Glycyrrhizin protects against sodium iodate‐induced RPE and retinal injury though activation of AKT and Nrf2/HO‐1 pathway

Abstract Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age‐related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI‐induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2‐related factor2 (Nrf‐2) and HO‐1, thereby protecting RPE from SI‐induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.

chronic or intense stress conditions, apoptosis occurs through various signalling pathways. A major objective for treating atrophic AMD involves reduction in retinal pigment epithelium (RPE) and photoreceptor death induced by various pathologic factors. Despite substantial progress in the development of new therapies for wet AMD using inhibition of vascular endothelial growth factor (VEGF), the severe visual impairment associated with geographic atrophy in dry AMD remains untreatable. 7 Recently, medicinal plants that produce ursolic acid have been reported to successfully prevent cell death in heart, liver and neurosystem. 8 Thus, it is necessary to investigate natural compounds derived from plants for the prevention of atrophic AMD.
Glycyrrhizin, also known as glycyrrhizic acid, is a substance extracted from a traditional Chinese medicinal herb, Glycyrrhiza. According to a previous report, glycyrrhizin reduced inflammatory injury in a model of inflammatory pain via suppression of NF-κB, TNF-α and intercellular adhesion molecules. 9 Besides anti-inflammatory, glycyrrhizin has also been reported to have anti-viral and hepatoprotective effects, and was given to patients with chronic hepatitis and human immunodeficiency virus infection. 10,11 Furthermore, glycyrrhizin possessed potential neuroprotective effect against ischaemia-reperfusion injury in a model of focal cerebral ischaemic-reperfusion injury. 12 However, whether glycyrrhizin prevents atrophic AMD is still unknown.
Sodium iodate increases reactive oxygen species (ROS) and induces consistent and selective damage to the RPE. 13 Exposure to sodium iodate (SI) results in a primary death of the RPE followed by a secondary death of the overlying photoreceptors, similar to what is observed in advanced atrophic AMD. Thus, SI has been widely used to study the molecular mechanism of cell death in atrophic AMD. [14][15][16][17] Hence, in this study, we investigated whether glycyrrhizin prevents SI-mediated RPE injury in vitro and retinal injury in vivo. Glycyrrhizin prevented SI-induced RPE and retinal injury through inhibition of ROS and decreased apoptosis, indicating a potential clinical usage of glycyrrhizin in protecting against atrophic AMD.
Human ARPE-19 cells were plated at a density of 1 × 10 4 cells/well in complete medium containing 10% fetal bovine serum (FBS) in 96-well plates. After cell attachment, the cells were pre-treated with a series of concentrations of glycyrrhizin (25-200 μmol) for 6 hours before adding SI (1200 μg/mL). After another 24 hours of cell culture, 20 μL of methylthiazolyldiphenyl-tetrazolium bromide (MTT) reagent was added to each well, and incubated for 4 hours at 37°C. After that, the cell culture medium was carefully aspirated, and 150 μL of DMSO was added to each well. The 96-well plates were shaken at room temperature for 10 minutes, and then the absorbance was measured on a microplate reader at 570 nm. Wells without cells were used as blank controls.

| Cell apoptosis assay
After pre-treatment of ARPE-19 cells for 6 hours with glycyrrhizin (50 μmol) in six-well plate, SI was added to the cell culture. After Cells were collected by centrifugation at 280 g for 5 minutes, followed by addition of 500 μL binding buffer, 5 μL Annexin V-FITC, and 5 μL PI. Subsequently, the samples were incubated at room temperature in the dark for 10 minutes, followed by flow cytometry within 1 hour. The percentages of early and late apoptotic cells were calculated.

| Measurement of ROS production
Dichloro-dihydro-fluorescein diacetate (DCFH-DA) method was used to assess the ROS levels in RPE cells. ARPE-19 cells were pre-treated with glycyrrhizin (50 μmol) for 6 hours before the addition of SI to the cell culture. After 24 hours of cell culture, the ARPE-19 cells were incubated with 2,7-dichlorofluorescin diacetate (20 μmoL) (ROS assay kit, E004; Nanjing Jiancheng Bioengineering Corporation, Nanjing, China) at 37°C for 60 minutes in the dark.
After that, the ARPE-cells were observed under an Olympus IX71 fluorescence microscope (Tokyo, Japan). Five high power fields were randomly selected. Fluorescence intensities of the sections stained for ROS were determined with the Image-Pro Plus software.

| Animals
All animal experiments were approved by the Experimental Animal  F I G U R E 2 Effects of glycyrrhizin on apoptosis of ARPE-19 cells in vitro. A, Hochest 33342 staining of ARPE-19 cells were treated with sodium iodate at a concentration of 50 μmol. a, sham; b, retinal pigment epithelium (RPE) treated with sodium iodate (SI); c, RPE treated with sodium iodate plus glycyrrhizin (SI + GA). B, Sodium iodate significantly increased apoptosis of ARPE-19 cells, compared normal control (*P < 0.01), while glycyrrhizin significantly inhibited apoptosis of ARPE-19 cells treated with sodium iodate, compared with sodium iodate control ( # P < 0.01). C, Flow cytometry analysis of apoptosis of ARPE-19 cells treated with SI at a concentration of 50 μmol. D, Sodium iodate significantly increased apoptosis of ARPE-19 cells compared normal control (*P < 0.01), while glycyrrhizin treatment significantly decreased the apoptosis of ARPE-19 cells treated with SI, compared with SI control ( # P < 0.01). E, Western blot detection of the effect of glycyrrhizin on cleaved caspase-3 in RPE treated with sodium iodate. Lane 1, sham; Lane 2, RPE treated with SI; Lane 3, RPE treated with SI + GA. F, Sodium iodate significantly increased expression of cleaved capase-3 in ARPE-19 cells compared with normal control (*P < 0.01), while glycyrrhizin significantly decreased the expression of cleaved-caspase 3 in ARPE-19 cells treated with sodium iodate, compared with the sodium iodate control ( # P < 0.01).

| Animal experimental setting
Thirty mice were randomly divided into three groups: sham (n = 10), SI (n = 10), and SI + glycyrrhizin treatment (n = 10). In the sham group, 0.1% DMSO in double distilled water (DDW) was injected intraperitoneally daily for 1 week before the mice were intravenously injected with DDW containing no SI one time, and intraperitoneal administration of 0.1% DMSO in DDW was continued until the mice were sacrificed. In the SI group, 0.1% DMSO in DDW was injected intraperitoneally daily for 1 week before the mice were intravenously injected with SI in DDW (30 mg/kg) one time, and intraperitoneal administration of 0.1% DMSO in DDW was continued until the mice were sacrificed. In the SI + glycyrrhizin group, glycyrrhizin in 0.1% DMSO (50 mg/kg/d) was injected intraperitoneally daily for 1 week before the mice were intravenously injected with SI in DDW (30 mg/kg) one time, and intraperitoneal administration of glycyrrhizin in 0.1% DMSO was continued until the mice were sacrificed.

| Micron IV imaging
Mice were anaesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). Pupils were dilated with topical administration of 2.5% phenylephrine containing 0.5% tropicamide, and the cornea was anaesthetized with 0.5% proparacaine. The retinas of the mice were imaged in vivo using the Micron IV retinal imaging camera system (Phoenix Research Laboratories, Pleasanton, CA). Optical coherence tomography was performed on days 0 and 28 to monitor the retinal morphology.

| Electroretinography
Mice were dark adapted overnight and anaesthetized by intraperitoneal injection of ketamine (100 mg/kg body weight) and xylazine (10 mg/kg body weight). Pupils were dilated with topical administration of 2.5% phenylephrine containing 0.5% tropicamide, and the cornea was anaesthetized with 0.5% proparacaine. Full-field electroretinography (ERG) was measured using a non-attenuated light stimulus. Photoreceptor responses

| Haematoxylin and eosin staining
Five mice in each group were sacrificed on day 28 after SI treatment, and eye samples were harvested, embedded in paraffin, and sliced into 4-μm-thick sections. Subsequently, the sections were stained with haematoxylin and eosin (HE). Images of retina at the same position in each section of the individual eye were selected and photographed under microscope.    ). B, Both SI and SI + GA significantly increased thinning of retina, compared with normal control (*P < 0.01), however, glycyrrhizin significantly inhibited thinning of retina after treatment of SI on day 28, compared with SI control ( # P < 0.05). C, Haematoxylin and eosin staining of retina on day 28 after SI treatment. a, sham; b, retina treated with SI; c, retina treated with SI + GA. Both SI and iodate and sodium plus GA had disorganization of Bruch's membrane, however, glycyrrhizin overtly alleviated the disorganization of Bruch's membrane caused by SI. D, Both SI and SI + GA significantly increased thinning of retina, compared normal control (*P < 0.01), however, glycyrrhizin significantly inhibited thinning of retina after treatment of sodium, compared with SI control ( # P < 0.05). E, Both SI and SI + GA significantly increased the drusen number of retina, compared with normal control (*P < 0.01), however, glycyrrhizin significantly decreased the drusen number of retina after treatment of sodium, compared with SI control ( # P < 0.01)

| Statistical analysis
All the values were presented as mean ± SD. One-way ANOVA followed by Bonferroni/Dunn test were performed for group comparisons. A P-value of <0.05 was considered statistically significant.

| Glycyrrhizin decreased the apoptosis of RPE treated with SI in vitro
Methylthiazolyldiphenyl-tetrazolium bromide assay was used to evaluate the effect of glycyrrhizin on the viability of ARPE-19 cells treated with SI. The results showed that glycyrrhizin at various concentrations of 25-100 μmol showed no toxicity on ARPE-19 cells and significantly increased its viability, compared with the SI control ( Figure 1A,B). Hochest 33342 staining showed that the apoptotic nuclei of ARPE-19 cells were overtly reduced at 50 μmol of glycyrrhizin after SI treatment (Figure 2A).

| Glycyrrhizin decreased ROS in RPE treated with SI in vitro
The DCFH-DA method was used to evaluate the effects of glycyrrhizin on ROS production in RPE after treatment with SI. The results showed that glycyrrhizin at 50 μmol significantly decreased the ROS levels in the ARPE-19 cells after SI treatment, compared with SI control ( Figure 3A,B).

| Glycyrrhizin decreased apoptosis of retina induced by SI in vivo
Terminal dUTP nick end-labelling staining was used to detect the effects of glycyrrhizin on apoptosis of retina on day 28 after treatment of SI in vivo. The results showed that glycyrrhizin significantly decreased apoptosis of retina treated with SI ( Figure 4A,B).

| Glycyrrhizin decreased thinning of retina and number of drusen induced by SI
Optical coherence tomography was performed to detect the effects of glycyrrhizin on the injury of retina induced by SI. The results showed that glycyrrhizin, at the end of the detection points, significantly decreased thinning of retina caused by SI treatment, compared with SI control (Figure 5A,B).
To further prove this, haematoxylin and eosin staining was performed to detect the effects of glycyrrhizin on the pathological changes of retina after SI administration. The results showed that at the end of detection points, glycyrrhizin significantly decreased the thinning of retina, especially outer nuclear layer (ONL) and inner nuclear layer (INL) (Figure 5C,D). The data also showed that the drusen number was significantly decreased by glycyrrhizin ( Figure 5C,E).

F I G U R E 6
Effects of glycyrrhizin on retinal function after sodium iodate (SI) treatment in vivo. A, Electroretinography measurement of retinal function. a, sham; b, retina treated with SI; c, retina treated with SI plus glycyrrhizin (SI + GA). B, Glycyrrhizin significantly inhibited the decreasing amplitude of a-wave treated with SI, compared with SI control (*P < 0.05). C, Glycyrrhizin significantly inhibited the decreasing amplitude of b-wave treated with SI, compared with SI control (*P < 0.05)

| Glycyrrhizin increased ERG amplitudes in SItreated mice
To determine whether glycyrrhizin had an effect on the retinal function after SI treatment, the full-field ERG response was detected. The results showed that glycyrrhizin significantly increased the amplitude of 'a-wave' that originates from the photoreceptors, compared with SI control (Figure 6A,B). Also glycyrrhizin significantly increased the amplitude of 'b-wave' that originates from the bipolar cells, compared with SI control (Figure 6A,C).

| Glycyrrhizin increased the phosphorylation of AKT in RPE treated with SI
Akt pathways play a major role in anti-apoptosis. Western blot was performed to detect Akt in ARPE-19 cells treated with SI at 24 hours.
Results revealed that glycyrrhizin significantly increased the phosphorylation of Akt in RPE treated with SI ( Figure 7A,B), indicating that Akt plays a crucial role in the anti-apoptosis of RPE by glycyrrhizin after SI treatment.

| D ISCUSS I ON
In this study, we found that glycyrrhizin inhibited SI-induced ROS in RPE, and decreased RPE cell apoptosis in vitro and retinal cell apoptosis in vivo. To the best of our knowledge, this is the first study that reported glycyrrhizin in the prevention of SI-induced RPE and retinal toxicity.
Although the mechanisms of toxicity of the eye by SI are not fully elucidated, inducing ROS and subsequently causing retinal apoptosis is one of the effects of SI on the eye. Thus, the strategy against the induction of ROS and apoptosis remained reasonable for the prevention of eye toxicity caused by SI. Several studies have showed that glycyrrhizin inhibited ROS-mediated photodamage in UV-B irradiated human skin fibroblasts, 18 and attenuated alcoholic acidinduced live injury. 19 This phenomenon suggested that glycyrrhizin might also reduce SI-induced eye injury. In this study, we found that glycyrrhizin significantly inhibited ROS in RPE treated with SI, and decreased SI-induced apoptosis of RPE in vitro and retinal cell apoptosis in vivo. We further confirmed that the thinning of retina was inhibited, the number of drusen was decreased and the retinal function was improved by glycyrrhizin. These indicated that glycyrrhizin prevented against SI-induced RPE and retinal injury through anti-ROS and apoptosis.
AKT plays an important role in cell apoptosis. 20 Numerous studies have showed that phosphorylation of AKT activated cell survival pathways, which in turn prevented the cells from apoptosis. [21][22][23][24][25] In this study, we found that glycyrrhizin significantly increased phosphorylation of AKT in RPE treated with SI, and significantly decreased apoptosis of RPE. This was in accordance with the studies that demonstrated glycyrrhizin-mediated protection in H9c2 cells 26 and neural cells 27 by increasing the phosphorylation of Akt.
Nrf2 is a crucial regulator of oxidative stress by the activation of endogenous antioxidant enzymes. 28,29 Normally, Nrf2 is bound to negative regulator Keap1 and is oriented toward the cytoplasm. In the oxidative stress state, Nrf2 dissociates from Keap1 and translocates into the nucleus, inducing the expression of HO-1 gene that acts as an antioxidative modulator. 30 Nrf2 protects against cell apoptosis by inhibiting MARKs and NF-κB, while HO-1 protects against apoptosis directly or by inhibiting ROS. 31 Our study demonstrated that glycyrrhizin increased the expression of Nrf2 and HO-1 and decreased ROS in RPE, and PI3K/AKT inhibitor LY294002 reversed the effect of glycyrrhizin on the pAKT, Nrf2 and HO-1 in RPE treated with SI. This suggested that the Nrf2/HO-1 signalling pathway participated in the protection of glycyrrhizin against SI-induced RPE and retinal apoptosis.
In summary, we found that glycyrrhizin preserves SI-induced RPE and retinal injury by inhibiting ROS and decreasing apoptosis.
The underlying mechanisms include the activation of Akt and Nrf2/ HO-1 pathway by glycyrrhizin, thereby preventing the retinal cells against the SI-induced toxicity. These results suggested that glycyrrhizin might serve as a putative tool for preventing atrophic AMD in clinical practice.