Ischemic stroke (IS) can disrupt various types of brain cells in the neurovascular unit (NVU) at both the structural and functional levels. Therefore, NVU is considered to be a more comprehensive target for the treatment of IS. It is necessary to develop drugs which targeted multiple mechanisms and cell types on NVU against IS. As a component of bile acid, cholic acid has been reported to be able to diffuse across phospholipid bilayers and further cross the blood-brain barrier (BBB). However, the effects exerted by cholic acid (CA) on the NVU after stroke remain unclear. Based on our previous research, we established and further supplemented the characteristics of the functional in vitro NVU model and its oxygen-glucose deprivation and reoxygenation (OGD/R) model. Then, we investigated the effect of CA on the maintenance of the in vitro NVU after OGD/R and further discussed the specific molecular targets that CA played a role in. For the first time, we found that CA significantly maintained BBB integrity, downregulated apoptosis, and mitigated oxidative stress and inflammation damage after OGD/R. Meanwhile, CA obviously increased the levels of brain-derived neurotrophic factor (BDNF), which were mainly secreted from astrocytes, in the coculture system after OGD/R. The results demonstrated that CA significantly increased the expression of TrkB, PI3K/Akt, MAPK/Erk, and CREB in neurons. These positive effects on the downstream proteins of BDNF were suppressed by treatment with ANA12 which is an inhibitor of TrkB. In conclusion, the present study demonstrates that CA exerted multiple protective effects on the NVU, mediated by increasing the release of BDNF and further stimulating the BDNF-TrkB-PI3K/Akt and BDNF-TrkB-MAPK/Erk signaling pathways in the context of OGD/R-induced injury. These findings indicate that CA possesses the effect of antagonizing multiple mechanisms of IS and protecting multiple cell types in NVU and may be useful as a treatment for IS.

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Background: Qinzhi Zhudan Formula (QZZD), optimized from Angong Niuhuang Wan, consists of Radix Scutellariae, Fructus Gardeniae and Pulvis Fellis Suis. We had investigated the neuroprotective effects of QZZD and its active components, and demonstrated that it could treat cerebral ischemia and dementia through multiple pathways and mechanisms. Nevertheless, toxicological data on this formula still remains limited. In the study, we sought to examine the toxicological effects of QZZD during the treatment and recovery periods.Methods: We investigated potential toxicities of QZZD in Sprague-Dawley (SD) rats via 28-day gavage administration. SD rats were randomly divided into control group and treatment groups of A (0.5 g/kg/d QZZD), B (1.5 g/kg/d QZZD), and C (5.0 g/kg/d QZZD). The 56-day course includes treatment period (administration with water or QZZD once a day for 28 consecutive days) and recovery period (28 days). The rats received daily monitoring of general signs of toxicity and mortality, as well as weekly determination of body weight and food consumption. Moreover, the complete blood cell count, biochemistry, coagulation, and urine indicators, organ weights, and histopathological report were analyzed respectively at the end of the treatment and recovery periods.Results: There was no death related to the active pharmaceutical ingredients of QZZD during the treatment period. The maximum no observed adverse effect level (NOAEL) was 0.5 g/kg/d, which is approximately 16.7 times of the equivalent dose of clinical dose in rats. In group TB (1.5 g/kg/d QZZD) and TC (5.0 g/kg/d QZZD), there were adverse effects of blue coloring of tail skin, weight loss, a significant increase of total bilirubin (TBIL), blackening of liver and kidney in gross examination, hyperplasia of bile duct and karyomegaly of hepatocytes in histopathological examination. Besides, in females rats, the food consumption was reduced, while in male rats, there was decrease in triglycerides (TG) and slight increase in white blood cell (WBC) count and neutrophils. In group TC (5.0 g/kg/d QZZD), the indicators of red blood cell (RBC) count, hemoglobin (HGB) and hematocrit (HCT) were decreased slightly, while the platelet count (PLT) was increased. However, these changes were not considered to be toxicologically significant because they resolved during the recovery period.Conclusion: Overall, QZZD exhibited a good safety profile. The maximum no observed adverse effect level was 0.5 g/kg/d, and no target organs toxicity were identified. The present findings might confirm the safety of QZZD in clinical practices.

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