| ObjectiveTo explore the effects of Liuwei Dihuang Pill(LW) on the age-related deterioration of learning and memory in SAM and find out if such changes are related the expression of cytokines(IL-2, IL-6);To discuss the content of "kidney" of TCM from a different angle. MethodsSixty 7-month-old male senescence-accelerated mice prone strain/8 (SAMP8) were assigned to the LW group, the huperzine (HUP) group and the model group by block randomization. Ten 7-month-old male senescence-accelerated mice resistant prone strain/R1(SAMR1) were used as the control group. Each group has been treated with the corresponding method such as LW(in the LW group), HUP(in the HUP group) and physiological saline(in the model group and the control group) for thirty days. After a month , we observed the ability of learning and memory of all the mice by the test of Morris water maze and got the data in the Place Navigation Training and the Spatial Probe Test. After finishing 5 days' Morris water maze test, we killed all the mice, collected their blood by excising the eyeballs and separated the hippocampus from the brain tissue. The blood were centrifuged and the serum were pipetted into other EP tubes. All these samples were deepfreezed for use. Enzyoned-linked immunosorbent assay (ELISA) was performed to assay the expression of IL-2 and IL-6 in all the samples. The protein level of the hippocampus samples were determined in a 96 well plate by a micromethod. The data were expressed by x|-±s and analysed statistically by SPSS11. 0. Results1 This is the first we apply the SAM to the research on Alzheimer' s disease. Because the Morris water maze equipment we have were primarilydesigned for the rats, we did a lot of work to have this equipment reconstituted for mice. The reconstitution included changes in the hardwares and the software. And after such reconstitution the water maze was proved to be applicable in test of learning and memory for mice.2 The Morris water maze test includes two parts- Place Navigation Training and the Spatial Probe Test. After the last day of the test, data of the escape latency, distance, average speed and primary angle were analysed.The data of Place Navigation Training were analysed by using Repeated Measures module in SPSS11.O, the data of Spatial Probe Test were analyzed by Multivariate module in SPSS11. 0. The results showed the following information: (1)Trend of the five-day changes: As time went on, the escape latencies seemed to be shorter;among these, the escape latency of the forth and the fifth day was significantly shorter than that of the first day (K0. 05);the escape lantency of the fifth day was significantly shorter than that of the first day (K0.05). All above indicats that the ability of learning and memorizing the position of the platform can be trained through a repeated way, after the first three days' training performance of the following day were improved remarkably and appeared to be stable. Trend of the changes in speed show an improvement on the second day and a decline since the third day, such changes became stable on the forth and the fifth day. For the SAM, the decline in the swimming speed indicates a related decline in physical vigor. Trend of the changes in distance show an increase on the second day and a decrease since the third day, such changes became stable on the forth and the fifth day. There were no obvious changes in the comparisons of primary speed. The comparison of the changes in speed, distance and primary speed show no significant difference among the five days. (2) Comparisons between the four groups: the escape latencies were sorted as below, control group< LW group< model group< HUP group, among which the control group' s escape latency was significantly shorter than other groups;the average speed were sorted as below, control group> model group>LW group> HUP group, among which the change between control group and HUP group was significant. (3) Daily Comparisons between each group: only the changes in escape latency showed a significant meaning. On the forth day , the escape latency of LW group was shorter than other SAMP8 group and the differences between LW group and control was not significant, which indicates that LW group is better in learning to find the platform thanother SAMP8 group. The performance of Spatial Probe Test showed no significant differencies between each group, which indicates that memory for the position of the platform had not been strengthened.3 Micromethod for protein measurement in the hippocampus. The result showed regression coefficient R as 0.987, which presents a good consistency among the points of the standard curve and an accuracy in the results.4 Assay of the expression of IL-2. Enzyoned-1inked immunosorbent assay(ELISA) was performed to assay the expression of IL-2. Serum IL-2 the readings were low, which led to non-detectable serum IL-2 concentrations. The concentrations(pg/ml) of IL-2 in hippocampus samples show no significant meaning among the groups.5 Assay of the expression of IL-6. Enzyoned-1inked immunosorbent assay(ELISA) was performed to assay the expression of IL-6. Serum IL-2 the readings were low, which led to non-detectable serum IL-2 concentrations. The concentrations(pg/ml) of IL-6 in hippocampus samples were sorted as the following, control> LW group> model group> HUP group, among which the readings of HUP group and model grouop were significantly lower the LW group, and which significant between LW group and control group .ConclusionsThe study showed that treating with LW can improve the learning and memory ability of SAM to some extent, but no superiority was found in this treatment compared with the HUP group. The different expression of IL-6 between SAMP8 and SAMR1 indicated that such changes may really exist. And LW can make the increased expression of IL-6 become lower, but whether the mechanism was to reduce inflammation directly or not is not clear. Such results at least show us that LW can improve learning and memory ability through an adjusting the immunization status way. IL-2 concentrations among the four group do not show obvious differencies, which may be accounted for other reasons such as different points that LW do to the central nervous system. |
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