The protective effect of luteolin on the depression-related dry eye disorder through Sirt1/NF-κB/NLRP3 pathway

Luteolin alleviated depression-like behaviors and dry eye symptoms in a model of depression-related dry eye disorder

To explore whether luteolin had an attenuated effect on CUMS-induced depression-like behaviors in mice, we performed SPT, OFT, TST and FST behavioral experiments. In the SPT experiment, the intake of sucrose in the CUMS-induced mice was significantly lower than that in control group, indicating that the CUMS-induced mice lacked pleasure (Figure 1A). While in the luteolin (5 mg/kg) (p < 0.05), luteolin (10 mg/kg) (p < 0.01), and escitalopram (p < 0.01) treatment groups, the preference for sucrose was significantly increased (Figure 1B). In TST, the immobility time of the mice in the model group was significantly longer than that in control group. Luteolin (10 mg/kg) administration (p < 0.01) could significantly reverse this change, which was similar with that of the positive drug escitalopram (p < 0.01) (Figure 1C). In FST, compared with CUMS group, luteolin (10 mg/kg) (p < 0.01), luteolin (5 mg/kg) (p < 0.05) significantly shortened the immobility time of mice (Figure 1D). CUMS stimulation did not significantly affect the distance and time in center zone compared with those of control group. Compared with the control group, CUMS stimulation had no effect on the movement distance and the time of staying in center zone Luteolin (5 mg/kg), luteolin (10 mg/kg), escitalopram treatment group compared with the depression-related dry eye disorder group also had no distinct difference. OFT data showed that luteolin did not affect the motor activity of mice. (Figure 1E, 1F).

Figure 1. Luteolin improved depressive behavior and dry eye symptoms in DEDC mice. (A, B) SPT, (C) TST, (D) FST, and (E, F) OFT. (G) Corneal epithelial defect fluorescence representative images of pyruvate sodium staining (H) Quantitative determination of corneal defects in mice (I, J) Phenol red line assay showing tear volume (K) The effect of luteolin on goblet cells in xerophthalmia and CUMS mice. Scale bar is 50 μm. Data analysis was performed with Mean ± SEM. ^#p < 0.05, ^##p < 0.01 compared with blank group; ^*p < 0.05, ^**p < 0.01 compared with model group, n = 9.

Besides, we also tested the effect of luteolin on dry eye symptoms. Sodium fluorescein is commonly used to measure the areas of corneal surface epithelial cells. Depression-related dry eye disorder group showed severe corneal impairment and ocular surface epithelium damage compared with those of control group. Luteolin (5 mg/kg) (p < 0.05) and luteolin (10 mg/kg) (p < 0.01) significantly attenuated corneal damage in the mice of depression-related dry eye disorder group, respectively (Figure 1G, 1H). Next, we used phenol red cotton thread to measure the tear secretion of mice [23]. Compared with the control group, the tear secretion of mice in the depression-related dry eye disease group was significantly reduced, but it was immensely increased in luteolin (5 mg/kg) group (p < 0.05) and luteolin (10 mg/kg) group (p < 0.01). (Figure 1I, 1J). In addition, the reduction in goblet cell density was positively associated with disease severity [10]. Conjunctival PAS staining showed that compared with the control group, conjunctival goblet cells in depression related dry eye disease group were significantly reduced (p < 0.01). The number of conjunctival goblet cells in the luteolin (5 mg/kg) group increased significantly (p < 0.05), and the luteolin (10 mg/kg) group had a better therapeutic effect (p < 0.01). Escitalopram, a positive drug for depression-like symptoms, did not significantly influence DED symptoms in mice. (Figure 1K).

The effect of luteolin on the inflammatory response in depression-related dry eye disorder mice

Previous studies displayed that the development of dry eye disease and depression was accompanied by inflammation in rodents [24] and humans [25]. The related pro-inflammatory factors IL-1β, IL-6, TNF-α and IL-18 were detected in mouse serum using enzyme-linked immunosorbent assay (ELISA). Compared with control group, the pro-inflammatory factors in the tears of model mice were significantly increased (p < 0.01). After the treatment with luteolin (5 mg/kg) (p < 0.05) and luteolin (10 mg/kg) (p < 0.01), the serum levels of pro-inflammatory factors IL-1β, TNF-α, IL-6 and IL-18 were significantly higher than those in the control group. The same trend was also shown for the above pro-inflammatory factors in tears (Figure 2). The results showed that luteolin could reverse and stabilize the elevated pro-inflammatory factors in mice with the depression-related dry eye disorder, and alleviate the inflammatory changes in mice.

Figure 2. The regulation of IL-1β, TNF-α, IL-18, and IL-6 in the serum and tears of mice with dry eye and CUMS mice. (A, B), (C, D) are the levels of IL-1β, TNF-α, IL-18, and IL-6 in serum. (E–H) are the contents of IL-1β, TNF-α, IL-18, and IL-6 in tear fluid. Data analysis was performed with Mean ± SEM. ^##p < 0.01 compared with blank group; ^*p < 0.05, ^**p < 0.01 compared with model group, n = 9.

The effects of luteolin on Sirt1/NF-κB/NLRP3 signaling pathway

The activity of Sirt1 in mouse hippocampal dentate gyrus was decreased after chronic stress. Additionally, inhibition of hippocampal Sirt1 function in mice on the basis of genetics and pharmacology can lead to the increase of depression-like behaviors [26]. Due to the reversal of depressive behaviors and dry eye symptoms in mice, we speculated that luteolin might act on Sirt1 protein to treat depression-related dry eye disorder in mice. To clarify the therapeutic effect of luteolin on CUMS-induced depression, we used Western Blot to detect the protein expression levels of Sirt1, NLRP3, Cleaved-caspase1 and Caspase1 in the mouse hippocampus (Figure 3A). The results showed that compared with the control group, the level of Sirt1 protein in the hippocampus of CUMS-induced mice was significantly decreased (P < 0.01). Both the luteolin (5 mg/kg) treatment group (p < 0.05) and the luteolin (10 mg/kg) (P < 0.01) treatment group significantly increased Sirt1 protein levels (Figure 3B). The protein expression levels of Ac-NF-κB, NF-κB, NLRP3, GSDMD-N, GSDMD, Cleaved-Caspase1 and Caspase1 were significantly up-regulated after CUMS stimulation (p < 0.01). The treatment with luteolin (5 mg/kg) and luteolin (10 mg/kg) could significantly reverse the down-regulations of protein expression of Ac-NF-κB, GSDMD-N and GSDMD (p < 0.01). In addition, NLRP3, Cleaved-Caspase1 and Caspase1 protein expression levels were remarkably down-regulated after luteolin (5 mg/kg) (p < 0.05) and luteolin (10 mg/kg) (p < 0.01) treatments (Figure 3C–3F). Similarly, we also detected the above proteins in mouse cornea tissue, showing the same change trend as in mouse hippocampus (Figure 3G–3L).

Figure 3. Luteolin could regulate the expressions of Sirt1, Nlrp3, AC-NF -κB, NF-κB, Nlrp3, GSDMD-N and GSDMD proteins in hippocampi and tears of mice. (A–F) Western Blot was used to detect the effects of luteolin on protein bands of Sirt1, Nlrp3, AC-NF-κB, NF-κB, GSDMD-N and GSDMD in the hippocampus of mice. (G–L) Western Blot analysis of the effects of luteolin on protein bands of Sirt1, Nlrp3, AC-NF -κB, NF-κB, GSDMD-N and GSDMD in mice cornea. (M) Molecular docking of sitrt1 and luteolin in discovery studio. (N) Molecular docking of sirt1 and Resveratrol by discovery studio. Data were analyzed by mean ± scanning electron microscopy. Compared with blank group, ^##P < 0.01; Comparison with model group ^*P < 0.05, ^**P < 0.01, n = 9.

As previously described, we performed further validation of the interaction between luteolin and Sirt1 protein by using molecular docking. We conducted molecular docking between Sirt1 protein and luteolin through Discovery Studio software, and the CDOCKER ENERGY of the two docking was −24.4965 kcal/mol, indicating that Sirt1 protein was related to luteolin and relatively closely bound. (Figure 3M). Luteolin might combine with Sirt1 at GLU208, ARG274, GLN294 and ARG446 via hydrogen bond. We hypothesized that luteolin played a role as a Sirt1 activator in the treatment of depressive-like behavior in mice. Surprisingly, the CDOCKER ENERGY of Sirt1 activator resveratrol and Sirt1 protein was −20.3636 kcal/mol, indicating that luteolin was more tightly connected to Sirt1 than resveratrol. However, the specific action relationship between luteolin and Sirt1 needs to be further investigated (Figure 3N).

The effects of luteolin on microglia

Iba1, a calcium-binding protein, is only expressed in microglia in the central nervous system (CNS) and is upregulated upon microglia activation [27]. Therefore, Iba1 immunofluorescence staining was used to detect the activation state of microglia. Considering the critical role of microglia in neuroinflammation, we examined microglia in mouse hippocampus by immunofluorescence. CUMS exposure significantly increased hippocampal microglia activation compared to controls (p < 0.01). The treatments with luteolin (5 mg/kg) (p < 0.05) and luteolin (10 mg/kg) (p < 0.01) hampered the upregulation of microglia induced by CUMS exposure (Figure 4A).

Figure 4. Effects of immunofluorescence on microglia in mice. (A) Effects of luteolin on microglia in different doses were determined by immunofluorescence. (B) Determination of microglia in the hippocampal area of mice after EX-527. (C) Effect of EX-527 on microglia activation. (D) Relative fluorescence intensity of Nlrp3. Data were analyzed by mean ± SEM. Compared with blank group, ^##P < 0.01; Comparison with model group ^*P < 0.05, ^**P < 0.01, n = 9.

In addition, the Sirt1 protein inhibitor EX-527 was further used to explore the role of Sirt1 in CUMS-exposed mice. The effect of luteolin (10 mg/kg) on microglia activation was undoubtedly abolished by EX-527, which was positive for Iba1. The number of cells increased, suggesting that luteolin reduces microglia activation by activating cells through the Sirt1 protein (Figure 4B, 4C).

EX-527 blocked luteolin mediated treatment of depression-like behaviors in mice

To further investigate the in vivo role of Sirt1 signaling in the antidepressant mechanism of luteolin, we further investigated the mechanism of luteolin against depression-related dry eye disorder using the SIRT1 selective inhibitor EX-527. As shown in the Figure 5A, Sirt1 inhibitor EX-527 inhibited luteolin (10 mg/kg) from improving sucrose preference in mice in SPT (p < 0.01). Unsurprisingly, EX-527 also inhibited the anti-depressive like effect of luteolin (10 mg/kg) in TST. The immobility time of mice was significantly longer when EX-527 and luteolin (10 mg/kg) were used together than when luteolin (10 mg/kg) was used alone (p < 0.01) (Figure 5B). These results revealed that the mechanism of luteolin against pleasure loss and behavioral despair in depressed mice depends on Sirt1 signaling. Give the above, after applying EX-527, we used Western Blot to detect the protein expressions of Ac-NF-κB, NF-κB, GSDMD-N and GSDMD in hippocampus. EX-527 resisted the decreased expression of Ac-NF-κB, NF-κB, GSDMD-N and GSDMD protein in hippocampus treated with luteolin (10 mg/kg) compared to the group treated with luteolin (10 mg/kg) alone (Figure 5C).

Figure 5. Role of Sirt1 in the reduction of CUMS-induced depressive behavior by luteolin. (A) Sucrose preference rate of mice was determined by SPT. (B) The immobility time of mice was measured by TST. (C) Western Blot analysis of the expression of AC-NF-κB, NF-κB, GSDMD-N and GSDMD protein in hippocampal tissue. (D) Nisi staining of mice treated with EX-527. Data were analyzed by means ± SEM. Compared with blank group, ^##P < 0.01; Comparison with model group ^*P < 0.05, ^**P < 0.01, n = 9.

Immunofluorescence showed that EX-527 inhibited the abolition of the relative fluorescence intensity of NLRP3 by luteolin (10 mg/kg) compared with luteolin (10 mg/kg) alone in the dry eye and depressive comorbid groups (p < 0.01) (Figure 4D).

Histological changed in the DG region of mice exposed to CUMS were visualized using Nissl staining. CUMS stimulation darkened the staining of positive cells, whereas after luteolin (10 mg/kg) treatment, nuclei and abundant Nissl vesicles could be seen. As shown in the Figure 5D, luteolin (10 mg/kg) increased the number of neurons in Nissl body, but EX-527 reversed this change, suggesting that luteolin had a protective role in the damage of cortical neurons induced by CUMS exposed mice protection, and Sirt1 was involved in this process.

EX-527 blocks the effect of luteolin in treating symptoms of dry eye disease in mice

To further elucidate the role of Sirt1 protein and dry eye-like symptoms in mice, we tested the tear secretion of mice in the dry eye and depression comorbidity group after the application of EX-527. EX-527 significantly reversed the increased tear secretion in the luteolin (10 mg/kg)-treated group compared with the depression and dry eye comorbid groups treated with luteolin (10 mg/kg) alone (P < 0.01) (Figure 6A). In addition, EX-527 and luteolin (10 mg/kg) inhibited the repairing effect of luteolin (10 mg/kg) on corneal defect (Figure 6B).

Figure 6. Effects of Sirt1 on luteolin in alleviating dry eye symptoms in DEDC mice. (A) Phenol red cotton thread testing mouse tears. (B) Sodium fluorescein staining. (C) Goblet cell staining. (D) Western blotting detects protein expression of AC-NF-κB, NF-κB and GSDMD-N, GSDMD in tear fluid. Data analysis was performed with mean ± SEM. ^##p < 0.01 compared with blank group; ^*p < 0.05, ^**p < 0.01 compared with model group, n = 9.

In addition, PAS staining of the conjunctival epithelium showed that EX-527 significantly inhibited the protective effect of luteolin on goblet cells of the conjunctival epithelium (Figure 6C).

Besides, the corneal Ac-NF-κB, NF-κB, GSDMD-N and GSDMD protein expressions were detected. Not surprisingly, compared with luteolin alone (10 mg/kg), the simultaneous use of EX-527 and luteolin (10 mg/kg) could abrogate the effect of luteolin on Ac-NF-κB, NF-κB, GSDMD-N and GSDMD protein (Figure 6D).

These results suggested that Sirt1 played an important role in the treatment of dry eye symptoms with luteolin (10 mg/kg).

Reagents

Luteolin (CAS: 491-70-3) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. (L812409, Shanghai, China). Escitalopram (CAS: 128196-01-0) was purchased from Shanghai McLean Biochemical Technology Co., Ltd. (E883441, Shanghai, China). EX-527 (CAS: 49843-98-3) was provided by Sigma Biotechnology Co., Ltd. (E7034, Sigma-Aldrich, USA).

Animals

Eight-week-old C57BL/6J male mice, weighing 18–22 g, were provided by Suzhou Spelford Biotechnology Co., Ltd (certificate number: SCXK(Jing)2019-0010). Animals were housed in normal-sized cages with free access to food and water under standard environmental conditions (22 ± 2°C, 12 h on/off light cycle, lights on at 8 am) and used for experiments after 3 days of acclimatization. All experimental procedures were approved by the Laboratory Animal Research Committee of Hunan University of Chinese Medicine and were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The approval number of the ethics committee is No.2022-5. All procedures were in accordance with the institutional guidelines for the use of laboratory animals, and every effort was made to alleviate the suffering of laboratory animals.

Groups and drug administration

According to the principle of random allocation, 40 mice were divided into five groups (8 mice in each group): control group, CUMS group, CUMS + Luteolin (5 mg/kg) group, CUMS + Luteolin (10 mg/kg) and CUMS + escitalopram group. Mariem Achour et al. [19] demonstrated that luteolin (10 mg/kg) reversed LPS-induced depression-like behaviors in mice. Luteolin (5 and 10 mg/kg) was used in this study. Luteolin and positive escitalopram (10 mg/kg) were dissolved in DMSO and normal saline. The concentration of DMSO was less than 0.1% (w/v). The drugs were orally treated once daily from 9:00 am to 10:00 am for 3 weeks.

CUMS procedure and experimental design

To explore the effects of luteolin on depression-related dry eye disorder, we made some modifications to the CUMS procedure described earlier [51]. We eliminated the use of moist litter and forced swimming in mice, retaining stressors such as food and water deprivation, constant light, 45° cage tilt, restraint, flashing lights, tail pinching, and space crowding. Meanwhile, a fan was placed above the mouse cage, blowing at a moderate speed (about 600 r/min) for 12 h a day to keep the humidity in the cage below 30–35%. After 4 weeks of stress, luteolin (5 mg/kg), luteolin (10 mg/kg) and escitalopram (10 mg/kg) were administered by gavage once daily for three weeks. Meanwhile, the control group and the CUMS group were gavaged with normal saline. Mice were then sacrificed after assessing behavioral testing and dry eye testing. In Experiment 2, EX-527 (10 mg/kg) was administered intravenously for two consecutive weeks starting at the fifth week.

Sucrose preference test (SPT)

The C57BL/6J mice undergoing the test were exposed to two bottles both containing 1% sucrose solution for 48 h to allow them to acclimatize, followed by 24 h of water and food deprivation. Subsequently, each mouse was exposed to two identical bottles (one containing pure water and the other containing 1% sucrose solution). After 6 h, the two bottle positions were swapped for 6 h and the bottles were weighed. The consumption of sucrose solution and pure water was determined by measuring the change in liquid consumption. Sucrose preference was defined as the ratio of sucrose volume to total sucrose volume and water consumption.

Forced swimming test (FST)

The C57BL/6J mice were placed individually in a glass container (20 cm diameter and 45 cm high) for 6 min in water 15 cm deep at 25 ± 1°C. The last 4 min of each mouse was assessed by an investigator unaware of the groups. The immobility was defined as no movement and/or the small movement required to keep the head above the water surface. Each mouse was provided with fresh water.

Tail suspension test (TST)

The C57BL/6J mice were individually suspended by tape approximately 20 cm from the ground and a transparent hollow anti-climbing tube (0.5 cm in diameter and 4 cm in length) was placed around their tails to prevent their tail-climbing behavior. The experiment lasted for 6 min and the time each mouse remained stationary (passively hanging and completely still) for the last 4 min was recorded and scored by a researcher who did not know the groups.

Open field test (OFT)

The C57BL/6J mice were placed in a (100 × 100 × 40 cm) box made of opaque plexiglass. Each mouse was gently placed in the center of the apparatus during the test and allowed to explore freely for 5 min. Thereafter, the distance the mice walked and the time spent in the center were monitored by ANY-maze software. After each mouse was tested, the apparatus was washed with 75% ethanol to remove the odor left by the mice.

Tear volume and corneal fluorescein staining

Tear secretion in mice was measured by the phenol red wire method at 9 am on day 28 and day 49, respectively. The silk threads were placed on the lateral eyes for 15 seconds, and the length of the wet red silk threads was recorded in millimeters. 5 μL (1%) of fluorescein dry was placed on the mouse eye surface to observe corneal epithelial disruption. Photographs of the eyes were taken with a digital camera. Each cornea was scored from 0 to 5 according to the Oxford University protocol.

Nissl staining

Three mice from each group were selected for cardiac perfusion with 4% paraformaldehyde after behavioral testing. The removed brain tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and sectioned. The slides were dehydrated in graded ethanol, soaked in xylene, and rehydrated in decreasing concentrations of ethanol. The slides were hydrated at 60°C for 30 min using 1% toluidine blue, dehydrated again with ethanol, xylene was removed, and finally sealed with neutral gel. The morphology of the hippocampus was observed under a light microscope.

Western blot

The mouse hippocampus/cornea was extracted with RIPA lysis solution (containing 1% PMSF, 2% phosphatase inhibitor), and the mouse hippocampus/cornea protein was extracted, and centrifuged at 10,000 r/min at 4°C for 10 min. The protein concentration in the Western Blot samples was determined according to the instructions of the BCA protein concentration assay kit. Proteins were separated using 8–12% sodium dodecyl sulfate-polyacrylamide gels. The samples were electro transferred onto PVDF membranes and blocked with 5% milk solution. Membranes were then incubated overnight at 4°C with the following primary antibodies: anti-Sirt1 (CST, #8469, 1:1000), anti-NLRP3 (CST, #15101, 1:1000), anti-Cleaved-caspase-1 (CST, #89332S, 1:1000) anti-GSDMD-N (CST, #93709, 1:1000), anti-Caspase 1 (Abcam, ab207802, 1:1000). After washing 3 times, they were incubated with horseradish peroxidase-labeled secondary antibody for 1.5 h at room temperature, and protein bands were detected by enhanced chemiluminescence system and analyzed by ImageJ software.

Conjunctival periodic acid-schiff (PAS) staining

4 μm sections were routinely dewaxed to water, then placed in 0.5% periodic acid oxidation solution at room temperature for 5–8 min, washed with tap water and twice with distilled water; the sections were soaked in Schiff reagent for 10–20 min at room temperature in a dark place, and rinsed with running water for 10 min; The sections were soaked in hematoxylin staining solution for 1 min, washed with tap water, differentiated with aqueous hydrochloric acid, washed with tap water, returned to blue with ammonia, and rinsed with running water. The sections were then placed in anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, anhydrous ethanol III for 5 min, xylene for 5 min, and xylene II for 5 min. Consequently, the microscopic examination, image acquisition and analysis were conducted.

Enzyme-linked immunosorbent assay (ELISA)

The Hippocampus/corneal homogenate was collected to be tested. The brain tissue was separated and homogenized, and the supernatant was harvested. The concentrations were determined by BCA commercial kit (Beyotime, Nanjing, China). The levels of TNF-α, IL-1β, IL-6, and IL-18 were detected by ELISA kit (Shanghai Zhuochai Biotechnology Co., Ltd, China) following the manufacturer’s instructions. The absorbance at recommended wavelength of each well was determined using Infnite F50 microplate reader (Tecan, Switzerland). The concentrations of cytokines were calculated from the standard curve.

Immunofluorescence analysis

The paraffin sections of 4 μm brain tissue were routinely dewaxed to water, microwave heated with sodium citrate antigen repair solution for 10 min for antigen repair, washed with PBS, and sealed with 10% sheep serum (Solebo, SL038) at room temperature for 1 h. The blocking buffer was vacuumed, and diluted IBA1 primary antibody (1: 400, Wako, 019-19741) and rabbit anti-NLRP3 (PA579740, 1:1,000, Thermo Fisher, USA). Then the sample was incubated overnight at 4°C, and rinsed with 1× PBS for three times prior to the incubation with the second antibodies including Goat anti-rabbit IgG-H&L (Alexa Fluor^® 594)(1:1000, Abcam, ab150080) and Goat Anti-Rat IgG H&L (Alexa Fluor^® 488) (1:1000, Abcam, ab150165) at room temperature in dark for 1 h. Each sample was incubated with DAPI (Solebo, C0065) at dark for 10 min. After staining, the staining solution was removed and rinsed with 1× PBS for three times. The slides were sealed quenchable sealing tablets and observed under Fluorescence microscope.

Molecular docking

The Sirt1 (PDB code:5BTR) and Luteolin (chemical book: #491-70-3)/Resveratrol (chemical book: #501-36-0) was prepared by Discovery Studio. The combining site was defined according to endogenous ligand. The protein ligand interaction was calculated using CDOCKER method. The interaction and CDOCKER ENERGY were observed.

Statistical analysis

The experimental data were expressed as Mean ± SEM. Statistical analysis was performed using one-way analysis of variance (ANOVA) and Dunnett’s multiple comparisons test with GraphPad prism 7 software, ^*p < 0.05 represents statistical difference.