A highly biosimilar synthetic Calculus Bovis enhances cerebral blood flow and provides neuroprotection against ischemic stroke.

Abstract

ETHNOPHARMACOLOGICAL RELEVANCE: Calculus Bovis (also known as ox bezoar, cattle gallstones, or Niu Huang in Chinese) has been used in traditional Chinese medicine (TCM) for centuries in China and Japan to treat a range of conditions, including high fever, convulsions, and stroke. However, the clinical use of natural Calculus Bovis (NCB) is limited due to its scarcity and high cost. As a result, several alternative substitutes have been developed in recent years, though their effectiveness remains unclear. AIM OF THE STUDY: This study aimed to evaluate the efficacy of a highly biosimilar synthetic Calculus Bovis (HBSCB) in mitigating ischemic brain injury and enhancing cerebral blood flow (CBF) following ischemic stroke. MATERIALS AND METHODS: HBSCB was synthesized using synthetic biology, modeled after the formation mechanism of NCB. Chromatographic fingerprinting was conducted to compare the composition of HBSCB with that of NCB. Male C57BL/6J mice, subjected to transient middle cerebral artery occlusion (tMCAO), were administered varying doses of HBSCB (35.95, 71.9, and 143.8 mg/kg) via intragastric gavage, 1 h post-tMCAO. Therapeutic efficacy was assessed through measurements of brain infarction, neurological deficits and forelimb grip strength. Based on the results, 71.9 mg/kg was identified as the optimal dosage. This dose was then administered at 1, 4, and 6 h post-tMCAO to determine the therapeutic time window of HBSCB. Finally, Fluoro-Jade B (FJB) staining, Nissl staining, and laser speckle contrast imaging of CBF were utilized to investigate the neuroprotective mechanisms of HBSCB in mitigating ischemic brain injury. RESULTS: HBSCB showed a high degree of similarity in key bioactive components with NCB, such as bilirubin and cholic acid. Administration of HBSCB at a dose of 71.9 mg/kg within 4 h post-tMCAO significantly reduced cerebral infarction and edema volumes, while also protecting neurons in the peri-infarct zone from cell death 3 days after tMCAO. Furthermore, HBSCB treatment notably alleviated neurological and motor deficits 1 day after tMCAO. These results demonstrate that HBSCB offers significant neuroprotective effects when administered within 4 h of tMCAO. Mechanistically, HBSCB significantly improved CBF in the ischemic region, which likely contributed to the restoration of energy levels and enhanced brain protection. CONCLUSIONS: The present study demonstrates that HBSCB enhances CBF and provides neuroprotection against ischemic brain injury. These findings support the potential of HBSCB as a viable alternative to NCB for the treatment of ischemic stroke.