Use of (−)-epigallocatechin-3-gallate on spinal cord injury

Introduction

Over the past decades, a large number of polyphenolic compounds with neuroprotective effects have been described. One of the main sources of these molecules is green tea, the most widely consumed beverage next to water. The chemical composition of green tea contains a number of bioactive components, mainly including polyphenols, caffeine, and amino acids ( ). Green tea polyphenols, generally known as catechins which make up 30% of the dry weight of green tea leaves, are the main bioactive constituents of green tea with a wide variety of beneficial health effects ( ). The main catechins in green tea include (+)-catechin, (−)-epicatechin, (+)-gallocatechin, (−)-epigallocatechin, (+)-catechin gallate, (−)-epicatechin gallate, (+)-gallocatechin gallate, and (−)-epigallocatechin-3-gallate (EGCG) ( ). EGCG, chemically (2 R ,3 R )-5,7-dihydroxy-2-(3,4,5trihydroxyphenyl)chroman-3-yl 3,4,5-trihydroxybenzoate ( Fig. 1 ), is the most abundant composition of the tea catechins, account for 65% of the total catechin content and is thought to be responsible for the majority of biological activity of green tea extracts ( ). In terms of bioavailability, EGCG is predominantly absorbed in the intestine and presented more than 77% in a free form in plasma as well as its metabolites excreted in urine after oral administration ( ; ; ). The half-life of EGCG in a purified form is around 3 h ( ). Meanwhile, it is well documented that EGCG is able to cross the blood-brain barrier (BBB) and can reach to nervous tissue even at a very low concentration, the first requirement of a dietary compound to apply neuroprotective effects ( ; ). Several experimental studies have shown that EGCG can afford neuroprotection against brain ( ) and spinal cord ( ) injuries, neurodegenerative diseases ( ), and peripheral nerve injuries ( ). These beneficial effects have been mainly attributed to free radical scavenging or anti-oxidant, anti-inflammatory, and anti-apoptotic properties of EGCG ( ; ; ). In this regard, it is well known that green tea catechins due to the hydroxyl groups on the B- and D-rings of the galloylated catechins can bind to the free radicals and neutralized those ( ). On the other hand, scavenging effects of EGCG lead to attenuation of nuclear factor kappa B (NF-κB) activity ( ), which regulates genes involved in inflammatory processes such as tumor necrosis factor alpha (TNFα), cyclooxygenase-2 (COX-2), and interleukin-1 beta (IL-1β), beside modulation of nitric oxide synthase isoforms ( ). Also, catechins have proven to modulate apoptosis at various points in the sequence and messengers, and so regulate the mitochondrial membrane permeabilization ( ; ).

In this chapter, we have reviewed the neuroprotective effects of EGCG and its molecular mechanisms responsible for the neuroprotection following spinal cord injury (SCI). Meanwhile, we also compared the neuroprotective effects of EGCG in SCI to other neurological disorders.

In vivo studies

SCI is a complex multifactorial process caused first by ischemia or mechanical trauma and then by various mechanisms of secondary injury ( ). The neurological outcome of SCI depends on the extent of secondary cellular, molecular, and biochemical cascades such as oxygen free radical-induced lipid peroxidation, inflammatory reaction, and apoptosis ( ; ; ). In recent years, much attention has been paid to secondary injuries, as they appear to be prone to therapeutic interventions that may include the use of natural anti-oxidants, anti-inflammatory, and anti-apoptotic agents ( ) such as EGCG ( Table 1 ). In this regard, our previous studies have shown that intraperitoneal injection of EGCG (50 mg/kg, i.p., immediately and 1 h after SCI) can protect spinal cord tissue and improve behavioral function after traumatic and ischemia-reperfusion injury (IRI) in rats ( ; ; ). In regard to mechanisms of neuroprotective effects of EGCG, our results revealed that malodialdehyde (MDA) levels as an index of tissue lipid peroxidation, an important pathologic event in post-traumatic neuronal degeneration, were significantly decreased in traumatized spinal cord tissue after EGCG treatment ( ). It is well documented that EGCG due to hydroxyl groups can bind to the free radicals and neutralize those, while can indirectly increase the body’s endogenous antioxidants ( ). We detected immunohistochemically anti-apoptotic properties of EGCG with decreasing pro-apoptotic protein Bax and increasing anti-apoptotic protein Bcl-2 staining in EGCG-treated rats, which was correlated with terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining method ( ). Also, our other study showed that EGCG treatment (50 mg/kg, i.p., immediately and 1 h after SCI) attenuated pro-inflammatory cytokines such as TNFα and IL-1β in traumatized spinal cord tissue ( ). In line with the anti-inflammatory properties of EGCG, documented that EGCG treatment (100 mg/kg, between 24 and 72 h after SCI) decreased spinal tissue edema through down-regulation of astrocyte expressing aquaporin-4 (AQP4), which plays a critical role in the transport of water from blood/CSF to spinal cord parenchyma, and through down-regulation of glial fibrillary acidic protein (GFAP) as a specific marker of astrocytes. Also, another study found that attenuated NF-κB pathway is a pivotal anti-inflammatory affect of EGCG (intra-spinal injection of 50 mg/kg EGCG immediately and then weekly for up to 28 days) after SCI ( ). Meanwhile, this study showed a significant increase in the gene expression of fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor (VEGF) after SCI. documented that EGCG (100 mg/kg, i.p., immediately after SCI) can protect secondary SCI by potential mechanism of regulating p38MAPK\NF -κB\AQP4 signaling pathway and thus reduce edema after SCI in rats. Also, another study documented that intrathecal administration of EGCG (10 or 20 mg/kg immediately after SCI) can significantly improve locomotors recovery, which may be related to the inhibition of Bcl-2-associated X protein (Bax) and to the up-regulation of brain-derived neurotrophic factor (BDNF) and glial cell line-derived neurotrophic factor (GDNF) ( ). Studies have shown that EGCG has beneficial effects not only on the tissue destruction but also on behavioral and functional outcome, so that intravenous infusion of EGCG (20 mg/kg/hour for continuous 36 h) in acutely (initiated within 4 h post-trauma 0 time) and chronically (initiated after 12 months of SCI onset) spinal cord injured rats improved significantly motor and sensory functions measured using Basso-Beattie-Bresnehan behavioral score test, Louisville forced swim test, and pain behavior assessment tests ( ). One of the common complications of SCI is neuropathic pain. About the effect of EGCG on neuropathic pain, documented that short time EGCG treatment (30 mg/kg i.p., 30 min after and daily during the first week post-SCI) reduced thermal hyperalgesia and gliosis via ras homologue gene family member A (RhoA) and fatty acid synthase (FASN) pathway. In this regard, it is mentioned that some biological properties of EGCG are attributed to its inhibitory action on FASN ( ). In another study, the synergistic effects of curcumin and EGCG in an animal model of acute SCI were investigated ( ). Results of this study revealed that combination of curcumin and EGCG reduced glial scar formation, increased axonal sprouting, and changed the amount of macrophage inflammatory protein 1-alpha (MIP-1α), IL-1β, interleukin-4 (IL-4), and interleukin-6 (IL-6). Recently, our laboratory assessed the neuroprotective effects of EGCG on spinal cord IRI in rats ( ). The level of MDA was significantly reduced in EGCG-treated rats. Attenuated caspase-3 ( Fig. 2 ), TNFα ( Fig. 3 ), and inducible nitric oxide synthase (iNOS) expression could be significantly detected in the EGCG-treated rats. Also, EGCG reduced the extent of degeneration of the spinal cord neurons, in addition to a significant reduction of motor deficit index (MDI). Overall, the behavioral, biochemical, and histopathological evidences demonstrated that pre- (50 mg/kg, i.p., before IRI) and post-ischemic (50 mg/kg, i.p., after IRI) treatment with EGCG had protective effects against spinal cord IRI in rats.