Shikonin attenuates blood-brain barrier injury and oxidative stress in rats with subarachnoid hemorrhage by activating Sirt1/Nrf2/HO-1 signaling

Abstract

Subarachnoid hemorrhage (SAH) is a serious intracranial hemorrhage characterized by acute bleeding into the subarachnoid space. The effects of shikonin, a natural compound from the roots of Lithospermum erythrorhizon, on oxidative stress and blood-brain barrier (BBB) injury in SAH was evaluated in this study. A rat model of SAH was established by endovascular perforation to mimic the rupture of intracranial aneurysms. Rats were then administered 25 mg/kg of shikonin or dimethylsulfoxide after surgery. Brain edema, SAH grade, and neurobehavioral scores were measured after 24 h of SAH to evaluate neurological impairment. Concentrations of the oxidative stress markers superoxide dismutase (SOD), glutathione (GSH), and malondialdehyde (MDA) in the brain cortex were determined using the corresponding commercially available assay kits. Evans blue staining was used to determine BBB permeability. Western blotting was used to quantify protein levels of tight junction proteins zonula occludens-1, Occludin, and Claudin-5. After modeling, the brain water content increased significantly whereas the neurobehavioral scores of rats with SAH decreased prominently. MDA levels increased and the levels of the antioxidant enzymes GSH and SOD decreased after SAH. These changes were reversed after shikonin administration. Shikonin treatment also inhibited Evans blue extravasation after SAH. Furthermore, reduction in the levels of tight junction proteins after SAH modeling was rescued after shikonin treatment. In conclusion, shikonin exerts a neuroprotective effect after SAH by mitigating BBB injury and inhibiting oxidative stress in the cerebral cortex.

Keywords: Antioxidants; Blood-brain barrier; Subarachnoid hemorrhage; Tight junctions; Traditional Chinese medicine.

Fig. 1. Chemical structure of shikonin and experimental design.

(A) Chemical structure of shikonin. (B) Experimental design. SAH, subarachnoid hemorrhage; DMSO, dimethyl sulfoxide.

Fig. 2. Shikonin alleviates neurological deficits and brain edema in rats after SAH.

(A, B) Beam balance test (A) and modified Garcia scoring system (B) were used to determine the neurological function of rats in the sham + DMSO group, sham + shikonin group, SAH + DMSO group, and SAH + shikonin group. (C) Brain water content of rats in the 4 groups was determined to evaluate the extent of brain edema. (D) Representative image of rat brains from the 4 groups. (E) SAH grade in the 4 groups. Values are shown as mean ± standard deviation. SAH, subarachnoid hemorrhage; DMSO, dimethyl sulfoxide. ***p < 0.001.

Fig. 3. Shikonin attenuates oxidative stress in the brain cortex of rats after SAH.

(A-C) Contents of MDA, GSH, and SOD in the brain cortex of rats were determined using the corresponding assay kits in the sham + DMSO group, sham + shikonin group, SAH + DMSO group, and SAH + shikonin group. (D) ROS content in the brain tissues of rats from each group was determined using a commercial assay kit. Values are shown as mean ± standard deviation. SAH, subarachnoid hemorrhage; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species. ***p < 0.001.

Fig. 4. Shikonin attenuates BBB permeability and upregulates tight junction proteins after SAH.

(A) Evans blue staining assay was used to determine the BBB permeability of rats in the sham + DMSO group, sham + shikonin group, SAH + DMSO group, and SAH + shikonin group. (B, C) Protein levels of tight junctions (ZO-1, Occludin, and Claudin5) in the 4 groups were determined using Western blotting. Values are shown as mean ± standard deviation. BBB, blood–brain barrier; SAH, subarachnoid hemorrhage; DMSO, dimethyl sulfoxide; ZO-1, zonula occludens-1. ***p < 0.001.

Fig. 5. Shikonin activates the Sirt1/Nrf2/HO-1 signaling in the brain tissues of rats with SAH.

(A) Western blotting to determine the protein levels of Sirt1, Nrf2, and HO-1 in the cerebral cortex of rats from the sham + DMSO, sham + shikonin, SAH + DMSO, and SAH + shikonin groups. (B-D) Quantification of the relative protein levels of Sirt1, Nrf2, and HO-1 with normalization to GAPDH. Values are shown as mean ± standard deviation. SAH, subarachnoid hemorrhage; DMSO, dimethyl sulfoxide. ***p < 0.001.