Effect of catalpol on remyelination through experimental autoimmune encephalomyelitis acting to promote Olig1 and Olig2 expressions in mice

Abstract

Background: Multiple sclerosis (MS) as an autoimmune disorder is a common disease occurring in central nervous system (CNS) and the remyelination plays a pivotal role in the alleviating neurological impairment in the MS. Catalpol, an effective component extracted from the Chinese herb Radix Rehmanniae, which has been proved protective in cerebral diseases.

Methods: To determine the protective effects and mechanisms of Catalpol on MS, the mice with experimental autoimmune encephalomyelitis (EAE) were induced by myelin oligodendrocyte glycoprotein (MOG) _35-55, as a model for human MS. Th17 cells were counted by flow cytometric (FCM). The expressions of nerve-glial antigen (NG) 2 and myelin basic protein (MBP) were measured by immunohistochemical staining. Olig1+ and Olig2+/BrdU+ cells were counted by immunofluorescence. Olig1 and Olig2 gene expressions were detected by real-time fluorescent quantitative reverse transcription (qRT) -PCR.

Results: The results showed that Catalpol improved neurological function, reduced inflammatory cell infiltration and demyelination. It could decrease Th17 cells in the peripheral blood. It increased the protein expressions of NG2 and MBP in mice brains, up-regulated markedly protein and gene expressions of Olig1 and Olig2 in terms of timing, site and targets.

Conclusions: These data demonstrated that Catalpol had a strong neuroprotective effect on EAE mice. Catalpol also plays a role in remyelination by promoting the expressions of Olig1 and Olig2 transcription factors.

Keywords: Catalpol; Experimental autoimmune encephalomyelitis; Multiple sclerosis; Olig1; Olig2; Remyelination.

Fig. 1

The neurological function scores of the mice. Results are expressed as means ± SE. The PA was given after the disease onset. The mice in the Catalpol treated groups were given oral suspensions of 80 mg/kg, 40 mg/kg, 20 mg/kg respectively once a day for 40 days. The scores were measured in NC (×), EAE (●), EAE+ PA (6 mg/kg, ◆), EAE + Catalpol–H (80 mg/kg, ▲), EAE + Catalpol–M (40 mg/kg,△) and EAE + Catalpol–L (20 mg/kg, ■). Note: ^# P < 0.05, ^## P < 0.01 vs. EAE; ^◆ P < 0.05, ^◆◆ P < 0.01 vs. EAE+ PA; ^△ P < 0.05 vs. EAE + Catalpol– M

Fig. 2

Observation of pathological changes in the brains of mice under the light microscope with H&E staining. A1 to F1 and A2 to F2 show the pathological changes in the brains of mice on Day 18 and Day 40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol–H (n = 4), M (n = 4) and L (n = 4) mice, respectively (scale bar 20 μm). Note: ^** P < 0.01 vs. NC; ^## P < 0.01 vs. EAE

Fig. 3

Observation of pathological changes in the spinal cord of mice under the light microscope with H&E staining. A1 to D1 and A2 to D2 show the pathological changes in the spinal cord of mice on Day 18 and Day 40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol – M (n = 4) mice, respectively (scale bar 20 μm)

Fig. 4

Observation of pathological changes (Area/Diameter) and remyelination (g-ratio) in the brains of mice under the TEM. a to f show the demyelination in the brains of mice on Day 40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol–H (n = 4), M (n = 4) and L (n = 4) mice, respectively (scale bar 1 μm). The EAE mice showed a fluffy layer structure, axonal edema and disintegration (arrows). Demyelination was lighter in PA and Catalpol-treated EAE mice (arrows). Note: ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. NC; ^# P < 0.05, ^## P < 0.01, ^### P < 0.001 vs. EAE

Fig. 5

Effect of Catalpol on the expression of CD4 + IL-17A + T cells in the peripheral blood on Day 18 and 40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol – M (n = 4) mice. Note: ^* P < 0.05 vs. NC; ^# P < 0.05, vs. EAE

Fig. 6

The protein expressions of NG2 in the brains of mice on Day 18 and Day 40. a to f show NG2 expressions in the cortex of mice on Day 40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol–H (n = 4), M (n = 4) and L (n = 4) mice, respectively (scale bar 20 μm). The protein expressions of NG2 in the cortex of mice were analyzed. Note: ^* P < 0.05 vs. NC; ^## P < 0.01 vs. EAE; ^◆ P < 0.05 vs. EAE + PA; ^▲ P < 0.05, ^▲▲ P < 0.01 vs. EAE + Catalpol–H

Fig. 7

The protein expressions of MBP in the brains of mice on Day 18 and Day 40. a to f show MBP expressions in the cortex of mice on Day40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol–H (n = 4), M (n = 4) and L (n = 4) mice, respectively (scale bar 20 μm). The protein expressions of MBP in the cortex, lateral ventricle and hippocampal DG region of mice were analyzed. Note: ^** P < 0.01 vs. NC; ^# P < 0.05, ^## P < 0.01 vs. EAE; ^◆ P < 0.05, ^◆◆ P < 0.01 vs. EAE+ PA;^▲ P < 0.05, ^▲▲ P < 0.01 vs. EAE + Catalpol–H

Fig. 8

Confocal images in the cortex of mice labeled with nucleus (blue) and Olig1 (red). Olig1 was located in nuclear of cells (arrows) and was also present in cytoplasm (arrowheads). A1 to F1 and A2 to F2 show the Olig1+ in the brains of mice on Day 18 and Day 40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol–H (n = 4), M (n = 4) and L (n = 4), respectively (scale bar 100 μm). The protein expressions of Olig1 in the brains of mice were analyzed. Note: ^** P < 0.01 vs. NC, ^## P < 0.01 vs. EAE;^◆◆ P < 0.01 vs. EAE+ PA;^▲ P < 0.05, ^▲▲ P < 0.01 vs. EAE + Catalpol–H;^△△ P < 0.01 vs. EAE + Catalpol– M

Fig. 9

Confocal images in the hippocampus DG of mice double labeled with nucleus (blue), Olig2 (green) and BrdU (red). Olig2 was expressed in cytoplasm of cells (arrows). A1 to F1and A2 to F2show the Olig2+/BrdU+ in the brains of mice on Day 18 andDay40 in NC (n = 4), EAE (n = 4), EAE+ PA (n = 4), EAE + Catalpol–H (n = 4), M (n = 4) and L (n = 4) mice, respectively (scale bar 50 μm). The protein expressions of Olig2/BrdU in the brains of mice were analyzed. Note: ^** P < 0.01 vs. NC, ^## P < 0.01 vs. EAE; ^◆◆ P < 0.01 vs. EAE+ PA; ^▲ P < 0.05, ^▲▲ P < 0.01 vs. EAE + Catalpol–H, ^△△ P < 0.01 vs. EAE + Catalpol–M

Fig. 10

A1 to F1 and A2 to F2 show the protein expressions of Olig1/Olig2 in the brains of mice on Day 18 and Day 40 in NC (n = 5), EAE (n = 5), EAE + PA (n = 5), EAE + Catalpol–H (n = 5), M (n = 5) and L (n = 5) mice, respectively. The protein expressions of Olig1/Olig2 in the brains of mice were analyzed. Note: ^# P < 0.05 vs. EAE; ^◆ P < 0.05 vs. EAE + PA; ^▲ P < 0.05 vs. EAE + Catalpol–H

Fig. 11

The gene expressions of Olig1 and Olig2 in the brains of mice on Day 18 and 40 in NC (n = 5), EAE (n = 5), EAE + PA (n = 5), EAE + Catalpol–H (n = 5), M (n = 5) and L (n = 5) mice, respectively. Note: ^# P < 0.05, ^## P < 0.01 vs. EAE; ^◆◆ P < 0.01 vs. EAE+ PA; ^▲▲ P < 0.01 vs. EAE + Catalpol–H, ^△△ P < 0.01 vs. EAE + Catalpol–M