Effect Of Blood-letting On Behaviors And NOS In CUMS Rats

Object:This study was to observe the influence of bloodletting on the behavior change in chronic unpredicted mild stress combined with isolation induced depression model rats as well as the possible nitric oxide synthase mechanism. We hope then generalize the application of bloodletting in clinical practice, provide a new therapy method for depression.Method:33 meal SD rats were chosen to be the experimental subjects according to the open-field test results after one week adaptive breeding. After that, we randomized these rats into control group(n=10) and model group(n=23).The model group rats got the CUMS combined with isolation for 21 days, while the control group had no intervention.20 successful modeling rats were randomized into model group(n=10) and bloodletting group(n=10) on the basis of horizontal motion path of open-field test. We did bloodletting treatment on the 10 rats(10 drops,3 times every week,14 days in total),the model group got no intervention. After the treatment,we evaluated the behavioristics of rats in these 3 groups according to the open filed test; the expression of eNOS, iNOS, nNOS mRNA in hippocampus of rats were tested by RT-PCR;the protein expression level of three NOS were tested by IHC and WB:IHC were used to observe the distribution of positive sediment and check the list of indicators by large area and the average optical density.;WB were used to observe the synthase electrophoresis stripe width, and carries on the gray level analysis.Results:1.The behavioristics test after molding:Compared with control group,the model group’s rats’horizontal motion path, rearing-movement times, explore the center-times and the number of grooming were markedly decreased(P<0.01); the defecation grain number of CUMS rats had no significant difference compaired with control group(P>0.05). Indicating CUMS combined with isolation can copy depression model successfully.2. The behavioristics test after treatment:Compared with control group, the model group’s rats’ horizontal motion path, rearing-movement times, explore the center-times and the number of grooming were declined(P<0.01), the number of grooming of bloodletting group also decreased(P<0.05);.Compared with the model group,the horizontal motion path, rearing-movement times, and explore the center-times of bloodletting group were all elevated(P<0.05). The defecation grain number of each group had no differences(P>0.05). Indicating bloodletting can improve the behavioristics of CUMS model rats.3.The test of the expression of NOS in rats’hippocampus of each group: 1) eNOS RT-PCR test:The expression of eNOS mRNA in hippocampus of model group rats were lower than control group,while bloodletting could increase the expression; IHC test: Compared with control group, the average optical and the expression area of eNOS in hippocampus were decreased in model group and bloodletting group(P<0.01). And the average optical and the expression area of eNOS in hippocampus were partial recoveried comparied with model group(P<0.001); WB test:Compared with control group,the gray value of eNOS/GAPDH in hippocampus of rats in CUMS model group and bloodletting group were decreased(P<0.05).Bloodletting group was still lower than the model group(P<0.01). Indicating bloodletting can elevate positive expression of eNOS of CUMS rats.2) iNOS RT-PCR test:The expression of iNOS mRNA in hippocampus of model group rats were higher than control group, while bloodletting could decrease the expression; IHC test:Compared with control group,the average optical and the expression area of iNOS in hippocampus were increased in model group(P<0.001) and bloodletting group(P<0.05),but the increasing amplitude of bloodletting group is not as much as model group(P<0.001); WB test:Compared with control group,the positive expression of iNOS/GAPDH in hippocampus of CUMS rats were markedly increased(P<0.01). Bloodletting decreased positive expression of iNOS/GAPDH of CUMS rats significantly(P<0.01), but still higher than control group(P<0.05). Indicating bloodletting can reduce the expression of iNOS of CUMS rats.3)nNOS RT-PCR test:The results were as same as the expression of eNOS mRNA; IHC test: Compared with control group,the average optical and the expression area of nNOS in hippocampus were decreased in both model group and bloodletting group rats(P<0.001),there were no differences between model group and bloodletting group(P>0.05). WB test:Compared with control group, the positive expression of nNOS/GAPDH in hippocampus of CUMS rats were decreased(P<0.001). The positive expression of nNOS/GAPDH in hippocampus of CUMS rats were increased in bloodletting group compared with model group(P<0.01), while had no statistical differences with control group(P>0.05). Indicating bloodletting can increase the expression of nNOS in hippocampus of CUMS model rats.Conclusion:1. CUMS combined with isolation can copy animal model of depression successfully, reducing horizontal motion path, rearing-movement times and explore the center-times of rats. Bloodletting can improve the behavioristics of CUMS rats effectively, increasing the motility and exploring ability in open-field test.2. The depressive behavior of rats induced by CUMS combined with isolation was related with the change of the NOS level in hippocampus:the expression of eNOS and nNOS were reduced in hippocampus of CUMS rats, while the expression of iNOS was increased. Bloodletting may play a role in anti-depression by regulating the level of NOS in hippocampus.