Inhibitory effect of matrine on blood-brain barrier disruption for the treatment of experimental autoimmune encephalomyelitis

Abstract

Dysfunction of the blood-brain barrier (BBB) is a primary characteristic of experimental autoimmune encephalomyelitis (EAE), an experimental model of multiple sclerosis (MS). Matrine (MAT), a quinolizidine alkaloid derived from the herb Radix Sophorae Flave, has been recently found to suppress clinical EAE and CNS inflammation. However, whether this effect of MAT is through protecting the integrity and function of the BBB is not known. In the present study, we show that MAT treatment had a therapeutic effect comparable to dexamethasone (DEX) in EAE rats, with reduced Evans Blue extravasation, increased expression of collagen IV, the major component of the basement membrane, and the structure of tight junction (TJ) adaptor protein Zonula occludens-1 (ZO-1). Furthermore, MAT treatment attenuated expression of matrix metalloproteinase-9 and -2 (MMP-9/-2), while it increased the expression of tissue inhibitors of metalloproteinase-1 and -2 (TIMP-1/-2). Our findings demonstrate that MAT reduces BBB leakage by strengthening basement membrane, inhibiting activities of MMP-2 and -9, and upregulating their inhibitors. Taken together, our results identify a novel mechanism underlying the effect of MAT, a natural compound that could be a novel therapy for MS.

Figure 1

MAT reduces clinical signs of EAE. (a) Structure of MAT. (b) Effect-versus-time curves of MAT effects on EAE. Clinical EAE was scored daily after immunization according to a 0–5 scale. (c) The mean maximum clinical scores are expressed as mean ± SD (n = 16 each group). ^△△ P < 0.01, compared to naive group; *P < 0.05, compared to vehicle-treated group.

Figure 2

CNS infiltration and demyelination. At day 17 p.i., spinal cord lumbar enlargements were harvested and transverse sections were stained with H&E and C-2R-B. (a) Digital images were collected under bright-field setting using a ×40 objective. (b) Quantitative analysis. Mean values and SD are shown (n = 8 each group). ^△△ P < 0.01, compared to naive group; **P < 0.01, compared to vehicle-treated group; ^# P < 0.05 and ^## P < 0.01, compared to DEX group; ^◊ P < 0.05 and ^◊◊ P < 0.01, comparison between MAT-L and MAT-H groups.

Figure 3

BBB integrity. Evans Blue was i.v. injected at day 17 p.i. and brains were harvested at 60 min to determine Evans Blue extravasation. Results are expressed as mean ± SD (n = 8 each group). ^△△ P < 0.01, compared to naive group; **P < 0.01, compared to vehicle group; ^## P < 0.01, compared to DEX group; ^◊ P < 0.05, comparison between MAT-L and MAT-H groups.

Figure 4

Collagen IV and ZO-1 mRNA expression. Spinal cords were harvested from treated and untreated EAE rats at day 17 p.i.; mRNA expression of collagen IV (a) and ZO-1 (b) was determined by RT-PCR, and an example of bands is shown. (c) Quantitative analysis. Results are expressed as mean ± SD (n = 8 each group). ^△△ P < 0.01, compared to naive group; **P < 0.01, compared to vehicle group; ^## P < 0.01, compared to DEX group; ^◊◊ P < 0.01, comparison between MAT-L and MAT-H groups.

Figure 5

Serum concentration of MMP-9 and TIMP-1. At day 17 p.i., sera were harvested from treated and nontreated EAE rats, with sera from naive rats serving as control. MMP-9 and TIMP-1 production was determined by ELISA. Results are expressed as mean ± SD (n = 8 each group). ^△△ P < 0.01, compared to naive group; **P < 0.01, compared to the vehicle group; ^## P < 0.01, compared to the DEX group; ^◊◊ P < 0.01, comparison between MAT-L and MAT-H groups.

Figure 6

Immunohistochemistry of MMP-2 and TIMP-2. At day 17 p.i., spinal cords were harvested after extensive perfusion. (a) MMP-2 and TIMP-2 expression was determined by immunohistochemistry. Magnification: ×40. (b) Quantitative analyses. Values represent mean ± SD (n = 8 each group). ^△△ P < 0.01, compared to naive group; *P < 0.05 and **P < 0.01, compared to the vehicle group; ^# P < 0.05, compared to the DEX group; ^◊ P < 0.05, comparison between MAT-L and MAT-H groups.