Abstract:Objective To explore the effect of proanthocyanidins B2 (PCB2) on cuprizone (CPZ)-induced demyelination in mice by targeting microglia to regulate the Toll-like receptor-4 (TLR4)/myeloid differentiation factor 88 (MyD88)/nuclear factor-κB (NF-κB) signaling pathway.Methods Forty male C57BL/6 mice were randomly divided into the normal group, PCB2 group, CPZ group and CPZ + PCB2 group. The mice in the CPZ group and the CPZ + PCB2 group were fed with a diet containing 0.2% CPZ for a total of 6 weeks to establish the acute demyelination model of mice, and the mice in the CPZ + PCB2 group were additionally treated with PCB2 for 2 weeks. The behavioral changes of mice were evaluated by using elevated plus maze and open field experiments. TrueGold myelin staining, myelin basic protein (MBP) and degraded myelin basic protein (dMBP) immunofluorescence staining were used to observe the loss of myelin in the corpus callosum. Immunofluorescence staining was used to observe the expression of microglial marker ionized calcium-binding adaptor molecule 1 (Iba1), M1-type polarization marker inducible nitric oxide synthase (iNOS), and M2-type polarization marker arginase 1(Arg1). The levels of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, IL-1β and IL-10 in brain tissue homogenates were determined by ELISA. The mRNA contents and protein expression levels of TLR4, MyD88 and NF-κB in brain tissue homogenates were detected by RT-qPCR and Western blotting.Results Compared with the normal group, the CPZ group showed increased total distance traveled in the open arms and increased number of entries into the open arms (P 0.05). There was no significant difference in the total distance traveled in the open arms or the number of entries into the open arms between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased total distance traveled in the open arms and decreased number of entries into the open arms (P 0.05). Compared with the normal group, the CPZ group showed increased total distance traveled in the open field and increased distance traveled in the center zone of the open field (P 0.05). There was no significant difference in the total distance traveled in the open field or the distance traveled in the center zone between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased total distance traveled in the open field and decreased distance traveled in the center zone of the open field (P 0.05). Compared with the normal group, the CPZ group showed a smaller corpus callosum area with TrueGold staining, which appeared lighter and sparser, along with decreased mean optical density values (P 0.05). There was no significant difference in TrueGold staining results between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited alleviated myelin loss in the corpus callosum with TrueGold staining, showing deeper staining and increased mean optical density values (P 0.05). Compared with the normal group, the CPZ group showed decreased MBP fluorescence intensity in the corpus callosum (P 0.05). There was no significant difference in MBP expression levels between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited increased MBP expression levels (P 0.05). Compared with the normal group, the CPZ group showed increased dMBP expression levels in the corpus callosum (P 0.05). There was no significant difference in dMBP expression levels between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased dMBP expression levels (P 0.05). Compared with the normal group, the CPZ group showed increased Iba1 expression in the brain (P 0.05). There was no significant difference in Iba1 expression between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased Iba1 expression in the brain (P 0.05). Compared with the normal group, the CPZ group showed increased Iba1/iNOS expression in the brain and increased mean fluorescence intensity of Iba1/iNOS-positive cells (P 0.05). There was no significant difference in Iba1/iNOS expression between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased Iba1/iNOS expression in the brain and decreased mean fluorescence intensity of Iba1/iNOS-positive cells (P 0.05). There was no significant difference in Iba1/Arg1 expression levels in the myelinated region among all groups (P 0.05). Compared with the normal group, the CPZ group showed elevated expression levels of TNF-α, IL-1β, and IL-6 (P 0.05) and decreased IL-10 expression levels (P 0.05). There was no significant difference in the expression levels of TNF-α, IL-1β, IL-6, or IL-10 between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased expression levels of TNF-α, IL-1β, and IL-6 (P 0.05) and increased IL-10 expression levels (P 0.05). Compared with the normal group, the CPZ group showed elevated mRNA expression levels of TLR4, MyD88, and NF-κB (P 0.05). There was no significant difference in the mRNA expression levels of TLR4, MyD88, or NF-κB between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased mRNA expression levels of TLR4, MyD88, and NF-κB (P 0.05). Compared with the normal group, the CPZ group showed elevated protein expression levels of TLR4, MyD88, and NF-κB (P 0.05). There was no significant difference in the protein expression levels of TLR4, MyD88, or NF-κB p65 between the PCB2 group and the normal group (P 0.05). Compared with the CPZ group, the CPZ + PCB2 group exhibited decreased protein expression levels of TLR4, MyD88, and NF-κB (P 0.05).Conclusion PCB2 may alleviate neuroinflammation and relieve CPZ-induced demyelination by inhibiting the TLR4/MyD88/NF-κB signaling pathway and the polarization of microglia to M1 type.
