Effects Of MANF Factor On The Proliferation And Apoptosis Of A2 Type Reactive Astrocytes

Objective:Astrocytes are the most abundant cell type in the central nervous system(CNS),they cover the entire CNS continuously,they are an integral part of the brain and spinal cord physiology,and perform many functions.Its main function is responsible for controlling neuronal and synaptic homeostasis by regulating ion homeostasis,removing or catabolizing metabolic neurotransmitters,releasing bioactive molecules(such as growth factors or glial transmitters),and playing a key role in maintaining redox status.These functions are essential for normal neuronal development,synapse formation and proper propagation of action potentials.When the CNS is stimulated by injury,astrocytes respond to this injury stimulus,a process known as reactive astrogliosis,which has become a pathological hallmark of structural CNS lesions.This remarkable ability to respond rapidly to the CNS environment allows it to play a key role in CNS defense and suggests that astrocytes are among the earliest cells to respond to CNS injury.This reactive proliferation of astrocytes activates into two types of cells with opposite functions,mainly related to the type of injury they have suffered,the A1 reactive astrocytes with a negative effect and the A2 reactive astrocytes with a positive effect.Mesencephalic astrocyte-derived neurotrophic factor(MANF)is a recently discovered neurotrophic factor,which is a neuroprotective molecule.There is evidence that MANF is critically involved in many neuronal activities.Therefore,the purpose of this experiment was to investigate the effect of MANF on proliferation and apoptosis of A2-reactive astrocytes in the spinal cord.Methods:The spinal cord tissues of SD rats were taken to culture astrocytes.After two weeks of culture,the cultured cells were separated and purified,and then the cultured cells were identified by immunofluorescence staining.Use IL-1β to stimulate astrocytes to prepare A2 RAS;And use the untreated group as a blank control.Immunofluorescence was used to identify A2 RAS.Divide the prepared cells into two groups(control group and MANF group).Treat the cells with different concentrations(0,0.5,1,2μg/ml)of MANF protein and different action times(12,24,36,48h),and use CCK-8 method detects the absorbance at 450 nm.The cells were treated with a certain concentration of MANF protein(1μg/ml)and divided into the control group and the MANF group.The Ed U method was used to verify the effect on the proliferation of A2 RAS.The TUNEL method was used to verify the effect on A2 RAS apoptosis.Result:Immunofluorescence results show that IL-1β can stimulate the transformation of astrocytes to type A2;The results of CCK-8 and Ed U proliferation experiments showed that after MANF protein intervention,the cell proliferation ability was significantly higher than that of the control group(P <0.05);TUNEL apoptosis experiment showed that after the intervention of MANF protein,apoptotic cells were significantly reduced.(P<0.05)Conclusion: MANF protein can promote proliferation and inhibit apoptosis of A2 RAS.