Elsevier

Journal of Ethnopharmacology

Volume 185, 5 June 2016, Pages 162-170
Journal of Ethnopharmacology

Protection of tauroursodeoxycholic acid on high glucose-induced human retinal microvascular endothelial cells dysfunction and streptozotocin-induced diabetic retinopathy rats

https://doi.org/10.1016/j.jep.2016.03.026Get rights and content

Abstract

Ethnopharmacological relevance

Tauroursodeoxycholic acid (TUDCA), one of the main ingredients from bear gall which hold “Clearing heat and detoxification, Removing liver fire for improving eyesight” functions, is formed by the conjugation of ursodeoxycholic acid (UDCA) with taurine. However, the limited information of TUDCA on protecting diabetic retinopathy (DR) has been known. The present study was conducted to evaluate the protection of TUDCA on high glucose-induced human retinal microvascular endothelial cells (HRMECs) dysfunction and streptozotocin (STZ)-induced diabetic retinopathy (DR) rats and the possible mechanism underlying was also explored.

Materials and methods

The proliferation of high glucose-induced HRMECs was determined by MTT assay. DR rats’ model was established by an administration of high-glucose-fat diet and an intraperitoneal injection of STZ (30 mg/kg). The cell supernatant and rats’ serum were collected for the assays of NO content by ELISA kits. Retinas were stained with hematoxylin and eosin (HE) to observe pathological changes. Immunohistochemical assay was applied to examine the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF in rat retinas. Furthermore, western blot analysis was carried out to examine the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF in high glucose-induced HRMECs.

Results

After treating with TUDCA, high glucose-induced HRMECs proliferation could be significantly inhibited. TUDCA (5.0 μM, 25.0 μM and 125.0 μM) could decrease NO content in high glucose-induced HRMECs. Furthermore, TUDCA (500 mg/kg/d and 250 mg/kg/d) also decrease NO content in serum of DR rats. Additionally, both immunocytochemistry analysis and western blot analysis showed that the over-expression of ICAM-1, NOS, NF-κB p65 and VEGF were significantly decreased by TUDCA.

Conclusion

The data indicated that TUDCA could ameliorate DR by decreasing NO content and down-regulating the protein expression of ICAM-1, NOS, NF-κB p65 and VEGF. Thus, our experimental results suggested that TUDCA might be a potential drug for the prevention and treatment of DR.

Introduction

Diabetes mellitus (DM) is a worldwide concern which has dramatically increased in recent years. Diabetic retinopathy (DR) is one of serious microvascular complication that affects the retina with symptoms appearing late after the onset of diabetes (Vlatka et al., 2015, Tapley et al., 2015). The change of blood composition in diabetic patients could cause abnormal function of vascular endothelium cells and then resulted in damaging blood retinal barrier. The studies have shown that several vascular, inflammatory and neuronal mechanisms are involved in the complex pathogenesis of DR (Semeraro et al., 2015). Therefore, the research about inflammatory and vascular related factors (ICAM-1, NOS, NF-κB p65 and VEGF) is highlighted.

Fel Ursi Pulvis getting from black bear (Selennarctos thibetanus Cuvier) or brown bear (Ursus arctos L.) in Ursidae family, a traditional Chinese medicine, has remarkable functions on “Clearing heat and detoxification, removing liver fire for improving eyesight” (Wang and Carey, 2014). Tauroursodeoxycholic acid (TUDCA), a major bile acid in bear gall, is formed by the conjugation of ursodeoxycholic acid (UDCA) with taurine and can be obtained by synthetic methods. (Gaspar et al., 2013, Xu et al., 2015). This endogenous hydrophilic bile acid, acts as a mitochondrial stabilizer and anti-apoptotic agent in several models of neurodegenerative diseases (Castro-Caldas et al., 2012, Yanguas-Casás et al., 2014). Multiple pharmacological activities of TUDCA have been shown to be neuroprotective in AD (Ramalho et al., 2013, Dionísio et al., 2015), prevention of acute kidney injury (Gupta et al., 2012), pancreatitis (Malo et al., 2010, Seyhun et al., 2011).

In recent years, there are some reports about the protection of TUDCA on retinopathy (Mantopoulos et al., 2011, Noailles et al., 2014). TUDCA could prevent vision loss in diseases and neurodegenerative diseases including retinitis pigmentosa (Mantopoulos et al., 2011, Noailles et al., 2014). Retinas of TUDCA-treated mice had thicker outer nuclear layers, more photoreceptor cells, and more fully-developed photoreceptor outer segments (Gaspar et al., 2013). However, whether TUDCA is effective on DR is unknown. Therefore, this study was conducted to evaluate the protection of TUDCA on high glucose-induced HRMECs dysfunction and STZ-induced DR rats and the possible mechanism was explored.

Section snippets

Chemicals and reagents

DMEM medium with high-glucose (4.5 g/L), DMEM medium with low-glucose (1.0 g/L) and trypsin were provided by KeyGEN Biotech Co., Ltd (Nanjing, China). NO (Lot: 20140715ELISA kits were purchased from Victoria Reagent Co., Ltd. (Shanghai, China). Fetal bovine serum (FBS) was purchased from Gibco/BRL (Grand Island, NY, USA). 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), and streptozotocin (STZ) were provided by Sigma (St. Louis, MO, USA).

High glucose-induced HRMECs proliferation

MTT assay was used to determine cell proliferation. As shown in Fig. 2, the viability of cells induced by high glucose-DMEM medium (4.5 g/L) was obviously higher than that in low glucose-DMEM medium (1.0 g/L) at 24 h, 48 h and 72 h respectively. However, the enhancement of the cell viability stimulated by high glucose-DMEM medium (4.5 g/L) was decreased in a time-dependent manner. Delightedly, the treatment with TUDCA (1.0 μM, 5.0 μM, 25.0 μM and 125.0 μM) could decrease significantly the cell viability

Discussion and conclusions

DR, a type of specific fundus lesions, is one of the leading causes of visual impairment in the working-age population of the world (Semeraro et al., 2015). DR is mainly due to cells in the retina at a high glucose environment for a long time, leading to a series of fundus diseases, such as apoptosis, migration and proliferation of endothelial cells, cell permeability disorder, and retinal detachment. The multiple biological activities of TUDCA have been revealed, however, its potential

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    Chun-fei Wang, Jia-rui Yuan and Dong Qin contributed equally to the work.

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