| Objective1.To investigate the effect and possible mechanism of Liuwei Dihuang Decoction on memory impairment in rats with chronic depression.2.To investigate the potential relationship between the changes of estrogen,GPR30,CREB and BDNF and memory impairment in rats with chronic depression.Methods1.In the first part of this study,chronic unpredictable mild stress was conducted to establish depression model in rats.Female Wistar rats were randomly divided into normal group(normal saline),chronic unpredictable mild stress(CUMS)model group(normal saline),low,medium and high dose Liuwei Dihuang Decoction(LWDHD)groups(2.60,7.81,23.50 g/kg/d).Except for the normal group,all the other groups were conducted by the chronic unpredictable mild stress for 5 weeks.Weights were recorded every week,changes in their behavioral tests were observed.In the experiment,the cut down of sucrose preference was the primary index of depression,because the sucrose preference experiment aimed to measure the degree of pleasure loss in CUMS rats;The open field experiment was used to observe the autonomic movement,exploratory behavior and tensity of the rats in the new environment in each group;the Morris water maze experiment aimed to assess the learning and memory ability of rats in each group;the level of G protein-coupled estrogen receptor(GPR30),cyclic adenosine monophosphate response element binding protein(CREB)and brain-derived neurotrophic factor(BDNF)mRNA in the rat hippocampus were determined by quantitative real time polymerase chain reaction(qRT-PCR);enzyme-linked immunosorbent assay(ELISA)method was conducted to measure the concentration of serum estrogen;the expression of GPR30,phospho-CREB/CREB and BDNF in hippocampus were detected by Western Blot.2.In the second part of this study,chronic unpredictable mild stress was conducted again to establish depression model in rats.In order to further observe the effect of estrogen/GPR30 on depressive behavior and learning and memory impairment.G15,the inhibitor of estrogen receptor GPR30,was added in the experiment.Female Wistar rats were all randomly divided into five groups,including normal group(normal saline),CUMS model group(normal saline),LWDHD(7.81 g/kg/d)group,LWDHD(7.81 g/kg/d)+G15(10μg/d)group and LWDHD(7.81 g/kg/d)+G15(40μg/d)group.Except for the normal group,all the other groups were conducted by the chronic unpredictable mild stress for 6 weeks.Weights were recorded every week,changes in their behavioral tests were observed.In the experiment,the sucrose preference,open field experiment and Morris water maze experiment were conducted to evaluate the behavior of rats in each group;the level of GPR30,CREB and BDNF mRNA in the rat hippocampus were determined by qRT-PCR;the concentration of serum estrogen were detected by ELISA method;Western Blot was conducted to detect the expression of GPR30,p-CREB/CREB and BDNF in rats hippocampus.Results1.When compared with normal group,the weight,activity and intreast of rats in CUMS group have significantly declined.Behavioral tests and ELISA results were showed below,compared with control group,the sucrose preference(P<0.01),rearing times(P<0.001)and total distance of open field experiment(P<0.05),Morris water maze latency(P<0.01)and serum estrogen concentration of CUMS rats(P<0.05)significantly declined.Low,medium and high dosage of LWDHD significantly improved the sucrose preference(P<0.01,P<0.01,P<0.01)and increased the rearing times(P<0.01,P<0.001,P<0.001)of CUMS rats in the open field experiment;medium and high dosage of LWDHD can significantly increased the total distance of CUMS rats in the open field experiment(P<0.01,P<0.05);Low and medium dosage of LWDHD can significantly shortened the latency of CUMS rats in the Morris water maze experiment(P<0.01,P<0.01);Low and medium dosage of LWDHD significantly upregulated the serum estrogen concentration(P<0.05,P<0.05)of CUMS rats.qRT-PCR results were showed below,the expression of GPR30,CREB and BDNF mRNA in hippocampus of CUMS rats decreased significantly(P<0.05,P<0.05,P<0.01),and the expression of GPR30 and CREB mRNA in hippocampus of rats in LWDHD low-dose group increased significantly(P<0.05,P<0.01),medium dosage of LWDHD significantly increased the expression of GPR30,CREB and BDNF mRNA in CUMS rats hippocampus(P<0.01,P<0.01,P<0.01);Western Blot results were showed below,the expression of GPR30 and BDNF in hippocampus of CUMS group significantly decreased(P<0.01,P<0.01),low and medium dosage of LWDHD significantly increased the expression of GPR30 in CUMS rats hippocampus(P<0.01,P<0.05),low and high dosage of LWDHD significantly increased the expression of BDNF in CUMS rats hippocampus(P<0.001,P<0.001,P<0.05).2.When compared with the normal group,the weight,activity and intreast of rats in CUMS group have significantly declined.Behavioral tests and ELISA results were showed below,compared with control group,the sucrose preference(P<0.01),rearing times(P<0.05)and total distance(P<0.05)in open field experiment,Morris water maze latency(P<0.01),times crossing the platform(P<0.05)and time-spending during the platform quadrant(P<0.01),and serum estrogen concentration(P<0.01)significantly declined.LWDHD(7.81 g/kg/d)significantly improved the sucrose preference(P<0.05),rearing times and total distance of CUMS rats in open field experiment(P<0.01,P<0.01);compared with LWDHD group,rearing times in open field experiment(P<0.05,P<0.001)of rats significantly lessened in LWDHD+G15 10 and 40μg groups.The total distance in open field experiment of rats significantly lessened in LWDHD+G15 high-dose group(P<0.01).The latency of Morris water maze experiment of rats in LWDHD(7.81 g/kg/d)group significantly shortened(P<0.05),and times crossing the platform and time-spending during the platform quadrant significantly increased(P<0.001,P<0.05).Compared with LWDHD group,the latency of platform searching of rats in LWDHD+G15 low-and high-dose group significantly prolonged(P<0.05,P<0.05),and times crossing the platform in LWDHD+G15 low-and high-dose group significantly increased(P<0.05,P<0.05).LWDHD(7.81 g/kg/d)significantly upregulated the serum estrogen concentration(P<0.05)of CUMS rats.qRT-PCR results were showed below,the expression of GPR30,CREB and BDNF mRNA in rats hippocampus from CUMS group decreased significantly(P<0.05,P<0.01,P<0.05),the expression of GPR30,CREB and BDNF mRNA in rats hippocampus from LWDHD(7.81 g/kg/d)group upregulated significantly(P<0.05,P<0.05,P<0.01).Compared with LWDHD group,the expression of GPR30,BDNF mRNA in rats hippocampus from LWDHD+G15 low-(P<0.05,P<0.01)and high-dose(P<0.05,P<0.01)groups significantly decreased;Western Blot results were showed below,the expression of GPR30,p-CREB/CREB and BDNF in rats hippocampus from CUMS group decreased significantly(P<0.05,P<0.05,P<0.05),the expression of GPR30,p-CREB/CREB and BDNF in rats hippocampus from LWDHD(7.81 g/kg/d)group upregulated significantly(P<0.01,P<0.05,P<0.05).Compared with LWDHD group,the expression of GPR30,p-CREB/CREB and BDNF in rats hippocampus from LWDHD+G15 low-dose group decreased significantly(P<0.01,P<0.05,P<0.01),the expression ofGPR30,p-CREB/CREB and BDNF in rats hippocampus from LWDHD+G15 high-dose group decreased significantly(P<0.05,P<0.05,P<0.05).Conclusion1.LWDHD has antidepressant effect,could reverse the depressive-like behavior of CUMS rats and improve their learning and memory disorders,among which the medium dose of LWDHD has the most significant effect.The mechanism may be related to increased expression of estrogen in rat serum and GPR30,CREB and BDNF in rat hippocampus.2.The mechanism of LWDHD on depression and improving learning and memory is related to the activation of estrogen/GPR30 and CREB/BDNF pathway. |
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