Berberine attenuates ischemia-reperfusion injury through inhibiting HMGB1 release and NF-κB nuclear translocation

Abstract

Inflammatory damage plays an important role in cerebral ischemic pathogenesis and represents a new target for treatment of stroke. Berberine is a natural medicine with multiple beneficial biological activities. In this study, we explored the mechanisms underlying the neuroprotective action of berberine in mice subjected transient middle cerebral artery occlusion (tMCAO). Male mice were administered berberine (25, 50 mg/kg/d, intragastric; i.g.), glycyrrhizin (50 mg/kg/d, intraperitoneal), or berberine (50 mg/kg/d, i.g.) plus glycyrrhizin (50 mg/kg/d, intraperitoneal) for 14 consecutive days before tMCAO. The neurological deficit scores were evaluated at 24 h after tMCAO, and then the mice were killed to obtain the brain samples. We showed that pretreatment with berberine dose-dependently decreased the infarct size, neurological deficits, hispathological changes, brain edema, and inflammatory mediators in serum and ischemic cortical tissue. We revealed that pretreatment with berberine significantly enhanced uptake of ¹⁸F-fluorodeoxyglucose of ischemic hemisphere comparing with the vehicle group at 24 h after stroke. Furthermore, pretreatment with berberine dose-dependently suppressed the nuclear-to cytosolic translocation of high-mobility group box1 (HMGB1) protein, the cytosolic-to nuclear translocation of nuclear factor kappa B (NF-κB) and decreased the expression of TLR4 in ischemic cortical tissue. Moreover, co-administration of glycyrrhizin and berberine exerted more potent suppression on the HMGB1/TLR4/NF-κB pathway than berberine or glycyrrhizin administered alone. These results demonstrate that berberine protects the brain from ischemia-reperfusion injury and the mechanism may rely on its anti-inflammatory effects mediated by suppressing the activation of HMGB1/TLR4/NF-κB signaling.

Keywords: HMGB1; NF-κB; TLR4; berberine; glycyrrhizin; inflammation; ischemic stroke.

Fig. 1

The change in rCBF in the MCAO region. a Representative images of cerebral blood flow for baseline, ischemia, and reperfusion during tMCAO. b Percent change in rCBF compared with baseline during occlusion and reperfusion (n = 5)

Fig. 2

The neuroprotective effect of berberine treatment in ischemic stroke. a Evaluation of the neurological deficit. Pretreatment with berberine significantly decreases neurological deficit scores (n = 12). b Above are the horizontal sections from mice in the vehicle group and berberine-H group. The uptake of ¹⁸F-FDG decreases from red to blue. The area indicated by the arrow in the diagram is the area of interest. c Quantitative ¹⁸F-FDG uptake in the different groups at 24 h after cerebral reperfusion (n = 3). Data are presented as the mean ± SD. *P < 0.01 versus the vehicle group

Fig. 3

Effects of berberine on infarct volume and brain edema in ischemic stroke (n = 5). a Representative images of TTC-stained brain sections of cerebral ischemia–reperfusion mice with berberine pretreatment, vehicle or sham. b Quantification of the infarct volume in the vehicle or berberine pretreatment groups. c Brain water content. Data are presented as the mean ± SD. ^#P < 0.01 versus the sham group; *P < 0.01 versus the vehicle group

Fig. 4

Representative images of ischemic brain tissue with hematoxylin and eosin staining (n = 3). Scale bar, 20 µm. Magnification of the microphotograph, × 400. Arrows indicate injured cells in the ischemic cortex region. a Sham group, b vehicle group, c berberine-L group, d berberine-H group. e Percentage of injured cells in the vehicle, berberine-L, and berberine-H groups in the cerebral cortex. Data are presented as the mean ± SD. *P < 0.01 versus the vehicle group

Fig. 5

Berberine improved brain injury by suppressing the inflammatory response through HMGB1/TLR4/NF-κB inhibition in mice after cerebral ischemia–reperfusion injury (n = 5). a Serum proinflammatory cytokine levels were measured using ELISA kits. b HMGB1, TLR4, and NF-κB mRNA expression in tMCAO-treated mice. c TNF-α, IL-1β, and IL-6 mRNA expression in tMCAO-treated mice. Data are presented as the mean ± SD. ^#P < 0.01 versus the sham group; ^※P < 0.05 versus the vehicle group; *P < 0.01 versus the vehicle group

Fig. 6

a Enlarged images from representative immunohistochemistry photographs of HMGB1, TLR4, and NF-κB in different groups (n = 3). The original images were placed on the top left corner of each photograph. Scale bar, 20 µm. Magnification of the microphotograph, × 400. b Number of cells with positive staining for HMGB1, TLR4, and NF-κB. Data are presented as the mean ± SD. ^#P < 0.01 versus the sham group; *P < 0.01 versus the vehicle group

Fig. 7

Effects of berberine on the expression of nHMGB1, cyHMGB1, nNF-κB, cyNF-κB, and TLR4 by western blot analysis at 24 h after cerebral ischemia–reperfusion injury (n = 3). Nuclear and cytosolic protein extracts were prepared and assayed for HMGB1 and NF-κB by western blot analysis. Total protein extracts were prepared for analysis of TLR4 expression. a Representative photographs of western blot for nHMGB1, cyHMGB1, nNF-κB, cyNF-κB, and TLR4. b Ratio of cyHMGB1 to nHMGB1. c Ratio of nNF-κB to cyNF-κB. d Ratio of TLR4 to β-actin. Data are presented as the mean ± SD. ^#P < 0.01 versus the sham group; *P < 0.01 versus the vehicle group