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Objective:To investigate the effect of aucubin on behaviors and excessive activation of astrocytic in attention deficit/hyperactivity disorder (ADHD) model mice.Methods:Twelve wild-type C57BL/6 pregnant mice (female, clean grade) were intraperitoneally administered with esketamine (15 mg/kg) to establish an ADHD model in offspring mice. The offspring mice were divided into control+ saline group, control+ aucubin group, Ketamine+ saline group and Ketamine+ aucubin group according to the nest matching principle with 15 in each group.At 14 days after birth, mice in the control+ aucubin group and Ketamine+ aucubin group were administered with aucubin (5 mg/kg, once a day) by gavage for 5 days. Mice in control+ saline group and Ketamine+ saline group were administered with equal volume of 0.9% sodium chloride solution. The offspring mice were housed with their mothers in the same cage until 21 days after birth. Twenty-one days after birth, the offspring mice were evaluated by open field test and elevated plus maze tests. Immunofluorescence assay was used to detect the expression of glutamate decarboxylase 2 (GAD2), γ- aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP) in the amygdala. The morphological changes of astrocytes were quantitatively analyzed by Sholl analysis. GraphPad Prim 9.0.1 software was used for statistical analysis. The comparison of multiple groups was conducted by one-way ANOVA or Kruskal-Wallis test.Results:(1)The results of behavioral experiments showed that the total distance traveled in the open field test and the residence time in open arm of the elevated plus maze were statistically significant ( F=236.90, H=39.92, both P<0.001). The total distance ((7 044±249)mm, (22 891±2 175)mm, P<0.05) and the residence time in open arm(12.69(9.86, 17.24)s, 2.72(0.57, 3.87)s, P<0.05) of mice in Ketamine+ saline group were both higher than those in control+ saline group.The total distance((22 891±2 175)mm, (8 252±839)mm, P<0.05) and the the residence time in open arm(5.45(1.13, 10.99)s, 12.69(9.86, 17.24)s, P<0.05) of Ketamine+ aucubin group were both lower than those of Ketamine+ saline group.(2)The immunofluorescence results showed that the levels of GAD2, GABA and GFAP intensity in amygdala of mice in the four groups were statistically significant ( F=145.50, 50.08, 53.83, all P<0.05). Compared with control+ saline group, the fluorescence intensities of GAD2 ((100.00±9.60)%, (24.86±4.14)%, P<0.05) and GABA ((100.00±16.84))%, (25.48±5.70)%, P<0.05) of Ketamine+ saline group were down-regulated, and the GFAP((100.00±18.02)%, (223.80±25.85)%, P<0.05) was up-regulated. Compared with Ketamine+ saline group, the fluorescence intensities of GAD2 ((24.86±4.14)%, (56.08±6.55)%, P<0.05) and GABA((25.48±5.70)%, (52.59±15.74)%, P<0.05) in Ketamine+ aucubin group were up-regulated, but the fluorescence intensity of GFAP ((223.80±25.85)%, (157.10±22.10)%, P<0.05) was down-regulated.(3)Sholl analysis indicated that the number of the intersections between the astrocyte processes or the branches of astrocyte processes was statistically significant in the 4 groups ( F=12.47, P<0.05). Compared with control+ saline group, the number of the intersections in Ketamine+ saline group((2.07±0.48), (1.67±0.72), P<0.05) increased. While the number of the intersections in Ketamine+ aucubin group was lower than that of Ketamine+ saline group ((1.20±0.78), (2.07±0.48), P<0.05). Conclusion:Aucubin administration can alleviate ADHD-like behaviors in offspring mice, and the mechanism may be associated with the inhibition of excessive astrocytic activation.
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Objective:To investigate the icariin on cognitive function and astrocytic pyroptosis in hemorrhagic shock resuscitation model mice.Methods:Forty-eight SPF grade C57BL/6 mice (male) were randomly divided into four groups ( n=12 in each group): Sham operation control group (Group C), hemorrhagic shock and resuscitation group (Group H), hemorrhagic shock and resuscitation plus icariin group (Group HI) and hemorrhagic shock resuscitation plus icariin and SSK1 group (Group HIS, SSK1 was a phosphorylation agonist of mitogen-activated protein kinase p38(p38MAPK). The mice in Group H, HI and HIS were subjected to hemorrhagic shock and resuscitation model by bleeding and retransfusion via left femoral vein; the mice in Group HI and HIS were administered with icariin (10 mg/kg) intragastrically for 7 days; the mice in Group C and H were administered with the same amount of normal saline containing dimethyl sulfoxide(DMSO). The mice in Group HIS were administered with SSK1 (0.5 mg/kg) intraperitoneally, but the mice in Group C, H and HI were only administered with the same amount of normal saline containing DMSO.At 15 days after resuscitation, novel objective recognition test and fear conditioning test were used to assess cognitive dysfunction of mice.Microtubule-associated protein 2(MAP2), a specific marker protein of neurons reflecting astrocytic pyroptosis in the hippocampus of mice, were detected by immunofluorescence assay so as to assess neuronal injury and astrocytic pyroptosis.The levels of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the hippocampus were evaluated by Western blot.SPSS 21.0 software was used for data analysis, multiple samples among groups were compared by one-way ANOVA, and SNK- q test was used for further pairwise comparison. Results:The results of new object recognition test showed that the difference of new object recognition index among the four groups was statistically significant ( F=50.75, P<0.05). The new object recognition indexes in H group(22.7±6.9), HI group(40.1±7.0) and HIS group (22.5±7.5) were significantly lower than that in C group (58.5±11.2). The index in HI group was higher than that in H group, while the index in HIS group was lower than that in HI group (all P<0.05). The results of the fear conditioning test showed that there was a statistically significant difference in the percentage of freezing time among the four groups of mice ( F=60.54, P<0.05). And the percentage of freezing time in H group((21.8±5.0)%), HI group ((38.4±7.4) %)and HIS group((21.3±4.2)%)were lower than that in C group((49.1±7.0)%), which in HI group was higher than that in H group ( P<0.05)and which in HIS group was lower than that in HI group(all P<0.05). The results of immunofluorescence showed that there were significant decreases of MAP2 intensity ((35.3±9.3)%, (63.3±6.1)%, (28.7±10.3)%) but increases of pyroptotic astrocytes ((24.5±4.2)%, (9.3±1.5)%, (22.1±3.3)%) in the H, HI and HIS groups compared with those of C group ((106.7±19.7) %, (3.4±2.0)%). There was an increase of MAP2 intensity but a decrease of pyroptotic astrocytes in the HI group compared with those in H group, and there was a decrease of MAP2 intensity but an increase of pyroptotic astrocytes in the HIS group compared with those of HI group (all P<0.05). The Western blot results showed that there were significant increases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the H, HI and HIS groups compared with C group, there were decreases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the HI group compared with H group, and there were increases of IL-1β, IL-18, the ratio of phosphorylated p38MAPK to total p38MAPK in the HIS group compared with those in HI group (all P<0.05). Conclusion:Icariin alleviates hemorrhage shock and resuscitation-induced cognitive dysfunction and astrocytic pyroptosis in mice, and the mechanism may be associated with inhibition of phosphorylated p38MAPK.
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Objective To investigate the effect of basic fibroblast growth factor (bFGF) on autophagy of nerve cells in rats after traumatic brain injury (TBI).Methods A total of 120 healthy adult male SD rats were randomly divided into sham group,TBI + vehicle group,and TBI + bFGF group by random number table method,with 40 rats in each group.PinPointTM Precision Cortical Impactor was used to simulate the pathological damage after TBI.In the sham group,the dura was exposed without impact.In the TBI + bFGF group,250 μg/kg of human recombinant bFGF was given in the nasal cavity 1 hour before the modeling,while in the sham group and TBI + Vehicle group,the same amount of saline was given in the nasal cavity 1 hour before the modeling.The necrotic cells were observed by propidium iodide(PI) staining 6 hours after injury.The effect of bFGF on the nerve function after TBI in rats was evaluated with modified neurological severity score (mNSS) 24 hours after injury.The water content of brain tissue was measured by dry and wet method 48 hours after injury.The ratio of Beclin-1,P62 protein and microtubule-associated protein 1 light 3 (LC3)-Ⅱ/Ⅰ protein was detected by western blot.The volume of brain injury was calculated by integral method of brain tissue section.The positive neuron specific nuclear protein (NeuN) cells were observed by immunofluorescence staining.The apoptotic cells were observed by TUNEL.Results Compared with the sham group [(4.0 ± 1.2) ％],the percentage of necrotic cells in TBI + vehicle group [(54.3 ± 10.1) ％] and TBI + bFGF group [(34.5 ± 10.5) ％] increased significantly (P ＜ 0.05),but the percentage of necrotic cells in TBI + bFGF group increased less than that in TBI + vehicle group (P ＜ 0.05).Compared with the sham group [(0.3 ± 0.5)points],the mNSS in the TBI + vehicle group [(5.8 ±0.8)points] and TBI + bFGF group [(4.7 ± 1.1) points] were significantly increased (P ＜ 0.05),but the mNSS of TBI + bFGF group was lower than that of TBI + vehicle group (P ＜ 0.05).Compared with the sham group [(76.7 ± 0.7) ％],the water content of brain tissue of TBI + vehicle group [(79.2 ± 0.5) ％] and TBI + bFGF group [(78.4 ± 1.0) ％] were significantly increased (P ＜0.05),but the water content of TBI + bFGF group was significantly lower than that of TBI + vehicle group (P ＜0.05).Compared with the sham group,protein expression of Beclin-1 and LC3-I/Ⅰ in TBI + vehicle group and TBI +bFGF group were significantly improved (P ＜ 0.05),P62 protein expression was significantly decreased (P ＜ 0.05),the volume of brain tissue injury was significantly increased (P ＜ 0.05),the number of positive NeuN cells increased significantly (P ＜ 0.05),and the number of apoptotic cells and apoptotic cells were significantly increased (P ＜0.05).Compared with the TBI + Vehicle group,the up-regulation of Beclin-1 protein and LC3-Ⅱ/Ⅰ protein ratio was obviously inhibited in the TBI + bFGF group (P ＜ 0.05),the down-regulation of P62 protien was significantly suppressed,the volume of brain tissue injury was significantly decreased,and the number of positive NeuN cells and apoptotic cells was significantly reduced (P ＜ 0.05).Conclusion The bFGF can significantly inhibit excessive autophagy in rats after TBI,reduce brain edema,reduce cell apoptosis and necrosis,and improve neural function.
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Compared with cultured fish, the fish oil of ocean wild fish contains much more Eicosapntemacnioc acid (EPA), Docosahexenoic acid (DHA), fat-soluble vitamin. To improve the utility value of oacean wild fish, small hairtail was used as raw material to investigate the technology of extracting fish oil with enzyme. The variables to affect the efficiency of extraction, extracting and centrifugation were selected as temperature, reaction time and pH value. Optimal technology conditions were determined by the response surface method: The liquid/solid ratio is 6, pH 7.3, enzyme amount of 1000 u/g raw material, agitation speed of 200 r/min, enzymolysis under 45 degrees C for 90 min. The optimum extraction conditions were as follows: 100 mL extractant (every 20 g surimi), pH4.0, extracted under 40 degrees C for 25 min. The optimal centrifuge conditions were: centrifuge speed of 3000 r/min (1865 g), centrifuged for 10 min. The oil extraction efficiency was 79.9%. This study developed the traditional technology of fish oil extraction, and improved the protection of the active components.