Cordyceps militaris Carotenoids Protect Human Retinal Endothelial Cells against the Oxidative Injury and Apoptosis Resulting from H2O2

Vision loss is primarily caused by age-related macular degeneration (AMD) due to oxidative retinal pigment epithelial (RPE) cell injury. Carotenoid utilization is deemed a possible strategy for treating AMD. Cordyceps militaris has advantages like immunomodulatory, anti-inflammatory, and antioxidative characteristics. This paper assessed the possible protective influence of carotenoids obtained by isolating and purifying the Cordyceps militaris (CMCT) into human RPE cells (ARPE-19) damaged by hydrogen peroxide (H2O2). The findings demonstrated that CMCT safeguarded the ARPE-19 cells against the damage and apoptosis caused by H2O2 and oxidative stress via Bcl-2 protein upregulation, as well as the expression of Bax and cleaved caspase-3 protein. In addition, CMCT treatment increased cell survival and restricted the generation of H2O2-induced reactive oxygen species (ROS) and the protein expression of NADPH oxidase-1 (NOX1). Additionally, the CMCT treatment of H2O2-induced ARPE-19 cells ameliorated high malondialdehyde (MDA) levels in oxidative stress-induced cells. The catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH) returned to standard levels, which were governed by the higher expression of nuclear Nrf2 protein in the ARPE-19 cells. Moreover, this study showed that CMCT safeguarded the ARPE-19 cells against the damage caused by oxidative stress via its antioxidant activity and antiapoptotic functionality, suggesting the potential therapeutic role of CMCT in AMD prevention and mitigation.


Introduction
Age-related macular degeneration (AMD) is affected by daily living habits and the environment [1,2]. It is estimated that up until 2020, AMD has affected 196 million people globally, a number anticipated to rise to 288 million by 2040 [3]. Furthermore, the pathogenesis of AMD is complicated, with no specific therapeutic strategy available. Increasing research demonstrates that injured retinal pigment epithelial (RPE) cells are vulnerable to oxidative stress. Additionally, reactive oxygen species (ROS)-induced RPE, in particular, is considered an initial pathological trigger of AMD occurrence [4][5][6]. RPE represents a pigment cell layer between the neural retina and the choriocapillaris and is responsible for external retinal protection [7]. ese cells display the most active cellular metabolism in ocular tissues and are essential for regenerating and repairing photoreceptor cells, and restricting the retinal entry of toxic plasma and molecules [7].
In addition, oxidative stress produces ROS in cells, causing cell damage and apoptosis [8]. Various clinical studies suggest that damage to RPE cells from prolonged exposure to oxidative stress eventually leads to AMD [9][10][11].
e RPE cell density decline over time increases the lipofuscin granules, impeding the protective effect against oxidative stress. Consequently, increasing interventions against oxidative stress in RPE cells may provide a therapeutic approach to slow down early AMD progression.
Extensive research data have demonstrated that natural antioxidants derived from plants prevent and ameliorate AMD by protecting RPE cells from oxidative stress injury [12,13]. Modulating retinal oxidation via daily dietary strategies is vital in slowing or attenuating AMD development. Recent research has indicated that the natural antioxidants found in legumes, plants, vegetables, fruit, and medicinal herbs, including flavonols, penicillin, carotenoids, vitamin E, vitamin C, resveratrol, polyphenols, and curcumin, are effective in reducing functional retinal damage and essential for retinal tissue microcirculation and protecting against oxidative stress changes [14][15][16]. is suggests that moderately consuming antioxidants via diet daily can help alleviate or delay the occurrence of AMD [17]. Some studies have indicated that specific carotenoids constitute macular pigments and protect the macula from oxidative damage. Chrysanthemum contains various natural bioactive components and pharmacological characteristics like antitumor, antioxidative, immunomodulatory, antiaging, and antibacterial activity [18], and is widely used in many Asian countries for medicinal and health purposes. Furthermore, Cordyceps militaris primarily consists of carotenoids, ergosterol, adenosine, cordycepin, proteins, ascorbic acid, phenols, and polysaccharides, providing it with unique medicinal properties [19]. In particular, carotenoids exhibit natural immunomodulatory, antioxidative, antitumor, antiaging, and antibacterial properties, as evidenced by several related studies [20]. However, the antioxidative effect of Cordyceps militaris carotenoids on RPE cells damaged by hydrogen peroxide remains known. erefore, this study investigates the protective effect of Cordyceps militaris carotenoids on undifferentiated human retinal epithelial cells (ARPE-19) cells to explore their activity against oxidative damage while discussing the possible mechanisms behind this effect.

Isolating and Purifying the Cordyceps militaris
Carotenoids. Fresh Cordyceps militaris was subjected to airdrying at 60°C until a steady weight was reached, it was then crushed and sieved. Next, a 2 g sample was mixed with acetone-60% ethanol (2 : 1, v/v) in 20 ml, followed by the addition of 0.5% compound enzymes at pH 4 for 45 min at 50°C. e sample was sonicated for 1.5 h, after which the supernatant was diluted 100 times via centrifugation at 4500 rpm for 10 min to obtain the subsequent Cordyceps militaris crude carotenoid extract (CMC). e CMC samples were purified further via adsorption using an HP-20 macroporous resin column (particle sizes: 0.3-1.2 mm) ≥ 90%, using a 60% ethanol eluent. e eluate was concentrated to a paste at lower pressure (60 rpm, 50°C/ min) using a rotary evaporator. e concentrated, dried CMCT was collected and stored at an ultra-low temperature for later use.

Analysis of the Carotenoids and Pigments.
e freezedried CMCT was dissolved in methanol in an ultrasonic bath, after which a 0.22 μm membrane was used to filter the sample solution while protected from light for carotenoid identification and subjected to ultra-performance liquid chromatography (UPLC). e analysis occurred according to a modified technique delineated before [21]. is process used a Waters C18 reversed-phase column (dimensions: 2.1 × 50 mm and 1.7 μm) at a steady 30°C temperature, 2 μl injection volume, and a flow rate of 0.25 ml/min. e elution process included a pure methanol solution for A, while B comprised a 0.1% formic acid solution. Furthermore, the program consisted of 0 min, 100% A; 4 min, 70% A; 16 min, 45% A; 25∼28 min, 10% A; 32∼40 min, 45% A, at a detection wavelength of 445 nm. High-resolution mass spectrometry conditions: electrospray ion source, positive ion mode, perimeter scan range m/z: 50-1000, drying temperature: 450°C, capillary voltage: 5500 V, drying gas flow rate: 40 L/min, and DAD detection wavelength range: 380 nm∼600 nm. Fourier transform infrared (FTIR) spectrometry: the CMCT-  Evidence-Based Complementary and Alternative Medicine potassium bromide mixture (2 : 1, v/v) was ground into a powder and pressed into tablets, after which the infrared absorption was detected in a wavelength range of 500-4000 cm −1 using the transparent potassium bromide tablets.

Oxidative Stress Induction Using H 2 O 2 .
e ARPE-19 cells were placed on 96-well plates at 1 × 10 5 cells/well. ey were left overnight and allowed to replicate and fuse. DMEM/ F12 containing H 2 O 2 (0-500 μM) was used to culture the cells for 12 h, while cells without H 2 O 2 served as a control.

MTT Assays.
e cell viability was ascertained via the MTT technique. To determine the performance of CMCT against the toxicity caused by H 2 O 2 , 96-well plates were inoculated with the ARPE-19 cells at 1 × 10 5 cell/well, treated with different CMCT (1-10 μg/ml) and H 2 O 2 concentrations for 12 h. Next, 20 μL of the MTT (5 mg/ml) was added to each well for a 4 h incubation period at 37°C. e aspiration of the culture solution from each well was followed by the addition of DMSO at 150 μL of DMSO and electric shaking for 10 min. e reaction was terminated via β-crystal dissolution. Using a microplate reader, the absorbance of the samples was immediately measured at 490 nm, while three replicates were obtained for each experiment. e results were illustrated as the absorbance value percentage vs. the control, i.e., the absorbance value of the plate wells, divided by the control percentage.

e Analysis of Cell Apoptosis via Annexin and Flow
Cytometry.
e apoptosis level was analyzed with an annexin V-FITC/PI apoptosis kit as per the instructions of the manufacturer. e ARPE-19 cells were inoculated into the culture plate at 1 × 10 5 cells/ml and subjected to treatment with and without CMCT, as well as H 2 O 2 for 12 h. e collected cells were rinsed two times using cold PBS, after which they were resuspended in a new medium and stained at room temperature for 15 min away from light with annexin V-FITC/PI. en, they were assessed via flow cytometry (BD Bioscience, USA), while a Cell Quest analysis tool (BD Biosciences, Franklin Lakes, NJ, USA) was used to calculate the degree of apoptosis. e results were as follows: normal cells: annexin-V-negative-PI-negative; early apoptotic cells: annexin-V-positive-PI-negative; late apoptotic or necrotic cells: annexin-V-positive-PI-positive. All experiments were repeated three times.

Measurement of the ROS in the Cells.
A ROS assay kit was used following the protocols of the manufacturer to detect the ROS levels in the ARPE-19 cells.
e DCF-DA fluorescent probe reacted with the ROS produced by cells to form DCF. e collected cells were subjected to a 30 min incubation period using 10 μM of DCFH-DA reagent in darkness at 37°C, after which ice-cold PBS (1 × 10 5 cells/ml) was used to rinse them twice, followed by resuspension in phosphate buffer. e fluorescence intensity was determined via flow cytometry at respective emission and excitation wavelengths of 525 nm and 488 nm.

Determination of the MDA, GSH, CAT, and SOD Levels.
e CMCT impact on the oxidative stress in the ARPE-19 cells was determined after the different treatments, using the SOD, MDA, CAT, and GSH oxidative biomarkers. e ARPE-19 cells were subjected to a 24 h incubation period in 6-well plates (1 × 10 6 cells/well), followed by applying different CMCT concentrations for 12 h and H 2 O 2 (400 μg/ml) for 12 h. Next, the collected cells were rinsed two times using ice-cold PBS. e oxidative biomarkers were then measured using the appropriate analytical kits using the protocols of the manufacturers.

Western Blot Analysis.
is analysis occurred according to a previously delineated method. Ice-cold PBS was used to wash the ARPE-19 cells twice. e cell lysates were extracted from the collected cells with RIPA lysis buffer, centrifuged at 4°C and 12,000 rpm for 15 min to obtain the supernatant, and measured via a BCA Protein Assay Kit to determine the protein level. Equal quantities of 30 μg of protein were relocated to polyvinyl fluoride (PVDF) membranes along with 12% sodium dodecyl sulfatepolyacrylamide for electrophoretic separation. e samples were exposed for 1 h to 5% skim milk and subjected to overnight incubation at 4°C with the β-actin (1 : 1000) Bc1-2 (1 : 200), Bax (1 : 500), caspase-3 (1 : 500), anti-Nrf2 (1 : 300), and NOX1 (1 : 500) primary antibodies. is was followed by room temperature incubation for 2 h using the secondary, horseradish peroxidase-conjugated antimouse antibody (1 : 2000). After repeatedly rinsing with PBST, the individual protein expression was observed via an ECL western blotting assay test kit as per the prescribed procedure.

Statistical Evaluation.
All experiments were performed at least three times, and the data after experiments were expressed as mean ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism software. Data were analyzed by one-way ANOVA. P < 0.05 was considered significant.

CMCT Preparation and Characterization.
e CMCTs were produced using a technique previously described with some modifications [22]. e CMCTs were obtained via crushing, acetone, and ethanol mixed solution extraction Evidence-Based Complementary and Alternative Medicine and complex enzyme precipitation at an extraction rate of 3888.34 μg/g. Chrysin carotenoids are intracellular pigments. e cell walls were disrupted and extracted with cellulase and pectinase (extraction ratio of 2 : 1), after which the CMC extract was dissolved using acetone and a 60% ethanol solution. ELISA was employed for absorbance determination at 445 nm (Figure 2(a)). e standard curve was configured as Y � 0.1093x + 0.0801 and R2 � 0.9992 (Y denoted the absorbance, while X signified the pigment level). e macroporous adsorbent resin was used to further separate the CMC, increasing the carotenoid purity for subsequent cellular experiments to 66.24% (Figure 2(b)). en, the CMCTs were exposed to concentrated sulfuric acid to obtain a blue-green color. Furthermore, exposure to an antimony trichloride chloroform solution yielded a green color, while the color values were consistent with those of olefin carotenoids. Spectroscopic and mass spectrometric analyses confirmed that the pigment purification exhibited general carotenoid characteristics. Five main pigment components were isolated, as shown in (Figure 2 e absorption peaks of the five carotenoids displayed identical absorption spectra and conjugation systems, which were consistent with the essential carotenoid characteristics. erefore, they are compatible with the basic attributes of carotenoids.

e Influence of CMCT Treatment on the ARPE-19 Cells.
e CMCT influence on cell survival was investigated via an MTTassay and presented as the survival rate of the untreated control samples. A slight increase in cell viability increase was evident in the cells subjected to different CMCT treatments at concentrations in a range from 1 μg/ml to 2.5 μg/ml (Figure 3(a)). However, the cell viability declined significantly after exposure to 5 μg/ml CMCT, while a 10 μg/ ml CMCT concentration decreased the cell survival rate to 79% in comparison with the untreated samples (P > 0.05). Consequently, 1 μg/ml and 2.5 μg/ml CMCT concentrations were utilized for the subsequent experiments. To determine the correct H 2 O 2 concentration for assessing the impact of CMCT on the cytotoxicity of H 2 O 2 , the ARPE-19 cell viability was examined after 12 h of exposure to H 2 O 2 (0-500 μM). e cell viability decreased dose-dependently from 100 ± 3.6% in the untreated group to 91.1 ± 1.94% at 50 μM, 78.76 ± 3.3% at 100 μM, 69.1 ± 2.9% at 200 μM, 63.5 ± 2.48% at 250 μM, 41.6 ± 1.85% at 400 μM, and 35.2 ± 1.3% at 500 μM, respectively (Figure 3(b)). erefore, an H 2 O 2 concentration of 400 μM was used during the experiments.
is study investigated if the target CMCT safeguarded ARPE-19 cells from destruction by H 2 O 2 . e cells were precultured for 12 h and 24 h in a medium containing CMCT (1 μg/ml, 2.5 μg/ml), which was changed to a new medium containing H 2 O 2 (400 μM), followed by culturing for 12 h. e results showed significant protection against H 2 O 2 -induced AEPE-19 cell death after 12 h CMCT treatment (1 μg/ml, 2.5 μg/ml) (P < 0.05). However, although 24 h CMCT treatment (2.5 μg/ml, 1 μg/ml) displayed only a slight protective effect, no substantial differences were apparent from the control group (P > 0.05). Some toxicity was evident in the cells after prolonged pretreatment. erefore, a 12 h incubation period was selected to assess CMCT protection against H 2 O 2 damage in the ARPE-19 cells. As illustrated in (Figure 3(c)), pretreatment with 2.5 μg/ml and 1 μg/ml CMCT significantly increased cell viability to 75.6 ± 4.7% and 69.4 ± 6.5%, respectively, displaying substantial variation from the control samples (P < 0.05).

CMCT Protection against the RPE Cell Death Caused by
RPE cell damage is usually due to oxidative stress. e ability of CMCT to protect against the ARPE-19 cell death caused by H 2 O 2 was examined by incubating the cells at 1 μg/ml and 2.5 μg/ml CMCT concentrations for 12 h, followed by H 2 O 2 for 12 h, after which they were observed via phase-contrast microscopy, as shown in (Figure 4(a)). e population dispersion of the stained annexin V-positive cells is demonstrated in (Figure 4(b)). H 2 O 2 (400 μM) treatment for 12 h significantly increased the apoptotic cell proportion to 63.87 ± 1.7% compared with the control group (5.16 ± 0.34%). However, CMCT (1 μg/ml and 2.5 μg/ml) pretreatment and H 2 O 2 exposure for 12 h significantly decreased the apoptosis induced by H 2 O 2 (27.21 ± 1.78% and 38.84 ± 1.58%, P < 0.05), when compared to the control cells (3.10 ± 0.67%).

e Impact of CMCT Treatment on the Production of Intracellular ROS and Expression of NOX1 Proteins in the ARPE-19 Cells.
Research has shown that the production of ROS exceeds the in vivo clearance limit due to oxidative stress. e subsequent imbalance in the oxidative and antioxidant systems leads to functional and morphological damage in the ganglion, endothelial, and RPE cells [23]. erefore, this study investigated whether CMCT affected the ROS levels in the ARPE-19 cells after stress. e cells were exposed to different CMCT concentrations (1 μg/ml, 2.5 μg/ml), as well as H 2 O 2 (400 μM) for 12 h, respectively, after which the ROS levels were measured directly via DCFH-DA staining. H 2 O 2 significantly elevated the production of intracellular ROS compared to the untreated samples ( Figure 5(a)). However, CMCT (2.5 μg/ml, 1 μg/ml) treatment for 12 h significantly reduced the upregulated ROS activity induced by H 2 O 2 (400 μM). According to ( Figure 5(b)), pretreatment with different CMCT concentrations (2.5 μg/ml, 1 μg/ml) attenuated the NOX1 protein expression induced by H 2 O 2 oxidative stress. Furthermore, some of the differences from the H 2 O 2 control were statistically significant (P < 0.01 or P < 0.05).

e Influence of CMCT on Regulating the Antioxidant Content of the ARPE-19 Cells Exposed to H 2 O 2 .
Since oxidative stress is crucial in the senescence state of normal ARPE-19 cells, this study examined the activity of MDA, an indicator of lipid oxidation, and GSH, CAT, and SOD, biomarkers of oxidative in the ARPE-19 cells subjected to various treatments. Compared with the untreated cells, H 2 O 2 pretreatment alone for 12 h yielded higher MDA levels but decreased the GSH, CAT, and SOD activity (Figures 6(a)-6(d)). CMCT treatment alone did not affect the MDA level and three oxidative stress biomarkers. However, after incubation with CMCT (2.5 μg/ml, 1 μg/ml) for 12 h, the MDA level decreased according to the concentration, while the SOD, CAT, and GSH activity wer restored to normal levels. e cellular response to oxidative stress is regulated by Nrf2 to maintain redox homeostasis. It

e Influence of CMCT on the Protein Expression Associated with ARPE-19 Cell Apoptosis after H 2 O 2 Exposure.
Apoptosis and cell damage are often the result of oxidative stress. erefore, this study explored whether CMCT effectively protected ARPE-19 cells against apoptosis and assessed the impact of CMCT on the expression of proteins associated with apoptosis. e results of the protein blot analysis are presented in (Figure 7). e ARPE-19 cells subjected to H 2 O 2 (400 μM) treatment for 12 h showed significantly higher downregulation in the protein expression of Bcl-2, as well as higher cleaved caspase-3 and Bax expression levels than the control group, which was consistent with the flow cytometry results. Moreover, CMCT (1 μg/ml, 2.5 μg/ml) pretreatment for 12 h dose-dependently reversed this, increasing the protein expression of Bcl-2 and significantly decreasing these levels in cleaved caspase-3 and Bax.

Discussion
Oxidative stress causes apoptosis in retinal endothelial cells. Excess ROS usually generates oxidative stress, leading to intracellular mitochondrial dysfunction and antioxidant system damage, ultimately causing degenerative diseases of the human retinal epithelium, such as AMD [24,25]. Oxidative stress is primarily caused by the discrepancy between biological scavenging ability and active free radical generation. Fat accumulation, nucleic acid molecule cleavage mutations, protein inactivation and degradation, and photosensitive cell regeneration and repair decline in conjunction with environmental changes and increased age [26]. Consequently, reducing the cellular oxidative stress is critical for slowing AMD progression and facilitating potential treatment options for vision loss.
Clinical trials and research data have shown that the consumption of lutein, vitamins E and C, flavonoids,   Evidence-Based Complementary and Alternative Medicine anthocyanins, zeaxanthin, and carotenoids can enhance cells and strengthen the antioxidant system to maintain good retinal function [27][28][29][30][31]. e minimal level of toxicity and powerful antioxidant capacity of the carotenoids found in natural plants have attracted considerable attention from researchers, rendering them effective as potential therapeutic agents. Singlet oxygen is quenched by carotenoids, scavenging free radicals to restrict lipid peroxidation reactions in vivo and blue light filtering, modulating light stress recovery time and neural processing speed [32]. Functions, Cordyceps militaris, as reported to contain many essential components required by the body, especially CMCT, has high antioxidant efficiency. As far as is known, this study is the first involving the antioxidant impact of CMCTon RPE cells. However, the mechanism underlying its anti-H 2 O 2 -induced oxidative stress effect remains unclear. erefore, this research investigated the possible effect of CMCT in protecting against the death of retinal endothelial cells caused by H 2 O 2 .
Several studies have shown that H 2 O 2 exposure induces ROS production while accelerating cell damage and apoptosis [33]. Since RPE cells are located behind photoreceptor cells and are mainly responsible for scavenging the oxidants generated by photoreceptor conversion, ARPE-19 cellular oxidative stress is used as a typical in vitro model to examine the functionality of human RPE cells.
is study also revealed the pathogenesis of AMD by inducing oxidative stress and cellular damage [34]. e survival rate of the ARPE-19 cell exposed to 400 μM H 2 O 2 decreased, while CMCT pretreatment (2.5 μg/ml, 1 μg/ml) substantially  Reports have indicated that protein restricts Bcl-2 and Bax. e increased protein expression of Bax and Bcl-2 alters the permeability of cellular mitochondrial membranes, subsequently inhibiting mitochondrial disruption and prompting the release of cytosolic c and cystathionine activation [35]. Moreover, caspase-3 selectively and efficiently cleaves proteins in various signal transduction pathways and is essential for controlling programmed cell death. Bcl-2 overexpression reportedly increases the survival of cells exposed to H 2 O 2 [36]. ROS generation is associated with the co-interaction between nanoparticles and redox proteins such as mitochondria and NOX1, and cell surface receptors [37]. NOX1 is reportedly a vital enzymatic regulator of redox reactions in vivo and an identified system for ROS production. As one of the primary sources of ROS, NOX1 is crucial for the formation of RPE cell lesions in humans [38]. In addition, the GSH, CAT, and SOD activity, MDA levels, and NOX1 protein expression were examined. is study showed that exposing ARPE-19 cells to H 2 O 2 significantly decreased the SOD, CAT, and GSH levels, while substantially increasing ROS and MDA production and NOX1 protein expression. However, CMCT treatment reversed these effects. Previous research indicated that the retinal cell apoptosis caused by oxidative stress was responsible for AMD pathogenesis, while the nuclear transcription factor, Nrf2, is crucial for oxidative stress regulation [39]. Nrf2 can upregulate antioxidant enzyme expression, scavenge free radicals, reduce intracellular oxidative stress levels, and decrease cell degeneration. It can also protect cells from oxidative damage by inducing ROS detoxification enzymes [40]. Moreover, Nrf2 is considered one of the most critical intracellular antioxidative stress mechanisms and plays a vital role in AMD treatment [41]. erefore, the impact of CMCT on the translocation of Nrf2 in the ARPE-19 cells was examined via Western blotting, indicating that the Nrf2 expression was upregulated after CMCT treatment. Furthermore, it highlighted the potential of CMCT to safeguard RPE cells against the oxidative stress resulting from H 2 O 2 by controlling NOX1 protein expression to reduce the ROS and MDA levels. Contrarily, the nuclear activation of Nrf2 protein increased the antioxidant enzyme activity of SOD, CAT, and GSH. Studies have shown that oxidative stress can reduce cell apoptosis, increase the total amount of apoptosis and ROS production, and prevent cells from oxidative stressinduced damage [42].
erefore, this study shows that CMCT treatment successfully safeguards ARPE-19 cells against the oxidative damage resulting from exposure to H 2 O 2 by inhibiting apoptosis, regulating ROS production and MDA formation, and increasing the activity of antioxidant enzymes. It also provides new insight into the ability of CMCT to help forestall and mitigate ocular diseases like AMD. Future research should explore the in vivo preventative and therapeutic effect of CMCT in AMD disease models to determine its complete mechanisms.
Data Availability e data supporting the findings of this study are available on reasonable request from the corresponding author.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Authors' Contributions
Lin Lan and Yufeng Li conceptualized and designed the study. Lin Lan, Shengyu Wang, Shuhua Duan, and Xiangyu Zhou collected and analyzed the data and experimental methods. Yufeng Li participated in drafting the manuscript and reviewing the final manuscript. All authors have read and approved the manuscript.