Effects And Mechanisms Of Alternate-day Fasting On Irradiation-induced Cognitive Dysfunction In Mice

Objective: To investigate the role of alternate-day fasting in irradiation-induced cognitive dysfunction and the possible mechanisms,and to find effective therapeutic interventions of irradiation-induced brain injury.Methods: 1)Animal grouping: Seven-week-old male c57BL/6J mice(n=45)were randomly divided into Control(Control),irradiation(IR)and irradiation+alternate-day fasting(IR+ADF)groups in accordance with the random number table method.2)Model establishment: The irradiation-induced cognitive dysfunction mouse model was established by using a single dose of whole-brain X-ray given to the mouse by the Siemens Oncor linear accelerator.3)Behavioral test:To observe the effect of alternate-day fasting on the basic condition of irradiation model mice by monitoring the body weight of animals in each group.The spontaneous locomotor activities of the mice were evaluated by the open field test,and the cognitive function of the mice were tested by the novel place recognition and novel object recognition tests.4)Morphological detection: Immunohistochemistry was used to detect the protein expression of GFAP(a marker of astrocyte)and Neu N(a marker of neuron)in the hippocampus tissues of mice in each group,and the morphological changes of individual astrocytes in each group were observed and counted by using the Sholl analysis;The co-localization of NLRP3,VDAC1 and cleaved caspase-3 proteins in astrocyte and microglia was observed with double immunofluorescent labeling.5)Biochemical detection: The expression of NLRP3inflammasome(NLRP3,ASC and caspase-1)and their downstream inflammatory factor(IL-1β and IL-18)were measured by Western Blot in hippocampus of mice in each group.Western Blot was used to analyze the expression changes of autophagy-related proteins(LC3II,ATG5,P62)and VDAC1 protein,and the expression changes of apoptosis-related protein(cleaved caspase-3).Results: 1)Compared with control group,the weight,total distance and average speed in the open field test of the IR group mice did not change significantly;Compared with IR group,the weight of IR+ADF group mice increased slowly(P <0.05),but the total distance and average speed in the open field test did not change significantly.2)Compared with control group,the discrimination ratio of IR group mice in the novel place recognition and new object recognition tests decreased significantly(P < 0.05);Compared with IR group,the discrimination ratio of IR+ADF group mice in the novel place recognition and new object recognition tests increased significantly(P < 0.05).3)Compared with control group,the number of GFAP-positive cells in the hippocampus of IR group was significantly increased(P <0.05),and the expression of GFAP protein was significantly up-regulated(P < 0.05);The intervention of ADF could significantly inhibit this change in IR mice(P < 0.05).4)Compared with control group,the soma of single GFAP cell in the hippocampus of IR group mice became larger and the processes became shorter.Sholl analysis proved that irradiation significantly decreased intersections of astrocytic processes with concentric circles in 24 μm and 27 μm.ADF treatment was useful for inhibiting activated astrocytes(P < 0.05).5)Compared with control group,the protein expression of NLRP3 inflammasome(NLRP3,ASC,caspase-1)and their downstream inflammatory factor(IL-1β and IL-18)in the hippocampus of IR group mice were significantly up-regulated(P < 0.05);The treatment of ADF can significantly down-regulate the expression of NLRP3 inflammasome(NLRP3,ASC,caspase-1)and their downstream inflammatory factor(IL-1β and IL-18)in IR mice(P < 0.05).The results of immunofluorescence showed that NLRP3 colocalized with GFAP.6)Compared with control group,the expression of autophagy markers(LC3II,ATG5)in the hippocampus of IR group decreased,the expression of anti-autophagy protein P62 increased,and the expression of VDAC1 protein decreased(P < 0.05);Compared with IR group,the expression of autophagy marker proteins ATG5 and LC3 II in the hippocampus of IR+ADF group increased,the expression of autophagy substrate P62 decreased,and the expression of VDAC1 protein increased(P < 0.05).Immunofluorescence study showed that VDAC1 was co-localized with GFAP and IBA-1.7)Compared with control group,the mean IOD of Neu N per unit area of hippocampus in IR group decreased significantly(P < 0.05);The treatment of ADF could increase the mean IOD of Neu N per unit area in IR mice(P < 0.05).8)Compared with control group,the expression of cleaved caspase-3 protein in hippocampus of IR group mice was significantly up-regulated(P < 0.05);Compared with IR group,the expression of cleaved caspase-3 protein in hippocampus of IR+ADF group was down-regulated(P < 0.05).The results of immunofluorescence showed that cleaved caspase-3 colocalized with Neu N.Conclusion: Our research demonstrated that ADF promoted the recovery of cognitive dysfunction after cerebral irradiation,and its mechanism may be related to upregulating the expression of VDAC1 protein in the hippocampus,promoting autophagy,inhibiting neuroinflammation mediated by NLRP3 inflammasome in the hippocampus,regulating the neuroinflammatory response of astrocytes,and ultimately reducing hippocampal neuronal loss.