Neuroprotective Effect of Zishen Huoxue Decoction treatment on Vascular Dementia by activating PINK1/Parkin mediated Mitophagy in the Hippocampal CA1 Region

Abstract

Ethnopharmacological relevance: Zishen Huoxue Decoction (ZSHXD) is a Traditional Chinese Medicine (TCM) prescription for the treatment of vascular dementia (VD). Although the clinical effects of ZSHXD have been demonstrated, the molecular mechanisms underlying the neuroprotective effects of ZSHXD remain unclear.

Aim of the study: To explore whether the neuroprotective effect of Zishen Huoxue Decoction (ZSHXD) treatment is associated with the PINK1/Parkin pathway-mediated mitophagy in hippocampal CA1 region of 2-VO model rats.

Materials and methods: Seventy-two male SD rats were randomly divided into the sham group, model group, Donepezil (0.45 mg/kg) group, ZSHXD low dose group (8.9 g/kg), ZSHXD medium dose group (17.8 g/kg), and ZSHXD high dose group (35.6 g/kg). Two-vessel occlusion (2-VO) rat model is established to evaluate the therapeutic effect of ZSHXD pretreatment. Hematoxylin-eosin (HE) staining is conducted to detect the morphological changes of neurons and the number of normal neurons in the hippocampal CA1 region. Then, the mitochondrial function and structure were reflected by the mitochondrial membrane potential (MMP) levels and transmission electron microscopy (TEM). Meanwhile, the expression of mitophagy related proteins mediated by PINK1/Parkin was detected by western blot (WB). After that, malondialdehyde (MDA) and superoxide dismutase (SOD) levels were measured by Elisa. At last, the apoptosis-related proteins Caspase-3、Bax、Bcl-2 were measured by WB.

Results: The results depict that ZSHXD has dose-dependently improved the cognitive function in 2-VO model rats. It has also been showed that ZSHXD can alleviate neuron damage, rescue the mitochondrial structural injury and dysfunction in hippocampal CA1 region. Besides, ZSHXD has increased the activity of SOD and decreased the activity of MDA. In addition, ZSHXD can inhibit apoptosis with Caspase-3, Bax decreasing and Bcl-2 increasing. Specially, the protection of ZSHXD showed in 2-VO model rats is along with the upregulation of PINK1, Parkin and LC3-Ⅱ/Ⅰ, and downregulation of p62 in the hippocampal CA1 region.

Conclusions: This study reveals that ZSHXD protects the 2-VO model rats from ischemic injury by activating the PINK1/Parkin-mediated mitophagy in the hippocampal CA1 region.

Keywords: 2-VO Model; CA1 Region; Mitophagy; Neuroprotection; Zishen Huoxue Decoction (ZSHXD).