| Objective: In this experiment, we selected the ovariectomized rats with D-galactose injection as the objects to observe the effects of Xie HaiZhou Fang to the expression of BDNF, NGF, TrkA, TrkB, p75, HMG-CoAR, CYP46 and ApoE genes and content of total cholesterol in hippocampus.Methods:1 Animals and groupsForty eight female SD rats aged 4 months were randomly divided into 2 groups: Sham operated group (Sham group, n=8) and Ovariectomized group (OVX group, n=40) after conventional feeding for two weeks. Rats in OVX group were carried out bilateral ovariectomy. Rats in Sham group were carried out the same operative procedure, but they were ablated some adipose tissue above ovaries only. After two weeks of ovariectomy, rats in Sham group were intraperitoneally injected with normal saline (NS) (1.67 ml/Kg.d), and rats in OVX group were intraperitoneally injected with D-galactose (66.7 mg/Kg.d). After 6 weeks' injection, rats in OVX group were randomly divided into the following groups: Pathological control group (PC group), Xie HaiZhou Fang group (HC group), MailLuoning group (MN group), Atrovastatins group (AT group) and VC+VE group (DV group). All drugs were administrated by intragastric administration for 4 weeks with the doses as follows: Sham group and PC group (distilled water, 5 ml/Kg.d), HC group (liquid extract of Xie HaiZhou Fang, 5 mg/Kg.d), MN group (Mailuoning injection, 1.8 ml/Kg.d), AT group (Atrovastatins, 7.2 mg/Kg.d) and DV group (VC 45 mg/Kg.d VE 27 mg/Kg.d). In the course of intragastric administration, rats in Sham group were unceasingly injected with NS (1.67 ml/Kg.d), and rats in the other groups were unceasingly injected with D-galactose (0.5 ml 66.7 mg/Kg.d). After 4 weeks' intragastric administration (namely 12 weeks after ovariectomy), all rats were sacrificed.2 Behavioral trainingThe Morris swim task was used to test the discrimination of the rats' spatial learning and memory ability 6 weeks after ovariectomy. All rats were given behavioral training consecutively for 4 days with the rats' escape latency measured by a stopwatch. Each rat was tested for four consecutive times every day and with an interval for 5 minute. The same Morris swim task was used again to test the discrimination of the rats' spatial learning and memory ability of each group 8 weeks after ovariectomy for 1 day. Then the average escape latency in the last three times was obtained as the index of spatial learning and memory in rats. One day before rats were killed, the same Morris swim task was used again to test the discrimination of the rats' spatial learning and memory ability of each group.3 Tissues and data determining3.1 Tissues obtainingTo anesthetize rats after behavioral training immediately, hippocampus was quickly separated and deeply freezed in liquid Nitrogen. Then tissues were stored at -80℃to extract total RNA and total cholesterol. Uterus was quickly separated and weighed by electronic balance.3.2 Assay of mRNA expressionTotal RNA of hippocampus was extracted by Trizol regent. The relative expression of BDNF,NGF,TrkA,TrkB,p75,HMG-CoAR,CYP46 and ApoE mRNA were measured by RT-PCR using the expression ofβ-actin mRNA as inner standard.3.3 Total cholesterol assayTotal cholesterol of the hippocampus was extracted with chloroform/methanol (2:1) and was determined by enzymatic- colorimetric assay. The values were expressed as mg of cholesterol/100 g wet weight of tissues (mg/100g).Results:1 The differential weights of uterus Compared with Sham group, the weights of rats' uterus in OVX group were lighter (P < 0.01). There was no difference among PC group, HC group, DV group, MN group and AT group (P > 0.05). 2 The performance of rats in the Morris water mazeCompared with Sham group, rats in OVX group held longer escape latency (P < 0.05). It showed that after ovariectomy and D-galactose injection, rats of this model showed impairments of spatial learning and memory. Rats in HC group and MN group held shorter escape latency than rats in PC group after administrating herbal compounds and MaiLuoning injection seperately(P < 0.05).3 Changes of the relative expression of BDNF mRNACompared with Sham group, the relative expression of BDNF mRNA of PC group was lower (P < 0.05). Compared with PC group, the relative expression of BDNF mRNA of HC group was higher (P < 0.05).4 Changes of the relative expression of TrkB mRNACompared with Sham group, the relative expression of TrkB mRNA of PC group was lower (P < 0.05). Compared with PC group, the relative expression of TrkB mRNA of HC group and DV group was higher (P < 0.05).5 Changes of the relative expression of NGF mRNACompared with Sham group, the relative expression of NGF mRNA of PC group was lower (P < 0.05). Compared with PC group, the relative expression of NGF mRNA of HC group and MN group was higher (P < 0.05).6 Changes of the relative expression of TrkA mRNACompared with Sham group, the relative expression of TrkA mRNA of PC group was lower (P < 0.05). There was no difference between PC group and HC group (P > 0.05).7 Changes of the relative expression of p75 mRNACompared with Sham group, the relative expression of p75 mRNA of PC group was lower (P < 0.05). Compared with PC group, the relative expression of p75 mRNA of HC group was higher (P < 0.05).8 Changes of the relative expression of CYP46 mRNACompared with Sham group, the relative expression of CYP46 mRNA of PC group was lower (P < 0.05). Compared with PC group, the relative expression of CYP46 mRNA of HC group, AT group and MN group was higher (P < 0.05).9 Changes of the relative expression of HMG-CoAR mRNACompared with Sham group, the relative expression of HMG-CoAR mRNA of PC group was higher (P < 0.05). Compared with PC group, the relative expression of HMG-CoAR mRNA of HC group, AT group, DV group and MN group was lower (P < 0.05).10 Changes of the relative expression of ApoE mRNACompared with Sham group, the relative expression of ApoE mRNA of PC group was higher (P < 0.05). Compared with PC group, the relative expression of ApoE mRNA of HC group, AT group, DV group and MN group was lower (P < 0.05).11 Changes of the content of total cholesterolCompared with Sham group, the content of total cholesterol of PC group was higher (P < 0.05). Compared with PC group, the content of total cholesterol of HC group and MN group was lower (P < 0.05).Conclusion:1 Rats of this model could mimic some changes of hippocampus at the early stage of Alzheimer's disease and could be used for medicine research.2 Xie HaiZhou Fang could play roles in protecting neurons by moderating the expression of some genes and the content of total cholesterol in hippocampus of this model. |
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