Mechanism Of Neuropeptide Y System In The Effects Of High Dose Caffeine And Theobromine On Anxious Behavior In Rats

Object:We designed this experiment to observe the effects of high dose Caffeine and Theobromine on the anxiety behavior in rats, and to explore the molecular mechanism of neuropeptide Y (NPY) in the potential effects.Methods:A total of 30 male Wistar rats, with body weight range from 260 to 300 g,were randomizedly divided into 5 groups on average. The 5 groups were as follows:NS (control), 30mg/kg Caffeine,60mg/kg Caffeine,30mg/kg Theobromine,60mg/kg Theobromine,. The agentia were intragastric administration. We used the elevated plus-maze(EPM) to test the anxiety behavior of rats 30mins after agentia administrated. To observe the effect of NS, Caffeine and Theobromine on the anxiety behavior. And we measured the level of NPY, Y1 and Y2 mRNA through fluorescent quantitation PCR. The results are compared with control group and we develop the role of NPY system in the effects of high dose Caffeine and Theobromine on the anxiety behavior in rats.Results:1. Locomotor activity results:60mg/kg Caffeine administration decreased the locomotor activity in rats (F=21.774, P=0.001) while 30mg/kg Theobromine administration increase the locomotor activity in rats (F=6.869, P=0.026).60mg/kg Caffeine administration shows decreased locomotor activity compared with 30mg/kg Caffeine (F=15.168, P=0.003). 60mg/kg Theobromine administration shows decreased locomotor activity compared with 30mg/kg Theobromine (F=12.331, P=0.006). Each group of 30mg/kg Caffeine (F=1.244, P=0.291)and 60mg/kg Theobromine (F=1.553, P=0.241)administration shows no significant impact on locomotor activity and no significant difference were found between the two groups (F=0.023, P=0.882).2. Anxiety behavior results in rats:The open arm entries %(F=5.336, P=0.044) and open arm time % (F=7.750, P=0.019) were decreased in 60mg/kg Caffeine administration group. The open arm entries% (F=5.022, P=0.049) are decreased in 60mg/kg Theobromine.60mg/kg Caffeine group shows significant less open arm entries% (F=7.569, P=0.020) than 30mg/kg Caffeine group. The open arm entries% (F=24.587, P=0.001) and open arm time % (F=17.065, P=0.002) of 60mg/kg Theobromine were lower than 30mg/kg Theobromine.3. fluorescent quantitation PCR results shows that when compared with the control group (NPY/GAPDH), NPY mRNA level are increased in 60mg/kg Caffeine group (F=13.961, P=0.004) and are decreased in 30mg/kg Theobromine group (F=7.597, P=0.020). NPY mRNA level are higher in 60mg/kg Caffeine group than in 30mg/kg Caffeine group (F=7.169, P=0.023). Y1 mRNA level are increased in 30mg/kg Caffeine group (F=32.963, P=0.000) and in 30mg/kg Theobromine group (F=10.541, P=0.009). It shows increased tendency of Y1 mRNA level in 60mg/kg Theobromine group (F=4.227, P=0.067)and Y2 mRNA level in 30mg/kg Theobromine group (F=4.532, P=0.059).Conclusion:This paper described that the effects of high dose Caffeine and Theobromine on anxious behavior in rats. We also explained the specific mechanism of the effects mentioned above through molecular biological methods.In the past, high dose caffeine was reported to have anxigenic effects. But the mechanism of it hasn't been discovered completely. Effects of high dose Theobromine on anxious behavior has not even been reported. Firstly, we studied the behavioral effects through EPM which was most widely used animal model for anxiety and we explained the specific effects of the two substance on anxious behavioral in rats. Secondly, we used the fluorescent quantitation PCR, an advanced molecular biological technology, to test the expression of NPY mRNA and Yl mRNA and Y2 mRNA and we found the molecular biological difference in Caffeine and Theobromine on different anxious effects. We explained the role of NPY system in high dose Caffeine and Theobromine on anxious behavior in rats first time.We have found:High dose Caffeine has anxigenic effects with dose-effects relationship. In addition, it decreases the locomotion activity of rats. We also found it can increase the expression of NPY mRNA and Yl mRNA with dose-effects relationship. But it has no effects on Y2 mRNA expression.High dose Theobromine has anxiolytic effects, and it could increase the locomotion activity of rats. But too high dose Theobromine makes rats anxious. We also found high dose Theobromine can decrease the expression of NPY mNRA which mediated by increasing the expression of Y2 mRNA. But too high dose Theobromine increased the expression of NPY mRNA and Y1 mRNA, and has no effects on Y2 mRNA. In summary, Caffeine and Theobromine are widely consumed in the world. The effects of them on anxious behavior and the specific mechanism of that are important. But the more researches are still needed. Our research has contributions to understand the effects of high dose Caffeine and Theobromine on anxious behavior and the specific mechanism of them. The results of this study are expected to guide the consuming of Caffeine and Theobromine, and the development of anxiolytic agents targeting on NPY system.