Astrocytes are divided into protoplasmic and fibrous types based on their morphologic characters and the distribution (Blackwood 1976; Hirano 1985; Privat and Rataboul 1987). The morphology of astrocytes are complex, different morphology of astrocytes can coexist in a given brain region (Matyash and Kettenmann 2010). Astrocytes can also express different functions in gap junction coupling, expression of transmitter receptors, membrane currents, and glutamate transporters (Matyash and Kettenmann 2010). The distribution of astrocytes is strongly determined by vasculature (Stone and Dreher 1987). Astrocytes which are closed to retinal vessels (Schnitzer 1987) will help maintain vascular integrity (Zhang and Stone 1997). Astrocytes enlarge their end-feet around blood vessels, participating in the formation of TJ of endothelial cells which could prevent BBB from damage, and also modulate the transport function of the cerebral endothelium (Montgomery 1994). Astrocytes are known to be the main target of ET-1 (Sasaki et al. 1997; Prasanna et al. 2003). Previous studies found that astrocytic ET-1 has deleterious effects on water homeostasis, cerebral edema, and BBB integrity, which contribute to severe ischemic brain injury (Lo et al. 2005). Disruption of BRB leads to the swelling of astrocytes associated with the overexpression of glial fibrillary acidic protein (GFAP) and aquaporin-4 (AQP4) under the ischemic insult (Kaur et al. 2007). Each astrocyte could form end-feet around at least one blood vessel, while blood vessels can affect the morphology of astrocytes (Bushong et al. 2002). Our previous study showed that LBP can decrease the number of proliferated astrocytes and reduce their end-foot edema around the blood vessel, which could protect the survival of neurons. Thus, LBP can induce the blood-vessel-associated astrocytes to show entirely different morphologic change. These changes included apparently more processes touching blood vessels without enlargement of their end-feet (Leung et al. 2009b; Lo et al. 2005; Yeung et al. 2009).

In normal retina, GFAP is expressed on astrocytes and the end-feet of Müller cells. When under the stress, the expression of GFAP will increase not only on astrocytes but also on the process of the Müller cells (Cheung et al. 2005b; Mori et al. 2009; Taylor et al. 2010; Hirrlinger et al. 2010). It was reported that the overexpression of GFAP could be as the indicator for the reduction of blood flow (Osborne et al. 1991). From our previous studies, LBP could reduce the expression of GFAP, which may be a possible mechanism for recovery of blood supply to the tissue (Mi et al. 2012b).

AQP4 is a member of water channel protein family which mainly expressed in astrocytes (Asai et al. 2013) and also a marker to reflect the capability to maintain the balance of water across the wall of blood vessels and to detect the functional change of astrocytes (Nagelhus et al. 1998). AQP4 is linked to the damage of BBB, suggesting the increase of permeability (Guo et al. 2006). It was reported that overexpression of AQP4 was related to the retinal swelling and involved in the process of neuronal death (Verkman et al. 2008). Previous studies demonstrated that pretreatment with LBP could reduce the expression of AQP4 in the animal model of ischemia in retina and brain (Li et al. 2011; Mi et al. 2012b; Yang et al. 2012).

ET-1, synthesized in vascular endothelial cells, is a potent vasoconstrictor. ET-1 regulates blood flow mainly via its two receptors, ET_A and ET_B (Narayan et al. 2003). ET_A is expressed on vascular smooth muscle cells and contracts blood vessels, while ET_B is mainly expressed in the endothelium and astrocytes and dilates blood vessels (Narayan et al. 2003; Masaki et al. 1994). Overexpression of ET-1 results in downregulating the level of occludin, the key protein located at TJ between blood vessel endothelial cells to maintain the integrity of blood-barrier (Leung et al. 2009a). Decreased expression of occludin has been reported in middle cerebral artery occlusion (MCAO)-induced ischemic brain and retina. However, LBP-pretreatment could maintain the level of occludin among blood vessels (Yang et al. 2012; Mi et al. 2012b). Further studies showed that LBP could reduce the ET-1-ET_A signal pathway while activate ET-1-ET_B pathway in the retinal blood vessels through downregulating the expression of ET_A and upregulating ET_B (Yorio et al. 2002). Not only endothelial cells, ET-1 is considered to stimulate contraction of pericytes (Ramachandran et al. 1993). Pericytes are considered to be other contractile cells to help regulating the blood flow (Wallow and Burnside 1980). Thus, through regulating the expression of ET_A and ET_B receptors, LBP could modulate the blood flow of retinal blood vessels (Mi et al. 2012a, b). Thus, LBP could protect endothelial cells and pericytes via downregulating ET-1.

Angiogenesis is the generation of new blood vessels from the original ones, playing an important role in the pathogenesis of diabetes. VEGF and TGF-β are angiogenin mediators. The high level of glucose in blood could result in the overexpression of VEGF (Martin et al. 2003) and TGF-β (Singh et al. 2013), contributing to vascular abnormalities in diabetes. LBP was shown to have a significant effect on reducing VEGF (Tian et al. 2013) and TGF-β (Singh et al. 2013). One recent study reported that a wolfberry contented formula diet (including Rhizoma dioscorea, L. barbarum, Prunella vulgaris and hawthorn) had effects on antidiabetes (Cheng et al. 2014).

RAGE is expressed on both smooth muscle cells and endothelial cells of blood vessel (Farmer and Kennedy 2009). It was reported that one role of RAGE was to transport Aβ across BBB to the brain tissue. Thereafter, Aβ could interact with RAGE resulting in the release of ET-1 from endothelial cells (Deane et al. 2003a), and contributing to vascular inflammation and endothelial dysfunction (Harja et al. 2008). Our previous study has proved that LBP can reduce the expression of RAGE (Mi et al. 2012b), therefore to inhibit the destructive effect of RAGE to blood vessels under ischemic insults.

Aβ is a ligand of RAGE, which can induce vasoconstriction, decrease cerebral flow, and enhance the effect of ET-1-induced vasoconstriction (Paris et al. 2000). The accumulation of Aβ transported from BBB is also toxic to vasculature (Zlokovic 2005). ET-1 could inhibit the cerebral blood flow (CBF) through activating Aβ-RAGE transportation across the BBB (Deane et al. 2003b). LBP could downregulate the expression of Aβ (Mi et al. 2012b) thus to inhibit the Aβ-induced vascular toxic effect (Ho et al. 2010).

AGE is another key ligand of RAGE. The accumulation of AGE will activate RAGE, leading to endothelial dysfunction (Xu et al. 2003). Besides, AGE-RAGE pathway could enhance the synthesis of ET-1 in the endothelial cells (Quehenberger et al. 2000). Amount of evidences showed that AGE could induce the diabetic vascular complication (Takeuchi et al. 2000; Yamagishi et al. 2002b). AGE could induce pericyte apoptosis and VEGF expression contributing to the destruction of retinal vasculature (Yamagishi et al. 2002a). Thus, accumulation of AGE and overexpression of VEGF will aggravate the disorder of the vascular system (Yokoi et al. 2005). In our previous study, it was demonstrated that LBP could downregulate the expression of AGE, which could inhibit the destructive effect of AGE to blood vessels (Mi et al. 2012b).