Effect And Mechanism of Endoplasmic Reticulum Stress Induced By Tunicamycin In Trabecular Meshwork Cells

Objective: To explore the role of endoplasmic reticulum stress in apoptosis of trabecular meshwork cells Methods: Human trabecular meshwork cells(HTMCs) were cultured in vitro. When the cells grew to logarithmic phase, they were digested and resuspended, and then cultured in 96-well plate. When the cells are close to fusion, the culture is continued for 24h in a serum-free medium, The HTMCs were treated with different concentrations of tunicamycin (0mg/L, 1.25mg/L, 2.5mg/L, 5mg/L, 10mg/L, and 20mg/L) for different durations (0h, 6h, 12h, 24h, 36h, and 48h). The cytoactive was detected by CCK-8, and the optimal intervention concentration of tunicamycin was determined. Then we use the 5mg/L tunicamycin to interfere with HTMCs for different durations (0h, 6h, 12h, and 24h). The apoptotic rate was detected by ow cytometry, and the expression levels of GRP78, CHOP, Bcl-2 and Caspase-3 were detected by RT-PCR and Western blot. Results: The number of RGCs in IRI group was signicantly lower than that in NC group (P < 0.05), demonstrated by HE staining. Western blotting results indicated that the protein expression of LC3 and Beclin-1 in the IRI group were signicantly elevated compared with those in the NC group (P < 0.05). However, with 3-MA treatment, the number of RCGs in 3-MA treated-IRI group was elevated and protein levels of LC3, Beclin-1 were downregultated, compared with those in the IRI group (P < 0.05). Further immunohistochemistry staining and Western blot showed that 3-MA treated-IRI group presented downregulated Caspase-3 and upregulated Bcl-2 protein expression with comparison of IRI group (P < 0.05). Western blot test results showed that compared with the control group, GRP-78 and CHOP protein expressions of the experimental groups (6h, 12h, 24h) with different intervention durations increased, the difference was statistically signicant (P <0.05). Compared with the control group, the expression level of Caspase-3 protein increased in the 12h group and the difference was statistically signicant (P <0.05). The increase in the expression level of the remaining experimental group was not obvious, and the difference was not statistically signicant (P> 0.05); 24h Compared with the control group, the expression level of BCL-2 protein was signicantly reduced, the difference was statistically signicant (P <0.05), the expression level of BCL-2 protein in the remaining experimental group was not signicantly reduced, and the difference was not statistically signicant (P> 0.05). Conclusion: Tunicamycin can increase the expression of endoplasmic reticulum stress proteins in trabecular meshwork cells and induce apoptosis. Our study demonstrated that endoplasmic reticulum stress played an important role in the apoptosis of trabecular meshwork cells.


Introduction
The glaucoma is an eye disease characterized by progressive loss of retinal ganglion cells and axons [1] .
The clinical symptoms of visual eld loss caused by elevated intraocular pressure make glaucoma become the irreversible blinding eye disease with the largest number of patients in the world,Besides, it is predicted that the global glaucoma patients will reach 111.8 million in 2040 [2] . Primary open-angle glaucoma is a common type of glaucoma, with hidden onset and extremely slow progression. Therefore, it's di cult to diagnose in disease early. When patients seek the treatment, the disease has often progressed to the advanced stage, at which time the disease has caused irreversible damage to the patients. Thus, research on the pathogenic mechanism, early accurate diagnosis and effective treatment of POAG is of great signi cance.
One of the anatomical characteristic changes of POAG is the degeneration of trabecular meshwork [3] .
Approximately eighty-ve percent of the aqueous humor from anterior chamber is discharged through the trabecular meshwork tissue [4] . The trabecular meshwork tissue consists of trabecular bundles formed by collagen bers and trabecular meshwork cells that cover the single-layer mesh-like structure. Trabecular meshwork cells are involved in the synthesis, secretion, and metabolism of the extracellular matrix of trabecular meshwork tissue [5,6] . Meanwhile, it contained mitochondria [7] , secondary lysosomes [8] and other organelles, which could eliminate cell debris, biological macromolecules and pigment particles attached to the trabecular meshwork tissue through phagocytosis [9,10] . The myo lament proteins contained in the trabecular meshwork cells can help the cells contract the mesh for keeping the patency of the aqueous humor and the stability of the intraocular pressure [11] . Therefore, the change in the number and activity of trabecular meshwork cells can affect the patency of aqueous humor out ow, interfere with changes in intraocular pressure. [12] It is well established that trabecular meshwork can be surgically removed to save vision in patients with advanced open-angle glaucoma [13] . However, the mechanism of apoptosis in trabecular meshwork in patients with open-angle glaucoma has not yet been elucidated. Recent studies have reported that endoplasmic reticulum(ER) stress may play an important role in the apoptosis of human trabecular meshwork cells HTM [14,15] . However, the relationship between ER stress signaling and Apoptosis in primary HTM cells is unknown. We proposed for the rst time that tunicamycin-induced ER stressmediated apoptosis in HTM cells is associated with activation of the CHOP/Bcl-2 signaling pathway.
Thus, in this experimental study, we aimed at exploring the relationship between CHOP /BCL-2 and apoptosis via establishing the model of ER stress-mediated apoptosis in HTM cells.

Materials And Methods
Primary Cell Culture and expansion Human trabecular meshwork cells (HTMCs) were bought from Beina Chuanglian Biotechnology Research Institute. HTMCs were cultures in Dulbecco's Modi ed Eagle Medium: F-12 (DMEM/F12) supplemented with 1% penicillin/streptomycin (100U/mL), and 20% fetal bovine serum (Invitrogen-Gibco) at 5% CO2 and 37°C. The culture medium is changed every 2 days, when the cells reach 80% con uence, cells were washed twice with PBS. The cells were digested by 0.25% pancreatin for 5min. Then add bovine serumcontaining medium to terminate digestion and replate through 1:2 dilutions.

Detection of Cell toxicity
HTMCs were seeded into 96-well plates (1.0 × 104 cells/well). 24 hours later, cells were treated with tunicamycin [dissolved in dimethyl sulfoxide (DMSO)] at nal concentrations of 1.25, 2.5, 5, 10 and 20mg/L as experimental group, while the cells without any treatment served as control group. Five wells were used for each dose. Subsequent to 6, 12, 24, 36 and 48h, 10µL of cell counting kit 8 (CCK8, TAKARA, Tokyo, Japan) for 2h. Ultimately, absorbance was measured at 490 nm by using a spectrophotometer. The inhibition rate was determined to gure out the suppressive effect of tunicamycin on HTMCs. The relative inhibition rate was calculated as a percentage, as follows: (1-Aexperiment/Acontrol) × 100%. Three independent experiments were performed.

Apoptosis assay
Cells were treated with tunicamycin at the concentrations of 5mg/L for 6, 12 and 24h, respectively. Then, we used an apoptosis detection kit (BD Co., Ltd., Nanjing, China) for an apoptosis assay. Cells were washed with PBS and resuspended gently with 1×Binding Buffer at a concentration of 1×106 cells/ml. Subsequently, pipette 100 ul solution (1×105 cells) into a 5 ml culture tube, and then add 5µl FITC annexin V and 5µl PI to the cells suspension. Add 400µl 1×Binding Buffer to each test tube and incubate them in the dark at room temperature (25 ° C) for 15 minutes. The apoptosis level of cells in each group was measured using FACSCalibur ow cytometer.
Quantitative RT-PCR Cells were treated as aforementioned in the previous paragraph (apoptosis assay).The western blotting experiment is the same. Cells were subsequently lysed in TRI Reagent (Total RNA Isolation Reagent,Biosharp, CN), and stored at -80℃. RNA was isolated following the manufacturer's instructions, including treatment with chloroform, precipitation and recovery with isopropanol, washing with ethanol, and nally adding DEPC water to dissolve RNA. Calculate the concentration of the isolated RNA sample at 260nm and 280nm by an ultraviolet spectrophotometer, and measure the OD260/OD280 ratio to judge the purity of the sample. If the ratio is between 1.8 and 2.0, the purity is acceptable. RNA was reverse transcribed into cDNA using Reverse Transcriptase kit (TAKARA, Japan). Quantitative PCR (qPCR) was performed using SYBR Green RT-PCR Reagents (TAKARA, Japan). The sequences of primers used in this study are presented in Table 1. Calculate the relative mRNA abundance by using the 2^(-ΔΔCt) method.
Gene expression was normalized to GAPDH. The sequences of the primers were as follows, The primers were designed using NCBI.

Western Blotting
The prepared cells were added with RIPA cell lysis buffer together with protease inhibitor and phosphorylase inhibitor and transferred to EP tubes which were placed on ice for 30 minutes, followed by cell lysis using a centrifuge and centrifugation at 13000 rpm for 5 minutes. The supernatant prepared for western blot analysis was then transferred to another sterile EP tube. The membranes were blocked with 10% fat-free milk and incubated with the primary antibody selected from ab108615, ab179823, ab32124, and ab184787 (Abcam, UK) overnight at 4 C. Goat anti-rabbit IgG (1:5000, ab6721, Abcam, UK) was used for the secondary antibody.

The effect of tunicamycin on the activity of HTMCs
CCK8 assay showed that the inhibitory effect of HTMCs treated with 1.25mg/L and 2.5mg/L tunicamycin was not obvious compared with control group 0mg/L (P > 0.05) .However,the inhibitory effect appeared a signi cantly increase when the treatment concentration was higher than 5mg/L.Moreover, under the 5mg/L of tunicamycin treatmnent, the HTMCs' proliferation was remarkably inhibited when treated duration last for 12 hours or more.The result showed that the inhibitory effect on HTMCs treated with tunicamycin of 12, 24, 36 and 48 hours (21.11 ± 1.23%, 31.34 ± 0.86%, 44.37 ± 1.01%, 51.47 ± 0.36%, respectively) were remarkably enhanced when compared to the control group 0 hours(P < 0.05; Fig. 1) 3.2The effect of tunicamycin on apoptosis of HTMCs According to the results of the above CCK8 assay,HTMCs were previously treated with 5mg/L tunicamycin for 0, 6, 12 and 24 hours and marked with Annexin V -FITC/ PI dye .The effect of tunicamycin on HTMCs apoptosis was analysed by ow cytometry analysis.Annexin V -FITC(+) and PI(-) cells were classi ed as early apoptosis,while Annexin V -FITC(+) and PI(+) cells wre classi ed as late apoptosis.The results showed that with the increase of treatment duration, the early apoptosis rate and late apoptosis rate increased gradually( Fig. 2 and Table 1).
Early apoptosis results: There was a statistical difference in the early apoptosis rate of each HTMCs groups ( F = 1024.457, P < 0.001 ).The early apoptosis rates of group 6,12,24 hours (7.43 ± 0.78%, 8.59 ± 0.89%, 34.22 ± 0.87%,respectively) were higher than negative control(4.68 ± 0.82%, P < 0.05),.The early apoptosis rate of 24h group was signi cantly higher than that of group 6 hours and group 12 hours ( P < 0.05 ),while there was no statistical difference in the early apoptosis rate between the other groups pairwise comparison ( P > 0.05 ).
Late apoptosis results: There was a statistical difference in the early apoptosis rate of each HTMCs groups ( F = 81.475, P < 0.001 ).The late apoptosis rates of group 6,12,24 hours (5.03 ± 0.90%,6.57 ± 0.93%, 10.53 ± 1.00%,respectively) were higher than negative control(0.52 ± 0.09%, P < 0.05).The late apoptosis rate of 24h group was signi cantly higher than that of group 6 hours and group 12 hours ( P < 0.05 ),while there was no statistical difference in the late apoptosis rate between the other groups pairwise comparison ( P > 0.05 ). Note :* represents the early apoptosis rate of each experimental group and the control group ( 0h ), the difference was statistically signi cant ( P < 0.05 ).
# represents the late apoptosis rate of each experimental group and the control group ( 0h ), the difference was statistically signi cant ( P < 0.05 ).

Effect of tunicamycin on ERS-related mRNA expression in HTMCs
HTMCs were previously treated with tunicamycin (0, 6, 12,24hours) and then extracted total RNA. The relative mRNA expression levels of HTMCs GRP-78,CHOP,Bcl-2,Caspase-3 were detected by RT-PCR.Cells treated for 0 hours was the negetive control group, and the rest was the experimental group.

Effect of tunicamycin on ERS-related protein expression in HTMCs
HTMCs were previously treated with tunicamycin (0, 6, 12,24hours) and then extracted total protein. The protein expression levels of HTMCs GRP-78,CHOP,Bcl-2,Caspase-3 were detected by Western Blot.Cells treated for 0 hours was the negetive control group, and the rest was the experimental group.

Discussion
Although it has been reported that endoplasmic reticulum stress plays an important role in the pathogenesis of glaucoma [16] , the role of ER stress in trabecular meshwork cells has not been reported yet. In present research, we investigated the effect of endoplasmic reticulum stress in trabecular meshwork cells. Tunicamycin is often used in establishing the model of endoplasmic reticulum stress [17,18] . Here, we found that the apoptosis of trabecular meshwork cells may go through affecting the interaction between CHOP and Bcl-2 protein which induced by tunicamycin.
Protein homeostasis plays an important role in the normal function of cells. In eukaryotic cells, endoplasmic reticulum is involved in the synthesis, folding and maturation of proteins [19,20] . When in some physiological or pathological conditions, such as nutritional de ciency, infection, chemical damage, DNA mutation and so on [21] , can trigger the abnormal function of endoplasmic reticulum (ER), which leads to the protein misfolding and accumulating [22] , which is called endoplasmic reticulum stress (ERS). Abnormal proteins accumulate in the endoplasmic reticulum and activate a series of protective intracellular signaling pathways, which are called the unfolded protein response (UPR). UPR includes reducing protein translation, activating chaperone transcription, inducing endoplasmic reticulum degradation of misfolded proteins and inducing apoptosis [23,24] . Whether UPR promotes cell survival or apoptosis depends on the duration and severity of ERS [25,26] . Under transient and mild ERS conditions, UPR promotes cell survival, whereas persistent and severe ERS promotes cell death by activating downstream apoptosis signaling molecules, including IRE1 / ASK1 / JNK pathway33, Caspase-12 kinase pathway34 and C/EBP-homologous protein or growth arrest and DNA damage-inducible gene153 (CHOP/GADD153) pathway [25,27,28] .
The 78-kDa glucose-regulated protein (GRP78), also known as binding-immunoglobulin protein (BiP) [29] , is localized in the ER. It can bind to unfolded or misfolded proteins in ER, and is considered to be a marker of the ER-stress cascade [30] . Chop is a transcription factor involved in ERS and has been con rmed as a proapoptotic protein [31] . Chop encoded by DDIT3 gene, induces the expression of proapoptotic genes such as DR5, TRB3, Bim and puma, and inhibit the expression of BCL2 [32] , thus triggering cell apoptosis during ER stress. [33] There are more and more evidences [34,35] suggested that the endoplasmic reticulum stress may play an important role in the pathogenesis of glaucoma. Ganglion cells undergo apoptosis in glaucoma leading to progressive loss of vision -the loss of RGCs is the principal endpoint in experimental glaucoma [36] .Peter et al [37] demonstrated that high level of ER-stress related-proteins can be detected in the human trabecular meshwork tissue. In the experiment conducted by Zode et al [38] , found that the reduction of ocular-speci c ER stress can rescue glaucoma in murine model of glucocorticoid-induced.
Besides, the deletion of CHOP in ER-stress pathway can reduce ER stress and prevented dexamethasoneinduced ocular hypertension.
Recently, accumulating evidence suggests that endoplasmic reticulum stress may play an important role in the survival and apoptosis of cell. however, the effect of endoplasmic reticulum stress for cell in vivo is controversial, some studies show that endoplasmic reticulum stress may play a protective role [39,40] . But, continuous endoplasmic reticulum stress can cause cells apoptosis [41,42] . In the current experiment, we found that Endoplasmic reticulum stress pathway activated by the tunicamycin induction might activate the apoptotic pathway and lead to the apoptosis of HTMCs successively. which is consistent with previous studies [43] .
In conclusion, our experiment show that under tunicamycin, ER stress can induce CHOP expression, whereas CHOP can lead cells to death by decreasing BCL-2 level and upregulation of CHOP may bring trabecular meshwork cells with harsh survival. These ndings highlight the importance of apoptosis regulated by ER stress in trabecular meshwork cells, disclose new mechanisms of POAG, and suggest CHOP as a potential anti-POAG target.

Declarations
Compliance with Ethical Standards: Disclosure of potential con icts of interest: The authors declare that they have no con ict of interest.
Research involving Human Participants and/or Animals: there is no human participants or animals involved in this study Informed consent: Not applicable.  Expression of ERS-related protein in HTMCs treated with tunicamycin at different time points Note:A-D represents GRP78, CHOP, Bcl-2, Caspase-3 , respectively.The ratios of above protein to β-actin indicate the relative expression levels of GRP78 , CHOP , Bcl-2 and Caspase-3 proteins.Compared with the control group ( 0h ),*P 0.05 E represents GRP78, CHOP, Bcl-2, Caspase3 proteins imprinting strip.