A Study On Expression Of α₇-nAChR In Astrocytes And Its Role In Reducing Inflammatory Factor Secretion Induced By Aβ

Alzheimer's disease (AD), a chronic, progressive and degenerative conditionof nerves, is characterized by intracranial deposition ofβ-amyloid protein (Aβ),neurofibrillary tangles and formation of senile plaques. Its chief pathologicalmanifestations include progressive degeneration of cholinergic neurons in cerebralcortex and hippocampus and deteriorated cognitive function. It has been knownthat the development and pathogenesis of AD are associated with cerebral chronicinflammation caused by genetic and environmental factors and progressivedegeneration of cholinergic system, but little is known about its specificetiopathogenesis. As a result, treatment of AD is still very difficult.The development of AD has been known to be related to central inflammationwhich is a response secondary to deposition of Aβand also a major factor for theprogressive degeneration of neuron. The cells involved in the inflammation areastrocytes, microgliocytes and oligodendrocytes which, once stimulated, canrelease a variety of cytokines, chemokines, alexins and their activators. The subsequent non-specific inflammatory cell infiltration can lead to chronicinflammation and harm the nervous system. Deposition of Aβnot only produces adirect toxic effect on nerve cells, but also activates gliacytes and oligodendrocytesin the nervous system to release inflammatory neurogens which thus trigger thesignal pathway to damage to neurons.It has been found that increase in cholinergic level and nicotinic acetylcholinereceptor agonist in the brain may have a therapeutic effect on AD, though thefunctional mechanism is not clear yet. There has been evidence that the cholinergicneurotransmitters released after peripheral vagus nerve being stimulated caninteract withα₇-nAChR receptors in macrophages to inhibit the synthesis andsecretion of inflammatory mediators of macrophages so that the peripheralinflammation is weakened. Although there are no peripheral macrophages in thecentral nervous system, there are a great number of astrocytes and microgliocyteswith similar function of peripheral macrophages, both of which are involved in thecentral inflammation induced by Aβ. Consequently, it is natural for us to askWhether astrocytes containα₇-nAChR or activiation ofα₇-nAChR in astrocytes maylead to reduced central inflammation induced by Aβ. On these grounds, the presentstudy, was designed to investigate the expression ofα₇-nAChR in the hippocampalastrocytes in rats, to observe the expression and secretion of Aβ-inducedinflammatory mediators from hippocampal astrocytes, and to explore the effect ofactivatedα₇-nAChR in hippocampal astrocytes on Aβ-induced inflammation.Methods:1. Culture ofhippocampal slicesNeonate rats of 10 days were used to harvest their hippocampus to be sliced.The rinsed hippocampal slices were put onto membrane inserters for solution-air culture. For the first 3 days, the slices were-cultured in air with 5ï¼…of CO₂, attemperature of 37℃and in solution exchanged once every day. Afterwards, thesolution was exchanged once every 3 days and the temperature lowered to 33℃2. Culture ofhippocampal astrocytesSterilized newborn SD rats were used to harvest their cerebrum promptly andaseptically. The hippocampus was resected and digested with trypsin forcentrifugalization. The cell suspension was prepared after removal of thesupernatant and inoculated into cell culture flasks for culture. After mixedcultivation of the cells for 8-10 days, the adherent cells were collected for furtherculture. The GFAP (glial fibrillary acidic protein) expression was detected throughthe antibody specific to astrocytes in order to assess the purity coefficients ofastrocytes.3. The expression of subunitsα₇-nAChR in hippocampal astrocytes in vivo andvitro.The hippocampus resected from rat cerebrum was frozen and cut into 30μmsections. Double staining of immunofluorescence was used to identify astrocyteswith -GFAP antibodies, and to observe the expression ofα₇-nAChR in astrocytes.The expression ofα₇-nAChR in astrocytes in vitro was assessed and analyzed incultured hippocampal slices and cultured astrocytes, using immunohistochemistry,immunocytochemistry, fluorescence double staining, ligand binding analysis andimmunofluorescence double labeling.4. Effect of activation ofα₇-nAChR in astrocytes on secretion of inflammatorymediators.Astrocytes were isolated and cultured. The supernatant fluids of culturedastrocytes with and withoutα₇-nAChR activated were collected separately. Theliquid phase chip technology was employed to investigate the changes of IL-1, IL-6, TNF (tumor necrosis factor), MIP-1 and RANTES in the supernatant todetermine the role ofα₇-nAChR in astrocytes in reducing the inflammation causedby Aβ.Results:1. Culture of hippocampal siicesThe initial structure of cultured hippocampal slices was obscure, but turned tobe obviously thinner and distinct as the culture time went by. Ten days afterculture when the hippocampal structure became most distinct, MTT stainingshowed blue-purple in almost all the parts of the hippocampal slice, whichindicates the vitality of cultured hippocampal slices.2. Culture and identification of astrocytesThe primary cultured cells were composed mainly of astrocytes, somemicrogliocytes and neuronal cells and a tiny amount of oligodendrocytes. Afterserial subcultivation, the neurons disappeared. Microscopical observation showedthat the purified astrocytes Obtained through vibration grew in single layer withoutoverlapping. Their morphology was irregular, with thick body, numerousprojections and multiple extensions. They tested positive for immunofluorescencedouble labeling by the probe of specific anti-GFAP antibody.3. The expression ofα₇-nAChR in astrocytesThe cells in hippocampal tissue which were positive for anti-GFAP stainingwere also positive for anti-α₇-nAChR staining, while only part of the cells whichwere positive for anti-α₇-nAChR staining were also positive for anti-GFAPstaining. The astrocytes derived from single cell culture in vitro could bind withFITC labeledα-BTX and positive for fluorescence staining. In addition, immunofluorescence double labeling showed that they were also positive foranti-GFAP and anti-α₇-nAChR stainings.4. Correlation between Aβand secretion of inflammatory factors in astrocytesWhen 200 nmol/LAβwas added into the cultured astrocytes, the contents ofIL-1α, IL-1β, IL-6, TNF-α, MIP-1 and RANTES changed differently. Aβ. showedlittle influence on the expression of IL-1α; its effect on IL-1βbecame obvious 48 hlater and the IL-1βincreased by 2.1 times and 6.3 times after 72 h and 96 hrespectively. Likewise, IL-6 increased by 2.1 times and 8 times at 72h and 96 hrespectively. Changes of TNF-a were not significant until 48 h after Aβaction, andTNF-αincreased by 4.7 times after Aβaction for 96 h. The changes of chemokineMIP-1 was the most significant. The increase of MIP-1 began at 8 h and went onin a time-dependent manner. It increased by 12 times at 96 h after Aβaction.RANTES, a chemokine too, did not increase as much as MIP-1. Aβaction for 96 honly resulted in an increase of RANTES by 4.80 times.5. Effect of activatedα₇-nAChR on secretion of inflammatory factors induced byAβfrom astrocytesTheα₇-nAChR in gliacytes pre-activated by nicotine could significantly reducethe Aβ-induced secretion of inflammatory mediators (except IL-1α) from gliacytes.The increases of IL-1β, IL-6, TNF-α, MIP-1 and RANTES were reduced from 6.3,8, 4.7, 12 and 4 times before nicotinic activation to 2.1, 3.0, 1.6, 4.8 and 1.5 timesafter nicotinic activation respectively. The differences before and after activationofα₇-nAChR were statistically significant.Conelusions:1.α₇-nAChR can be normally expressed in astrocytes in vivo and vitro.2. The activatedα₇-nAChR in astrocytes can reduce the Aβinduced release of inflammatory cellular factors IL-1β, IL-6 and TNF-αfrom astrocytes, andweaken the increase in Aβ-induced secretion of cellular chemokines fromastrocytes.