Changes In Contents Of HCNP And Genes Expression Of Cholinergic Nerve System In Hippocampus And Prefrontal Cortex Of Ovariectomized Rats

Objective: In this experiment,we selected the ovariectomized rats as the object to observe the effect of estrogen on HCNP,ChAT,M1 AChR,α₇ nAChR and NR₁.Methods:1 Animals and groups30 female SD rats aged 4 months were randomly divided into 3 groups:sham operated group (Sham group),ovariectomized group (OVX group),ovariectomized and Estradiol Benzoate supplied group (OVX+EB group).Sham group was ablated some adipose tissue above ovaries,OVX group was ablated two sides ovaries,OVX+EB group was supplied by Estradiol Benzoate (100ug/kg.2d) after 10 days of ovariectomy.Postoperative rats were fed by routine method and then sacrificed 4 months later.2 Tissues and data determiningTo anesthetize rats,dorsal hippocampus and prefrontal cortex were quickly separated and deep freezed in liquid Nitrogen.Then tissues were stored at -80℃to extract protein and total RNA. Uterus was quickly separated and weighed by electronic balance.2.1 Interest protein determiningThe protein of dorsal hippocampus and prefrontal cortex were extracted.Protein quantity was measured by Bradford, expressed in mg/ml.The relative protein content of C-HCNPpp and N-HCNPpp were measured by western-blotting using andβ-actin as inner standards.2.2 Assay of mRNA expressionDorsal hippocampus and prefrontal cortex total RNA were extracted by Trizol regent.The relative mRNA contents (HCNPpp,ChAT,M1 AChR,α₇ nAChR and NR₁)were measured by RT-PCR usingβ-actin as inner standard.Results:1 The differential weight of rat's uterusCompared with Sham and OVX+EB, the weight of uterus of OVX was lighter (p<0.01), and there was no difference between Sham and OVX+EB group (P>0.05).2 The relatibe amount of HCNPpp in hippocampusCompared with Sham and OVX+EB group, the relative amount of C-HCNPpp was higher, and there was no difference between Sham and OVX+EB group (P>0.05).There was no difference among three groups.3 The amount of HCNPpp in prefrontal cortexCompared with Sham and OVX+EB group,the relative amount of C-HCNPpp was higher,and there was no difference between Sham and OVX+EB group (P>0.05).There was no difference among three groups.To the expression of N-HCNPpp, there was no difference among three groups(P>0.05).Sham group,OVX group and OVX+EB.It suggested that estrogen may be have no effect on N-HCNPpp.4 The relative expression of HCNPpp mRNA in hippocampusCompared with Sham, the relative expression of HCNPpp of OVX and OVX+EB was not different(P>0.05).Compared with OVX+EB,the relative expression of HCNPpp mRNA of OVX was lower (P<0.05).5 The relative expression of HCNPpp mRNA in prefrontal cortexThere was no difference among Sham,OVX+EB and OVX group (P>0.05).6 The relative expression of ChAT mRNA in hippocampusCompared with Sham and OVX+EB group, the relative expression of ChAT receptor mRNA of OVX was lower (P<0.05).There was no difference between Sham and OVX+EB (P>0.05).7 The relative expression of ChAT mRNA in prefrontal cortexThere was no difference among Sham,OVX+EB and OVX group (P>0.05).8 The relative expression of M1 AChR mRNA in hippocampusCompared with Sham,the relative expression of M1 AChR mRNA of OVX was higher (P<0.05), and the relative expression of M1 AChR mRNA of OVX+EB was higher(P<0.01), and there was no difference between OVX and OVX+EB group(P>0.05).9 The relative expression of M1 AChR mRNA in prefrontal cortexThere was no difference among Sham,OVX+EB and OVX group (P>0.05).10 The relative expression ofα₇ nAChR mRNA in hippocampusCompared with Sham and OVX+EB group, the relative expression ofα₇nAch receptor mRNA of OVX was lower (P<0.05).There was no difference between Sham and OVX+EB.11 The relative expression ofα₇ nAChR mRNA in prefrontal cortexCompared with Sham and OVX+EB group,the relative expression ofα₇nAch receptor mRNA of OVX was lower (P<0.05).There was no difference between Sham and OVX+EB group.12 The relative expression of NR₁ mRNA in hippocampusCompared with Sham and OVX+EB group, the relative expression of NR₁ mRNA of OVX was lower (P<0.05).There was no difference between Sham and OVX+EB group.13 The relative expression of NMDA receptor mRNA in prefrontal cortexThere was no difference among Sham,OVX+EB and OVX group (P>0.05).Conclusion:1 The deficiency of estrogen in female rats led to the increasment of the relative amount of HCNP in CA1/CA2 of hippocampus and prefrontal cortex of ovariectomized rats.2 The expressions of ChAT,α₇ nAChR mRNA in CA1/CA2 region of hippocampus were lower in ovariectomized rats.It indicated that estrogen may regulate the expression of ChAT, α₇ nAChR mRNA in CA1/CA2 region of hippocampus, which seemed to be one of the molecular mechanisms of AD that was caused by estrogen deficiency.3 The expressions of NR₁ mRNA in CA1/CA2 region of hippocampus were lower in ovariectomized rats.It indicated that estrogen may regulate the expression of NR₁ mRNA in CA1/CA2 region of hippocampus, which seemed to be one of the molecular mechanisms of AD that was caused by estrogen deficicency.