Qixiong Zuogui Compound affects neurological function in aged middle cerebral artery occlusion/reperfusion rats by regulating bone marrow mesenchymal stem cell-derived exosomes

To study the effects of Qixiong Zuogui Compound (QXZG) on the neural function of the aged middle cerebral artery occlusion/reperfusion (MCAO/R) rat model by regulating exosomes derived from the bone marrow mesenchymal stem cell (MSC).

Male SPF grade SD rats were randomly divided into five groups using a random number table. Rats were divided into five groups (n = 15): young sham-operated, aged sham-operated, aged MCAO/R, blank serum-treated exosome, and medicated serum-treated exosome groups. The aged rat model was established by intraperitoneal injection of D-galactose (500 mg/kg, once daily for 8 consecutive weeks) in all groups, except for the young sham-operated group, which received the same volume of normal saline as a control condition. After the final injection, the aged MCAO/R group, blank serum-treated exosome, and medicated serum-treated exosome groups underwent MCAO/R model induction using the modified suture method, whereas the sham-operated groups underwent vascular and nervous isolation without occlusion. The exosomes were isolated by differential centrifugation and were characterized using transmission electron microscopy, nanoparticle tracking analysis, and Western blotting. At 24 h and 72 h after surgery, rats in the blank serum-treated exosome and medicated serum-treated exosome groups were injected through the tail vein with an equal volume of blank serum-treated or serum-treated exosomes containing 10% QXZG, respectively. All rats were euthanized at Day 7 after surgery. Cerebral infarct area, neurological deficit severity, and learning and memory ability were assessed by 2,3,5-triphenyltetrazolium chloride staining, Zea-Longa scoring, and the Morris water maze test, respectively. Brain histopathological morphology, Nissl body number, and neuronal dendritic spine density were observed by HE staining, Nissl staining, and Golgi-Cox staining. The expression of neuronal nuclei (NeuN) and microtubule-associated protein 2 (MAP-2) were detected by immunofluorescence and Western blotting.

Compared with the young sham-operated group, the aged sham-operated group exhibited multiple deficits, including impaired learning and memory (prolonged escape latency, reduced time spent in the target quadrant and the number of platform crossings), decreased Nissl body, and dendritic spine density, with downregulated NeuN and MAP-2 protein expressions (P<0.05). Compared with the aged sham-operated group, the aged MCAO/R group exhibited exacerbated neurological deficits, as evidenced by higher neurological deficit scores and further impaired cognitive performance (prolonged escape latency, reduced time spent in the target quadrant, and fewer platform crossings). These functional impairments were accompanied by more severe neuropathological changes, including neuronal necrosis in the infarcted tissue, decreased Nissl body, reduced dendritic spine density, and downregulated NeuN and MAP-2 protein expressions (P<0.05). In comparison with the aged MCAO/R group, both the exosomes treated with blank serum group and the exosomes treated with medicated serum group demonstrated significant therapeutic effects characterized by reduced neurological deficit scores, improved brain histopathology, increased Nissl body, shortened escape latency, prolonged time spent in the target quadrant, increased platform crossings, elevated dendritic spine density, and upregulated NeuN and MAP-2 protein expressions (P<0.05). Notably, the improvements in the medicated serum-treated exosome group were significantly superior to those observed in the blank serum-treated exosome group (P<0.05).

QXZG may ameliorate cerebral histopathological damage and neuronal injury and improve learning and memory abilities as well as neurological function in aged MCAO/R rats, by regulating bone marrow MSC-derived exosomes.