GPR120/FFAR4 protects retinal vascular endothelial cells against high glucose injury via suppressing ROS-ERS mediated apoptosis

Abstract

Aim: To evaluate the role of reactive oxygen species-endoplasmic reticulum stress (ROS-ERS) in the cellular protection of G protein-coupled receptor 120 (GPR120/FFAR4) against high glucose (HG) induced human retinal vascular endothelial cell (HRVEC) injury and its underlying mechanisms.

Methods: HRVECs were divided into the control group, GW9508 (an agonist of GPR120) group, HG group, and HG+GW9508 group. The cell proliferation and apoptosis were assessed by cell counting kit-8 and annexin V-FITC/PI apoptosis detection kit, respectively. Western blotting analysis was performed to assess the protein expressions of Bax, Bcl-2, activating transcription factor 6 (ATF6), PKR-like endoplasmic reticulum kinase (PERK), and inositol-requiring enzyme 1 (IRE1). The ROS assay kit was used for the detection of ROS production. Then the cells were transfected with siRNA of GPR120 and the ROS level and protein levels of ATF6, PERK, and IER1 were compared.

Results: GW9508 promoted the proliferation of HRVECs, which was significantly reduced by the stimulation of HG. GW9508 remarkably reduced the apoptosis rate of HRVECs under HG and the expression of proapoptotic protein Bax, while increased the expression of antiapoptotic protein Bcl-2. Under HG condition, a significant increase of ROS production was noticed in HRVECs, and GW9508 treatment greatly decreased it. The over-expressions of ERS-related proteins ATF6, PERK, and IER1 under HG were down-regulated by GW9508 treatment. After successfully transfected with siGPR120, the effects of GW9508 on the production of ROS as well as the expressions of ATF6, PERK, and IER1 were reversed.

Conclusion: GPR120 protects HRVECs against HG induced apoptosis, and suppressing ROS-ERS pathway is one of the mechanisms involved. Activation of GPR120 may be considered as a potential therapeutic target for diabetic retinopathy.

Keywords: GPR120/FFAR4; apoptosis; high glucose; oxidative stress; retinal vascular endothelial cells.

Figure 1. Activation of GPR120 by GW9508 inhibits injury of HG-treated HRVECs

A: Proliferation of HRVECs in four groups (control, GW9508, HG, and HG+GW9508) was assessed by CCK-8 assay; B: Apoptosis of h HRVECs in the four groups was tested by flow cytometry; C: Expression of apoptosis-related proteins Bax and Bcl-2 in HRVECs in four groups was evaluated by Western blotting. n=3, ^aP<0.05 vs control group; ^bP<0.05 vs GW9508 group; ^cP<0.05 vs HG group. GPR120: G protein-coupled receptor 120; HG: High glucose; HRVECs: Human retinal vascular endothelial cells.

Figure 2. Activation of GPR120 by GW9508 restrains HG-induced reactive oxygen species overproduction in HRVECs

DCFH-DA staining assay was performed and reactive oxygen species levels in HRVECs in four groups (control, GW9508, HG, and HG+GW9508) were measured. n=3, ^aP<0.05 vs control group; ^bP<0.05 vs GW9508 group; ^cP<0.05 vs HG group. GPR120: G protein-coupled receptor 120; HG: High glucose; HRVECs: Human retinal vascular endothelial cells.

Figure 3. Activation of GPR120 by GW9508 restrains HG-induced endoplasmic reticulum stress in HRVECs

A: Expression of endoplasmic reticulum stress-related proteins (ATF6, PERK, and IER1) in HRVECs of four groups (control, GW9508, HG, and HG+GW9508) was detected by Western blotting; B-F: Statistical comparison of protein expressions of ATF6, p-PERK, PERK, p-IRE1, and IRE1 among the four groups. n=3, ^aP<0.05 vs control group; ^bP<0.05 vs GW9508 group; ^cP<0.05 vs HG group. GPR120: G protein-coupled receptor 120; HG: High glucose; HRVECs: Human retinal vascular endothelial cells; ATF6: Activating transcription factor 6; PERK: PKR-like endoplasmic reticulum kinase; IRE1: Inositol-requiring enzyme 1.

Figure 4. Expression of GPR120 in HRVECs was successfully silenced by transfection with siGPR120

Expression of GPR120 protein in HRVECs of two groups (NS and siGPR120) was detected by Western blotting. n=3, ^aP<0.05 vs NS group. GPR120: G protein-coupled receptor 120; NS: Negative control; HRVECs: Human retinal vascular endothelial cells.

Figure 5. Knockdown of GPR120 attenuates the effect of GW9508 on HG-induced ROS overproduction in HRVECs

DCFH-DA staining assay was performed and ROS levels in HRVECs in five groups (NS, siGPR120, HG+NS, HG+GW9508+NS, and HG+GW9508+siGPR120) were measured. n=3, ^aP<0.05 vs NS group; ^bP<0.05 vs HG+NS group; ^cP<0.05 vs HG+GW9508+NS group. GPR120: G protein-coupled receptor 120; ROS: Reactive oxygen species; HG: High glucose; NS: Negative control; HRVECs: Human retinal vascular endothelial cells.

Figure 6. Knockdown of GPR120 attenuates the effect of GW9508 HG-induced ERS in HRVECs

A: Expression of ERS-related proteins (ATF6, PERK, and IRE1) in HRVECs of five groups (NS, siGPR120, HG+NS, HG+GW9508+NS, and HG+GW9508+siGPR120) was detected by Western blotting; B-F: Statistical comparison of protein expressions of ATF6, p-PERK, PERK, p-IRE1, and IRE1 among the five groups. n=3, ^aP<0.05 vs NS group; ^bP<0.05 vs HG+NS group; ^cP<0.05 vs HG+GW9508+NS group. GPR120: G protein-coupled receptor 120; ERS: Endoplasmic reticulum stress; HG: High glucose; NS: Negative control; HRVECs: Human retinal vascular endothelial cells; ATF6: Activating transcription factor 6; PERK: PKR-like endoplasmic reticulum kinase; IRE1: Inositol-requiring enzyme 1.