| Background:Depressive disorder is an emotional disorder, which the main clinical features are significant and long-lasting depression. The World Health Organization estimates that depressive disorder will become the second largest human disabling disease by2020. Although many hypotheses have been proposed the etiology of depressive disorder, the etiology and pathogenesis of depressive disorder remains unclear. A large number of studies show that cytokines play an important role in the occurrence and development process of the depressive disorder.The hippocampus is an important part of the limbic system, which is an important structural basis of emotion, learning, memory and other Central nervous system activity. Many imaging and autopsy studies have shown that patients with depressive disorder and animal models of depressive disorder have reduced of the hippocampal volume, reduced in the number of neurons and changed in the structure of the dendrites of hippocampal neurons. The cytoskeleton is the basis of the brain structure and the function of the hippocampal neurons. Microtubules and microfilaments are the main component of the neuronal cytoskeleton. In different stimulation both inside and outside of cells occur rapid polymerization/depolymerization, which due to the polar structure and the dynamic instability of microfilaments and microtubules, and maintain the structure and function of the cytoskeleton by the conversion of polymerization and depolymerization.Microtubule system is an architecture backbone of cytoskeleton. Microtubules are formed by the polymerization of tubulin, a heterodimer of two subunits designated a and ?,which molecular weight is about55kD. Normal tubulin exists in the form of a heterodimer, and is both linked to the manner polymerized to form microtubules fibril. Microfilaments also are ubiquitous in eukaryotic cell by solid-shaped fiber, which exists in two forms, the monomer (G-actin)and fibrous (F-actin). Microfilament have kinds of functions with contraction, support, non-muscle movement and information conduction.Cytokines are bioactive signaling molecules secreted by immunocompetent cells of the regulations of the immune response. Cytokines divided into inflammatory cytokines and anti-inflammatory cytokines according to the different role of cytokines in the inflammatory response. Inflammatory cytokines involved in the reaction process of the inflammation, such as IL-1, IL-6and IFN-y and TNF-alpha. Anti-inflammatory cytokines have anti-inflammatory functions by inhibiting the cell activation and inflammatory regulon generated to suppress the immune response, such as IL-4, IL-10, IL-13and other cytokines. Peripheral cytokines are produced by immune activation when the body's immune system is activated by physical stress, psychological stress and immune intervention, which increases monocyte-macrophage system activities, and elevates IL-1, IL-6, TNF-a and other proinfiammatory cytokines level. The study found that various cytokines exist in the central nervous system. Astrocytes and microglial cells produce a variety of cytokines under various stress and injury stimulus, such as IL-1, IL-6, TNF-a and of IFN-y, and cause a series of physiological and biochemical changes of the body. Genetic, animal behavior and clinical studies have shown that is closely related to with serum TNF-a, IL-1and inflammatory cytokine content changes and increase the risk of depressive disorder, cognitive impairment, and reduce the treatment effect of depressive disorder. The clinical studies about chronic non-infectious autoimmune disease, such as multiple sclerosis, rheumatoid arthritis, allergic diseases, are often accompanied by depressive symptoms. Depression patients increase the level of plasma IL-1?, IL-6and IFN-?, and increase IL-1? in the cerebrospinal fluid of major depressive disorder patients. Depression severity is related to the increase of IL-1? in the cerebrospinal fluid of depression patients. The relationship between cytokines and animal depressive disorder studies found that lipopolysaccharide (LPS) stimulation increases production and secretion of proinfiammatory cytokines such as IL-1, IL-6, TNF-a. In the immune activation model of LPS, LPS stimulation makes the experimental rats show reduced activity, loss of appetite, exploration the surroundings of declining interest, diminished sex drive, and increased temperature. Antidepressant treatment effectively inhibits LPS-induced rat thrill loss.In short, cytokines play an important role in the formation and aggravation of many physical diseases and depressive disorder pathophysiological basis. Animal experiments and clinical studies between cytokines and depressive disorder have been clearly demonstrated that cytokines have an important role in the development of depressive disorder; however, cytokines cause depression-like behavior, what is the molecular mechanism of its occurrence? Whether cytokines affect hippocampal neuronal cytoskeleton, and abnormal changes of hippocampal structure and function caused occurrence of depressive symptoms? What are the signal transduction mechanisms? The studies show that the relationship of cytokines effects hippocampal neuronal cytoskeleton and its signal transduction mechanism. Therefore, in order to clear the relationship preinflammatory cytokines, IL-1? and hippocampal neurons, to explore IL-1? effects morphological changes of hippocampal neuronal cytoskeleton, and microtubule and microfilament. To understand the pathogenesis of depressive disorder and to discover new depressive disorder therapeutic targets have important significances.ObjectiverTo investigate effect of IL-1? on hippocampal neuron survival and neuron morphology and a-tubulin and F-actin cytoskeletal protein expression change after IL-1? or IL-lra treatment primary cultured hippocampal neurons.Methods:(1) Primary hippocampal neuron culture, purification and identification;(2) Experimental groups:(i) control group:control+saline;(ii)0.11ng/ml experimental group:IL-1? concentration was0.01ng/ml;(iii)0.1ng/ml experimental group:IL-lp treatment concentration was0.1ng/ml;(iv) lng/ml experimental group:IL-1? concentration was ng/ml;(v)10ng/ml experimental group:IL-1? concentration was10ng/ml;(vi)20ng/ml experimental group:IL-1? concentration was20ng/ml;(vii)50ng/ml experimental group:IL-1? treatment concentration was50ng/ml;(viii)100ng/ml experimental group:IL-1? treatment concentration was100ng/ml.(3) Different concentrations of IL-1? treated hippocampal neurons for12hours after culturing6days, and then detected neuronal activity.(4) Cell immunochemical detected hippocampal neuron morphology.(5)The application of immunofluorescence staining detected distribution and arrangement of IL-1? on hippocampal neurons cytoskeletal, a-tubulin and F-actin.(6) Western blot detected expression changes of IL-1? on hippocampal neurons cytoskeletal a-tubulin and F-actin.Results:(1)IL-1? at a concentration of0.01,0.1and lng/ml promote neuronal development and growth. IL-1? at concentration of0.1ng/ml was the most significant role, which showed neuronal dendrites elongation, branchs and spines increase; Hippocampal neurons were inhibited gradually with raising the concentration of IL-1?. The branchs of spinous decrease, shorten, dendritic atrophy, even dendritic fracture, spinous process disappear and neuronal death.When the concentration of IL-1? at0.01,0.1and lng/ml, the second and the third projections increased, a number of neurons protrusions compared with the control group showed no significant difference (P>0.05), the second and the third projections changes compared with the control group has significantly difference (P<0.01); when IL-1? concentration increased from10to 100ng/ml with a number of protrusions reduced, the concentration of IL-1? at20,50,100ng/ml compared with control group has a statistically significant difference (P<0.05), the second and the third projections change with the control group has significantly difference (P<0.01). IL-1ra can inhibit IL-1? role.(2) When IL-1(3concentration at0.01,0.1and1ng/ml had obvious promoting effect on hippocampal neuronal activity. IL-1? at0.1ng/ml has the highest role on hippocampal neuron activity (P<0.01). IL-1? on hippocampal neuron activity was inhibited gradually with the concentration of IL-1?increasing (P<0.01).(3) IL-6content increased gradually in culturing supernatant with IL-1? concentration increasing. IL-6content was significant difference compared with the control group of IL-1? concentration at20,50and100ng/ml(P<0.05); TNF-a content showed a rising trend with IL-1? concentration increasing in culturing supernatant. TNF-a content significant difference compared with the control group of IL-1? concentration at10,20,50and100ng/ml (P<0.01).BDNF secretion increased gradually at low concentrations of IL-1?, which was significant statistical difference compared with the control group (P<0.01). BDNF secretion significantly decreased with IL-1? concentration increased was significant difference compared with the control group (P<0.01).(4)Immunofluorescence staining showed that neurons protruding quantity increased compared with the control group when IL-1? concentration at0.01,0.1,1ng/ml treated hippocampal neurons.IL-1? treating concentration at0. lng/ml increased obviously. The first neurons spines were not significantly increased, and the second, the third neurons spines significantly; morphological features of hippocampal neurons changed when IL-1? concentration increased to10,20,50ng/ml. Neurons spines started to decrease, the second and the third protrusions become shorter small, or even spines disappeared.(5)IL-1? on hippocampal neurons cytoskeletal protein a-tubulin and F-actin expression experimental results shows that IL-1? concentration at0.01ng/ml a-tubulin and F-actin expression increased compared with the control group.0.1ng/ml protein expression comparison with0.01ng/ml, and cytoskeletal protein expression comparison with the control group were statistically significant difference at0.1ng/ml of IL-1? (P<0.01). IL-1? concentration at lng/ml cytoskeletal protein expression was statistically significant difference compared with control group (P<0.05). With the higher concentration of IL-1?, cytoskeletal protein expression compared with the control group decreased. The20,50,100ng/ml treatment group compared with control group were statistically significant difference (P<0.05).Conclusion:Low concentrations of IL-1(3promoteed survival of hippocampus neurons and the dendritics spines of growth, development, and increased IL-6, TNF-a, BDNF secretion, the cytoskeletal protein a-tubulin and F-actin expression; high concentrations of IL-1? inhibited the survival of hippocampal neurons and the dendritics spines of growth, development and increased IL-6, TNF-a and inhibited the secretion of BDNF, and reduceed expression of the cytoskeletal protein a-tubulin and F-actin. |
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