Sirtuin 6 protects human retinal pigment epithelium cells from LPS-induced inflammation and apoptosis partly by regulating autophagy

Abstract

Lipopolysaccharides (LPS)-induced retinal inflammation is an important factor in retinal diseases. This study was aimed to investigate the effect of Sirt6 on LPS-induced retinal injury. ARPE-19 cells were incubated with LPS to induce inflammation. The cell viability was determined using CCK-8 assay. The mRNA level and protein expression of corresponding genes was detected using qRT-PCR and western blot, respectively. The production of inflammatory cytokines was measured using ELISA kit. The levels of oxidative stress-related factors were measured using their detection kits. Cell apoptosis was observed using TUNEL assay. The results showed that Sirt6 was downregulated after LPS treatment. Sirt6 strengthened LPS-induced autophagy by promoting the expression of LC3II/I, beclin1 and ATG5. Sirt6 treatment significantly inhibited LPS-induced inflammation, oxidative stress and cell apoptosis, which was then partly abolished by 3 MA. These results suggest Sirt6 to be an important regulator for LPS-induced inflammation, oxidative stress, and apoptosis partly by regulating cell autophagy.

GRAPHICAL ABSTRACT

A schematic model of the role and potential mechanism of Sirt6 in LPS-induced retinal 20 diseases.

Sirtuin 6, human retinal pigment epithelium cells, inflammation, apoptosis, autophagy

Retinal pigment epithelium (RPE), located between the choroid and the neural retina, is the monolayer of pigmented epithelial cells that play important roles in large range of retinal pathologic processes such as secretion of various cytokines and growth factor, and transportation of water and nutrients to the retina [1,2]. RPE cells were closely involved in different pathologic processes of the retina and act a critical role in retinal diseases such as diabetic retinopathy, age-related macular degeneration (AMD), retinitis pigmentosa, and uveitis [3–6]. Inflammation is a common factor in the pathogenesis of these diseases. The inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) are secreted from monocyte macrophage under normal physiological condition but were also produced by other cells such as RPE cells under a pathological condition. The previous studies suggest that TNF-α and IL-6 were involved in the pathogenesis of AMD and propelled AMD progression [7,8], thus controlling the production of these inflammatory cytokines might be an effective targets for disease intervention.

Sirtuin 6 (Sirt6), a member of the sirtuin family of adenosine diphosphate (ADP)-ribosyltransferase and NAD(+)-dependent protein deacetylase, is a nuclear localized protein that regulates many biological processes such as transcription, inflammation, carcinogenesis, metabolism and so on, and influences a wide range of pathophysiological process including Alzheimer’s disease, periodontitis, diabetes mellitus and cardiovascular diseases [9–13]. A recent study reported an elevated expression of Sirt6 in RPEs mice aged, indicating that the aberrant Sirt6 might be a reason that triggers RPE dysfunction [8].

Autophagy is a lysosomal degradation pathway commonly found in eukaryotes. Autophagy has been suggested to be vital in cellular physiological and pathological event, as autophagy is responsible for the lysosomal digestion of damaged organelles and abnormal proteins to demand the metabolism requirement and maintain homeostasis [14,15]. Autophagy is critical for the maintenance of RPE homeostasis, and aberrant autophagy level might induce RPE damage and contribute to the progression of retinal diseases [16,17]. Interesting, a recent study showed that Sirt6-mediated activation of autophagy was involved in inflammatory response in RPEs, suggesting a close connection among Sirt6, autophagy and inflammation in RPEs [8]. However, until now, there are only little reports concerning about whether Sirt6 involves in the occurrence and development of retinal diseases through regulating autophagy, and the knowledge in this term was incomplete. Therefore, the goal of the present study was to investigate more about the role of Sirt6 in the occurrence and development of retinal diseases and explore the potential mechanism, to gain a clear understanding of Sirt6 in retinal diseases.

Materials and methods

Cell culture and treatment

Human retinal-pigmented epithelium cells (ARPE-19) were maintained from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% fetal bovine serum (FBS; GIBCO, Thermo Fisher Scientific, Inc.,Waltham, MA, USA), 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C under 5% CO₂. For Sirt6 overexpression, the pcDNA-Sirt6 and the empty vector (negative control, NC) were transfected into ARPE-19 cells using Lipofectamine 3000 (Invitrogen, USA) in accordance with the manufacturer’s protocols. 48 h after transfection, cells were harvested for qPCR analysis. For lipopolysaccharides (LPS) treatment, ARPE-19 was induced by different concentrations of LPS (2.5 μg/mL, 5 μg/mL, and 10 μg/mL) for 24 h. Cell viability and Sirt6 expression were measured to select the appropriate concentration for next research.

Cell viability assay

Cell viability was detected using a cell-counting kit-8 (CCK-8) assay kit (Dojindo Laboratories, Kumamoto, Japan). ARPE-19 cells were seeded onto 96-well plates and incubated for 24 h, followed by incubation with CCK-8 solution for another 4 h. The absorbance at 450 nm was detected under a microplate reader.

Enzyme-linked immunosorbent assays (ELISA)

Concentrations of inflammatory cytokines including TNF-α, IL-6, IL-4, and IL-1β from cell culture media were measured using corresponding ELISA kits (R&D Systems, Minneapolis, USA) according to the manufacturer’s protocols.

Determination of reactive oxygen species (ROS) level

The ROS level was determined by a fluorescence probe DCFH-DA (Sigma, St.Louis, MO). After treatment for 24 h, cells were incubated with DCFH-DA at a final concentration of 10 μM at 37°C for 30 min according to the manufacturer’s instructions. The fluorescence intensity was observed under an Olympus BX51 fluorescence microscope.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)

Total RNA was extracted using Trizol reagent (Invitrogen) in accordance with manufacturer’s protocols. The exacted RNA was then reverse transcribed to cDNA using the PrimeScript RT reagent kit (Takara, Tokyo, Japan). qPCR was then conducted using Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Inc.) using SYBR Green Master Mix (Thermo Fisher Scientific, Inc.) in accordance with manufacturer’s protocols. Relative expression of genes were normalized to that of GAPDH using 2^−ΔΔCt method.

Western blot

After treatment, the total proteins were extracted using ice-cold RIPA lysis buffer with the complete protease inhibitor cocktail. The concentration of extracted protein was determined using a BCA Protein Assay kit (Beyotime Institute of Biotechnology, Haimen, China). Then, the protein samples were subjected to 10% sodium dodecysulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). Membranes were blocked with 5% skimmed milk for 2 h at room temperature, followed by incubation with primary antibodies at 4°C overnight and subsequent incubation with horseradish peroxide-conjugated secondary antibody for 2 h at room temperature. Protein bands were detected by ECL detection (Millipore, Darmstadt, Germany) and were quantified by Image J software (National Institutes of Health).

TUNEL assay

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was conducted to detect cell apoptosis. Cells were fixed in 4% paraformaldehyde for 30–60 min, followed by incubation with 1% Triton X-100 on the ice for 2 min and subsequent incubation with 0.3% H₂O₂ in methanol at room temperature for 20 min.

After washing with PBS for three times, cells were incubated with TUNEL detection solution at 37°C for 60 min and added with stop solution, followed by incubation with DAB solution and staining with hematoxylin and eosin for observation.

Statistical analysis

Data were obtained from three independent experiments and were presented as mean ± SD. The statistical analysis were performed using GraphPad Prism (GraphPad Software, San Diego, CA) and SPSS (SPSS, Inc., Chicago, IL, USA). Group comparisons were determined by one-way analysis of variance with Tukey’s post hoc test. P value <0.05 was considered statistically significant.

Results

Expression of Sirt6 is downregulated after LPS treatment in ARPE-19 cells

In the present study, ARPE-19 was stimulated by different concentrations of LPS (2.5 μg/mL, 5 μg/mL, and 10 μg/mL) for 24 h, and the expression of Sirt6 and cell viability was examined. As shown in Figure 1(a), the protein expression of Sirt6 was downregulated under LPS treatment with a concentration-dependent manner. Cell viability was declined when treated with LPS at 5 μg/mL and 10 μg/mL (Figure 1(b)). The results suggested that Sirt6 was downregulated in LPS-induced ARPE-19 cell, and LPS (10 μg/mL) was used for further study.

Figure 1.

The expression of Sirt6 after LPS treatment.

(a) ARPE-19 was induced by different concentrations of LPS (2.5 μg/mL, 5 μg/mL, and 10 μg/mL) for 24 h. The protein expression of Sirt6 was examined using western blot. (b) After LPS treatment for 24 h, Cell viability was measured using CCK-8 assay. Data were presented as mean± SD. *, ***p < 0.05, 0.001 vs. 0 μg/mL of LPS.

Sirt6 strengthens cell autophagy induced by LPS in ARPE-19 cells

To investigate the role of Sirt6 in the occurrence and development of retinal diseases, ARPE-19 cells were transfected with pcDNA-Sirt6 to overexpress Sirt6, qRT-PCR showed that the expression of Sirt6 was significantly increased (Figure 2(a)). Then, we detected expression of Sirt6 in transfected ARPE-19 cells with LPS treatment. qRT-PCR and western blot results showed that mRNA level and protein expression of Sirt6 was downregulated by LPS, while was significantly upregulated after cell transfection (Figure 2(b,c)). The western blot results also showed that the protein expression of LC3II/I, beclin 1, ATG5 were significantly upregulated in LPS-induced ARPE-19 cells, and the effect was then strengthened by Sirt6 overexpression (Figure 2(d)). These findings suggested that Sirt6 could strengthen LPS-induced cell autophagy in ARPE-19 cells.

Figure 2.

Effect of Sirt6 on cell autophagy in LPS-induced ARPE-19 cells.

(a) Cells were transfected with pcDNA-Sirt6, and the expression of Sirt6 was detected by qRT-PCR. *** p < 0.001 vs. NC. (b, c) Cells were induced with LPS with or without transfection with pcDNA-Sirt6. The mRNA level and protein expression of Sirt6 were detected using qRT-PCR and western blot, respectively. (d) The expression of autophagy-related proteins including LC3II/I, Beclin1, and ATG5 was determined using western blot. Data were presented as mean± SD. *, **, ***p < 0.05, 0.01, 0.001 vs. control; #, ##, ###p < 0.05, 0.01, 0.001 vs. LPS+NC.

Sirt6 inhibited inflammation induced by LPS in ARPE-19 cells

To investigate the anti-inflammatory activity of Sirt6 in ARPE-19 cells, we examined the production of TNF-α, IL-6, IL-1β, and IL-4. As shown in Figure 3(a-d), LPS-induced excessive production of TNF-α, IL-6, IL-1β, as well as the declined level of IL-4, which was restored after Sirt6 was overexpressed, but was enhanced by co-treatment with LPS and autophagy inhibitor 3 MA. Besides, 3 MA itself induced the upregulated production of TNF-α, IL-6, IL-1β, and downregulated production of IL-4. In addition, as Sirt6 could promote cell autophagy, autophagy inhibitor 3 MA was used to inhibit cell autophagy, and the results showed that the regulatory effect of Sirt6 on inflammatory cytokines production was reversed, indicating that the anti-inflammatory activity of Sirt6 was partly depending on cell autophagy activity. These findings suggested that Sirt6 could inhibit LPS-induced inflammation partly by promoting autophagy.

Figure 3.

Effect of Sirt6 on inflammation in LPS-induced ARPE-19 cells.

(a) Cells were treated with both of LPS and 3 MA or alone. Besides, cells transfected with pcDNA-Sirt6 were induced by LPS with or without 3 MA. The concentration of IL-1β, (b) TNF-α, (c) IL-6, and (d) IL-4 were determined using their corresponding ELISA kit. Data were presented as mean± SD. **, ***p < 0.01, 0.001 vs control; ###p < 0.001 vs LPS+NC; $, $$$p < 0.05, 0.001 vs. LPS. &, &&, &&&p < 0.05, 0.01, 0.001 vs. LPS+Sirt6. ^^, ^^^ p < 0.01, 0.001 vs. LPS+3 MA.

Sirt6 inhibited oxidative stress induced by LPS in ARPE-19 cells

Next, we investigated whether Sirt6 plays a role in oxidative stress in LPS-induced ARPE-19 cells. As shown in Figure 4(a), the level of intracellular ROS in the ARPE-19 cells under LPS induction or 3 MA treatment was markedly higher in comparison with that of the control cells. Overexpression of Sirt6 markedly reduced the activity of ROS induced by LPS, which was then reversed by 3-MA. Similarly, we also examined the activity of MDA, SOD, and CAT. The results showed that LPS and 3 MA induced the obvious upregulation of MDA and the downregulation of SOD and CAT level, which were then reversed by Sirt6 overexpression, but were enhanced by 3 MA (Figure 4(b-d)). Finally, co-treatment with LPS, Sirt6, and 3 MA significantly reversed the effects of co-treatment with LPS and Sirt6 or co-treatment with LPS and 3 MA.

Figure 4.

Effect of Sirt6 on oxidative stress in LPS-induced ARPE-19 cells.

(a) Cells were treated with both of LPS and 3 MA or alone. Besides, cells transfected with pcDNA-Sirt6 were induced by LPS with or without 3 MA. ROS activity was determined by a fluorescence probe DCFH-DA. (b–d) The level of MDA, SOD, and CAT were determined with their corresponding detection kits. Data were presented as mean± SD. *, ***p < 0.05, 0.001 vs. control; ###p < 0.001 vs. LPS+NC; $ p < 0.05 vs. LPS. &&&p < 0.001 vs. LPS+Sirt6. ^^^ p < 0.001 vs. LPS+3 MA.

Sirt6 inhibited cell apoptosis induced by LPS in ARPE-19 cells

Finally, we investigated the role of Sirt6 in LPS-induced cell apoptosis in ARPE-19 cells. As shown in Figure 5(a), compared to the control group, the apoptotic cells in LPS group or 3 MA group shrink in size and exhibited nuclear shrinkage. Besides, the nuclear was stained brown in LPS group, suggesting that LPS or 3 MA induced a markedly apoptosis of ARPE-19 cells. The effect that induced by LPS was restored by Sirt6, which was then weakened by 3 MA, suggesting that Sirt6 alleviated LPS-induced cell apoptosis, but the anti-apoptotic effect of Sirt6 was weakened by 3 MA, indicating that inhibition of autophagy partly contributed to the exacerbation of cell apoptosis. Further, cell apoptosis-related proteins were detected to examine the change of cell apoptosis. As shown in Figure 5(b), the protein expression of Bcl-2 was significantly decreased while the protein expression of Bax, Bin, and cleaved caspase-3 were significant increased upon LPS treatment or 3 MA treatment, compared to that in the control. LPS-induced changes of these apoptosis-related proteins were reversed by Sirt6, and the effect of Sirt6 was then weakened by 3 MA. Besides, there was no obvious change between LPS group and LPS+3 MA group. These results suggested Sirt6 could inhibit LPS-induced cell apoptosis, which was weakened when autophagy activity was inhibited.

Figure 5.

Effect of Sirt6 on cell apoptosis in LPS-induced ARPE-19 cells.

(a) Cells were treated with both of LPS and 3 MA or alone. Besides, cells transfected with pcDNA-Sirt6 were induced by LPS with or without 3 MA. TUNEL assay was conducted to determine cell apoptosis. (b) The protein expression of Bcl-2, Bax, Bin and Cleaved caspase-3 were measured using western blot. Data were presented as mean± SD. *, ***p < 0.05, 0.001 vs. control; ###p < 0.001 vs. LPS+NC; $ p < 0.05 vs. LPS. &, &&, &&&p < 0.05, 0.01, 0.001 vs. LPS+Sirt6. ^^, ^^^ p < 0.01, 0.001 vs. LPS+3 MA.

Discussion

Millions of people around the world suffer from retinal diseases such as AMD and diabetic retinopathy, troubling lots of people in daily life. Accumulating evidence reveals that inflammation, oxidative stress, and cell apoptosis of RPE cells are crucial aspects of the pathophysiology of these retinal diseases [18]. Bacterial endotoxins such as LPS can result in cell apoptosis in human RPE cells by inducing inflammatory cytokines production and free radical secretion [19]. Just as the results shown in the present study, LPS triggered serious inflammation characterized by elevated levels of IL-1β, TNF-α, IL-6, and decreased level of IL-4, and obvious oxidative stress characterized by elevated levels of ROS, MDA, and decreased expression of SOD and CAT. Besides, LPS also caused an obvious apoptosis of RPE cells. On the other hand, overexpression of Sirt6 alleviated cell injury caused by LPS. These results demonstrated that Sirt6 has protective potential in RPE cells.

Accumulating evidence has documented the protective effect of Sirt6 on different diseases and could produce significant anti-inflammatory, antioxidant and anti-apoptotic effects. Zhang W et al. reported that Sirt6 could protect the brain from cerebral ischemia/reperfusion injury by suppressing oxidative stress via NRF2 activation [20]. Sirt6 has been found to interact with the nuclear factor-kappa B (NF-κB) subunits RelA and p65, and thereby inhibit the proinflammatory NF-κB signaling pathway and exert anti-inflammatory activity [21]. Moreover, Sirt6 was also shown to be a targeted protein for miR-351-5p to regulate intestinal mucosal oxidative stress, inflammation, and apoptosis [22]. In the present study, the results exhibited that overexpression of Sirt6 significantly inhibited inflammation, oxidative stress, and apoptosis, exerting a protective effect in RPE cells, which was in accordance with the previous reports.

Autophagy is a very active catabolic process in RPE cells. Cell survival and death are complex processes involving apoptotic and autophagic pathways. Once autophagy is initiated, the cytosolic form of microtubule-associated protein 1 light chain 3 (LC3I) is processed and transformed by the addition of a group of phosphatidylethanolamine to form LC3II. LC3I and LC3II have been commonly used to monitor autophagy, as the conversion of LC3I to LC3II reflected the number of autophagosomes and autophagic flux [23–25]. ATG5 is an E3 ligase crucial for the conjugation of LC3 to phagophore membranes during the induction of autophagy [26]. Beclin-1 is essential for forming the autophagosome as its regulation on synthesizing autophagosome membrane synthesis and engulfing cytoplasmic material [24]. Recent studies have shown that Sirt6 was closely related to the autophagy. Activation of Sirt6 was demonstrated to induce a time-dependent activation of autophagy in several human tumor cell lines, and the autophagy-related cell death resulted from sustained activation of Sirt6 was attenuated using an autophagy inhibitor, indicating that activation of Sirt6 resulted in autophagy induction [27]. Besides, Sirt6 could regulate autophagy to exert different functions. Wang L et al. demonstrated that the expression of Sirt6 in melanoma played a critical role in regulating melanoma growth through an autophagy-dependent manner [28]. Feng Y et al. pointed it out that autophagy was modulated by Sirt6 in amyloid-β stimulated RPEs and Sirt6-autophagy pathway is potential harnessed to treat RPE-derived ocular inflammation [8]. In the present study, we determined that Sirt6 increased autophagic markers (LC3II/LC3I, Beclin-1, and ATG5) and promoted autophagy. These results suggested that LPS-induced autophagy in the ARPE-19 cells by upregulating LC3II/LC3I, Beclin-1, and ATG5, which increased the potency of Sirt6 for protecting ARPE-19 cell from inflammation and oxidative stress-induced cell apoptosis.

As a recycling process, autophagy has been described as a cellular pro-survival process, and impaired autophagy in RPE leads to cell transcytosis and exocytosis and early signs of degeneration [25,29]. The previous study has demonstrated that impaired autophagy caused RPE apoptosis that in turn modulated macrophage-inflammasome activation and resulted in inflammation in vivo and in vitro [30]. Thus, maintaining autophagy is important for RPE health. In the present study, autophagy acted as a protective role in LPS-induced retinal injury. In the early stage of LPS stimulation in ARPE-19 cells, LPS caused significant cell damage, including inflammation and apoptosis. At the same time, responding to this stimulation, the autophagy was activated, which in turn and enhanced, which might serve as a protective role for the cells, just as reported in previous study [25]. Then, overexpression of Sirt6 further strengthened the activity of autophagy. Autophagy removed the defective organelles and injured cells to maintain the stability of cellular environment. Therefore, Sirt6-induced high active autophagy might be an important factor for alleviating retinal injuries and decreasing cell apoptosis.

Furthermore, oxidative stress is also a recognized risk factor for retinal diseases, which is usually associated with the generation of ROS. Recent evidence showed that the enhancement of autophagy had a protective effect against H₂O₂-induced ROS generation in RPE cells, but the autophagy inhibitor 3 MA rendered cells more susceptible to H₂O₂-induced cell toxicity and cell death [31]. Therefore, autophagy played a protective role in RPE cells under oxidative stress. In agreement with the previous study, our study also demonstrated that the autophagy enhanced by Sirt6 protected ARPE-19 cells against oxidative stress.

Conclusion

In conclusion, Sirt6-mediated autophagy and inhibited LPS-induced inflammation, oxidative stress, and cell apoptosis in ARPE-19 cells. These findings have therapeutic implications for retinal diseases.

Author’s contributions

JL conceived and designed the experiments. JL and DL performed the experiments and wrote the manuscript. All readers read and approved the final manuscript.

Disclosure statement

The authors declare no competing interest.