Objective:To investigate the effects of baicalein on the protein kinase B(AKT)/glycogen synthase kinase 3β(GSK3β)pathway and the expression of tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1 β)in lipopolysaccharide(LPS)-activated BV2 microglial cells.Methods:BV2 microglial cells were cultured and divided into control group,LPS-induced group,and LPS+Baicalein group.Molecular docking was conducted to verify the bind-ing affinity of baicalein to AKT.Western blot and immunofluorescence staining were used to assess the expression and phosphorylation levels of AKT,GSK3β,TNF-α,and IL-1β in activated BV2 cells.Results:Baicalein exhibited a strong binding affinity for AKT.Western blot results showed that LPS stimulation led to increased TNF-α and IL-1 βexpression and decreased phosphorylation of AKT and GSK3β in BV2 cells(P＜0.05).After Baicalein treatment,TNF-α and IL-1 β expression significantly decreased,while AKT and GSK3β phosphorylation levels increased compared to the LPS group(P＜0.05).Immunofluorescence staining results were consistent with those of Western blot.Conclusion:Baicalein inhibits the expression of TNF-α and IL-1 β in activated microglia,potentially through activation of the AKT/GSK3β pathway.

Objective:To investigate the mechanism of recombinant human mesencephalic astrocyte-derived neurotrophic factor (rhMANF) in spinal cord injury (SCI) repair promoted by A2 reactive astrocyte polarization.Methods:One hundred and twenty female SPF SD rats were randomly divided into sham-operated group, SCI group, SCI+control group and SCI+rhMANF group ( n=30 in each group). SCI models were prepared by heavy drop method in the later 3 groups, and 10 μL sterile saline or 10 μL sterile saline+5 μg rhMANF were injected intrathecally in the later 2 groups 30 min after modeling. Basso-Beattie-Bresnahan (BBB) scale was used to evaluate the motor function in each group 1, 3, 7, 14, 21 and 28 days after injection. After behavioral assessment 3 days after injection, the protein expressions of ReIB, p52, phosphorylated (p)-ReIB and p-p52 in the spinal cord tissues were detected by Western blotting, and the expressions of anti-inflammatory cytokine and neurotrophic factor in the spinal cord tissues were detected by ELISA. After behavioral assessment 14 days after injection, immunofluorescent staining was performed to detect the expressions of neuronal nuclear antigen (NeuN), Syn and S100A10 in the spinal cord tissues. After behavioral assessment 28 days after injection, HE staining and uranyl acetate-lead citrate double staining were used to observe the pathological changes of the spinal cord under light microscope and electron microscope, respectively. Results:On 14, 21, and 28 days after injection, the BBB score in the SCI+rhMANF group was significantly higher than that in the SCI group and SCI+control group ( P<0.05). On 3 days after injection, the p-ReiB and p-p52 protein expressions in the SCI+rhMANF group (1.17±0.02 and 1.00±0.07) were significantly higher than those in the SCI group (0.74±0.01 and 0.42±0.11) and SCI+control group (0.79±0.00 and 0.64±0.02, P<0.05); the SCI+rhMANF group had significantly increased interleukin (IL)-4, IL-10, IL-13, neurotrophin-3, transforming growth factor-β and granulocyte colony-stimulating factor expressions ([217.58±16.06] pg/mg, [276.53±15.00]) pg/mg, [178.88±7.03] pg/mg, [172.61±16.43] pg/mg, [241.00±15.80] pg/mg, and [166.63±14.61] pg/mg) compared with the SCI group ([132.15±18.86] pg/mg, [173.48±18.24] pg/mg, [109.01±3.79] pg/mg, [104.64±18.21] pg/mg, [138.09±9.93] pg/mg, and [91.26±11.09] pg/mg), and SCI+control group ([137.80±27.70] pg/mg, [185.78±19.20] pg/mg, [112.44±13.51] pg/mg, [93.13±22.09] pg/mg, [159.48±32.50] pg/mg, and [112.67±18.32] pg/mg, P<0.05). On 14 days after injection, the immunofluorescent staining intensities of NeuN/S100A10, NeuN/Syn in the SCI+rhMANF group (2.51±0.24/2.85±0.27 and 2.48±0.35/1.92±0.32) were significantly higher than those in the SCI group (0.99±0.11/1.00±0.18 and 1.00±0.19/1.00±0.08) and SCI+control group (1.39±0.09/0.93±0.20 and 1.26±0.35/0.94±0.19, P<0.05). Light microscopy showed that the spinal cord nerve tissues in the SCI group and SCI+control group had loose structure, with edema and vacuolar degeneration; those in the sham-operated group and SCI+rhMANF group had dense structure, with round and cone-shaped neurons and large and round nucleus, and without vacuolar degeneration. Transmission electron microscopy showed intact structure of myelin sheath and axon in the sham-operated group, loose and shrunked spinal cord nerve cells (chromatin condensation, and cell membrane bleb formation) in the SCI group and SCI+control group, and relatively complete cell structure in the SCI+rhMANF group. Conclusion:The rhMANF can activate ReIB/P52 nuclear translocation phosphorylation, up-regulate the anti-inflammatory factor and neurotrophic factor expressions, induce the A2 astrocyte polarization, and promote the synaptic growth and spinal cord injury recovery.

Objective:To investigate the effects of baicalein on the protein kinase B(AKT)/glycogen synthase kinase 3β(GSK3β)pathway and the expression of tumor necrosis factor-α(TNF-α)and interleukin-1 β(IL-1 β)in lipopolysaccharide(LPS)-activated BV2 microglial cells.Methods:BV2 microglial cells were cultured and divided into control group,LPS-induced group,and LPS+Baicalein group.Molecular docking was conducted to verify the bind-ing affinity of baicalein to AKT.Western blot and immunofluorescence staining were used to assess the expression and phosphorylation levels of AKT,GSK3β,TNF-α,and IL-1β in activated BV2 cells.Results:Baicalein exhibited a strong binding affinity for AKT.Western blot results showed that LPS stimulation led to increased TNF-α and IL-1 βexpression and decreased phosphorylation of AKT and GSK3β in BV2 cells(P＜0.05).After Baicalein treatment,TNF-α and IL-1 β expression significantly decreased,while AKT and GSK3β phosphorylation levels increased compared to the LPS group(P＜0.05).Immunofluorescence staining results were consistent with those of Western blot.Conclusion:Baicalein inhibits the expression of TNF-α and IL-1 β in activated microglia,potentially through activation of the AKT/GSK3β pathway.

Objective:To investigate the mechanism of recombinant human mesencephalic astrocyte-derived neurotrophic factor (rhMANF) in spinal cord injury (SCI) repair promoted by A2 reactive astrocyte polarization.Methods:One hundred and twenty female SPF SD rats were randomly divided into sham-operated group, SCI group, SCI+control group and SCI+rhMANF group ( n=30 in each group). SCI models were prepared by heavy drop method in the later 3 groups, and 10 μL sterile saline or 10 μL sterile saline+5 μg rhMANF were injected intrathecally in the later 2 groups 30 min after modeling. Basso-Beattie-Bresnahan (BBB) scale was used to evaluate the motor function in each group 1, 3, 7, 14, 21 and 28 days after injection. After behavioral assessment 3 days after injection, the protein expressions of ReIB, p52, phosphorylated (p)-ReIB and p-p52 in the spinal cord tissues were detected by Western blotting, and the expressions of anti-inflammatory cytokine and neurotrophic factor in the spinal cord tissues were detected by ELISA. After behavioral assessment 14 days after injection, immunofluorescent staining was performed to detect the expressions of neuronal nuclear antigen (NeuN), Syn and S100A10 in the spinal cord tissues. After behavioral assessment 28 days after injection, HE staining and uranyl acetate-lead citrate double staining were used to observe the pathological changes of the spinal cord under light microscope and electron microscope, respectively. Results:On 14, 21, and 28 days after injection, the BBB score in the SCI+rhMANF group was significantly higher than that in the SCI group and SCI+control group ( P<0.05). On 3 days after injection, the p-ReiB and p-p52 protein expressions in the SCI+rhMANF group (1.17±0.02 and 1.00±0.07) were significantly higher than those in the SCI group (0.74±0.01 and 0.42±0.11) and SCI+control group (0.79±0.00 and 0.64±0.02, P<0.05); the SCI+rhMANF group had significantly increased interleukin (IL)-4, IL-10, IL-13, neurotrophin-3, transforming growth factor-β and granulocyte colony-stimulating factor expressions ([217.58±16.06] pg/mg, [276.53±15.00]) pg/mg, [178.88±7.03] pg/mg, [172.61±16.43] pg/mg, [241.00±15.80] pg/mg, and [166.63±14.61] pg/mg) compared with the SCI group ([132.15±18.86] pg/mg, [173.48±18.24] pg/mg, [109.01±3.79] pg/mg, [104.64±18.21] pg/mg, [138.09±9.93] pg/mg, and [91.26±11.09] pg/mg), and SCI+control group ([137.80±27.70] pg/mg, [185.78±19.20] pg/mg, [112.44±13.51] pg/mg, [93.13±22.09] pg/mg, [159.48±32.50] pg/mg, and [112.67±18.32] pg/mg, P<0.05). On 14 days after injection, the immunofluorescent staining intensities of NeuN/S100A10, NeuN/Syn in the SCI+rhMANF group (2.51±0.24/2.85±0.27 and 2.48±0.35/1.92±0.32) were significantly higher than those in the SCI group (0.99±0.11/1.00±0.18 and 1.00±0.19/1.00±0.08) and SCI+control group (1.39±0.09/0.93±0.20 and 1.26±0.35/0.94±0.19, P<0.05). Light microscopy showed that the spinal cord nerve tissues in the SCI group and SCI+control group had loose structure, with edema and vacuolar degeneration; those in the sham-operated group and SCI+rhMANF group had dense structure, with round and cone-shaped neurons and large and round nucleus, and without vacuolar degeneration. Transmission electron microscopy showed intact structure of myelin sheath and axon in the sham-operated group, loose and shrunked spinal cord nerve cells (chromatin condensation, and cell membrane bleb formation) in the SCI group and SCI+control group, and relatively complete cell structure in the SCI+rhMANF group. Conclusion:The rhMANF can activate ReIB/P52 nuclear translocation phosphorylation, up-regulate the anti-inflammatory factor and neurotrophic factor expressions, induce the A2 astrocyte polarization, and promote the synaptic growth and spinal cord injury recovery.