Protocatechuic acid promotes cell proliferation and reduces basal apoptosis in cultured neural stem cells

Abstract

Protocatechuic acid (PCA), a phenolic compound isolated from the kernels of Alpinia oxyphylla, showed anti-oxidant neuroprotective property in our previous study. However, it is still unknown whether PCA have effects on the cultured neural stem cells (NSCs). In this study, we investigated the roles of PCA in the survival and apoptosis of rat NSCs under normal conditions. NSCs obtained from 13.5-day-old rat embryos were propagated as neurospheres and cultured under normal conditions with or without PCA for 4 and 7 days. The cell viability was determined by the cell counting kit-8 (CCK-8) test, while cell proliferation was assayed by bromodeoxyuridine (BrdU) labeling. PCA increased the cellular viability of NSCs and stimulated cell proliferation in a dose- and time-dependent manner. Apoptotic cells were detected after 4 days by observing the nuclear morphological changes and flow cytometric analysis. Compared with the control on both culture days, treatment with PCA effectively reduced the levels of apoptosis of NSCs. At the same time, the reactive oxygen species (ROS) level in NSCs was depressed. In addition, PCA also significantly decreased the activity of elevated caspase-3, indicating that PCA may inhibit apoptosis of NSCs via suppression of the caspase cascade. These results suggest that PCA may be a potential growth inducer and apoptosis inhibitor for NSCs.

Introduction

Neural stem cells (NSCs), isolated from embryonic brain, are a heterogeneous population of mitotically-active, self-renewing and multipotential cells, and show complex patterns of gene expression that vary in space and time of normal development (Gage, 2000, Temple, 2001, Reh, 2002, Ivanova et al., 2002). Recently, it has been reported that NSCs exist not only in the developing mammalian nervous system but also in the adult nervous system where NSCs have been found in the two principal adult neurogenic regions: the hippocampus and the subventricular zone. Moreover, it has been revealed that NSCs in adult hippocampus have the potential to become functional neurons, indicating that the plasticity of adult NSCs may contribute to the regenerative potential of the adult nervous system (Song et al., 2002, van Praag et al., 2002). Therefore, NSCs possess the utilizing potential to develop the transplantation strategies and screen the candidate agents for neurogenesis in neurodegenerative diseases (Goldman, 2005, Martino and Pluchino, 2006).

Extensive literature suggests that in vitro, NSCs form aggregates, or neurospheres, in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) (Reynolds and Weiss, 1996). These neurospheres are composed of a heterogeneous mixture of stem cells and progenitor cells, but only NSCs can retain the ability to form neurospheres over 6 months for >50 passages in culture (Gobbel et al., 2003, Kallos et al., 2003). Recently, it has been shown that mouse neural precursor cells growing in neurospheres undergo spontaneous apoptosis in vitro (Milosevic et al., 2004). Moreover, Brand and van Roessel (2003) show that region-specific apoptosis limits neural stem cell proliferation. It is well established that apoptotic cell death occurs not only during development, but also under pathological conditions as a consequence of cellular damage (Nagata, 1997, Du et al., 2000, D’Sa-Eipper and Roth, 2000, Cheng et al., 2001, Esdar et al., 2001). Therefore, to optimize NSC expansion for therapeutic applications (Kallos et al., 2003), drug testing, and research it is important to further understand the basic cell biology of NSCs such as proliferation (including cell division and death) and differentiation characteristics of these cells.

Protocatechuic acid (PCA, Fig. 1), one of the major benzoic acid derivatives from vegetables and fruits and also naturally present in many Chinese herbal medicines such as Salvia miltiorrhiza (Danshen) (Chan et al., 2004) and Hibiscus sabdariffa L. (Liu et al., 2002), shows wide but contradictory biological effects. Studies with laboratory animals have demonstrated recently PCA to show strong anti-oxidant and antitumor promotion effects (Ueda et al., 1996, Tseng et al., 1996, Tseng et al., 1998) and induce apoptosis in some cancer cells (Tseng et al., 2000, Kampa et al., 2004). It has been revealed that PCA may be a candidate chemical for the treatment of oxidative stress-induced neurodegenerative diseases such as Parkinson’s disease (An et al., 2006, Guan et al., 2006a, Guan et al., 2006b, Liu et al., 2008). Although information about its neuroprotective property has been widely reported, little is known about the effect of PCA on the cultured NSCs. Therefore, in present research, we investigated the roles of PCA in the proliferation and apoptosis of rat NSCs under normal conditions.