Screening triterpenoid compounds with potential to treat retinal diseases

Abstract

Retinal degeneration is a leading cause of impaired vision or even blindness all around the world, unfortunately, most retinal diseases are irreversible, and there is no ideal treatment to cure the diseases, therefore it is important to protect retina from disease progression. In the recent years, natural compounds have shown great pharmacological potentials. Pentacyclic triterpenoids (PTs) are a group of compounds widely existing in nature, they have shown diverse pharmacological effects in vitro and in vivo. In this thesis, we evaluated the antioxidative effect of betulin, betulinic acid and their 18 derivatives (groups were modified at C3 and C28 positions of betulinic acid skeleton) in human retinal pigment epithelium (RPE) and human Müller cells. RPE is a monolayer of cells between the outer neural layer of photoreceptors and choroid, responsible for mediating visual cycle and nourishing photoreceptors. Hypoxia in RPE cells is likely to cause retinal diseases such as the age-related macular degeneration (AMD), which is the leading cause of blindness in old people worldwide. In Chapter 2, we established the hypoxia model in ARPE-19 cells with CoCl2, which induced a dramatic reduced cell viability, increased reactive oxygen species (ROS) level, induced apoptosis and necrosis. H7 pretreatment could significantly reversed the above damage. In addition, H7 could inhibit the activation of Akt, Erk1/2 and JNK pathways stimulated by CoCl2. We also verified the same signaling pathways in human primary RPE cells. Müller cells are predominant neuron-supporting glial cells, they play important roles in maintaining the retinal homeostasis, releasing trophic factors and supporting other retinal cells. One of the key functions is to uptake the neurotransmitter glutamate and convert it into glutamine, therefore high concentrations of glutamate will lead to pathological changes of Müller cells, which may promote progressions of some retinal degenerations like diabetic retinopathy (DR) and glaucoma. In Chapter 3, we tested the antioxidative effect of betulinic derivatives in MIO-M1 cells against glutamate-induced excitotoxicity. The betulinic derivatives H3, H5, H7 and H11 could significantly increase cell survival and reduce necrosis level after glutamate treatment. H3, H5 and H7 could effectively inhibit ROS production. H5 and H7 showed the effects by inhibit the activation of Akt, Erk1/2 and JNK pathways induced by glutamate, while H3 only inhibited Erk1/2 phosphorylation. Studies in Chapter 2 and Chapter 3 indicate that these betulinic derivatives protected RPE and Müller cells by deactivating Akt, Erk1/2 and JNK. However, the effective concentrations of betulinic derivatives in Chapter 2 and Chapter 3 are hard to reach in the retina by systematic administration. Liposome is a widely-used approach to increase drug delivery efficacy and therapeutic effects, therefore we encapsulated the active betulinic derivatives (H3, H5 and H7) into Compritol 888 ATO or Compritol HD5 ATO liposomes and evaluated the cytoprotective effect of each liposome-based formulations in ARPE-19 and MIO-M1 cells. We found that H3 and H5 Compritol 888 ATO liposomes, as well as H5 Compritol HD5 ATO liposomes increased cell viability in glutamate-treated MIO-M1 cells. H5 Compritol 888ATO liposome, and Compritol HD5 ATO liposomes with H3, H5, H7 significantly attenuated ROS production caused by CoCl2. Furthermore, Compritol 888 ATO liposomes particularly enabled encapsulated compounds to release sustainably. Studies in this Chapter indicated that liposomes are a promising drug delivery carrier for betulinic acid derivatives to achieve an improved cellular protective effect against oxidative stress in human RPE and Müller cells.