| Objective:In this experiment, we selected the ovariectomized rats injected with D-gal as the subject to observe the effect of mailuoning, traditional Chinese medicine compound,western medicine vitamin C+E and atorvastatin recipe on the expression of Sirt1 gene.Methods1 Animals and groupsForty-eight female SD rats aged four months were randomly divided into two groups: sham operated group (Sham group, eight rats),ovariectomized group (OVX group, forty rats). Sham group was ablated some adipose tissue above ovaries; OVX group was ablated two sides ovaries. Rats in OVX received D-gal (20 mg in 0.5 ml 0.9% saline intraperitoneal injection everyday); rats in Sham received saline (0.5 ml 0.9% saline intraperitoneal injection everyday), after two weeks of operation. The rats in OVX were randomly divided into five groups again after six weeks of intraperitoneal injection: one group was administered orally with mailuoning injection at 1.8 ml/kg.d (ODM group); the second group was administered orally with traditional Chinese medicine compound recipe 16.83 g/kg.d (ODZ group); the third group was administered orally with V.C 45 mg/kg.d and V.E 27 mg/kg.d (ODV group); the fourth group was administered orally with atorvastatin for 7.2 mg/kg·d ( ODA group ); the last group was administered orally with distilled water 1.5 ml/kg.d (OD group). The Sham group was administered orally with distilled water for 1.5 ml/kg·d, too. OVX group and Sham group continued to receive D-gal and saline intraperitoneal injection respectively. After surgery, all rats were killed after 12 weeks of regular feeding. 2 Behavioral testsFour weeks after intraperitoneal injection, behavioral tests were carried out to evaluate learning and memory abilities by Morris water maze . Four escape trails were given to all mice per day for four consecutive days. The same procedure was repeated for one day , after six weeks intraperitoneal injection and four weeks of medicine administered orally. The escape latency was recorded.3 Tissues and data determiningTo anesthetize rats, right temporal cortex were quickly separated and deep freezed in liquid Nitrogen. Then tissues were stored at -80℃to extract total RNA and protein. Uterus was quickly separated and weighed by electronic balance. Right temporal cortex total RNA were extracted by Trizol regent. The relative Sirt1 mRNA contents were measured by RT-PCR using GAPDH as inner standard. Protein quantity was measured by Lowry, expressed in mg/ml. The relative protein contents of Sirt1 were measured by western-blotting using GAPDH as inner standards.Results1 Impairments of behavioral performanceThe escape latency in the Sham group was 8.7±4.1. The escape latency in the OVX+D-gal groups was 15.2±8.1. Data from Morris water maze showed that the escape latency was longer in the OVX+D-gal groups compared with the Sham group (P<0.05). The escape latency in the ODM+ODZ+ODV+ODA was 9.1±6.5. The escape latency in the OD group was 16.7±12.3. The escape latency was shorter significantly in the ODM+ODZ+ODV+ODA groups compared with the OD group (P<0.05).2 The relative expression of Sirt1 mRNA in right temporal cortexThe relative expression of Sirt1 mRNA of OD was 0.464±0.063. The relative expression of ?Sirt1 mRNA of Sham was 0.624±0.030. Compared with Sham, the relative expression of Sirt1 mRNA of OD was lower (P<0.05). The relative expression of Sirt1 mRNA of ODM was 0.968±0.127. The relative expression of Sirt1 mRNA of ODZ was 0.859±0.119. The relative expression of Sirt1 mRNA of ODV was 0.790±0.076. The relative expression of Sirt1 mRNA of ODA was 0.603±0.091. Compared with OD, the relative expression of Sirt1 mRNA of ODM,ODZ,ODV and ODA was higher (P<0.05).3 The relative expression of Sirt1 protein in right temporal cortexThe relative expression of Sirt1 of OD was 0.581±0.199. The relative expression of Sirt1 of Sham was 0.906±0.085. Compared with Sham, the relative expression of Sirt1 of OD was lower (P<0.05). The relative expression of Sirt1 of ODM was 2.023±0.288. The relative expression of Sirt1 of ODZ was 1.818±0.287. The relative expression of Sirt1 of ODV was 1.605±0.225. The relative expression of Sirt1 of ODA was 1.033±0.091. Compared with OD, the relative expression of Sirt1 of ODM,ODZ,ODV and ODA was higher (P<0.05).Conclusion1 The right temporal cortex of rats with the target protein expression of Sirt1 mRNA and protein levels were reduced.2 Mailuoning, traditional Chinese medicine compound, vitamins (C+E), atorvastatin can increase the expression of Sirt1 mRNA and protein levels in varying degrees, suggesting that increased expression of Sirt1 could be one of the molecular mechanisms of neuroprotection of these drugs on AD rat model. The specific regulation mechanism still need further study. |
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