The Influence Of Fluoxetine On MicroRNA Levels From Chronic Unprediced Mild Stress Rats And Mechanism

ObjectivesMajor depression is among the most disabling medical disorders that affects the lives and functioning of many worldwide. Although genetic and familial studies strongly suggest that a neurobiological basis may underlie the pathophysiology of depression, its etiology is still poorly understood. In recent years, there is a growing appreciation that these illnesses can best be conceptualized as genetically influenced disorders of synapses and circuits, rather than simply as deficits or excesses in individual neurotransmitters.In this context, it is noteworthy that microRNAs also regulate brain development, dendritic spine morphology, and neuroplasticy, that is, certain processes that are hypothesized to be associated with Major depression neuropathology. Moreover, miRNA identified as targets for the actions of chronic lithium and VPA are known to play diverse and intriguing roles in brain function.The prime example of a microRNA, which is involved in remodeling of neuronal structures in consequence of synaptic activity, is miRNA-134. This microRNA regulates the size of dendritic spines, which are the post-synaptic sites of excitatory synaptic transmission in rat hippocampal neurons. MiRNA-134 represses the translation of the Limk1-mRNA, a protein kinase that influences dendritic spine development. The second miRNA-143 represses the translation of the BCL-2-mRNA, which has been emerged as a key mediator for protecting against neuron death. The last one is miRNA-34a,One of miRNA-34a’s target mRNA is GRM7, which has been involved in neuron survival.Given that miRNA-134、miRNA-143、miRNA-34a has important roles at multiple steps of synaptic development, and Major depression has been associated with abnormalities in the formation, maturation or plasticity of synapses, we hypothesize that miRNA-134、miRNA-143、miRNA-34a regulates distinct sets of target genes may be involved in the pathophysiology related to depression episodes. Futhermore, in malignant states, circulating miRNAs from the serum or plasma of patients have been shown to be stable and highly predictive kinds of diseases. Therefore, in this study, our goals were to investigate the changes of plasma and brain miRNA-134、miRNA-143、miRNA-34a and their target levels after CUMS and to analyze the effects of fluxetine medication on its levels.Methods:1. Groups.40 adult male Sprague-Dawley rats were housed with ad lib access to food and water, and maintained on a 12-h light/dark cycle(lights on at 7:00 a.m.), at 22℃with low humidity. After 1 week of habituation, Rats were randomly divided into four groups of 10 animals each. The groupⅠ(Con) received saline (1 ml/kg), groupⅡ(FLX) received Fluoxetine (10 mg/kg) + saline (1 ml/kg), groupⅢ(CMS) received CMS+ saline (1 ml/kg) and groupⅣ(CMS+ FLX) received CMS+ Fluoxetine (10 mg/kg) + saline (1 ml/kg). Fluoxetine (10 mg/kg) were intraperitoneally administered with a volume of 10 ml/kg repeatedly. The repeated treatment was performed once a day. For the CMS+ FLX and FLX rats, the administration of antidepressants was from day 1 to day 21. The weight of rats and consumption of saccharose was monitored during the periods of stress as an index.2. CUMS. Rats were subjected to various mild stressors for 3 weeks: cage tilting (45°) for 24 h, damp sawdust (200ml water in a cage) for 24 h, predator sounds record of an adult cat sounds,15 min), swimming in 25℃water for 15 min,24 h of food deprivation immediately followed by 1 h of restricted access to food (5 micropellets), 24 h of water deprivation immediately followed by 1 h exposure to an empty bottle, nip trail for lmin, shaking for 15 min (120rpm rocking bed), immobilising for 1 h (in a 25 cm×8cm cylindrical plastic rodent restrainer) and alterations of the light/dark cycle. Animals were exposed to stressors singly. Stressors were never presented simultan-eously.3. The open field test was performed as usually conducted to measure spontaneous activity in rodents.4. HE and nissi staining analysis neurons in hippocampus of rats. 5. Real time PCR examination of MiR-34a、miR-134、miR-143 in brain and plasma.6. Western blot examination of GRM-7、Limkl、bcl-2 protein. RT PCR examination of GRM-7、Limk1、bcl-2 protein mRNA.7. Pathological analysis and Immunofluorescence examination of GRM-7、Limk1、bcl-2 positive cells’number in neuralstemcell(NSCS).Results1.Effects on sucrose consumption、body weight and open-field testSucrose consumption and body weight、crossing、number of rears and the urine or excrement frequency was measured 2 times during the experimental period.the 3 weeks of chronic stress-induced marked decrease in the sucrose consumption、body weight crossings and rears of open field activity but a increase in the urine or excrement frequency in the stressed rats compared to the non-stressed group(P<0.05). Post hoc analysis showed that no behavior differences were found between control and animals treated with fluoxetine for 3 weeks. In contrast, the behaviors of sucrose preference and open field were different between CMS group and other groups(P<0.05). As that suggests, after 3 weeks of fluoxetine treatment period, behavioral changes of stressed rats were reversed. (P<0.05)2. HE and nissi staining analysis neurons in hippocampus of rats.HE and nissi staining show that, Compared to the rats of the CON group, the rats of the CMS group showed markedly decreased neuron numbers in the hippocampus of rats. (P<0.05)3.Real time PCR examination of MiR-34a、miR-134、miR-143 in brain and plasma.CMS significantly upregulated MiR-34a、miR-134、miR-143 levels in hippocampus but not in cortex and thalamus(p<0.001), an effect prevented by the concomitant administration of Fluoxetine (CMS+Flu) (p<0.001). Administration of Flu alone (under stress-free conditions) show a significant decrease in MiR-34a、miR-134、miR-143 levels in hippocampus. (p<0.001).CMS significantly upregulated MiR-34a levels and downregulated miR-134 in plasma (p<0.05), an effect prevented by the concomitant administration of Fluoxetine (CMS+Flu) (p<0.05). Administration of Flu alone (under stress-free conditions) downregulated MiR-34a levels and upregulated miR-134 in plasma(p<0.05).4.Western blot examination of GRM-7、Limk1、bcl-2 protein. RT PCR examination of GRM-7、Limk1、bcl-2 mRNA.CMS significantly downregulated GRM-7、Limk1、bcl-2 mRNA and protein levels in hippocampus (p<0.001), an effect prevented by the concomitant administration of Fluoxetine (CMS + Flu) (p<0.001). Administration of Flu alone (under stress-free conditions) show a significant increase in GRM-7、Limk1、bcl-2 mRNA and protein levels in hippocampus. (p<0.001).5. Pathological analysis and Immunofluorescence examination of GRM-7、Limk1、bcl-2 positive cells’number in neuralstemcell(NSCS).We found that collocallsation of BRDU-Limk1(% of brdu-ir cell)、BRDU-GRM-7(% of brdu-ir cell) and BRDU- bcl-2 (% of brdu-ir cell) is decreased after CUMS, an effect prevented by the concomitant administration of Fluoxetine (CMS + Flu) (p<0.001). Administration of Flu alone (under stress-free conditions) show a significant increase in BRDU-Limk1(% of brdu-ir cell)、BRDU-GRM-7(% of brdu-ir cell) and BRDU- bcl-2 (% of brdu-ir cell) in hippocampus. (p<0.001).Conclusion1. In agreement with previous studies, the present study confirmed that CMS caused reduction in sucrose consumption, motor and exploratory activities. Furthermore, the stressed animals treated with fluoxetine showed a significant increase in sucrose Consumption, motor and exploratory activities.2. These behavioral changes were accompanied with increased MiR-34a、miR-134、miR-143 levels and decreased GRM-7、Limk1、bcl-2 mRNA and protein levels, respectively. These findings could be indicative of hippocampal MiR-34a、miR-134、miR-143 and their target changes which might in turn contribute to the behavioral changes. 3. Our findings support the possibility that decreased expression of GRM-7、Limk1、bcl-2 contributes to the deficiency in hippocampus neurogenesis that underlie the chronic mild stress. The concomitant administration of Fluoxetine can prevented this effect.4. This findings indicate that plasma miRNA-134 and 34a levels in the CUMS rat may reflect associated pathophysiological processes as well as the behaviors of depressive symptoms. Plasma miRNA-134 and 34a in CUMS may be considered as a potential peripheral marker that can respond to CUMS and associate with effective antidepressant treatment. Whether it represents a pathologic or a clinical response remains to be determined.