Improvement Of Mongolian Zhenbao Pills On Hippocampus Oxidative Stress And Synaptic Plasticity In Sleep Deprivationinduced Rats

Objective sleep deprivation can lead to cognitive impairment,including increasing oxidative stress and impairing synaptic plasticity function in hippocampus.Through the treatment of sleep deprivation rats,we analyzed whether the Mongolian Medicine Zhenbao pills could improve the level of oxidative stress by increasing the levels of superoxide dismutase（SOD）and glutathione（GSH）,and whether improve synaptic plasticity by increasing the expression of brain-derived neurotrophic factor（BDNF）and postsynaptic density protein95（PSD95）in hippocampus.Methods Seventy-eight SPF male Sprague-Dawley rats were randomly divided into six groups（n=13 per group）:Control group（0.9%normal saline）,sleep deprivation group（0.9%normal saline）,melatonin group（1.5mg/100g）and Mongolian Medicine Zhenbao Pills（20mg/100g,40mg/100g and 80mg/100g three groups）.From the first day,gavage was given once a day for 15 consecutive days.On the fifth day,the modified multiple platform method was used for sleep deprivation in 7 consecutive days.Morris water maze was used to evaluate the spatial memory and learning ability after sleep deprivation.The general condition was observed during the whole experiment.The levels of SOD and GSH were measured by enzyme activity in hippocampus.The number of neurons in hippocampal CA3region was observed by Nissl staining.The Integral optical density（IOD）of BDNF and PSD95protein in hippocampal CA3 region were compared by immunohistochemical staining.The expression levels of BDNF and PSD95 proteins in the hippocampus were detected by Western blotting.m RNA expression levels of BDNF and PSD95 were detected by RT-q PCR.Results1.General condition:on the first day of sleep deprivation,the rats all were at good mental state,Clean and neat fur,frequent grooming and normal diet.On the 7th day of sleep deprivation,control group were in the same state as usual.In the model group,the hair of the rats was unkempt,biting each other and self-harming.In the high dose group,there was no obvious decreasing in feeding,scattered hair,less frequency of combing hair and also no biting and attacking behavior.In the melatonin and middle-dose groups,the hair was scattered,the frequency of grooming and feeding were decreased,also and there was no biting and aggressive behavior.In the low-dose group,the hair was scattered,the frequency of combing hair and feeding were decreased,also and the biting and aggressive behaviors were occasionally observed.2.The weight:beginning with sleep deprivation,there was no statistical difference each group（P>0.05）.On the third day of sleep deprivation,the control group was significantly more than others five group（P<0.01）.On the 5th day of sleep deprivation,compared with the model group the middle-dose was significantly more（P<0.01）and the high-dose was significantly more（P<0.01）.On the 7th day of sleep deprivation,compared with model group the weight of the melatonin,middle-dose and high-dose groups were significantly different（P<0.01）.3.Morris Water Maze:compared with the control group,the escape latency in the model group was significantly long（P<0.01）.Compared with the model group,melatonin,low,middle and high-dose groups were significantly short（P<0.01）.Compared with the control group,there was no significant difference in melatonin,middle and high-dose group（P>0.05）.Compared with the control group,the escape latency in the low-dose group was significantly long（P<0.01）.The escape latency of low-dose group was longer than melatonin,middle-dose and high-dose group(i_low-j_melatonin=16.74,P=0.002;i_low-j_middle=17.33,P=0.002;i_low-j _high=23.37,P=0.0001).Exploration experiment result:compared with the control group,the time in the target area was short,the first time of crossing the platform was long and numbers of crossing the platform was low in the model group（P<0.01）.Compared with the model group,the time in the target area was long,the first time of crossing the platform was short and numbers of crossing the platform was more in melatonin and middle-dose group（P<0.01）.Compared with the model group,the time in the the target,the first time of crossing the platform and numbers of crossing the platform were no statistical difference in low and high-dose group.（P>0.05）.Compared with the control group,the time in the target was short,the first time of crossing the platform and numbers of crossing the platform was short in the middle group（P<0.05）.Compared with the control group,the time in the target,the first time of crossing the platform and numbers of crossing the platform was no statistically difference（P>0.05）.There was no statistically significant difference in melatonin,low,middle and high groups.（P>0.05）.4.Oxidative stress enzyme:compared with control group,the levels of SOD and GSH were significantly less in model group（P<0.01）.Compared with the model group,the levels of SOD and GSH were more in melatonin,middle and high-dose groups（P<0.05）.Compared with the model group,the levels of SOD and GSH were no different significantly in the low group（P>0.05）.Compared with control group,the levels of SOD and GSH in the melatonin group were significantly high（P<0.05）and the levels of SOD and GSH were less in low-dose group（P<0.05）.there was no difference between middle and high-dose group（P>0.05）.The level of SOD in low-dose group was low in melatonin group,middle-dose group and high-dose group(i_low-j_melatonin=-28.55,P=0.001;i_low-j_middle=-22.84,P=0.0001;i_low-j_high=-27.22,P=0.0001).The level of GSH enzyme in low-dose group was low in melatonin,middle-dose and high-dose group(i_low-j_melatonin=-10.01,P=0.007492;i_low-j_middle=-13.41,P=0.000746;i_low-j_high=-10.31,P=0.007).There was no significant difference in melatonin,middle and high-dose group（P>0.05）.5 Nissl staining:compared with the control group,the number of neurons in CA3 region of the model group was less（P<0.05）.Compared with the model group,melatonin group and high-dose group were significantly more（P<0.01）.Compared with the model group,middle and low had no significantly difference.Compared with the control group,the number of neurons were significantly more in middle-dose group（P<0.01）.Compared with the control group,there was no difference in high-dose group and melatonin group（P>0.05）.The number of neurons in CA3 region of high dose group was higher than melatonin,low-dose and middle-dose group(i_high-j_melatonin=28.13,P=0.001025;i_high-j_low=35.38,P=0.00004;i_high-j_middle=18.38,P=0.022884).There was no significant difference in melatonin,low and middle-dose group（P>0.05）.6.Immunohistochemistry:Compared with control group,BDNF IOD（P<0.05）and PSD95 IOD（P<0.01）were less in model group.Compared with model group,BDNF and PSD95 IOD in the model group was more in melatonin,middle and high-dose groups（P<0.01）.Compared with model group,there had no significantly difference in low dose group（P>0.05）.PSD95 IOD in low-dose group were lower than middle and high dose groups(i_low-j_melatonin=-2.259000,P=0.000038;i_low-j_middle=-2.283333,P=0.000017;i_low-j_high=-2.46,P=0.000012)。BDNF IOD in low dose group were lower than melatonin,middle and high dose groups(i_low-j_melatonin=-2.20,P=0.000741;i_low-j_middle=--2.00,P=0.602716;i_low-j_high=-2.46,P=0.000012).There was no significant difference in Melatonin group,middle and high dose group（P>0.05）.7.Western blotting analysis:The expression of BDNF and PSD95 in the model group was significantly lower than that in the control group（P<0.05）.Compared with the model group,the expression of BDNF and PSD95in the Melatonin group,middle and High Dose Group was significantly high（P<0.05）,the low-dose group was not significantly different（P>0.05）.Compared with the control group,there was no significant difference in melatonin group,low,middle and high-dose groups（P>0.05）.The expression of BDNF relative protein in low dose group was lower than that in melatonin group,middle and high dose group(i_low-j_melatonin=-0.30,P=0.010317;i_low-j_middle=-0.25,P=0.026277;i_low-j_high=-0.23,P=0.034091).There was no significant difference in the expression of BDNF relative protein in Melatonin Group,middle and high dose group（P>0.05）.The expression of PSD95 in low dose group was lower than that in melatonin group,middle and high dose group(i_low-j_melatonin=-0.23,P=0.025317;i_low-j_middle=-0.22,P=0.031277;i_low-j_high=-0.24,P=0.015091).There was no significant difference in melatonin,middle and high dose group（P>0.05）.8.RT-q PCR:Compared with the control,the expression of BDNF and PSD95 m RNA was low in the model（P<0.01）.Compared with the model group,the expression of BDNF and PSD95 m RNA was high in middle and high-dose groups（P<0.01）.Compared with the model group,there was no significantly difference in low-dose group（P>0.05）.Compared with the control group,the expression of BDNF m RNA in melatonin group and high-dose Group was significantly high（P<0.01）,the expression of PSD95 m RNA was significantly high in middle-dose Group（P<0.01）,and there was not significant difference in low dose group（P>0.05）.The relative expression level of BDNF m RNA was lower than melatonin group,middle and high dose group(i_low-j_melatonin=-0.25,P=0.000007;i_low-j_middle=-0.38,P=0.000023;i_low-j_high=-0.38,P=0.000012).There was no significant difference in melatonin group,middle and high dose group（P>0.05）.The relative expression of PSD95m RNA in low-dose group was lower than that in melatonin group,middle-dose group and high-dose group(i_low-j_melatonin=-0.31,P=0.053695;i_low-j_middle=-0.52,P=0.004136;i_low-j_high=-0.47,P=0.007391).There was no significant difference in relative expression of PSD95 m RNA in melatonin,middle and high-dose group（P>0.05）.The relative expression of PSD95 m RNA in low-dose group was lower than melatonin,middle-dose and high-dose groups(i_low-j_melatonin=-0.31,P=0.053695;i_low-j_middle=-0.52,P=0.004136;i_low-j_high=-0.47,P=0.007391).There was no significant difference in relative expression of PSD95 m RNA in melatonin,middle and high-dose groups（P>0.05）.Conclusion 1.Mongolian Medicine Zhenbao pill improves spatial learning and memory in sleep-deprived rats.2.Mongolian Medicine Zhenbao pill can increase the levels of SOD and GSH 3.Mongolian Medicine Zhenbao pill can increase the levels of BDNF and PSD95.4.The Mongolian Medicine Zhenbao pill may improve the learning and memory in sleep-deprived rats hippocampus by increasing SOD and GSH levels to reduce oxidative stress and increasing the expression of BDNF and PSD95.