Cerebral IL-1β,IL-6 Interacting With The Surppressive Regulatory Molecules In BALB/c Mice With Brain Asymmetry

Background and Objective:Brain asymmetry is thought to be one of the biological characteristics in humans or non-human primates, which is evidenced on the basis of anatomical, neurochemical and functional data. Thus far, many studies have indicated that brain asymmetry could involve in the modulation of immune reactivity mediated by HPA axis and sympathetic nervous system. Acting as important messengers, cytokines in neuro-immuno-endocrine network are also revealed to depend on the brain lateralization. A good case in point is that cytokines in cortex and hippocampus exhibit asymmetric. It is well known that cytokines interacting with relevant regulatory molecules play a potent role in the cross-talk of the neuro-immuno-endocrine system. A family of cytokine-inducible proteins named as suppressors of cytokine signaling (SOCS) appears to form part of a negative feedback loop in inhibition of the JAK/STAT signaling cascade, in that cytokine-triggered signal pathway could be tightly controlled to avoid detrimental consequences of excessive stimulation. Moreover, the suppressive molecules such as TGF-β1 also contribute to the balance of the cytokine network. Here, concerning the important correlation of cytokines with brain lateralization, in present study, we aim to investigate the cytokine (mainly refers to IL-1β, IL-6) levels in the subcortical structures related to brain asymmetry; and to explore the SOCS-3, TGF-β1 expression and distribution in brain tissue, therefore, the integrity between cytokines and relevant suppressive regulatory molecules in BALB/c mice with brain asymmetry could be further elucidated. Materials and Methods:Female BALB/c mice (4-week-old) were used. By paw preference test,right-pawed, left-pawed and ambidextrous mice were classified into three groups according to their RPE (right paw entry) score. Mice were assigned to right-pawed group if RPE was equal to or greater than 30, to left-pawed group if RPE was equal to or less than 20, and to ambidextrous group if the score was between 21 and 29. Subsequently, each experimental mouse was challenged by 0.5ug/0.5mL lipopolysaccharide (LPS, Escherichia coli,026:B6, Sigma) intraperitoneally, and every control was received an equivalent injection of 0.5mL sterile saline. The mice were sacrificed by decapitation 2h after the LPS injection. Trunk blood samples were collected and the prepared plasma was used to detect the levels of corticosterone by EIA. Then cortice, hippocampi and hypothalami were rapidly separated on ice and homogenized for detection of IL-ip, IL-6 levels by ELISA. Meantime, another portion of cortex, hippocampus, and hypothalamus of BALB/c mice with brain asymmetry were selected to isolate the total RNA and then to determine SOCS-3, TGF-pi mRNA by RT-PCR. Their protein levels were investigated by Western blot as well. As for the rest mice, the whole brains were taken out and embedded in paraffin, finally, immunohistochemistry SABC were performed on the paraffin sections for detection of SOCS-3, TGF-pi expression and distribution. Results:1. Plasma corticosterone levels in BALB/c mice with brain asymmetry: In physiological condition, left-pawed, right-pawed and ambidextrous mice showed almost at the same level. As to the LPS-challenged mice, corticosterone levels were elevated greatly, particularly in right-pawed and ambidextrous. Levels of the two groups were higher than that of left-pawed (P<0.05).2. IL-ip, IL-6 levels of hypothalamus in BALB/c mice with brain asymmetry: (D In hypothalamus, IL-ip of the normal control were at almost the same levelin right-pawed and left-pawed groups, both showed higher level than ambidextrous. When treated with LPS, right-pawed, left-pawed were elevated and higher than ambidextrous (PO.05).? As such, for IL-6, the result revealed higher level in left-pawed mice if compared with right-pawed mice, and there was significance difference in statistics(PO.05). After challenged by LPS, all three groups were upregulated and showed higher level than their corresponding normal control (PO.05).3. SOCS-3 > TGF-p 1 expression in brain of lateralized BALB/c mice? Concerning the expression of SOCS-3 mRNA in cortex and hippocampus, in basicline, SOCS-3 expression was low and exhibited lower in left-side than in right-side of such regions for left-pawed mice (i><0.05). Moreover, left-pawed was at lower expression than right-pawed in left-side of cortex and hippocampus (PO.05). In hypothalamus, left-pawed showed lower level than right-pawed mice (PO.05). In turn, LPS could elicit the expression of SOCS-3 in these three regions, but no significant differences were found among left-pawed, right-pawed and ambidextrous mice. For determination of SOCS-3 protein, the results of Western blot indicated SOCS-3 expression was low and exhibited lower in left-side than in right-side of such regions for left-pawed mice, left-pawed was at lower expression than right-pawed in left-side of cortex and hippocampus as well. In hypothalamus, the same trend of SOCS-3 protein as mRNA level was observed. However, that LPS notably up-regulated the levels of SOCS-3 protein was only found in cortex.?TGF-P 1 mRNA expression of cortex in left-side was higher than right-side in left-pawed mice, which revealed a significance in statistics (PO.05); Likewise, the same trend was also found in right-pawed while no difference was indicated statistically. Comparing left-pawed with right-pawed, there was a higher expression of left-side of the left-pawed (PO.05). In hippocampus and hypothalamus, no differences of lateralized mice were found. Furthermore, LPS did not alter a lot of the expression of TGF-p 1. As to the protein level, Western blot revealed almost the coincident results as mRNA, but no significance was shown.4. The distribution of SOCS-3, TGF-p 1 in brain of BALB/c mice: Results of immunohistochemistry showed SOCS-3 and TGF-P 1 existed1 in brain extensively but lowly expressed in physiological condition. As expected, LPS significantly promoted the positive signals of SOCS-3 but a little of TGF-P 1. Concerning their location, most of positive signals were distributed in the nuclear of neurons except for a little in the cytoplasma. In other resident cells including glia cells and vascular endothelia,positive results could be discovered as well. In neocortex, extremely conspicuous positive signal was found particularly in external granular layer. As for hippocampus, both pyramidal neurons and granule cells in the dentate gyrus were stained in brown color that referred to positive results. Conclusion:1. Brain asymmetry can influence the distribution of IL-ip, IL-6 in hypothalamus and their activities if subjected to LPS. That is, right-pawed and left-pawed mice show higher IL-1(3 than ambidextrous; For IL-6, left-pawed mice have higher level if compared with right-pawed mice. With the challenge by LPS, all three groups represent different reactivities to LPS.2. The heterogeneities of IL-ip, IL-6 expressed in brain are tightly correlated with the asymmetric function of HPA axis. The activity and reactivity of HPA axis in left-pawed mice is less sensitive than right-pawed mice.3. The suppressive molecules including SOCS-3, TGF-pi are distributed extensively in brain of BALB/c mice, particularly in cortex and hippocampus. SOCS-3, TGF-pi expression also depends on brain asymmetry, which counterbalances the effects of asymmetrically distributed cytokines.4. The asymmetric expression of SOCS-3 and TGF-pi in such three regions attributes to the asymmetrically distributed cytokines in part. The integrity of IL-ip, IL-6 and the suppressive regulatory molecules plays a potent role in the control of brain asymmetry over immune functions.5. The left-handedness(left-pawed) and right-handedness(ritht-pawed) population show different characteristics in immunoendocrine functions, and the right-handedness are more active to maintain homeostasis if confronted with various stressors. On the ground of our study, it is speculated that the left-handedness population prone to immune disorders may partly account for some insufficiency of cytokine network and neuroendocrine loop.