The protective roles of L-borneolum, D-borneolum and synthetic borneol in cerebral ischaemia via modulation of the neurovascular unit

Objective: Borneol has been used to treat stroke in China since ancient times. In our previous research, we demonstrated the effect of borneol on cerebral ischaemia injury via meta-analysis. The neurovascular unit (NVU) is the structural basis of the preservation of the brain microenvironment and is believed to be a promising target in treating stroke. In this research, we explored the roles of three kinds of borneol, namely, L-borneolum (B1), D-borneolum (B2) and synthetic borneol (B3), in the NVU with permanent middle cerebral artery occluded (pMCAO) rats. Methods: The Longa scoring method was used to evaluate nerve function deficits in the pMCAO rats. Awakening time, brain water content, brain index and brain edema rate were also measured. TTC staining was used to calculate the cerebral infarction rate. The morphology of the ischaemia penumbra brain tissue was observed via HE staining, and the neuronal denatured cell index (DCI) was calculated. An enzyme-linked immunosorbent assay (ELISA) was used to determine the levels of vascular endothelial growth factor VEGF and TNF-alpha in the serum. Moreover, the ultrastructures of the neurons and of the blood-brain barrier (BBB) were observed using transmission electron microscopy. The expression levels of Claudin-5, Bcl-2 and Bax in the ischaemia penumbra of pMCAO rats were detected using real-time PCR and immunohistochemistry. Results: Pretreatment with B1, B2 and B3 delayed the recovery time (P < 0.01). B1 remarkably ameliorated neurological deficits 24 h after cerebral ischaemia (P < 0.05). Moreover, B1 and B3 were both able to ameliorate brain edema and the area of cerebral infarction. In addition, B1, B2 and B3 all increased serum VEGF levels and decreased serum TNF-a levels (P < 0.01). For the ultrastructure determination, the BBB and the nerve centre were significantly improved by B1, B2 and B3. The mechanistic exploration revealed that B2 and B3 protected the brain by reducing the Bax/Bcl-2 ratio (P < 0.05, P < 0.01, respectively). Immunohistochemistry suggested that B1, B2 and B3 could also enhance the expression of Claudin-5 (P < 0.01). Conclusion: The three kinds of borneol demonstrated different protective effects on cerebral ischaemia injury. L-Borneolum displayed the most prominent anti-cerebral ischaemia effect among them. The mechanism was most likely executed via anti-apoptosis and anti-inflammation effects and maintenance of the stability of the BBB and TJs to comprehensively improve NVU function.