Baicalin Promotes CNS Remyelination via PPARγ Signal Pathway

(A) A schematic drawing of demyelination/remyelination mice model. Male, 8- to 10-wk-old C57BL/6 mice were fed with cuprizone for 6 weeks to achieve complete demyelination, followed by feeding normal chow in another 2 weeks. Mice were randomly divided into normal chow + PBS groups (NC, n = 6 and PBS-treated groups (CPZ + PBS, n = 12) and BA-treated (CPZ + BA, n = 12), all groups receive BA or PBS treatment starting at CPZ model beginning until the 6 + 3 weeks. A schematic diagram of corpus callosum lateral to lateral ventricle and location observed by paraffin section staining (LFB). (B) LFB and FluoroMyelin staining at different time point (Scale bar, 200 μm). Slices were assessed and scored in a blinded fashion for demyelination: 3, large (confluent); 2, a few areas of demyelination; 1, rare foci; 0, none. The quantification of the mean density of FluoroMyelin staining was performed using ImageJ. (C) Representative electron micrographs at 6 + 2 weeks (Scale bar, 2 μm for the upper row, and 1 μm for the insets in the lower row, rad arrows indicate typical myelin structure in each group). (D) The quantification of myelin G-ratio value (axon diameter/fiber diameter) of NC, CPZ + PBS, and CPZ + BA group. Scatter plot of G-ratio value. Data are shown as mean values ±SEM (n = 6 each group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with control group, one-way analysis of variance with Tukey multiple comparisons test. BA = baicalin; NC = naïve chow; PBS = phosphate-buffered saline.

CPZ mice were treated with BA or PBS as shown in Figure 2A. (A) Double immunostaining of Olig2 (red) and APC (green). Scale bar, 100 μm for the upper row, and 50 μm for the insets in the lower row. The quantification of Olig2 positive cells and quantification analysis of the percentage of APC positive cells and Olig2 positive cells measured at random areas using Image-Pro. Date are mean ± SEM (n = 3 each group). (B) GFAP (green) immunostaining at corpus callosum section of all group mice at 6 + 2 weeks (Scale bar, 100 μm). The mean of GFAP-positive cell density was measured in corpus callosum using ImageJ. Data are mean ± SEM (n = 3 each group). (C.a-c) Behavioral assessment (beam walking test (C.a), wire hang test (C.b), and rotating rod test (C.c) of CPZ-induced demyelination mice. (D) The expression of cytokine genes was determined using real-time RT-PCR analysis at CPZ-induced 5 weeks. The expression of myelin-associated protein genes and neurotrophic factors was determined using real-time RT-PCR analysis at CPZ-induced 6 + 1 week. Data are shown as mean values ±SEM (n = 6 each group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group, one-way analysis of variance with Tukey multiple comparisons test. BA = baicalin; CPZ = cuprizone.

(A) Three-dimensional structure diagram of PPARγ docked with BA. (B) MST affinity determination of BA and PPARγ. (C) HEK293T cells were transfected with PPRE×3-TK-luciferase vector, a PPRE-dependent luciferase reporter constructs. After 24 hours of transfection, cells were cultured with BA of 10 μg/mL for 6 hours, and the activity of luciferase was monitored in cell lysates by a ONE-Glo EX Luciferase Assay System kit (Promega). (D) BA enhanced oligodendrocyte differentiation in primary OPC cultures. Primary OPCs, prepared from brains of newborn C57BL/6 mice, were cultured in the differentiation medium with or without BA (1, 5, or 10 μg/mL) for 5 days, followed by MBP immunofluorescence staining. Primary OPCs were cultured in a differentiation medium with BA (10 μg/mL) alone or pretreated for 30 minutes with GW-9662 (PPARγ-specific antagonist) before addition of BA or GW-9662 (10 nM) alone for 5 days, followed by MBP immunofluorescence staining. Nuclei were stained with DAPI (blue). Scale bar, 50 μm. A quantitative analysis was performed for numbers of MBP⁺ mature oligodendrocytes. Data are shown as mean values ± SEM and analyzed by two-tailed Student t-test (n = 5 each group). *p < 0.05, **p < 0.01, and ***p < 0.001 compared with the control group. BA = baicalin; MBP = myelin basic protein; OPC = oligodendrocyte precursor cell; PPARγ = peroxisome proliferator-activated receptor γ; PPRE = peroxisome proliferator element.