Tanshinone IIA attenuates the cerebral ischemic injury-induced increase in levels of GFAP and of caspases-3 and -8

Highlights

• Two hours of ischemia, increased levels of caspases-3 and -8, and GFAP, in the cortex.

• Tanshinone IIA reduced indices of inflammation and cell death.

• Tanshinone IIA treatment reduced infarct size after middle cerebral artery occlusion.

• Tanshinone IIA may effect neuroprotection by reduction of inflammation and cell death.

Abstract

Tanshinone IIA (TSA) is a lipid soluble agent derived from the root of Salvia miltiorrhiza (Danshen). This plant is a traditional Chinese herb, which has been used widely in China especially for enhancing circulation. However mechanisms underlying its efficacy remain poorly understood. The present study was designed to illuminate events that may underlie the apparently neuroprotective effects of TSA following ischemic insult. Adult Sprague–Dawley rats were subjected to transient focal cerebral ischemia by use of a middle cerebral artery occlusion model. They were then randomly divided into a sham-operated control group, and cerebral ischemia/reperfusion groups receiving a two-hour occlusion. Further subsets of groups received the same durations of occlusion or were sham-operated but then received daily i.p. injections of high or low doses of TSA, for seven or 15 days. Hematoxylin and eosin staining revealed lesions in the entorhinal cortex of both rats subject to ischemia and to a lesser extent to those receiving TSA after surgery. Levels of glial fibrillary acidic protein (GFAP), caspase-3 and caspase-8, were quantified by both immunohistochemistry and Western blotting. TSA treatment after middle cerebral artery occlusion, markedly reduced infarct size, and reduced the expression of caspase-3 and caspase-8. These changes were considered protective and were generally proportional to the dose of TSA used. These results suggest that TSA may effect neuroprotection by way of reduction of the extent of cell inflammation and death within affected regions.

Introduction

Stroke is one of the leading causes of death worldwide and can also be a source of permanent disability in survivors. Cerebral ischemic stroke accounts for approximately 80% of all strokes (Chen et al., 2012, Donnan et al., 2008, Feigin et al., 2003). The most common cause of ischemic stroke is the occlusion of a blood vessel by a thrombus, resulting in an immediate loss of the normal supply of oxygen and glucose to cerebral tissue (Genoverse et al., 2011). A variety of therapeutic strategies are currently being considered as a means of minimizing the neuronal damage resulting from ischemia, leading to increasing study of the pathological mechanisms underlying stroke.

The diterpenoid Tanshinone IIA (TSA) is an important component of Chinese medicine, made from the dried rhizome of danshen, Salvia miltiorrhiza. This herb is traditionally used for the treatment of cardiovascular and cerebrovascular diseases, including ischemic stroke (Yang et al., 2008, Xu et al., 2009). TSA is a derivative of phenanthrenequinone;1,6,6-trimethyl-8,9-dihydro-7H-naphtho[8,7-g][1]benzoxole-10,11-dione (Fig. 1). Many plant-derived terpenoids have been found to possess anti-inflammatory and anti-neoplastic properties, and TSA has been reported to modulate many kinds of biological activities, including the quenching of reactive oxygen species, and antagonism of calcium-promoted events. Such molecular changes may account for the ability of TSA to promote microcirculatory function, inhibit neutrophil migration, and exert anti-tumor effects (Han et al., 2008). TSA is able to traverse the blood-brain barrier although the brain content is limited to 30% of the plasma concentration (Chen et al., 2007), and this could relate to its effects in reducing the volume of cerebral infarction and maintenance of neuronal function (Lam et al., 2003). TSA has been shown to have protective effects against focal cerebral ischemia/reperfusion (I/R) injury in animal models (Tang et al., 2010, Liu et al., 2010) and has been proposed as a potential therapeutic agent in heart disease, liver disease and cancer treatment (Dai et al., 2012).

The present study was intended to evaluate the potentially protective effects of TSA in a stroke model and illuminate on the mechanisms that could underlie this. A suture-occlusion method was used to block the middle cerebral artery, followed by release of the block thus initiating reperfusion. Levels of the pro-inflammatory indicators such as glial fibrillary acidic protein (GFAP) were increased after I/R together with levels of two caspases associated with apoptosis; caspase-3 and capsase-8. Treatment with TSA after the surgical procedure partially reversed all of the changes caused by I/R. This provides a mechanistic basis accounting for the utility of TSA on the treatment of stroke.