| Brachionus manjavacas is a common marine zooplankton with good adaptability,which is widely distributed all over the world.Its aging characteristics are considered to be one of the potentially valuable model organisms used for aging biological research.Berberine is an amine compound,commonly known as berberine(Berberine),can activate AMPK,maintain the balance of energy supply and demand,and play a vital role in extending life.Rapamycin is an immunosuppressive agent,it has been widely used in regulation of aging organism.In recent years,these two drugs have attracted much attention in the research of aging biology.In this thesis,the common seawater rotifers were used as the test animals,and four different experiments were designed(the effect of berberine and rapamycin on the lifespan and reproduction of rotifers;effects of combination of berberine and rapamycin on longevity and reproduction of rotifers;the combined effect of berberine and rapamycin on the expression of rotifer heat shock protein;the effect of different concentrations of berberine and rapamycin on the expression of AMPK-m TOR related genes),to explore the effects of berberine and rapamycin on the life span of Brachionus manjavacas,and preliminarily investigate the changes of the expression of HSP genes(HSP40,HSP70 and HSP90)after the two drugs on rotifers at different treatment times and the regulation of upstream and downstream gene expression of AMPK-m TOR.This experiment can provide a theoretical basis for the study of the specific aging mechanism of rotifers.The main research contents and results of this paper are as follows:1.The effect of berberine and rapamycin on the lifespan and reproduction of rotifersDuring the experiment,the density of Nannochloropsis salina to be fed is 3.0×106 cell/ml,the temperature is 25±1℃,the ratio of day to night time is L:D=16:8.1)Berberine concentration experiment: Design a blank control group(ASW),6 concentration gradient groups were designed,which were 5,10,20,30,40 and 50 μM respectively.2)Rotifers is significantly extended,and the pre-reproductive period is prolonged by about 1.7 times.When the concentration is 10 μM,the lifespan of rotifers has the maximum value,the progeny period is prolonged by about 2.3 times,and the number of offspring increases.When the concentration reaches 40 μM,it will have the effect of inhibiting the lifespan of rotifers,but the prereproductive period is not shortened,and it is extended about 84%.The prereproductive period of the 50 μM berberine treatment group prolonged about 44%,and the number of offspring increased significantly.3)Rapamycin concentration experiment: Design a blank control group(ASW),8 concentration gradient groups were designed,respectively 2.0,4.0,6.0,8.0,10.0,12.0,14.0 and 16.0 μM.When the concentration is 2.0,4.0,6.0 μM,the average life span of rotifers is prolonged.When the concentration is 4.0 μM,the rotifers reach the maximum average life span,which is nearly 36% longer than the average life span of the blank control group.When the concentration reaches 12.0 μM,it will have the effect of inhibiting the lifespan of rotifers,and as the concentration gradually increases,the inhibitory effect of the lifespan of rotifers is more obvious.At the concentration of 4.0 μM,the pre-reproductive period was prolonged more than 2 times than the control group.The lowconcentration treatment group had no effect on the number of offspring of rotifers;the high-concentration treatment group would significantly inhibit the number of offspring of rotifers.2.Effects of the combined action of berberine and rapamycin on the lifespan and reproduction of rotifersSet up a blank control group,and then set three rapamycin concentrations of 2.0,4.0 and 6.0 μM,respectively,and interact with the two berberine concentration groups of 5.0 and 10 μM for experiments.The experimental results show that: the combined concentration treatment group with 2.0 μM rapamycin concentration and 5.0 μM berberine and the combined treatment group with 4.0 μM rapamycin concentration and 10.0 μM berberine concentration,the average life span of rotifers was prolonged and sexual maturity time and the pre-reproductive period was prolonged significantly.The average number of rotifers in the combined treatment group of 4.0 μM rapamycin concentration and 10.0 μM berberine concentration was 24.22±0.33 ind.,and the number of offspring increased significantly.3.Effects of the combined action of berberine and rapamycin on the expression of heat shock protein genes in rotifersDifferent concentrations of solutions were treated with 100 rotifers/ml Brachionus manjavacas for 1,3,5,7,9,and 11 days,three in parallel for each group,and the total RNA of each group of samples was collected and extracted,and then the changes in gene expression of HSP40,HSP70 and HSP90 were detected by fluorescent quantitative PCR.The experimental results showed that the HSP40 gene expression of 5th,7th and 9th instar rotifers decreased.The expression level of BMhsp70 gene m RNA decreases during the aging process of rotifers,and the expression level of BMhsp90 gene m RNA first decreases and then increases.Treatment of rotifers at various concentrations for 1 day and 3 days reduced the expression of HSP40 and HSP90 genes,but treatments for 1,3,5,7,and 9 days would instead promote the expression of HSP70 genes in rotifers.4.The effect of different concentrations of berberine and rapamycin on the expression of AMPK-m TOR related genesChanges in the amount of expression of some genes related to AMPKm TOR pathway(PKA,S6 K,TSC1,TSC2,LKB1,ULK1)under different concentration treatment groups(single berberine solution,single rapamycin solution and mixed solution of berberine and rapamycin).The experiment found that the solution treatment groups of various concentrations had no significant effect on the expression of each gene of the newly hatched rotifer B.manjavacas.The main effect of TSC complex is TSC2.On 1,3,5,and 7 days,the expression of TSC2 gene of rotifers in different treatment groups increased,but the expression of TSC1 gene was not affected.The PKA gene and LKB1 gene were not regulated by rapamycin,and the expression levels of the other concentrations decreased at first and then increased.Under different concentrations of berberine,the expression of AMPK gene in rotifers decreased at first and then increased on the first,third,fifth,seventh,and ninth different concentrations of rapamycin,the expression of TOR gene in rotifers decreased first and then increased on the 1,3,5,7 and 9 different concentration treatment groups,the effects of ULK1 gene and S6 K expression in rotifers were more obvious. |
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