Mechanism of Zishen Huoxue Formula Improves Mitochondrial Function of Vascular Dementia Model Rats via Sirt1/PGC-1α Signal Pathway

OBJECTIVE To explore the improvement effect and potential mechanism of Zishen Huoxue Formula on mitochondrial function of neurons in 2-VO rats.

METHODS SPF SD rats, 6−8 weeks old, were selected to construct vascular dementia rat model by 2-vessel occlusion(2-VO) and were randomly divided into sham group, 2-VO group, donepezil group(0.00045 g·kg⁻¹), and Zishen Huoxue Formula low-dose(8.9 g·kg⁻¹), medium-dose(17.8g·kg⁻¹) and high-dose(35.6 g·kg⁻¹) groups. After 4 weeks of intervention, the water maze test detected learning and memory ability. Hematoxylin-eosin staining was used to observe the pathological changes in the CA1 region of the hippocampus. Fluoro-JadeB(FJB) fluorescence staining was used to observe neuronal degeneration. Transmission electron microscopy was used to observe the ultrastructure of mitochondria, Flameng scoring method was used for semi-quantitative analysis of mitochondrial damage. The kit was used to detect adenosine triphosphate content in hippocampal tissue. Dihydroethidium fluorescence staining was used to detect the content of reactive oxygen species. IF staining was used to detect the expression of peroxisome prolifertor receptor γ coactivator α(PGC-1α). Western blotting was used to detect the expressions of PGC-1α, silent information regulator 2 homolog 1(Sirt1), B-cell lymphocytoma-2(Bcl-2) gene and Bcl-2-related X protein(Bax).

RESULTS Compared with the sham group, the 2-VO group had a longer escape latency(P<0.001), a decrease in the number of platform crossings(P<0.001), a shorter stay time in the target quadrant(P<0.001), and significantly impaired learning and memory ability. The number of neurons in the CA1 region of the hippocampus was reduced and the morphology was abnormal. The number of FJB-positive neurons increased(P<0.001). The mitochondrial ultrastructure was damaged and the Flameng scores significantly increased(P<0.001). The ATP content in the hippocampus decreased(P<0.001), and the ROS content in the CA1 region of the hippocampus increased significantly(P<0.001) and the expression level of PGC-1α decreased(P<0.001). The protein expressions of Sirt1 and PGC-1α were down-regulated(P<0.001), and the Bcl-2/Bax ratio was down-regulated(P<0.001); compared with the 2-VO group, the escape latency of the 2-VO rats in the low, medium and high-dose groups was significantly shortened(P<0.01 or P<0.001), and the residence time of the target quadrant was prolonged(P<0.01 or P<0.001), and the number of platform crossings of 2-VO rats in the medium and high-dose groups was significantly increased(P<0.01 or P<0.001). The pathological morphology of neurons in the hippocampus CA1 region was improved, the number of FJB-positive cells decreased(P<0.01 or P<0.001). The mitochondrial ultrastructure damage was improved and the Flameng scores significantly decreased(P<0.001). The ATP content in hippocampal tissues increased(P<0.001), and the ROS content in hippocampal CA1 region decreased(P<0.01 or P<0.001) and the expression level of PGC-1α increased(P<0.01 or P<0.001). The protein expressions of Sirt1 and PGC-1α were up-regulated(P<0.01 or P<0.001), and the Bcl-2/Bax ratio increased(P<0.001).

CONCLUSION Zishen Huoxue Formula may improve mitochondrial morphology and function, reduce neuronal deformation damage and apoptosis by activating the Sirt1/PGC-1α signaling pathway, and promote the improvement of cognitive function.