The current study has found that BSYQG treatment significantly reduces apoptosis of neurons, astrocytes, and endothelial cells in the ischemic brain after MCAO. So, it can be protected from apoptosis in the neurovascular unit in rats after MCAO. We also showed that the expression of thrombin was upregulated after 2h of ischemic/reperfusion. Importantly, BSYQG treatment significantly decreased brain thrombin activity at 4h and 6h after MCAO. Our study demonstrates that the treatment of BSYQG on the MCAO animal model plays a pivotal role in protecting the NVU by inhibiting the thrombin.
The NVU is a multicellular and multi-component network that is necessary for brain health and equilibrium, and its disturbance is the prime pathophysiological mechanism of ischemic stroke. Thrombin plays a vital role in the mechanisms and pathways with brain injury following ischemic stroke [11]. In ischemic stroke, the higher expression of thrombin is due to breakdown blood-brain-barrier (BBB) [29] and facilitates severe neurovascular unit injury [13]. High thrombin concentration also has a role in brain injury, particularly in the production of cerebral edema and the expansion of the ischemic region after a stroke [30]. It can cleave fibrinogen into insoluble fibrin to generate thrombus, is involved in the death and survival of brain cells, and may act as a "gas pedal" in hemostasis [31–33]. Furthermore, neuronal edema, excitotoxicity, and neuroinflammatory can cause neuron and astrocyte death followed by the high concentration of thrombin after ischemic stroke [34]. This study found that the number of apoptotic neurons, astrocytes, and endothelial cells in the brain cortex rose considerably following cerebral ischemia-reperfusion in the ischemic cortex compared to the sham group. Also, the quantity of thrombin in the ischemic brain was significantly higher at 4 hours than at 0.5 and 6 hours following ischemia/reperfusion.
Bu-Shen-Yi-Qi (BSYQ), comprised of three traditional Chinese herbal medicines: Astragalus membranaceus (Huangqi), Epimedium brevicornu Maxim (Yinyanghuo), and Rehmannia glutinosa (Dihuang), is a compound traditional Chinese herbal prescription used to be proved as anti-inflammatory and neuroprotective effects [21–23]. In the previous study of the effect of BSYQ components, the following chemical components were extracted from BSYQ: Catalpol, Leonuride, Calycosin-7-ο-β-D-glucoside, Hyperoside, Acteoside, Formononetin-7-ο-β-D-glucoside, Epimedin A, Calycosin, Epimedin B, Epimedin C, Icariin, Formononetin, Astragaloside IV, Astragaloside II, Baohuoside-I, Astragaloside I [35]. The primary chemical constituents in the effective BSYQ were determined by HPLC. The highest concentrations were Icariin and epimedin C followed by catalpol, epimedin B, astragaloside IV, and baohuoside-I [35]. Icarlin is a key active ingredient of Epimedium grandiflorum, a traditional Chinese medicinal plant. Icarlin has been shown to have a neuroprotective effect, preventing brain damage caused by ischemia/reperfusion [36, 37]. Catapol, the main component of Rehmannia glutinosa, can ameliorate impaired neurovascular units in the ischemic region and promote angiogenesis to replenish lost vessels and neurons [38]. Astragaloside IV, a primary bioactive compound of Radix Astragali, could decrease the neurological score, reduce the brain's infarct volume, and alleviate cerebral injury after cerebral injury [39]. According to our previous study, the two dosages of BSYQ (3g/kg, 6g/kg) enhanced neurological dysfunction, lowered the extent of cerebral infarction, boosted neuron survival, and decreased neuron apoptosis following cerebral I/R [24]. There was also a dose-response association between the low and high doses. In the study, we found BSYQ (6g/kg) treatment could significantly attenuate cell apoptosis in NVU and protect neurons and astrocytes in the ischemic brain. Furthermore, 6 hours after MCAO, BSYQG treatment significantly lowered brain thrombin activity.
In conclusion, we have demonstrated that BSYQ can inhibit thrombin activity and hence ameliorate ischemic I/R injury in the NVU. In addition, a traditional Chinese medicine compound prescription is a mixture of multiple traditional medicines, and its chemical composition can impact the total medicinal effect by not only enhancing but also limiting the effects of each other. As a result, the protective mechanisms of BSYQ on ischemic injury remain to be further clarified.