the SE+ZD group was significantly decreased(P<0.05).Compared with the SE group,the opening score of the SE+ZS group was significantly increased(P<0.05).4.Novel Object Recognition test:(1)P42,there was no significant difference in the recognition index between ZD group and Control group,ZS group and Control group,SE+ZD group and SE group,SE+ZS group and SE group.Compared with the Control group,the recognition index of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the recognition index of the SE+ZD was significantly decreased(P<0.05).Compared with the ZS group,the recognition index of the SE+ZS was significantly decreased(P<0.05).(2)P56,compared with the Control group,the recognition index of the ZD group was significantly decreased(P<0.05).Compared with the Control group,the recognition index of the ZS group was significantly increased(P<0.05).Compared with the Control group,the recognition index of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the recognition index of the SE+ZD group was significantly decreased(P<0.05).Compared with the ZS group,the recognition index of the SE+ZS group was significantly decreased(P<0.05).Compared with the SE group,the recognition index of the SE+ZD group was significantly decreased(P<0.05).Compared with the SE group,the recognition index of the SE+ZS group was significantly increased(P<0.05).5.Passive avoidance test:(1)P42,there was no significant difference in latency time between ZD group and Control group,ZS group and Control group,SE+ZD group and SE group,SE+ZS group and SE group.Compared with the Control group,the latency time of the SE group was significantly shortened(P<0.05).Compared with the ZD group,the latency time of the SE+ZD was significantly shortened(P<0.05).Compared with the ZS group,the latency time of the SE+ZS was significantly shortened(P<0.05).(2)P56,compared with the Control group,the latency time of the ZD group was significantly shortened(P<0.05).Compared with the Control group,the latency time of the ZS group was significantly prolonged(P<0.05).Compared with the Control group,the latency time of the SE group was significantly shortened(P<0.05).Compared with the ZD group,the latency time of the SE+ZD group was significantly shortened(P<0.05).Compared with the ZS group,the latency time of the SE+ZS group was significantly shortened(P<0.05).Compared with the SE group,the latency time of the SE+ZD group was significantly shortened(P<0.05).Compared with the SE group,the latency time of the SE+ZS group was significantly prolonged(P<0.05).6.Seizure threshold and survival rate:?Seizure threshold:Compared with the Control group,the seizure threshold of the ZD group was significantly shortened(P<0.05).Compared with the Control group,the seizure threshold of the ZS group was significantly prolonged(P<0.05).Compared with the Control group,the seizure threshold of the SE group was significantly shortened(P<0.05).Compared with the ZD group,the seizure threshold of the SE+ZD group was significantly shortened(P<0.05).Compared with the ZS group,the seizure threshold of the SE+ZS group was significantly shortened(P<0.05).Compared with the SE group,the seizure threshold of the SE+ZD group was significantly shortened(P<0.05).Compared with the SE group,the seizure threshold of the SE+ZS group was significantly prolonged(P<0.05).?Survival rate:the Control group(83.3%),the ZD group(66.7%),the ZS group(91.7%),the SE group(50.0%),the SE+ZD group(38.1%),the SE+ZS group(60.0%).7.Serum zinc concentration:(1)P43,there was no significant difference in the serum zinc concentration between ZD group and Control group,ZS group and Control group,SE+ZD group and SE group,SE+ZS group and SE group.Compared with the Control group,the serum zinc concentration of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the serum zinc concentration of the SE+ZD was significantly decreased(P<0.05).Compared with the ZS group,the serum zinc concentration of the SE+ZS was significantly decreased(P<0.05).(2)P57,compared with the Control group,the serum zinc concentration of the ZD group was significantly decreased(P<0.05).Compared with the Control group,the serum zinc concentration of the ZS group was significantly increased(P<0.05).Compared with the Control group,the serum zinc concentration of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the serum zinc concentration of the SE+ZD group was significantly decreased(P<0.05).Compared with the ZS group,the serum zinc concentration of the SE+ZS group was significantly decreased(P<0.05).Compared with the SE group,the serum zinc concentration of the SE+ZD group was significantly decreased(P<0.05).Compared with the SE group,the serum zinc concentration of the SE+ZS group was significantly increased(P<0.05).8.Neo-Timm's staining analysis:There were significant differences in the semi-quantitative score of aberrant mossy fiber sprouting in the hippocampal CA3 area and dentate gyrus between the six groups(P<0.05).There was no significant difference in the germination between ZD group and Control group,ZS group and Control group.Compared with the Control group,the germination of the SE group was significantly increased(P<0.05).Compared with the ZD group,the germination of the SE+ZD was significantly increased(P<0.05).Compared with the ZS group,the germination of the SE+ZS was significantly increased(P<0.05).Compared with the SE group,the germination of the SE+ZD group was significantly increased(P<0.05).Compared with the SE group,the germination of the SE+ZS group was significantly decreased(P<0.05).9.Western-blot analysis:? the expression of GPR39 in cerebral cortex and hippocampus:Compared with the Control group,the protein level of GPR39 in cerebral cortex and hippocampal of the ZD group was significantly decreased(P<0.05).Compared with the Control group,the protein level of GPR39 of the ZS group was significantly increased(P<0.05).Compared with the Control group,the protein level of GPR39 of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the protein level of GPR39 of the SE+ZD group was significantly decreased(P<0.05).Compared with the ZS group,the protein level of GPR39 of the SE+ZS group was significantly decreased(P<0.05).Compared with the SE group,the protein level of GPR39 of the SE+ZD group was significantly decreased(P<0.05).Compared with the SE group,the protein level of GPR39 of the SE+ZS group was significantly increased(P<0.05).?the expression of KCC2 in cerebral cortex and hippocampus:Compared with the Control group,the protein level of KCC2 in cerebral cortex and hippocampal of the ZD group was significantly decreased(P<0.05).Compared with the Control group,the protein level of KCC2 of the ZS group was significantly increased(P<0.05).Compared with the Control group,the protein level of KCC2 of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the protein level of KCC2 of the SE+ZD group was significantly decreased(P<0.05).Compared with the ZS group,the protein level of KCC2 of the SE+ZS group was significantly decreased(P<0.05).Compared with the SE group,the protein level of KCC2 of the SE+ZD group was significantly decreased(P<0.05).Compared with the SE group,the protein level of KCC2 of the SE+ZS group was significantly increased(P<0.05).?the expression of CB-D28K in cerebral cortex and hippocampus:Compared with the Control group,the protein level of CB-D28K in cerebral cortex and hippocampal of the ZD group was significantly decreased(P<0.05).Compared with the Control group,the protein level of CB-D28K of the ZS group was significantly increased(P<0.05).Compared with the Control group,the protein level of CB-D28K of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the protein level of CB-D28K of the SE+ZD group was significantly decreased(P<0.05).Compared with the ZS group,the protein level of CB-D28K of the SE+ZS group was significantly decreased(P<0.05).Compared with the SE group,the protein level of CB-D28K of the SE+ZD group was significantly decreased(P<0.05).Compared with the SE group,the protein level of CB-D28K of the SE+ZS group was significantly increased(P<0.05).?the expression of MBP in cerebral cortex and hippocampus:There was no significant difference in the protein level of MBP in cerebral cortex and hippocampal between ZD group and Control group,ZS group and Control group.Compared with the Control group,the protein level of MBP of the SE group was significantly decreased(P<0.05).Compared with the ZD group,the protein level of MBP of the SE+ZD group was significantly decreased(P<0.05).Compared with the ZS group,the protein level of MBP of the SE+ZS group was significantly decreased(P<0.05).Compared with the SE group,the protein level of MBP of the SE+ZD group was significantly decreased(P<0.05).Compared with the SE group,the protein level of MBP of the SE+ZS group was significantly increased(P<0.05).Conclusion:1.Zinc deficiency diet and zinc supplement diet intervention for 2 weeks had no significant effect on body weight,neurobehavior and cognition,and serum zinc concentration.2.Zinc deficiency intervention for 4 weeks can aggravate the weight loss,neurobehavior and cognitive impairment,the decline in serum zinc concentration and seizure threshold,and increase the abnormal germination of mossy fibers,which can further reduce the expression of GPR39,KCC2,CB-D28K,MBP in cerebral cortex and hippocampus.3.Zinc supplement intervention for 4 weeks can significantly improve weight loss,improve neurobehavior and cognitive impairment,increase serum zinc concentration,increase the seizure threshold,and inhibit the abnormal germination of mossy fibers.It may be related to correcting abnormal expression of GPR39,KCC2,CB-D28K,MBP in cerebral cortex and hippocampus. |
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