| Objective: Type 2 diabetes mellitus(T2DM)has become a global public health problem due to its high prevalence.T2 DM patients have a high risk of developing mild cognitive impairment and dementia.The mechanism of cognitive impairment in T2 DM patients is unclear,and there is no effective treatment for those disorders.The decrease of the expression of nicotinic acetylcholine receptors(n ACh Rs)in the neurons of brain tissue is closely related to the cognitive dysfunction,and enhancing n ACh Rs function is closely related to cognitive improvement.At present,the expression of n ACh Rs in T2 DM brain and its relationship with cognitive function are not clear.In this study,we selected brain tissues of T2 DM patients,the T2 DM animal model db/db mice,cells co-culture model of lipopolysaccharide(LPS)-induced microglias(rat primary cultured microglia or human microglia cell line HMC3)and neurons(rat primary cultured neuron or human neuroblastoma cell SH-SY5Y)in vitro to explore(1)the expression of n ACh Rs subunit proteins in brain tissue of T2DM;(2)the effects of using ?7 n ACh Rs agonist / antagonist on the spatia learning and memory of db/db mice,as well as on neuroinflammation,insulin resistance and cell energy pathway in mice brain tissue and cultured cells;the molecular mechanism of enhancing the function of ?7 nicotinic acetylcholine receptors to ameliorate spatial learning and memory impairment in diabetic mice.Methods: 1.The alterations in brains of T2DM:(1)Research objects: the samples of hippocampus,frontal and temporal cortex with T2 DM patients and normal human(no-DM,NDM)brains,brains of db/db mouse and db/m mouse.(2)Determination methods: immunofluorescence was used to detect the expression of n ACh R ?7,?4,?2 subunit proteins in the brain tissue of T2 DM patients and controls;Morris water maze(MWM)was used to detect the spatial learning and memory of animals;West boltting(WB)and RT-q PCR were used to detect the expression levels of n ACh R ?7,?4,?2 subunit protein and m RNA in the mouse brain tissue,respectively.2.Intervention experiment combined using of selective ?7 n ACh Rs agonist/antagonist:(1)Research objects: male db/db mice and db/m mice,Cell coculture models of lipopolysaccharide(LPS)-induced microglia(rat primary cultured microglia or human microglia cell line HMC3)and neuron(rat primary cultured neuron or human neuroblastoma cell SH-SY5Y)in vitro.(2)Experimental protocol and treatment: 10 weeks age of db/db mice and db/m mice were given intraperitoneal injection of MLA(a selective ?7 n ACh Rs antagonist,1mg/kg),PNU-282987(a selective ?7 n ACh Rs agonist,0.5mg/kg)and normal saline(NS)once a day for 8 weeks,respectively.The cells co-culture models were treated with MLA+PNU-282987+LPS,PNU-282987+LPS and LPS for 26 hours,respectively.(3)Determination method: fasting blood glucose was measured by fast blood glucose meter;spatial learning and memory was detected by MWM;the protein expression levels of JAK2/p-JAK2,STAT3/p-STAT3,IRS-1/p-IRS-1(Ser636),PI3K/p-PI3K(110),Akt/p-Akt(Thr308),HK2,PFK-2 were detected by WB;the levels of glucose-6-phosphate,fructose-1,6-diphosphate,trans-aconitic acid,fumaric acid,malic acid and ATP in mice hippocampus were detected by high performance liquid-mass spectrometry(HPLCMS);nuclear translocation of NF-?B(p65)were detected by immunofluorescence;levels of TNF-? and IL-1? in culture medium were detected by ELISA Kit;the mitochondrial membrane potential were measured by JC-1 Kit.Results: 1.The alterations in the brains of T2DM:(1)the levels of n ACh R ?7 and ?4 subunit protein were significantly decreased in the hippocampus of T2 DM patients and db/db mice comparison to controls.(2)The spatial learning and memory of db/db mice were significantly worse than that of db/m mice.2.Intervention experiment combined using of selective ?7 n ACh Rs agonist/antagonist:(1)Animal experiments: in db/db mice,1)the protein levels of p-JAK2,p-STAT3 were significantly increased,the nuclear translocation of NF-?B were reduced,the protein levels of TNF-?,IL-1β were significantly decreased,the protein levels of p-PI3K(110),p-Akt(Thr308)were significantly increased in the mice brain with PNU-282987 treated group,as compared to control group;While those in the mice brain with MLA or with NS treated group were no significant difference as compared to control group.2)the protein levels of pIRS-1(Ser636)were significantly decreased,the protein levels of p-PI3K(110),p-Akt(Thr308)were significantly increased in the mice brain with PNU-282987 treated group,as compared to control group;While those in the mice brain with MLA or with NS treated group were no significant difference as compared to control group.3)In the hippocampus of db/db mice,the protein levels of HK2 and PFK-2 and the levels of glucose-6-phosphate,fructose-1,6-diphosphate,trans aconitic acid,fumaric acid,malic acid and ATP in PNU-282987 treated group were significantly higher than those in MLA or in NS treated group.4)In db/db mice,the spatial learning and memory of PNU-282987 treated group were significantly better than that of other treated groups.(2)Cell experiments: the co-culture model of rat hippocampal neurons and microglia,SH-SY5 Y and HMC3 cells were showed that the protein levels of p-JAK2 and p-STAT3 were significantly increased,the protein levels of TNF-? and IL-1β were significantly decreased,the protein levels of p-PI3K(110)and p-Akt(Thr308)were significantly increased,the protein levels of HK2 and PFK-2 were significantly increased,and mitochondrial membrane potential were significantly higher in the PNU-282987 treated group as compared to the LPS treated group.These effects were eliminated with MLA pretreated and then with PNU-282987 treated.Conclusion: 1.Lowered levels of n ACh R ?7 and ?4 subunit in the hippocampus of T2 DM patients and experimental animals;2.Enhancing ?7 n ACh Rs function reduces inflammatory response and improves insulin sensitivity by the activation of the cholinergic anti-inflammatory,and consequently improves energy metabolism of hippocampal neurons in db/db mice and in vitro co-cultured neurons;3.Enhancing ?7 n ACh Rs function ameliorates spatial learning memory impairment via improve the energy metabolism in hippocampus of db/db mice;4.The lower cognitive function of T2 DM may be related to the lower expression of n ACh Rs in hippocampus. |
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