Maresin-1 inhibits high glucose induced ferroptosis in ARPE-19 cells by activating the Nrf2/HO-1/GPX4 pathway

Background Maresin-1 plays an important role in diabetic illnesses and ferroptosis is associated with pathogenic processes of diabetic retinopathy (DR). The goal of this study is to explore the influence of maresin-1 on ferroptosis and its molecular mechanism in DR. Methods ARPE-19 cells were exposed to high glucose (HG) condition for developing a cellular model of DR. The CCK-8 assay and flow cytometry were used to assess ARPE-19 cell proliferation and apoptosis, respectively. Furthermore, the GSH content, MDA content, ROS level, and Fe2+ level were measured by using a colorimetric GSH test kit, a Lipid Peroxidation MDA Assay Kit, a DCFH-DA assay and the phirozine technique, respectively. Immunofluorescence labelling was used to detect protein levels of ACSL4 and PTGS2. Messenger RNA and protein expression of HO-1, GPX4 and Nrf2 was evaluated through western blotting and quantitative real time-polymerase chain reaction (qRT-PCR). To establish a diabetic mouse model, mice were intraperitoneally injected 150 mg/kg streptozotocin. The MDA content, ROS level and the iron level were detected by using corresponding commercial kits. Results Maresin-1 promoted cell proliferation while reducing the apoptotic process in HG-induced ARPE-19 cells. Maresin-1 significantly reduced ferroptosis induced by HG in ARPE-19 cells, as demonstrated as a result of decreased MDA content, ROS level, Fe2+ level, PTGS2 expression, ACSL4 expression and increased GSH content. With respect to mechanisms, maresin-1 treatment up-regulated the mRNA expression and protein expression of HO-1, GPX4 and Nrf2 in HG-induced ARPE-19 cells. Nrf2 inhibitor reversed the inhibitory effects of maresin-1 on ferroptosis in HG-induced ARPE-19 cells. In vivo experiments, we found that Maresin-1 evidently repressed ferroptosis a mouse model of DR, as evidenced by the decreased MDA content, ROS level and iron level in retinal tissues of mice. Conclusion Maresin-1 protects ARPE cells from HG-induced ferroptosis via activating the Nrf2/HO-1/GPX4 pathway, suggesting that maresin-1 prevents DR development. Supplementary Information The online version contains supplementary material available at 10.1186/s12886-023-03115-9.


Background
Diabetes retinopathy (DR), a well-known sort of diabetes complication, is the primary cause of visual loss and even blindness [1,2].DR is a progressive, permanent degeneration of the retinal microvasculature induced by persistent hyperglycemia [3,4].It is characterized by retinal edema, neuronal dysfunction and disruption of bloodretinal barrier [5,6].At present, DR can be alleviated via blood glucose control, but whereas blindness will inevitably occur in a considerable portion of DR patients [7,8].Thus, a better understanding of DR pathogenesis is necessary to seek novel therapeutic tactics.
Ferroptosis is a novel programmed mode of cell death driven by iron-dependent lipid peroxidation [9,10].It is caused by an imbalance between production and degradation of lipid active oxygen as well as the damage to the antioxidant capacity in cells, eventually leading to membrane rupture and cell death [11,12].The increased malonialdehyde (MDA) content, reactive oxygen species (ROS) level and Fe 2+ level as well as decreased GSH content are major features of ferroptosis [13].Recent researches have associated ferroptosis to the pathogenic process of diabetic complications, including DR [14][15][16][17].Ferroptosis in pigmented epithelium of retina cells, in particular, is a major pattern of oxidative stress-mediated retinal pigment epithelium (RPE) cell death [18][19][20].Thence, pharmacological regulation of ferroptosis has emerged as a prospective therapy option for DR [21,22].
With the discovery of arachidonic acid-derived protectins, lipoxins and resolvins, specialized pro-resolving lipid mediators (SPMs) called maresins are discovered in lipid mediators and inflammatory exudates [23].Maresin-1 is the first maresin to be discovered, which is a derivative of n-3 unsaturated fatty acids [24].It is believed to have anti-inflammatory and anti-oxidative stress characteristics in acute renal failure and acute hepatic injury [25,26].Maresin-1, for example, alleviates the damage induced by high hyperglycemia (HG) to mice renal mesangial cells by controlling fibrosis and inflammation [27].Maresin-1 alleviates inflammation and oxidative stress in diabetic kidney disease (DKD) [28].Maresin-1, in particular, suppresses HG-induced ferroptosis of osteoblasts in type 2 diabetic osteoporosis (T2DOP) [29].However, it is uncertain whether maresin-1 inhibits HGinduced cell ferroptosis in DR.
Nuclear factor erythroid 2-related factor 2 (Nrf2) is considered to be a redox-sensitive transcription factor [30]. Overproduction of intracellular ROS can promote Nrf2 translocation to the nucleus, where Nrf2 binds to downstream antioxidant response elements to induce transcriptional activation of antioxidant enzymes such as HO-1, therefore relieving cellular redox imbalance [31][32][33].The pathway of Nrf2/HO-1 has been found to have a protective impact on the retina in DR [34].Moreover, an antioxidant named phospholipid glutathione peroxidase 4 (GPX4) is used in catalytic process of peroxides for example organic hydro peroxides and hydrogen peroxides, and it prevents ferroptosis [35].Notably, recent investigation indicated that maresin-1 may protect against ferroptosis in acute liver injury when the pathway of Nrf2/HO-1/GPX4 is activated [36].Therefore, we tested whether maresin-1 suppressed ferroptosis in a DR cell model through activating the pathway of Nrf2/HO-1/ GPX4.
In this study, we treated an adult retinal pigment epithelial cell line (ARPE-19) with HG to establish a cell model of DR, and we intraperitoneally injected streptozotocin to mice for establish a mouse model of DR.Then we explored the effect of maresin-1 on ferroptosis in these models and associated molecular pathways.

Cell culturing and treatments
ARPE-19 cells were bought from the American Type Culture Collection (ATCC; Manassas, VA, USA).These cells had normal karyology, and formed polarized epithelial monolayers on porous filter supports.Meantime, ARPE-19 cells had functional and structural properties resembling those of RPE cells in vivo [37].ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 (Hyclone, Logan, UT, USA) containing 1% penicillin-streptomycin (Life Technologies, Carlsbad, CA, USA) and 10% fetal bovine serum (FBS; Life Technologies).These cells were put into the incubator having 37 °C temperature, 95% humidity and 5% CO 2 .The culture medium was renewed 2-3-times every week until cells reached 80% confluence.ARPE-19 cells between passages 23 and 25 were used for the following experiments.
ARPE-19 cells before exposing to HG for 24 h, they were treated with maresin-1 for thirty minutes.
In order to explore the involvement of the Nrf2/HO-1/ GPX4 pathway, Nrf2 inhibitor (ML385; MCE, Shanghai, China) was used to block Nrf2 pathway activation.ARPE-19 cells were first treated with 5 μM ML385 for 48 h, followed by above mentioned maresin-1 treatment and HG treatment.

Quantitative real time-polymerase chain reaction (qRT-PCR)
Total RNAs from ARPE-19 cells were extracted using TRIzol (TRI) reagent and extracted RNAs were quantified by using Nano Drop.After that, reverse transcription of RNA (200 ng) was performed to generate cDNA according to the guidelines of a Prime script RT reagent kit (Takara, Dalian, China).To conduct qRT-PCR, a SYBR Green PCR kit (Takara) was used, with GAPDH as the endogenous control.The following were reaction conditions: after 30 s at 95 °C, 40 cycles of 5 s at 95 °C and 35 s at 60 °C were performed.Primers were obtained from Guangzhou Boxin Biotechnology (Table 1).Relative mRNA expression of Nrf2, HO-1 and GPX4 was determined by using delta-delta CT method (2 −ΔΔCt method).

Cell counting Kit-8 (CCK-8) test
CCK-8 assay was performed to assess proliferation of ARPE-19 cells.The 96-well plates were used for cell culture (1 × 10 4 cells in each), which were incubated for 48 h.Then these cells were incubated with CCK-8 solution (10 μL) for 1 h.Finally, a microplate reader (Bio-Rad, CA, USA) was used to measure the optical density at 450 nm.

Flow cytometry analysis
Apoptosis of ARPE-19 cells was evaluated by using a FITC Annexin V Apoptosis Detection Kit I (BD Biosciences, Franklin Lakes, NJ, USA).In brief, trypsin was used to harvest ARPE-19 cells, which were subsequently washed in phosphate buffered saline (PBS).These cells were re-suspended in 1 × Binding Buffer to produce a cell suspension of 1 × 10 6 cells/mL.After transferring 100 μL cell solution to a culture tube, PI (5 μL) and FITC Annexin V (5 μL) were added.Following fifteen minutes in the dark, 1 × Binding Buffer (400 μL) was mixed and stained cells were evaluated by using Flow Cytometer (BD Biosciences).

Examination of cell morphology
The morphological changes of ARPE-19 cells were evaluated using an inverted IX71 microscope (Olympus, Tokyo, Japan).

Determination of Fe 2+
The phirozine technique was used to quantify the Fe 2+ level in ARPE-19 cells.Disintegration of ARPE-19 cells was conducted with trypsin.After centrifugation, these cells were washed twice with PBS and were lysed with RIPA lysate for 30 min.Then total proteins were extracted after centrifugation.After that, the supernatant was added to the 96-well plate (50 μL/well) and the same volume of hydrochloric acid was added.Following 30 min of reaction, the iron probe (100 μL) was added to incubate for 1 h in the dark.The absorbance at 562 nm was measured to evaluate the level of Fe 2+ .

Detection of GSH, MDA and ROS
A colorimetric GSH test kit (Nantong, Jiangsu, China) was used to measure the GSH level.Lipid peroxidation MDA assay kit was used to assess the MDA content.The level of intracellular ROS was detected using 2' ,7'-dichlorodihydrofluorescein diacetate (DCFH-DA) assay.
Mice were assigned into three groups (n = 6 mice/ group): the control group (normal mice), the DR group (mice injected with STZ) and the DR + Maresin-1 group.

Measurement of ferroptosis factors in vivo
The content of MDA in retinal tissues was detected via a MDA assay kit from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).The level of ROS in retinal tissues was determined using an oxidation-sensitive fluorescent probe DCFH-DA with a ROS Assay kit (Jianglaibio, Shanghai, China).
For detecting the iron level, retinal tissues were homogenized in Iron Assay Buffer at 4 °C.Then the total iron in tissue homogenate was determined through an Iron Assay Kit (ab83366, Abcam).The absorbance at 593 nm was measured using a microplate reader and the iron level was normalized to the protein concentration.

Statistical analysis
Statistical analysis was conducted through GraphPad Prism 8.0 (San Diego, CA, USA).Data from three replicates were presented as mean ± standard deviation.Difference comparisons between two groups of data were conducted via Student's t test.Difference comparisons among multiple groups of data were conducted using one-way ANOVA, accompanied by Tukey's test.P < 0.05 indicated significant differences.
The influence of maresin-1 on cell morphology was investigated.ARPE-19 cells in the NG group were found to have a long spindle shape, monolayer extended adherent development, mosaic pattern, clear border, regular shape, uniform distribution, and good condition (Fig. 1C).ARPE-19 cells in the HG group, on the other hand, displayed deformation, an unclear border, disorganized organization, low cell density, and an unsatisfactory growth state (Fig. 1C).The number of round shriveled cells in HG-induced ARPE-19 cells considerably lowered after treatment of maresin-1 (10, 100, and 1000 nmol/L) (Fig. 1C).

Maresin-1 represses ferroptosis in a mouse model of DR
To validate the effect of maresin-1 on ferroptosis in DR in vivo, a mouse model of DR was established via intraperitoneal injection of STZ.We discovered that the MDA content, ROS level and iron level were markedly boosted in retinal tissues of DR mice compared to normal mice (p < 0.01, Fig. 4A C).More importantly, the MDA content, ROS level and iron level in retinal tissues of DR mice were significantly decreased by maresin-1 administration (p < 0.01, Fig. 4A C).

Discussion
With increasing incidence of DR, there is growing recognition of the importance of retinal protection in early diabetes [41,42].In consideration of sensibility of RPE cells to hyperglycemia [43,44], we constructed a DR cell model by exposing ARPE-19 cells to HG to simulate their hyperglycemia condition.In this cell type, we discovered reduction of cell viability, increase of apoptosis and abnormal cell shape in comparison with NG-induced ARPE-19 cells.Ferroptosis is a different sort of cell death that varies from typical cell apoptosis [11].Here, in HGinduced ARPE-19 cells, we observed increases of the Fe 2+ level, the ROS level and MDA content as well as a decrease of the GSH level.Also, increased protein levels of PTGS2 and ACSL4 were observed.In addition, we found that the MDA content, ROS level and iron level were markedly increased in retinal tissues of STZinduced DR compared to normal mice.These data definitely confirmed the occurrence of ferroptosis in this DR cell model.
Maresin-1 has previously been found to affect inflammatory regression, wound healing and tissue homeostasis [45][46][47].Although the influence of maresin-1 on DR has not been investigated, the influences of maresin-1 on several diabetic disorders have been considerably documented [27,28].Maresin-1, for example, inhibits fibrosis and inflammation to protect mouse renal mesangial cells from HG damage [27].Maresin-1 alleviates DKD by decreasing inflammation and oxidative stress [28].It is noteworthy that maresin-1 regulates HG-induced ferroptosis of osteoblasts in T2DOP, which is associated with decreases in Fe 2+ , ROS, and MDA levels as well as an increase in GSH [29].Consistently, we also discovered that maresin-1 increased the GSH level, and reduced the Fe 2+ level, the ROS level and MDA content in HGinduced ARPE-19 cells.At same time, we found that maresin-1 reduced the PTGS2 and ACSL4 expression in HG-induced APRE-19 cells.In vivo experiments, we discovered that the MDA content, ROS level and iron level in retinal tissues of DR mice were markedly decreased Nrf2 has been found to control antioxidant protein production and to protect cells from ROS damage [48,49].In particular, the Nrf2/HO-1 pathway has been explored in diabetic complications [50,51].In diabetic kidneys, HMGB1 down-regulation protects mesangial cells from glucose-induced ferroptosis through activation of the Nrf2/HO-1pathway [14].This pathway prevents HG-induced oxidative stress, thereby protecting the retinal pericytes [52].In the present study, we discovered that the mRNA expression and protein expression of Nrf2, HO-1 and GPX4 were up-regulated by maresin-1 treatment in HG-induced ARPE-19 cells, suggesting activation of the Nrf2/HO-1/GPX4 pathway.At the same time, Nrf2 inhibitor reversed the inhibitory effects of maresin-1 on ferroptosis in HG-induced ARPE-19 cells.Similarly, a previous research has demonstrated that maresin-1 activates the Nrf2/HO-1/GPX4 pathway to mitigate ferroptosis-induced liver injury [36].Our data implied that maresin-1 impaired HG-induced ferroptosis in ARPE-19 cells by activating the Nrf2/HO-1/GPX4 pathway.
There was a limitation in our study.For observing the effect of osmotic control conditions on the experiment, the mannitol control group should be established.In future studies, to assess the effect of maresin on ARPE-19 cells under high glucose exposure, the mannitol isotonic control group should be considered to negate osmotic and metabolic effects.

Fig. 4
Fig. 4 Maresin-1 represses ferroptosis in a mouse model of DR. (A) The content of MDA in retinal tissues was detected via a MDA assay kit from Nanjing Jiancheng Bioengineering.(B) The level of ROS in retinal tissues was determined using an oxidation-sensitive fluorescent probe DCFH-DA with a ROS Assay kit (Jianglaibio, Shanghai, China).(C) The total iron in tissue homogenate was determined through an Iron Assay Kit.** p < 0.01, *** p < 0.001.N = 6 (mice)

Table 1
Primer sequences for qRT-PCR in this study °C, humidity at 55 ± 5%, with a 12/12 h light/dark cycle), which had free access to water and food.The animal experiment was approved by the Animal Care and Use Ethics Committee of Beijing Viewsolid Biotechnology Co. LTD (VS232176245).All methods were carried out in accordance with relevant guidelines and regulations.All methods are reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).