Rhein and rhubarb similarly protect the blood-brain barrier after experimental traumatic brain injury via gp91phox subunit of NADPH oxidase/ROS/ERK/MMP-9 signaling pathway

Abstract

Oxidative stress chiefly contributes to the disruption of the BBB following traumatic brain injury (TBI). The Chinese herbal medicine rhubarb is a promising antioxidant in treating TBI. Here we performed in vivo and in vitro experiments to determine whether rhubarb and its absorbed bioactive compound protected the BBB after TBI by increasing ZO-1 expression through inhibition of gp91^phox subunit of NADPH oxidase/ROS/ERK/MMP-9 pathway. Rats were subjected to the controlled cortical impact (CCI) model, and primary rat cortical astrocytes were exposed to scratch-wound model. The liquid chromatography with tandem mass spectrometry method showed that rhein was the compound absorbed in the brains of CCI rats after rhubarb administration. The wet-dry weights and Evans blue measurements revealed that rhubarb and rhein ameliorated BBB damage and brain edema in CCI rats. Western blots showed that rhubarb and rhein downregulated GFAP in vitro. RT-PCR, immunohistochemistry, Western blot and dichlorodihydrofluorescein diacetate analysis indicated that rhubarb prevented activation of gp91^phox subunit of NADPH oxidase induced ROS production, subsequently inhibited ERK/MMP-9 pathway in vivo and in vitro. Interestingly, rhein and rhubarb similarly protected the BBB by inhibiting this signaling cascade. The results provide a novel herbal medicine to protect BBB following TBI via an antioxidative molecular mechanism.

Figure 1. Ultra performance liquid chromatography-electrospray ionization-tandem mass (UPLC-ESI-MS/MS) analysis for determining the rhein content in CCI rats after rhubarb administration.

(A) The chemical formula of rhein derived from rhubarb. (B) LC-MS/MS spectra of rhein, [M-H]⁻ was dominant and used as the precursor ion to obtain the spectra. The mass transitions of rhein were m/z 283.06 → 239.0. (C) Representative multiple reaction monitoring (MRM) chromatogram of blank brain tissue from a CCI rat. (D) Representative MRM chromatogram of rhein originated from its parent herbal medicine rhubarb. (E) Representative MRM chromatogram of blank brain tissue from CCI rat spiked with rhein. (F) Representative MRM chromatogram of rhein detected in the brain tissue of CCI rat after rhubarb administration.

Figure 2. Rhubarb and its absorbed compound rhein attenuate TBI induced BBB permeability and brain edema formation.

(A) Controlled cortical impact (CCI) models were induced in rats and brain tissues were obtained for the subsequent analyses. (B) Effects of rhubarb and rhein on the brain water content in the ipsilateral hemispheres. Compared with the Sham group, the brain water content in the Vehicle group was significantly increased at 6, 12 and 24 h post-CCI. CCI rats that were treated with rhubarb (6 g/kg and 12 g/kg) and 12 mg/kg rhein showed a marked reduction in brain edema at 12 and 24 h post-CCI compared to the Vehicle group. (C) The effects of rhubarb and rhein on BBB integrity in the injured hemispheres were assessed using the EB dye extravasation method. Compared with the Sham group, the amount of intracerebral EB dye was significantly increased in the Vehicle group at 6, 12 and 24 h post-CCI. Rhubarb (3 g/kg, 6 g/kg and 12 g/kg) and rhein (12 mg/kg) significantly reduced the quantity of extravasated EB dye at 12 and 24 h post-CCI compared to the Vehicle group. Values are expressed as mean ± SD. n = 6/group, ^Δp < 0.01 vs. the Sham group. *p < 0.05 and ^#p < 0.01 vs. the Vehicle group.

Figure 3. Rhubarb and rhein decrease MMP-9 expression and increased the ZO-1 level in CCI rats at 24 h post-CCI.

(A) Immunohistochemical examinations of the MMP-9 and ZO-1 positive cells. (B) Expression levels in the cortexes of CCI rats were reported as integrated optical density scores. The rhubarb treatments (6 g/kg and 12 g/kg) significantly decreased MMP-9 expression and increased ZO-1 expression compared with the Vehicle group following the cortical contusion. Similarly, 12 mg/kg rhein markedly attenuated MMP-9 expression and increased ZO-1 expression compared with the Vehicle group following the cortical contusion. Scale bar = 200 μm. (C) RT-PCR and Western blot analyses of brain MMP-9 and (D) ZO-1 expression at 24 h post-CCI. Rhubarb (3 g/kg, 6 g/kg and 12 g/kg) and rhein (12 mg/kg) significantly alleviated the MMP-9 mRNA levels and aggravated the ZO-1 mRNA levels compared with the Vehicle group. In agreement with the RT-PCR results, rhubarb (3 g/kg, 6 g/kg and 12 g/kg) and rhein (12 mg/kg) significantly alleviated the levels of the MMP-9 protein and aggravated the levels of the ZO-1 protein compared with the Vehicle group. The values are expressed as the mean ± SD, n = 6/group, ^Δp < 0.01 vs. the Sham group. *p < 0.05 and ^#p < 0.01 vs. the Vehicle group.

Figure 4. Rhubarb and rhein block TBI-induced activation of the gp91^phox subunit of NADPH oxidase/ROS/ERK pathway at 24 h post-CCI.

(A) Rhubarb (6 g/kg and 12 g/kg) and rhein (12 mg/kg) significantly decreased MMP-9 expression and increased the ZO-1 mRNA levels compared with the Vehicle group at 24 h. (B) The analysis of the relative fluorescence intensity showed an increase in ROS production in the ipsilateral cortexes of the Vehicle group compared with the Sham group. The increase was markedly reduced in injured cortexes of CCI rats that were treated with rhubarb (6 g/kg and 12 g/kg) and rhein (12 mg/kg). (C) Representative Western blot analysis of the gp91^phox expression and the ERK1/2 phosphorylation in the ipsilateral cortexes from CCI rats. (D) Quantification of gp91^phox expression and ERK1/2 phosphorylation. Rhubarb (3 g/kg, 6 g/kg and 12 g/kg) and rhein (12 mg/kg) suppressed the upregulation of gp91^phox expression and ERK1/2 phosphorylation in the ipsilateral cortexes from CCI rats. The values are expressed as the mean ± SD, n = 6/group, ^Δp < 0.01 vs. the Sham group. *p < 0.05 and ^#p < 0.01 vs. the Vehicle group.

Figure 5. Rhubarb and rhein exert similar pharmacological actions as the NADPH inhibitor apocynin by inhibiting the gp91^phox subunit of NADPH oxidase/ROS/ERK/MMP-9 signaling pathway in CCI rats.

(A) Inhibition of NADPH oxidase with apocynin (50 mg/kg) significantly reduced ROS generation in injured brain cortexes of CCI rats compared with the Vehicle group. Rhubarb and rhein exerted inhibition similar to the NADPH inhibitor apocynin by decreasing ROS production. (B) Representative WB analysis of gp91^phox, p-ERK1/2, ERK1/2, MMP-9 and ZO-1 expression in the ipsilateral cortexes from CCI rats. (C) Quantification of the WB showed that inhibition of NADPH oxidase with apocynin (50 mg/kg) significantly downregulated gp91^phox and (D) MMP-9 expression, (E) suppressed ERK1/2 phosphorylation, (F) and increased the ZO-1 levels. The values are expressed as the mean ± SD, n = 6/group, ^Δp < 0.01 vs. the Sham group. *p < 0.05 and ^#p < 0.01 vs. the Vehicle group.

Figure 6. The rhubarb and rhein treatments increase ZO-1 expression in scratch-wounded rat astrocytes by inhibiting the gp91^phox subunit of NADPH oxidase/ROS/ERK/MMP-9 pathway.

(A) Microscopic images of normal and scratch-wounded rat astrocytes. (B) The WB analysis showed that rhubarb and rhein reinforced neuroprotection by significantly downregulating the GFAP protein levels. (C) ROS production was increased within 24 h in the Vehicle group compared with the Control group. Rhubarb and rhein markedly reversed this trend in the scratch-wounded rat astrocytes. (D) The RT-PCR analysis showed that rhubarb and rhein significantly alleviated gp91^phox and (E) MMP-9 mRNA levels, and (F) aggravated the ZO-1 mRNA levels compared with the Vehicle group. (G) Representative WB analysis of gp91^phox, p-ERK1/2, ERK1/2, MMP-9 and ZO-1 expression in the scratch-wounded rat astrocytes. (H) Quantification of the WB indicated that rhubarb and rhein significantly decreased the gp91^phox, (I) phosphorylated ERK1/2 and (J) MMP-9 levels, and subsequently increased (K) ZO-1 expression in the scratch-wounded rat astrocytes. The values are expressed as the mean ± SD, n = 6/group, ^Δp < 0.01 vs. the Control group. *p < 0.05 and ^#p < 0.01 vs. the Vehicle group.

Figure 7. Schematic depiction of the mechanism by which rhubarb and its bioactive component rhein may protect the BBB after TBI via the gp91^phox subunit of NADPH oxidase/ROS/ERK/MMP-9 pathway.

Trauma is responsible for inducing sudden biochemical changes that occur at the time of impact. During TBI, gp91^phox is immediately upregulated. Subsequently, ROS overproduction by NADPH oxidase contributes to BBB damage and brain edema formation through the activation of downstream signaling molecules. After TBI, ROS activates ERK1/2, inducing increased MMP-9 synthesis and ZO-1 degradation. Inhibition of gp91^phox-triggered ROS production may help prevent the disruption of the BBB and brain edema post-TBI. In the present study, rhubarb is reported to be an effective inhibitor of gp91^phox-derived ROS production as a treatment for TBI. The underlying mechanisms include ROS-mediated ERK1/2 induction, followed by MMP-9 degradation and increased ZO-1 expression. Furthermore, rhein exerted similar neuroprotective effects as its parent herb rhubarb through the signaling pathway described above.