| ObjectiveTo explore the role of microglial polarization mediated by SIRT1/HMGB1/NF-κB signaling pathway on learning and memory impairment induced by perinatal decabromodiphenyl ether(BDE-209)exposure,and neuroprotective effects of melatonin(MT),hoping to provide experimental evidence for prevention and elucidating the mechanisms of BDE-209 neurotoxicity.MethodsThree-month-old Sprague-Dewley(SD)rats(female 40,male 20)were mated at 2:1 ratio,and pregnant rats were randomly divided into five groups:control,30 mg/kg BDE-209,100 mg/kg BDE-209,MT(10 mg/kg),and MT+100mg/kg BDE-209(MT+BDE-209),MT was administrated via intraperitoneal injection 2 hours before BDE-209 intragastric administration from gestational day 15(GD15)to delectation(postnatal day 21,PND21),the control group treated with normal saline and corn oil instead.Spontaneous behavior and learning and memory ability were evaluated using open field at PND28 and Morris water maze test during PND30 ~ 35.The offspring rats’ brain were collected at PND21 and PND35,hippocampal neuron impairment was estimated by Nissl stainng and TUNEL apoptosis assay;microglial polarization was analyzed by flow cytometry,cytokines such as Tumor necrosis factor alpha(TNF-α)、Interleukin-1β(IL-1β)、Arginase-1(Arg-1),and Interleukin-10(IL-10)contents and m RNA level was detected by ELISA and Real-Time PCR respectively,Sirtuins 1(SIRT1)and Ionized calcium binding adapter molecule 1(Iba1)protein level were evaluated by immunofluorescence staining,finally,SIRT1 、 HMGB1 、 NF-κB and phosphorylated-p65 prootein expression and cytoplasmic/nuclear distribution by western blot.Results1.BDE-209 or MT treatment affected spontaneous behavior and learning and memory of offspring rats.Open field test showed,the central area time and distance of BDE-209-exposed rats were significantly lower than the control group(P<0.05).Compared with the 100 mg/kg BDE-209 group,the central area time and distance in the MT+BDE-209 group were increased(P<0.05),but there was no significant difference in the total distance among the groups.On the 1st-2nd day of Morris water maze,there was no significant difference in escape latency and swimming distance among the groups(day1 F=1.486,P=0.209;day2 F=2.015,P=0.095).On the 3rd to 5th day of training,the escape latency of offspring in 30 mg/kg and 100 mg/kg BDE-209 group were significantly longer than the control group(P<0.01),the swimming distance also increased significantly(P<0.05).The latency and swimming distance in MT+BDE-209 group were less than those in 100 mg/kg BDE-209 group(P<0.01).In spatial exploration test,compared with the control group,the times of crossing the platform and the stay time in the target quadrant of the offspring in the 30 mg/kg and 100 mg/kg BDE-209 group were decreased(P<0.01),and the number of crossings and the residence time in the target quadrant were increased in the MT+BDE-209 group compared with the 100 mg/kg BDE-209group(P<0.01).In nissl staining and TUNEL aopotosis assay,hippocampus neurons and nissl body of 100 mg/kg BDE-209-group rats was reduced(P<0.01),more positive apoptotic cells(P<0.01),nissl body was increase and positive apoptotic cells was decrease in the MT+BDE-209 group compared with the 100 mg/kg BDE-209 group(P<0.01).2.BDE-209 or MT treatment affected microglial polarization in hippocampus of offspring rats.The surface receptor CD206 and CD86 of microglia recognized as CD11 bhigh CD45low was analyzed by flow cytometry,and results showed the mean fluorescence intensity of CD206 in BDE-209-exposed rats was obvious decreased while CD86 increased(P<0.01),in MT+BDE-209 group,the level of CD206 increased while CD86 decreased(P<0.01).3.BDE-209 or MT treatment affected cytokines content and m RNA transcription in hippocampus of offspring rats.As ELISA and Real-Time PCR examination showed,both content and m RNA level of TNF-α and IL-1β in BDE-209-group rats were significantly elevated(P<0.01),whereas Arg-1 and IL-10 were obvious lower(P<0.05),content and m RNA level of TNF-α and IL-1β in MT+BDE-209-group rats were decrease(P<0.01),Arg-1 and IL-10 were increase(P<0.01).4.BDE-209 or MT treatment affected SIRT1/HMGB1/NF-κB signaling pathway in hippocampus of offspring rats.Heightened Iba1 meanwhile weakened SIRT1 fluorescence intensity was observed in 100 mg/kg BDE-209-exposed rats compared with control in immunofluorescence colocalization(P<0.01),Iba1 level decrease and SIRT1 increase in MT+BDE-209-group(P<0.05).Western blot showed: in 100 mg/kg BDE-209 group,the level of Iba1 protein was significantly up-regulated,SIRT1 was significantly decreased,pp65(Ser536)and p-Ik B α(Ser32)were significantly increased(P<0.01),cytoplasmic HMGB1 protein level was significantly increased,nuclear SIRT1 was significantly decreased,and p-p65 was significantly up-regulated(P<0.01).Compared with 100 mg/kg BDE-209 group,the expression of Iba1 protein decreased,the total protein and nuclear protein of SIRT1 increased significantly,p-p65(Ser536)and p-Ik B α(Ser32)decreased significantly in MT+BDE-209 group(P<0.01),and the cytoplasmic transfer of HMGB1 protein and nuclear metastasis of p-p65 decreased significantly(P<0.01).Conclusion1.Peranatal BDE-209 exposure caused exploratory behavior reduction and learning and memory impairment of offspring rats;2.BDE-209 promoted microglial pro-inflammatory activation(M1 subtype polarization),which contributed to learning and memory deficit;3.SIRT1/HMGB1/NF-κB signaling pathway modulated microglial M1/M2polarization;4.MT treatment promoted microglial polarization to M2 subtype,alleviated BDE-209-induced learning and memory impairment. |
