Experimental Study Of Galectin-1 In Improving Neuroinflammation And Neuron Damage In Alzheimer’s Disease

Background:Alzheimer’s disease（AD）is a chronic neurodegenerative disease which is high incidence in the elderly.The pathological features of AD are deposition ofβ-amyloid protein（Aβ）,neurofibrillary tangles and neurons loss in the brain.Although AD has been studied for more than 100 years,the specific mechanisms that lead to its neuronal damage remain unclear.With the progress of research,numerous experimental evidences have shown that central nervous inflammation is an important pathway to mediate neuron damage in AD.Therefore,limiting AD-related neuroinflammation will be conducive to alleviating neuron loss and alleviating AD symptomsMicroglia is the innate immune cells of the central nervous system（CNS）.Under physiological condition,microglia cells are in the"resting state",play the functions of neuroimmunity and monitoring,and maintain the stability of the central environment.Under the stimulation of pathological factors such as Aβdeposition and craniocerebral injury,microglia can be activated rapidly from the"resting state"to the"active state"to perform immune defense functions.Activated microglia shows opposite functional states:the"classically activated"（M1）and the"alternatively activated"（M2）states.M1 microglia release inflammatory cytokines such as interleukin-1β（IL-1β）to mediate neuronal damage,while M2 microglia release anti-inflammatory cytokines such as brain-derived neurotrophic factor（BDNF）to combat inflammatory spread and promote neuronal repair and survival.Obviously,the induction of microglia to M2-type differentiation and activation will be beneficial to the improvement of central nervous inflammation and the reduction of AD-related neuronal damage.Galectin-1（Gal-1）belongs to the galactosin-lectin family of endogenous lectins.It has a characteristic sugar recognition domain and can bind to sugar or lactose ligands commonly found in cell membrane/nucleus to participate in the regulation of cell proliferation,differentiation,chemotaxis,apoptosis and cellular immune function.Studies have shown that Gal-1 can selectively promote the apoptosis of T-helper cells 1 and 17,reduce the release of pro-inflammatory cytokines from T-cells,and induce the production of anti-inflammatory cytokine IL-10,thus alleviating the spread of peripheral inflammation.Importantly,researchers have reported that intracerebral injection of recombinant Gal-1 alleviates neuronal injury and improves CNS inflammation,accompanied by a decrease in the number of M1-type microglia,suggesting that the central anti-inflammatory effect of Gal-1 may be related to microglia activation and transformation.Based on the above research,we suspect:Gal-1 May participate in the microglia activation regulation,in the role of Gal-1,increased to M2 activated microglial cells,M1 microglia function relatively weak,which makes the brain proinflammatory factor release and anti-inflammatory factors increase,so as to alleviate the inflammation of the central nervous,reducing neuron injury,improve symptoms AD.To verify the research hypothesis,this research with Aβ_1-42 segments of toxicity research animal model of brain injection preparation of AD brain injection Gal-1 to intervene at the same time,the detection of different concentrations under the action of Gal-1 animal cognition function,damage of neurons and microglia activation,central inflammatory cytokines and inflammation related NF-κB and PPARγsignal protein expression changes,discusses Gal-1 improve AD possible mechanisms of neuron injury.Research methods:SD rats were divided into five groups.There are 10 animals in each group,half of them are male and other are female:（1）Control group;（2）AD model group;（3）Gal-1 L experimental group;（4）Gal-1 M experimental group;（5）Gal-1 H experimental group.The control group was given A single injection of PBS,and the AD model group,the Gal-1 L group,the Gal-1 M group and the Gal-1 H group were given A single injection of Aβ_1-42 fragment 10μg.Subsequently,5μL of normal saline was injected into the brain of control group and AD model group,and 1μg,5μg and 10μg of Gal-1 were injected into the brain of Gal-1 L group,Gal-1 M group and Gal-1 H group,respectively.Normal saline and Gal-1 were injected into the brain for 14 consecutive days,once a day.After the intervention,in Morris water maze experiment evaluation rat spatial learning ability,Nissl staining and TUNEL staining to detect neuronal injury and apoptosis of rats,real-time PCR detection of rat hippocampus M1 and M2 microglia markers（CCL2,CCL3,Arg1,Ym1）,enzyme-linked immunosorbent determination in the rat hippocampus proinflammatory factor（IL–1β,TNF-α）and anti-inflammatory factor（IL-4,IL-10）.NF-κB pro-inflammatory signaling pathway related proteins（NF-κB,IκB kinase）and PPARγanti-inflammatory signaling proteins（PPARγ）in rat hippocampus were detected by Western Blot.Results:1.Morris water maze experiment results show that the injection of Aβinto the brain of rats can significantly damage their spatial learning ability,while high concentration of Gal-1 can significantly alleviate the learning and memory damage caused by Aβ,suggesting that Gal-1 can improve the cognitive impairment of AD rats;2.The results of Nissl staining showed that the hippocampal neurons of the control animals were deeply stained and filled with blue-purple Nissl bodies,while the neurons of the AD model animals were light stained and the intracellular Nissl bodies were rare,showing significant neuronal damage.Compared with the AD group,the number of nissl body in the Gal-1 M group and the Gal-1 H group were increased and the neuronal staining was more deeper,suggesting that the intervention of Gal-1 significantly improved the neuronal damage induced by Aβ,while the neuronal protective effect in the Gal-1 L group was not shown.3.The loss of hippocampal neuronal apoptosis was detected by TUNEL assay.The results showed that AD model showed significant neuronal apoptosis.Compared with the model group,neuronal apoptosis was reduced in the Gal-1 M group and Gal-1 H group,which confirmed that Gal-1 could reduce the loss of AD-related neuronal damage,while no improvement of neuronal apoptosis was shown in the Gal-1 L group.4.The results of real time PCR showed that the expression of M1 microglia markers,CCL2 and CCL3 in hippocampus of rats increased under the action of Aβ,while the expression of M2 microglia markers,Arg1 and Ym1 decreased.The expression of Arg1and Ym1 was up regulated under the action of high concentration Gal-1.5.The data of ELISA experiment showed that the expressions of IL-1βand TNF-αare increased in hippocampus of AD model rats,and the expressions of IL-4 and IL-10 are reduced.However,the above changes induced by Aβcan be alleviated by the treatment of high concentration Gal-1.The results suggested that aβcaused central inflammation imbalance in the brain of animals,and Gal-1 could improve the imbalance of inflammation to some extent.6.The results of WB showed that NF-κB and IκB kinase are increased in the brain of AD model rats,while down regulated PPAR-γexpression in PPARγanti-inflammatory signaling pathway.The above conditions can also be reversed under the action of Gal-1,suggesting that Gal-1 may play an important role in improving the pivot validation through NF-κB Pro inflammatory signaling pathway and PPARγanti-inflammatory signaling pathway.Conclusion:1.Gal-1 improved AD-related cognitive memory impairment and neuron damage loss,showing a clear anti-AD effect;2.Gal-1 inhibited the activation of M2-type microglia induced by AD,decreased the expression of IL-1βand TNF-α,increased the expression of IL-4 and IL-10,which had a significant effect of improving central nervous inflammation.3.Gal-1 improves the activation and differentiation of AD-related microglia,which may be achieved by regulating NF-κB,IκB and PPARγsignaling pathways.