| PartⅠThe Role of Tp73 in Cognitive Dysfunction in Alcohol-dependent RatsBackgrounds Alcohol Dependence（AD）is a mental disease caused by long-term drinking,which is the third global public health problem after cardiovascular disease and tumor.The cognitive impairment such as distraction and memory decline caused by alcohol dependence is the key problem in the treatment of alcohol dependence.Hippocampus is an important brain region related to cognitive function,and it is also an important region of the brain affected by alcohol.Tumor protein 73（Tp73）,a member of the Tp53 family,is closely related to apoptosis and plays an important role in neural development.However,the relationship between alcohol dependence and cognitive dysfunction has rarely been reported.Objectives To investigate the changes of Tp73 and cognitive function in alcohol-dependent model rats;To analyze the relationship between cognitive dysfunction induced by alcohol dependence and Tp73.Methods 1.Thirty SPF Wistar male rats were randomly divided into control group（Con）and alcohol-dependent group（AD）.Rats in AD group were intraperitoneally injected 10%（W/V）alcohol solution at 1g/kg/d.Rats in Con group were injected with equal volume of normal saline,and the rats in the two groups were trained with conditional place preference（CPP）to establish an alcohol dependent rat model.2.The changes of cognitive function,emotion and motor ability of rats in the two groups were observed by behavioral test.The morphological changes of hippocampal neurons in the two groups were detected by Nissl staining.The expression levels of Tp73 and apoptosis related molecules were detected by Western blot.3.Thirty-six male Wistar rats were randomly divided into 3 groups,namely,siRNA control virus group（Con+ Con-AAV,CCA）,alcohol-dependent +siRNA control virus group（AD+ Con-AAV,ACA）and alcohol-dependent +Tp73 siRNA virus group（AD+ AAVi-Tp73,AAT）,12 in each group.The hippocampus CA1 and CA3 of rats were injected with Tp73-siRNA or control siRNA adeno-associated virus using stereotactic brain injection technique.After 21 days of virus treatment,rats in each group were trained with CPP.Then,The changes of cognitive function,emotion and motor ability of rats in the three groups were observed by behavioral test.The morphological changes of hippocampal neurons in the three groups were detected by Nissl staining The expression levels of Tp73 and apoptosis related molecules were detected by Western blot.Results 1.Behavioral,pathological and molecular biological tests of alcohol dependence model rats（1）Behavioral ressults ssuggest that there was no difference in basic value between Con group and AD group（P > 0.05）.Compared with Con group,the test values and scores of CPP in AD group were significantly higher（all P < 0.05）,indicating that the AD group had obvious CPP effect,and the alcohol-dependent rat model was successfully constructed.Compared with Con group,the discrimination index of AD group was significantly reduced in the objective recognition test（P < 0.05）,the total movement distance in the open field experiment was significantly decreased（P < 0.01）,the time and times of the elevated cross maze entering the open arm were decreased（P < 0.05）,and the exploration time of the new heteroarm in the Y maze experiment was decreased（P < 0.01）.（2）Nissl staining showed that compared with Con group,the number of surviving neurons in hippocampal CA1 and CA3 areas in AD group decreased（all P < 0.05）,the arrangement of cell bodies was loose and the staining was pale,but there was no significant change in DG area.（3）Compared with Con group,the protein expression levels of TP73 in hippocampus,prefrontal cortex and striatum in AD group were significantly increased after modeling（all P < 0.05）.The expression levels of Bax and Caspase-3 in the hippocampus increased,while the expression levels of Bcl-2 decreased,and the ratio of Bax/Bcl-2 decreased（all P < 0.05）.2.Behavioral,pathological and molecular biological detection of in alcohol-dependent model rats after adeno-associated virus intervention（1）Behavioral ressults ssuggest that compared with Con+ Con-AAV group,the total movement distance and central area exploration time of rats in AD+ Con-AAV group were decreased（P < 0.05）,the time to enter the open arm of the elevated cross maze was decreased（P < 0.05）,and the exploration time to the new heteroarm of rats in Y maze was decreased（P < 0.05）.However,in AD+ AAVi-Tp73 group,the total movement distance and central area exploration time,the time of the elevated cross maze entering the open arm,and the exploration time of Y maze to the new heteroarm increased in the open field experiment（all P < 0.05）.（2）Nissl staining showed that compared with Con + Con-AAV group,the number of neurons in hippocampal CA1 and CA3 areas in AD + Con-AAV group decreased（all P < 0.05）,the arrangement of cell bodies was loose and the staining was pale,but there was no significant change in DG area.Compared with AD + Con-AAV group,the structure of hippocampal CA1 and CA3 in AD + AAVi-Tp73 group recovered and the number of neurons increased after AAV intervention（all P < 0.05）.（3）Compared with AD+ Con-AAV group,the protein expression levels TP73 in hippocampus of AD+ AAVi-Tp73 group was significantly decreased（P < 0.01）,the level of Bcl-2 protein was significantly increased（P < 0.01）,and the ratio of Bax/Bcl-2 was increased（P < 0.01）.However,the expression levels of Bax and Caspase-3 did not change significantly.Conclusion 1.Alcohol-dependent rats had cognitive impairment,and Tp73 expression increased in hippocampus and other brain regions 2.Tp73 participates in the regulation of cognitive impairment in alcohol dependent rats by regulating the apoptosis of hippocampal neurons.Part II Targeted Regulation of Cckbr by miR-34b-3pBackgrounds miRNA was found to be a new target for alcohol dependence therapy.The role of miR-34 family in brain development has been confirmed.The up regulation of miR-34 a and miR-34 c in hippocampus is related to the cognitive impairment caused by alcohol dependence,but the research on the relationship between miR-34 b and alcohol dependence is limited.In the study of other diseases,it is found that overexpression of miR-34b-3p will inhibit cell proliferation,block cell cycle and increase apoptosis,while the use of miR-34b-3p inhibitor will improve the survival rate of cells.In addition,the excessive use of alcohol will cause cell apoptosis during the development of the central nervous system.Therefore,this study speculates that the abnormal expression of miR-34 b is related to the apoptosis of central nervous system cells induced by alcohol.Objectives This study aims to explore the role and mechanism of hippocampal miR-34b-3p in alcohol dependence,further explore the regulatory effect of miR-34b-3p on Cckbr,and verify the relationship between miR-34b-3p and apoptosis.Methods 1.In this study,the expression of miR-34b-3p and its target gene Cckbr in alcoholdependent rats was detected by Western Blot and real-time PCR using the previously established alcohol-dependent rat model.2.In this study,the mimic of miR-34b-3p was transfected into PC12 cells to observe the effect of elevated expression of miR-34b-3p on Cckbr.The interaction between miR-34b-3p and Cckbr was determined by double luciferase experiment.3.The concentration gradient of miR-34b-3p mimic transfection was set,and the change of cell survival rate was detected by CCK-8 kit.4.Detect the effect of miR-34b-3p mimic transfection on the expression of apoptosis related proteins Bax and bcl-2.Results 1.Western blot and Real Time PCR results showed that the expression of miR-34b-3p increased and the expression of ckbr decreased in the hippocampus of alcohol dependent rats（all P < 0.05）.2.After PC12 cells were transfected with miR-34b-3p mimic,the protein expression level of Cckbr decreased compared with negative control（NC）（P < 0.05）.Double luciferase experiment showed that miR-34b-3p regulated the expression of Cckbr by targeting the site of its 3 ’untranslated region.3.The survival rate of PC12 cells decreased with the increase of miR-34b-3p mimic transfection concentration（all P < 0.05）.4.After transfection of miR-34b-3p mimic into PC12 cells,compared with NC group,the expression level of Bax increased and the expression level of Bcl-2 decreased（all P < 0.05）.Conclusion In alcohol dependent rats,the expression of miR-34b-3p in hippocampus was up-regul ated and the expression of Cckbr was decreased;miR-34b-3p targeted regulation of Cckbr expression;miR-34b-3p promotes apoptosis. |
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