Citation: Ye
Q, Lin YN, Xie MS, Yao YH, Tang SM, Huang Y, Wang XH, Zhu YH. Effects of
etanercept on the apoptosis of ganglion cells and expression of Fas, TNF-α, caspase
DOI:10.18240/ijo.2019.07.05
·Basic Research·
Effects of etanercept on the apoptosis
of ganglion cells and expression of Fas, TNF-α, caspase
Qin Ye1, Yu-Ni Lin2, Mao-Song Xie1, Yi-Hua Yao1, Shu-Min Tang1, Yan Huang3, Xiao-Hui Wang1, Yi-Hua Zhu1
1Department of Ophthalmology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian Province, China
2XiaMen Haicang Hospital, Xiamen 361026, Fujian Province, China
3Fujian Medical University, Fuzhou 350005, Fujian Province, China
Correspondence to: Xiao-Hui Wang and Yi-Hua Zhu. Department of Ophthalmology, the First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian Province, China. wxhfyyk@163.com; zhuyihua209@163.com
Received:
Abstract
AIM: To evaluate the effects of etanercept on the expression of Fas, tumor
necrosis factor-alpha (TNF-α) and caspase
METHODS: A total of 60 Sprague-Dawley (SD) rats were randomly
and evenly divided into 3 groups with 20 rats each, including control group,
and diabetic groups with or without treatment. Streptozotocin (STZ)-induced
diabetic rats were established for diabetic groups. Blood glucose and body
weight were measured weekly. All the rats were sacrificed at the 12wk after
treatment. The expressions of Fas, TNF-α and caspase
RESULTS: The expressions of Fas, TNF-α and caspase
CONCLUSION: Etanercept can effectively reduce the expression of Fas, TNF-α and caspase-8, as well as the retinal leakage and retinal cell apoptosis in diabetic rats.
KEYWORDS: etanercept; ganglion cells; Fas; tumor necrosis factor-alpha; caspase-8; apoptosis; diabetes; rat
DOI:10.18240/ijo.2019.07.05
Citation: Ye
Q, Lin YN, Xie MS, Yao YH, Tang SM, Huang Y, Wang XH, Zhu YH. Effects of
etanercept on the apoptosis of ganglion cells and expression of Fas, TNF-α, caspase
INTRODUCTION
Diabetic retinopathy (DR) is a common severe complication of diabetes mellitus (DM). With the increasing number of DM patients, DR has become a major issue threatening the vision of people in China. However, the pathogenesis of DR is complicated. To date, animal models of DM indicated the pathogenesis from the perspective of metabolism, hemodynamics, angiogenin and apoptosis. Researchers revealed that a variety of cytokines constitute a network of cytokines involved in the pathogenesis of DR, including vascular endothelial growth factor (VEGF), interleukin (IL) -2, tumor necrosis factor-alpha (TNF-α), IL-1, interferon-gamma (IFN-γ), etc[1-3]. Furthermore, Fas, TNF-α, and caspase-8 are important cytokines in the apoptotic signaling pathway, which taken together cause apoptosis of retinal cells and trigger a series of inflammatory responses in DR patients.
Etanercept is one of competitive inhibitors of cell surface TNF receptor, inhibiting the biological activity of TNF, via which further regulate the other downstream molecules (such as cytokines, adhesion molecules and proteases) to control the biological response. In the current study, we aimed to observe the effect of etanercept on the expression of Fas, TNF-α and caspase-8, as well as on the retinal leakage and cell apoptosis of retina in diabetic rats, to further understand the mechanism and provide supports and guidance for clinical treatment.
MATERIALS AND METHODS
Ethical Approval Experimental protocols were approved by the animal care and Ethics Committee at Fujian Medical University according to the Association for Research in Vision and Ophthalmology under the guidelines with the Animal Welfare Act (www.nal.usda.gov/awic/animal-welfare-act).
Establishment of Diabetic Animal Model The animal center of Fujian Medical University provided
60 male Sprague-Dawley (SD) rats aged 2mo and weighed 180
Real-Time
Quantitative Reverse Transcription-Polymerase Chain Reaction of
Retina RNA was extracted from
retinal tissue samples of 6 rats randomly selected from each group. RNA purity
was determined through ultraviolet (UV) absorption method, cDNA was synthesized
by reverse transcription, which was amplified through real-time quantitative
polymerase chain reaction (RT-PCR). RT-PCR primers were designed by Primer
Premier 5.0 shown in Table 1. The annealing temperature was
Table 1 Real-time PCR primer sequences
Gene |
Primer sequence |
Fas |
|
|
|
TNF-α |
|
|
|
Caspase-8 |
|
|
|
GAPDH |
|
|
Western Blot Six rats were taken from each group and their eyeballs
were collected under anesthesia. Subsequently, the anterior segments of the eye
and the retinal tissues were removed from eyeballs. After the eyeballs from
both eyes were weighed with a precision balance, a 10% homogenate was prepared
in an ice bath with 0.05 mmol/L of ice-cold phosphate buffer [pH=7.8,
containing 0.01 mmol/L of ethylene diamine tetraacetic acid (EDTA)] for
detection. Frozen retinal tissues were taken out from refrigerator and added
with lysate (containing 5% protease inhibitor). After being fully cracked,
tissues were centrifuged for 5min with 12 000 rpm. And then the proteins were
added by the loading buffer solution according to the ratio of 4:1. After
Evans Blue Leakage of Retina After anesthesia of 4 rats in each group, the amount of retinal Evans blue (EB) leakage was measured to understand the degree of damage of the blood-retinal barrier (BRB)[4]. The absorbance (OD) of samples at 620 and 740 nm was measured with a spectrophotometer. Each sample was measured 3 times and averaged. Net OD value =OD620-OD740. A standard curve for the concentration of EB dye in formamide was established. Statistical analysis showed a highly linear correlation between EB concentration and net OD (r=0.996, P=0.001). Y=0.0702X+0.0125; where Y represents the net OD value and X represents the EB concentration. Based on the OD of each group, the EB mass concentration was calculated, and the retinal EB leakage amount (ng) was obtained by multiplying the concentration by 300 μL. Finally, the EB leakage (ng) was normalized by the dry weight of the retina (mg) and the result was expressed as ng/mg.
TUNEL Method: The Number of Apoptotic Cells TUNEL Assay Kit was used to detect the apoptosis (Roche,
Switzerland). Four rats were taken from each group. After anesthesia, the
eyeballs were removed for frozen sections, and the tissues sections were
mounted on slide coated with polylysine and then fixed. The slide was immersed
in trypsin for 40min, and washed with phosphate buffer saline (PBS) for 3min×3
times. Place the slides in 3% H2O2 and incubate in a wet
box for 10min to eliminate endogenous peroxidase activity. Totally 50 μL of TUNEL reaction mixture was added to the sample, and
reacted in a dark and wet box at
Statistical Analysis All analyses were performed using SPSS 17.0 software (USA). The mean±standard deviation of PCR products, Western blot data and EB leakage in each group were calculated. One-way analysis of variance (ANOVA) and subsequent least significant difference (LSD) pairwise comparison tests were used to compare the difference among three groups. A P-value of 0.05 or less was considered statistically significant.
RESULTS
Changes of Fasting Blood Glucose and Weight in Rats Compared with the control group, the blood glucose of diabetic groups no matter treated or not was significantly increased, and the body weight was significantly decreased at each time point (P<0.05). After treated with etanercept, the weight of the treatment group significantly increased compared with non-treatment group (P<0.05; Table 2). However, there was no significant difference in decreasing of blood glucose between the treatment groups and non-treatment group (P>0.05; Table 3).
Table 2 Weight of each group at each time point in rats mean±SD, g
Groups |
Sample size |
Weights of rats at each time point |
|||
Before model establishment |
4wk |
8wk |
12wk |
||
Control |
20 |
220.4±4.6 |
301.7±4.9 |
352.6±5.0 |
405.9±6.9 |
Treatment group |
20 |
220.3±5.1 |
250.6±4.1 |
281.7±4.3 |
302.4±5.1 |
Non-treatment group |
20 |
221.4±5.5 |
281.5±5.2 |
312.8±4.6 |
334.3±4.1 |
F |
0.305 |
591.973 |
1181.246 |
1886.506 |
|
P |
0.739 |
<0.001 |
<0.001 |
<0.001 |
|
Table 3 Blood glucose of each group at each time point in rats mean±SD, mmol/L
Groups |
Sample size |
Blood glucose at each time point |
|||
Before model establishment |
4wk |
8wk |
12wk |
||
Control |
20 |
5.2±0.3 |
5.4±0.5 |
5.5±0.3 |
5.7±0.3 |
Treatment group |
20 |
5.2±0.5 |
25.2±1.1 |
26.8±1.2 |
27.0±1.0 |
Non-treatment group |
20 |
5.3±0.5 |
25.5±1.0 |
26.2±1.2 |
27.0±1.0 |
F |
0.229 |
3109.582 |
2933.011 |
4247.192 |
|
P |
0.796 |
<0.001 |
<0.001 |
<0.001 |
|
Real-Time Quantitative Reverse Transcriptase-Polymerase Chain Reaction of Retina The results of RT-PCR were shown in Figure 1. There were significant differences in the expression of Fas, TNF-α and caspase-8 between different groups (F value of Fas=67.626, P<0.01; F value of TNF-α=113.645, P<0.01; F value of caspase-8=72.136, P<0.001). The expression of diabetic groups no matter treated or not was significantly higher than the control group (P<0.01), however, the expression of treatment group was lower than the non-treatment group (P<0.01).
Figure 1 Relative expression levels of Fas, TNF-α and caspase-8 mRNA in the retina of rats There were significant differences in the mRNA of Fas, TNF-α and caspase-8 between different groups. aP<0.01.
Results of Quantitative Western Blot The results of Western blot quantitative detection are shown in Figure 2. There were significant differences in the expression levels of Fas, TNF-α and caspase-8 between groups (F value of Fas=6.726, P<0.01; F value of TNF-α=12.695, P<0.01; F value of caspase-8=25.039, P<0.01). The expression levels of the non-treatment group and the treatment group were stronger than those of the control group (P<0.01), and the expression level of the treatment group was lower than that of the non-treatment group (P<0.01).
Figure 2 The relative protein
expression levels of Fas, TNF-α and caspase
Retinal Evans Blue Leakage Results The average leakage of EB in the control group was 2.43±0.24 ng/mg, the average leakage of EB in the non-treatment group was 7.93±0.31 ng/mg, and the average leakage of EB in the treatment group was 4.87±0.24 ng/mg. There was a statistically significant difference in the average leakage of EB between the groups (F=854.966, P<0.01). The average leakage of the non-treatment group was significantly higher than that of the control group (P<0.01), while the average leakage of the treatment group (P<0.01) and the non-treatment group (P<0.01) were higher than that of the control group.
Detection of Number of Retinal Apoptotic Cells by TUNEL Method The number of retinal apoptotic cells in control group was 4.6%±0.5%, in the non-treatment group was 16.3%±1.1%, and in the treatment group was 8.6%±0.7%. There was a statistically significant difference in the number of apoptosis between the groups (F=417.326, P<0.01). Compared with the control group, the number of apoptotic cells in the non-treatment group (P<0.01) and treatment group (P<0.01) was significantly higher. However, the number of apoptotic cells in the treatment group was significantly reduced than in the non-treatment group (P<0.01).
The number of apoptotic cells in the treatment group and non-treatment group was significantly higher than that in the control group, and it was lower in the treatment group than the non-treatment group (Figure 3). The number of apoptotic cells in the model group and the treatment group were significantly higher than those in the control group, and the number of apoptotic cells in the treatment group was significantly lower than that in the model group.
Figure 3 Detection of retinal apoptotic cells by TUNEL assay in each group A: Control group; B: Model group; C: Treatment group; D: The apoptosis rate in each group.
DISCUSSION
In this study, the diabetic rat model was successfully established by intraperitoneal injection of STZ. The blood glucose of the modeled rats all met the modeling requirements of DR. All DR model rats had symptoms of polydipsia, polyphagia, polyuria and weight loss. Rats had obvious signs of white opacity of the lens at 10wk, indicating that metabolic cataracts caused by hyperglycemia have occurred. At the 4th, 8th and 12th week, the fasting blood glucose of the non-treatment group and the treatment group were significantly higher than the control group, but there was no significant difference in fasting blood glucose between the non-treatment group and the treatment group at each time point, indicating that etanercept had no effect on lowering blood glucose.
Current study showed that the
expression levels of the three cytokines Fas, TNF-α, and caspase
In this study, the EB leakage measurement method was used to evaluate the retinal leakage in each group. The EB leakage in the non-treatment group and the treatment group was significantly higher than that in the control group, indicating an increase of leakage in the late stage of DR in rats. The EB leakage in the treatment group was significantly lower than that in the non-treatment group, indicating that etanercept can reduce the retinal leakage of DR caused by the apoptosis and inflammation, thereby reducing the damage of retina. The abnormal cellular metabolism in the hyperglycemic environment would lead to ischemia and hypoxia of the retina. It would break the balance between retinal vascular factors and inhibitors, which increases the distribution of TNF-α on the surface of DR vascular endothelial cells, and destroys the tight junction between the cells and the pigment epithelial cells resulting in the destruction of the BRB[3,8], which further increase the vascular permeability. Moreover, TNF-α can increase the expression of vascular endothelial VEGF which would interfere with the interaction between peripancreatic cells and capillary endothelial cells, causing neovascularization and increasing the capillary permeability, and inducing a series of pathological changes, such as an increase of retinal leakage in DR patients. In addition, TNF-α can increase the reactivity of target cells to VEGF, TGF-β, IGF-1 and etc., so as to increase the expression of VEGF, leading to retinal neovascularization and BRB destruction. TNF-α also can directly damage BRB and improve the permeability of retinal blood vessels[9]. Furthermore, TNF-α can also activate microglia, which would also involve in the destruction of the BRB[10].
TUNEL is a common pathological method for detecting apoptosis. In this study, frozen sections were cut to assess the apoptosis. We found that the number of apoptotic cells in the non-treatment group and the treatment group was significantly higher than that in the control group, indicating a serious apoptotic in the retina of diabetic rats. It suggests that apoptosis plays a crucial role in the pathogenesis of DR. The number of apoptotic cells in the treatment group was significantly lower than that in the non-treatment group, indicating that etanercept can effectively reduce the retinal apoptosis response in diabetic rats. Previons studies found that the apoptosis of retinal ganglion cells (RGC) appeared at 4wk of STZ rats established, the mean of the initials RGC followed by the apoptosis of photoreceptor cells and neuronal apoptosis[11-15]. The potential mechanisms of apoptosis were as follows: 1) TNF-α binds to its receptor, activates the death receptor pathway, and trigger the apoptosis[16-17], which would also induce the death of ganglion cell death[18]. 2) Death factors such as FasL and TNF bind to their related receptors such as Fas and TNFR, and then form a death-inducing signaling complex through the death domain structural protein and apoptosis promoters including caspase-8, caspase-12 etc., resulting in caspase cascade reaction and causing the apoptosis. 3) TNF-α can also alter the expression of vascular adhesion molecules, allowing lymphocytes and macrophages to reach the target site, stimulate the inflammatory cycle, and induce apoptosis by releasing cytotoxic substances. 4) TNF-α induces nuclear factor-κB (NF-κB) activation, increases the expression of apoptotic bodies and causes apoptosis of perivascular cells in the retina, leading to retinal microcirculatory disorders[19]. 5) The interaction of vascular endothelial apoptotic factor (Fas) and apoptotic factor ligand (FasL) adhering to the surface of leukocytes triggers a series of pathological changes such as vascular endothelial cell injury and apoptosis[19].
The pathogenesis of DR is complicated and chronic inflammation and immune response play a crucial role in the whole process of DR. Fas, TNF-α and caspase-8 are three important factors in the apoptotic signaling pathway. They work together and cause apoptosis of retinal cells and trigger a series of inflammation in DR. Etanercept is a TNF-α inhibitor that is a human tumor necrosis factor receptor p75Fc fusion protein produced by the expression system of Chinese hamster ovary cell. The extracellular ligand binding site of tumor necrosis factor receptor 2 (TNFR2/p75) is linked to the Fc fragment of human IgG1, which form the dimer. Etanercept is a competitive inhibitor of TNF receptors on the cell surface that inhibits the biological activity of TNF and thereby blocks the cellular response of TNF. It may also be involved in the regulation of biological responses controlled by other downstream molecules (such as cytokines, adhesion molecules or proteases) induced or regulated by TNF. Some researchers have studied the therapeutic effect of etanercept on experimental autoimmune uveitis in rats, confirming that etanercept can effectively relieve the ocular inflammation of uveitis, delay the onset time, and reduce the TNF-α and IL-6 levels in the eye of rats. The role of TNF-α-mediated apoptosis in the early stage of DR and the long-term histopathological changes, confirming that etanercept can reduce apoptosis and reduce risk of DR[20].
In summary, the mRNA and protein levels of Fas, TNF-α and caspase
ACKNOWLEDGEMENTS
Foundations: Supported by National Natural Science Foundation of China (No.81270999); the Key Project of Miaopu of Fujian Medical University (No.2015MP004); the Qihang Funds of Fujian Medical University (No.2018QH1063).
Conflicts of Interest: Ye Q, None; Lin YN, None; Xie MS, None; Yao YH, None; Tang SM, None; Huang Y, None; Wang XH, None; Zhu YH, None.
REFERENCES