USP48 deubiquitination stabilizes SLC1A5 to inhibit retinal pigment epithelium cell inflammation, oxidative stress and ferroptosis in the progression of diabetic retinopathy.

BACKGROUND: Diabetic retinopathy is one of the complications of diabetes mellitus. The aim of this study was to explore the effects of ubiquitin-specific protease 48 (USP48) and its underlying mechanisms in the development of diabetic retinopathy. METHODS: CCK-8 assay, EdU assay, and flow cytometry were used to measure the proliferative ability and the apoptotic rate of ARPE-19 cells, respectively. ELISA kits were utilized to assess the levels of inflammatory cytokines. The levels of Fe2+, ROS and MDA were detected using the corresponding biochemical kits. The protein expression of USP48 and SLC1A5 was examined through western blot. The mRNA level of SLC1A5 was determined using RT-qPCR. The interaction relationship between USP48 and SLC1A5 was evaluated using Co-IP assay. RESULTS: High glucose (HG) treatment significantly inhibited cell proliferation and elevated cell apoptosis, inflammation, ferroptosis and oxidative stress in ARPE-19 cells. HG treatment-caused cell damage was hindered by USP48 or SLC1A5 overexpression in ARPE-19 cells. Fer-1 treatment improved HG-caused cell damage in ARPE-19 cells, which was blocked by USP48 knockdown. Moreover, USP48 knockdown decreased SLC1A5 expression. SLC1A5 downregulation reversed the improvement effects of USP48 upregulation on cell damage in HG-treated ARPE-19 cells. CONCLUSION: USP48 overexpression deubiquitinated SLC1A5 to elevate cell proliferation and suppress cell apoptosis, inflammation, ferroptosis and oxidative stress in HG-triggered ARPE-19 cells, thereby inhibiting the progression of diabetic retinopathy.

Pelargonidin alleviates acrolein-induced inflammation in human umbilical vein endothelial cells by reducing COX-2 expression through the NF-κB pathway.

Acrolein, a common environmental pollutant, is linked to the development of cardiovascular inflammatory diseases. Pelargonidin is a natural compound with anti-inflammation activity. In this study, we aimed to explore the effects of pelargonidin on inflammation induced by acrolein in human umbilical vein endothelial cells (HUVECs). MTT assay was utilized for assessing cell viability in HUVECs. LDH release in HUVECs was measured using the LDH kit. Western blot was used to detect the protein expression of p-p65, p65 and COX-2. Inflammation was evaluated through determining the levels of PGE2, IL-1ß, IL-6, IL-8 and TNF-α in HUVECs after treatment. COX-2 mRNA expression and COX-2 content were examined using RT-qPCR and a human COX-2 ELISA kit, respectively. Acrolein treatment at 50 µM resulted in a 45% decrease in the viability and an increase in LDH release (2.2-fold) in HUVECs. Pelargonidin at 5, 10, 20, and 40 µM alleviated acrolein-caused inhibitory effect on cell viability (increased to 1.3-, 1.5-, 1.8-, and 1.9-fold, respectively, compared to acrolein treatment group) and promoting effect on LDH release (decreased to 82%, 75%, 62%, and 58%, respectively, compared to acrolein treatment group) in HUVECs. Moreover, pelargonidin or pyrrolidine dithiocarbamate (PDTC; an NF-κB pathway inhibitor) inhibited acrolein-induced activation of the NF-κB pathway. Acrolein elevated the levels of PGE2, IL-1ß, IL-6, IL-8 and TNF-α (from 40.2, 27.3, 67.2, 29.0, 24.8 pg/mL in control group to 224.0, 167.3, 618.3, 104.6, and 275.1 pg/mL in acrolein treatment group, respectively), which were retarded after pelargonidin (decreased to 134.8, 82.3, 246.2, 70.2, and 120.8 pg/mL in acrolein + pelargonidin treatment group) or PDTC (decreased to 107.9, 80.1, 214.6, 64.0, and 96.6 pg/mL in acrolein + PDTC treatment group) treatment in HUVECs. Pelargonidin inactivated the NF-κB pathway to reduce acrolein-induced COX-2 expression. Furthermore, pelargonidin relieved acrolein-triggered inflammation through decreasing COX-2 expression by inactivating the NF-κB pathway in HUVECs. In conclusion, pelargonidin could protect against acrolein-triggered inflammation in HUVECs through attenuating COX-2 expression by inactivating the NF-κB pathway.