| ObjectiveTo discuss the effect of Sini Powder on behavior,hippocampal neurons,peripheral and central immunologic changes of different sexual adolescent rats suffering from chronic unpredictable mild stress.MethodsFour-week old Wistar rats(half male and female)were randomly divided into blank group without chronic unpredictable mild stress and stress group to suffer from chronic unpredictable mild stress(CUMS)for 21 days according to weight.Then based on weight again,the stress group was randomly divided into the model group,the lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group,which continued receiving CUMS for another 21 days.Meanwhile the 3 medicated groups were given aqueous solution of the corresponding drug by gavage once a day and the blank controlled group and the model group were given saline.After the last administration,all the rats were weighed,their sucrose preference were tested,and the open field test was done.The hippocampal DG subregion of rats from all the groups were observed by HE staining.Both pro-inflammatory cytokines(TNF-?,IL-1 ?,IL-6,IFN y)and anti-inflammatory cytokines(IL-4,IL-10)in plasma and hippocampus were tested by Merck milliplex map.The expression of CD11b(marker of activated microglia),glial fibrillary acidic protein(GFAP,marker of activated astrocyte),nuclear factor ?B p65(NF-?B p65)and Phospho-NF-?B p65 in hippocampus of rats were detected by Western blot.GFAP and NF-?B p65 in the hippocampal DG subregion were also observed after immunohistochemistry.When the data from different sexual rats in the same group presented similar and the same changing trend among groups,the different sexual data in the same group would be merged to analyze statistically.Otherwise the data would be analyzed according to different sex.Results1.The effect of Sini Powder on behavior of adolescent rats suffering from CUMS1.1 General conditionBefore suffering from CUMS,all the rats were healthy,clean,active and ate well.After the first CUMS for 21d,the rats which suffered from stress became anxious,ate less,their hair became messy and shriveled.When being gavage and stressed,they reacted violently,became easily scared,defensive and irritable.Then after the second CUMS for another 21d,the model group became fatigue,thin,easily lost hair,and preferred hiding.But the lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group represented active with glossy hair.The blank group kept well.1.2 WeightThe weight of the male model group was lower significantly than that of blank group(P<0.05).But the male model group did not significantly differ from the lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group respectively(P>0.05).The weight of the female model group was lower significantly than that of blank group(P<0.05).But the female model group did not significantly differ from the lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group respectively(P>0.05).1.3 Sucrose preference testAll groups did not differ significantly from each other in sucrose preference test(P>0.05).1.4 Open field testIn the open field test,the total distance of the model group was lower significantly than that of blank group(P<0.05).The lower dosage of Sini Powder group and the fluoxetine group were higher significantly than the model group(P<0.05).The higher dosage of Sini Powder group was higher than the model group,but not significantly(P>0.05).2.The effect of Sini Powder on hippocampal neurons of adolescent rats suffering from CUMSIn HE staining of the hippocampal tissue section,all the male groups were not significantly different from each other in DG;while the neurons of the female model group decreased and loosely arranged,but Sini powder and Fluoxetine could treat this pathology.3.The effect of Sini Powder on peripheral immunologic changes of adolescent rats suffering from CUMS3.1 TNF-? in plasmaTNF-? in plasma from all male groups did not differ significantly from each other(P>0.05).TNF-a in plasma from the female model group was lower significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group,the higher dosage of Sini Powder group and the fluoxetine group were higher significantly than the model group respectively(P<0.05).3.2 IL-1? in plasmaIL-1? in plasma from all the groups did not differ significantly from each other(P>0.05).3.3 The level of IL-6,IFN y,IL-4 and IL-10 in plasma was too low to be detected by Merck milliplex map.4.The effect of Sini Powder on hippocampal immunologic changes of adolescent rats suffering from CUMS4.I TNF-? in hippocampusTNF-? in hippocampus from the male model group was lower significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group,the higher dosage of Sini Powder group and the fluoxetine group were not different significantly from the model group respectively(P>00.05).TNF-?in hippocampus from all female groups did not differ significantly from each other(P>0.05).4.2 IL-1? in hippocampusIL-1? in hippocampus from the male model group was higher significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group and the higher dosage of Sini Powder group were lower significantly than the model group respectively(P<0.05).The fluoxetine group was lower than the model group,but not significantly(P>0.05).IL-1? in hippocampus from all female groups did not differ significantly from each other(P>0.05).4.3 IL-6 in hippocampusIL-6 in hippocampus from the model group was higher significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group and the higher dosage of Sini Powder group were lower significantly than the model group respectively(P<0.05).The fluoxetine group was lower than the model group,but not significantly(P>0.05).4.4 IFN? in hippocampusIFN? in hippocampus from the model group was higher significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group were lower significantly than the model group respectively(P<0.05).4.5 IL-4 in hippocampusIL-4 in hippocampus from the model group was lower significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group was higher significantly than the model group(P<0.05).The higher dosage of Sini Powder group and the fluoxetine group were higher than the model group respectively,but not significantly(P>0.05).4.6 IL-10 in hippocampusIL-10 in hippocampus from the male model group was higher significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group,the higher dosage of Sini Powder group and the fluoxetine group were lower significantly than the model group respectively(P<0.05).IL-10 in hippocampus from all female groups did not differ significantly from each other(P>0.05).4.7 CDllb in hippocampusCDllb in hippocampus from all male groups did not differ significantly from each other(P>0.05).CDllb in hippocampus from the female model group was lower significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group and the higher dosage of Sini Powder group were higher significantly than the model group respectively(P<0.05).The fluoxetine group did not differ significantly from the model group(P>0.05).4.8 GFAP in hippocampusBy Western blot,GFAP in hippocampus from the male model group was lower significantly than that from blank group(P<0.05).The model group did not differ significantly from the lower dosage of Sini Powder group,the higher dosage of Sini Powder group or the fluoxetine group(P>0.05).GFAP in hippocampus from the female model group was higher significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group,the higher dosage of Sini Powder group and the fluoxetine group were lower significantly than the model group respectively(P<0.05).By immunohistochemistry,GFAP was expressed in the hippocampal DG subregion of all the groups,mainly distributing in cytoplasm and dendrites of astrocyte,which could be seen sandy and starlike or radial by microscope.Thick dendrites with different length could be seen in the hippocampal DG subregion of the male blank group that was stained diffusely.In the male model group,the number of staining cells decreased and the cells stained less distributed more sparsely with dendrites shorter and thinner than the blank group.The male model group did not significantly differ from the lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group respectively.The staining cells were small with slim dendrites in the hippocampal DG subregion of the female blank group.In the female model group,the hippocampal DG subregion was stained more and the dendrites of the staining cells became thicker,branching more and longer than the blank group.The gliosis of astrocyte was improved in the lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group.4.9 NF-?B p65 in hippocampusBy Western blot,NF-?B p65 in hippocampus from all the groups did not differ significantly from each other(P>0.05).By immunohistochemistry,NF-?B p65 was expressed in the hippocampal DG subregion of all the groups,which could be seen sandy by microscope.NF-? Bp65 was stained in the cytoplasm of both male and female blank groups,but not in the intranuclear.The stained NF-?B p65 could be seen in the cytoplasm of both male and female model groups,as well as in the intranuclear of some cells,which meant NF-?B p65' s translocation into the nuclear.The stained NF-?B p65 could be also seen in the cytoplasm of the lower dosage of Sini Powder group,the higher dosage of Sini Powder group,and the fluoxetine group,while their numbers of cells with stained NF-KB p65 in the nuclear were less than that of the model group respectively,which implied NF-?B p65' s translocation was suppressed.4.10 Phospho-NF-?B p65 in hippocampusPhospho-NF-?B p65 in hippocampus from all the male groups did not differ significantly from each other(P>0.05).Phospho-NF-?B p65 in hippocampus from the female model group was higher significantly than that from blank group(P<0.05).The lower dosage of Sini Powder group,the higher dosage of Sini Powder group and the fluoxetine group were lower significantly than the model group respectively(P<0.05).Conclusion1.Only chronic unpredictable mild stress in adolescence may not induce characteristic performance of depressive disorders in animals,but can result in some abnormal autonomic behavior.That also leads to neuronal impairment in female,the "biological scar",which has potential and long-lasting influence.2.Only chronic unpredictable mild stress in adolescence has more effect on central immunity than peripheral.3.Only chronic unpredictable mild stress in adolescence can induce central immune disorders,including immune activation and suppression,for example,the state of microglial and astrocyte change,cytokines increase or decrease,and NF-?B signaling pathway is activated,etc,which are distinct in different gender.The neural "biological scar" resulting from stress may be related to central immune disorders.4.Sini Powder can regulate immune activation and suppression simultaneously resulting from chronic unpredictable mild stress in adolescence.Sini Powder can improve immune disorders and restore balance,and treat neural impairment,which indicates didirectionally regulated effects of Chinese medicine.These results can provide experimental evidence for Sini Powder used in treating the early stage of chronic stress impairment.Innovation point1.Animal experiments about chronic stress mainly correlated to adults and males before.Both male and female rats in adolescence are used in this paper to study the potential biological changes caused by chronic unpredictable mild stress in adolescence and their distinction between different gender,which can provide experimental evidence for prevention and early treatment to mental illness.2.Chronic unpredictable mild stress in adolescence was mainly studied in respects of neurogenesis and neuroendocrine to explore neural impairment before.Based on the " inf lammatory-neuroprogressive" hypothesis in depression,this paper studies the immune disorders caused by chronic stress in adolescence,especially the central immune imbalance,which enriches the studying range of chronic stress in adolescence.3.The results in this paper indicate that Sini Powder has didirectionally regulated effects on immune disorders caused by chronic unpredictable mild stress in adolescence,which can provide experimental evidence for Sini Powder used in treating the early stage of chronic stress impairment. |
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