Cordyceps Militaris Carotenoids Protect ARPE-19 Cells From Oxidative Stress Damage Induced By Hydrogen Peroxide

A retinal disorder known as age-related macular degeneration (AMD) can be very damaging, which may result in vision loss. Cordyceps militaris has antioxidant, anti-inammatory, anticancer activity and immunomodulatory functions. carotenoids were considered potential therapeutic agents for the treatment of age-related macular degeneration. In this study, we evaluated for the rst time the protective effect of puried carotenoids (CMCT) extracted from Cordyceps militaris on hydrogen peroxide (H 2 O 2 )-induced damage to human retinal pigment epithelial cells (ARPE-19). The pretreatment of ARPE-19 cells with CMCT (1, 2.5μg/ml) for 12h after exposure to H 2 O 2 (400μM) greatly improved cell viability and injury tolerance. and reduced reactive oxygen species production (ROS) and decreased malondialdehyde (MDA) formation. Furthermore, Bax and Caspase-3 showed increased protein expression and less protein expression of Bcl-2 in cellular oxidative stress by ow cytometry analysis but showed the opposite expression by adding CMCT. Besides, cells were treated with H 2 O 2 and then CMCT restored catalase (CAT), superoxide dismutase (SOD) and glutathione (GSH) enzyme activities to normal levels. Our results suggest that CMCT can protect RPE cells from oxidative stress damage by regulating oxidative activity and anti-apoptotic function. It indicates that CMCT has a potential therapeutic role in oxidative stress-directed protection and prevention of AMD.


Introduction
Age-related macular degeneration (MDA) has become a common blinding disease with increasing age [1] , and was driven by other lifestyle and environmental factors [2,3]. By 2020, it is estimated that 196 million people worldwide will be affected by age-related macular degeneration, with the number increasing to 288 million by 2040 [4]. As a public illness its pathogenesis is complex and no targeted countermeasures have been developed, while it can be classi ed into early, intermediate and late stages according to macular characteristics [5][6][7]. In AMD's pathophysiology, oxidative stress in the retinal pigment epithelium was considered a crucial role, more importantly, according to reports in the retinal pigment epithelium (RPE) where oxidative damage and mechanism malfunctions appear in the early stages of AMD. Protecting the damage to the retinal pigment by oxidative stress thus plays a signi cant role in stopping or slowing the macular degeneration pathological phase [8,9]. This RPE is situated at the interface between the neural and choriocapillaris retinas. It was composed of a single layer of pigment epithelial cells, which are arranged very regularly and form part of the retina's external protection [10]. Drusen will store lipid materials amassing under the retinal pigment epithelium (RPE). What's more, pale-yellow stains on the retina may be shown. It plays an important role as the most active cell in the cellular metabolism of ocular tissues, promotes the regeneration and repair of photoreceptor cells, and prevents toxic molecules and blood from entering the retina to promote cellular metabolism [11][12][13]. Because of the particular structure and function of RPE under prolonged exposure to photo-oxidative effects, RPE has a higher sensitivity to stress and is more susceptible to damage to generate oxidative substances ROS, which leads to retinal damage over, peroxidation including continuous degradation of lipid-rich photoreceptor outer mesenchyme discs, the intracellular formation of lipofuscin and deposition of toxic waste such as extracellular vitreous membrane coelomic [14,15]. An increasing number of reports have demonstrated that oxidative stress causes in vivo ROS production in cells showing retinal cell damage and apoptosis ultimately leading to AMD [16,17].With increasing age, RPE cell density decreases and the number of lipofuscin granules increases, weakening the protective mechanisms that cause oxidative cell damage. Therefore, it may be possible to slow down AMD progression by reducing the protection of RPE cells from stress damage.
A large amount of research data proves that the bene cial effects of natural antioxidants extracted from plants are related to bioactive photochemical, by protecting the retinal epithelium from oxidative stress and thus for treating AMD [18]. Such substances extracted can attenuate oxidative stress by protecting the RPE from damage and regulate the imbalance between free radicals thus protecting cells. For example, avonols, cyanidins, curcumin, polyphenols, resveratrol, vitamin C, vitamin E and cryptochrome are widely distributed in (plants, fruits, vegetables, legumes and medical herbs) and have the ability to mitigate age-related changes in functional retinal damage, microcirculation of the retinal tissue and defense from oxidative stress [19][20][21]. Daily natural vegetative antioxidant supplementation in moderation has become a topic of research interest for alleviating or slowing down AMD development.
Carotenoids, lutein and zeaxanthin are potential mechanisms for the antioxidant and free radical scavenging properties of these natural extracts in preventing AMD pathology [22]. At the same time, Cordyceps militaris has multiple pharmacologically active functional components, many studies have reported anti-in ammatory, antioxidant anti-aging, anti-tumor, hypotensive and vasodilator activities, free radical scavenging, antibacterial, anti-brotic, improved insulin secretion and anti-diabetic properties of Cordyceps militaris [23]. In particular, Cordyceps militaris was currently identi ed as the strain with the highest carotenoid content among the large-use herbal fungi reported. In addition to acting as antioxidants, carotenoids were known to function as prooxidants in high oxygen stress, high concentrations of carotenoids and unbalanced intracellular redox conditions [24]. However, the protective

Isolation and puri cation of Carotenoids from Cordyceps militaris
The fresh fruit bodies of Cordyceps Sinensis was air-dried at 60°C at a constant weight, and then the dried fruit bodies were ground into powder by passing through a 60mesh sieve. Weigh (2g) with acetoneethanol (2:1,20ml), then add complex enzyme (0.5%) to adjust the pH of the extraction system to 4. The enzymatic digestion time was 45 min at 50°C, and then the mixture was sonicated for 1.5h. The supernatant was taken by centrifugation for 10 min at 4500 rpm and 100 times diluted. The resulting solution was the crude extract of Cordyceps carotenoids (CMC).
The crude extract CMC pigment supernatant was loaded onto an HP-20 macroporous resin adsorption column for further puri cation by complete adsorption (particle size 0.3-1.2 mm) ≥ 90%, and the eluate was desorbed with 60% ethanol as the carotenoid puri cation (CMCT) pigment puri cation solution. The eluent was placed on a rotary evaporator and concentrated under reduced pressure to a paste (60 rpm for min at 50℃), collected the vacuum freeze-dried CMCT purify, which were used for further studies.
Bintong Total Antioxidant Capability Kit (T-AOC) to detected the total antioxidant activitied of pigments.

Antioxidant analysis and UV spectrum analysis
The DPPH was removed by colorimetric method using pyrogallol auto-oxidation to remove O 2 in combination with sodium salicylate complexation to remove OH. The total antioxidant activity of the extracted pigments was determined by colorimetric method (T-AOC) test kit and using a microplate reader, the absorbance was determined. (Bio-Tek, Winooski, VT, USA) In the wavelength range of 380-600 nm, in the wavelength range for UV-Vis spectral scanning.

Determination of pigment composition
The composition of carotenoids of Cordyceps Sinensis was determined by ultra-performance liquid chromatography (UPLC). The carotenoids of Cordyceps Sinensis were dissolved in pure methanol through a 0.22µm lter protected from light. A Waters C18 reversed-phase column with reversed-phase speci cations (2.1×50mm, 1.7μm) was used, the column temperature was held constant at 30°C, and the ow rate was (0.25Ml/min), the injection volume (2µl), the elution gradient temperature (32-40min), and the detection wavelength was 445nm. The drying temperature for HRMS detection was (450℃). The DAD wavelength was (380nm-600nm) The drying gas ow rate was 40L/min. For HRMS detection. The pigment was mixed with potassium bromide at 1:100 to a powder to a wavelength of 500-4000 cm -1 .

ARPE-19 Cell Culture
Human retinal epithelial cells ARPE-19 cells were purchased from the American (ATCC) model culture collection and Dulbecco's Modi ed Eagle Medium (DMEM)/F12 preserved the ARPE-19 cells. Taken 10% heat-inactivated fetal bovine serum (Los Angeles, U.S.A.) 100 U/ml of penicillin (Los Angeles, U.S.A.) and 100 µg/ml of 10% streptomycin (SV3010 Los Angeles, U.S.A.) Cells were kept in an incubator and monitored in a humidi ed environment at 37 ℃ and 5% CO 2 and RPE cells were selected during the rst 5-7 passages when they reached 90% con uence passed with 0.25% Trypsin (Biotechnology Hyclone Inc Los Angeles, U.S.A), every 3-4 days, and placed into appropriate culture plates for each experiment.
Induction of oxidative stress using H 2 O 2 .
Oxidative stress tests in serum-free or serum-containing media were conducted. After fresh serum-free base cell processing, ARPE-19 cells were plated on 96-well plates at a concentration of 1-10 5 cells/mL and enabled cell replication to cling to the bottom of the board and fusion overnight. Add DMEM/F12 medium with a nal concentration of H 2 O 2 (0-500μM) and incubate for 12h. Untreated cells served as controls to obtain nal concentrations depending on the experiments, followed by a 12h exposure of different concentrations of CMCT.

MTT assays
The MTT test was used as the cell viability test measure. Brie y, the viability of ARPE-19 was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (St. Louis, MO, USA). The spread of cells ARPE-19 cells was plated at 1×10 5 cell/mL at 96-well plates. ARPE-19 cells cultivated in DMEM/F12 were pretreated for 12 hours at various CMCT concentrations. The medium was discarded, washed twice with PBS, and continuously exposed to H 2 O 2 for 1h. After various treatments, MTT solutions were prepared in advance, stored in keeping in darkness, and then added to 96-well plates with 20μm of 5mg/mL MTT solution per well to re ect the mitochondrial activity of the cells through the transformation and dissolution of eszopiclone MTT methyl crystals to form particles, and combed at 37°C for 4h. After incubation the medium solution in each well was discarded 150μl of dimethyl sulfoxide (DMSO) was added, and the reaction was stopped and placed on an electric shaker for 10 min. The wells' crystals were then fully dissolved. The absorption of each well with a microplate reader at 492 nm was calculated (Model Devices SpectraMaxi3X; American Valley Molecular Instruments, Inc.), and all experiments were performed in triplicate. The average of the results was read for calculation. The absorbance values represent cell viability normalized to the untreated control and represent 100% cell viability the cells' relative viability as a percentage of the control.

Detection of cell death by ARPE-19
Apoptosis assays were evaluated by Annexin V-FITX/PI kit according to the manufacturer's instructions.
ARPE-19 cells were incubated on 1 × 10 5 -well plates with or without CMCT treatment for 12h after exposure to H 2 O 2 for 12h. Afterward, cells were collected by washing twice in ice-cold PBS liquid by centrifugation at 4°C for 5 min and resuspended in fresh medium and 5μl Annexin V-FITX was added for staining in the dark for 15 min. Immediately after staining, Flow cytometry was used to analyze the cells, and the number of apoptotic cells was measured using Cell Quest analysis tools. Results were expressed as Annexin-V negative-PI negative for normal cells, Annexin-V positive-PI negative for cells in early apoptosis, and Annexin-V positive-PI positive for cells in late apoptosis or necrotic cells and all assay data were performed in triplicate.

Intracellular ROS levels are measured
A uorescent probe DCFH-DA staining determined the number of reactive oxygen species in cells. Brie y, ARPE-19 cells in 94-well plates were re-stained with H 2 O 2 (400μM) for 12h, extended and excess liquid discarded then ARPE-19 cells were pretreated with CMCT for 12h. Cells were incubated with 10μM DCFH-DA reagent from the kit for 30min at 37°C in the dark, and serum-free cell culture medium was washed twice to remove excess DCFH-DA and resuspended in PBS. The cells' absorbance wavelength was recorded immediately with a microplate reader. Flow cytometry was used to record the intracellular uorescence strength during therapy at 488 nm excitation and 525 nm emission wavelengths. GSH activity assay, the same operation was used to prepare 20μl of cell-free supernatant in the sample for SOD activity determination. The detection kit was used to analyze the total protein, and the supernatant was centrifuged for glutathione determination. (4℃ 15000*9 10min). Read the absorbance at a wavelength of 405nm according to the manufacturer's instructions. The quanti cation of intracellular glutathione was based on its use solution as a standard.
The impact of CMCT on the identi cation of oxidation biomarkers in cells was calculated using the MDA activity assay. Brie y, cells were treated with H 2 O 2 (100μM) for 1h and CMCT overnight. After completion of treatment, the cells were then washed with PBS, and the oxidation biomarkers were obtained by centrifugation and identi ed using the test set as directed by the manufacturer. For both antioxidant enzymes and MDA amounts, nmol per mg of protein has been expressed.

Western blot analysis
Cells were pretreated. Brie y, two washes of ice-cold PBS were performed on the cells. After treatment, the cells were collected that the cells were laced with RIPA buffer for nuclear tissue. At 4°C for 15min the lysate was centrifuged at 12,000 × g and the supernatant were extracted. The protein content in the samples was detected in the test kit and according to the directions of the manufacturer. 20μg protein samples were electrophoresed on polyacrylamide (PAGE) 10% sodium dodecyl sulfate (SDS), moved to the membrane of polyvinyl uoride (PVDF), and sealed with 5% skim milk at a temperature of 4°C. Incubate the antibody characterizing overnight, wash with PBS and then incubate the eggs at room temperature for 1h. with the following secondary antibody. The ECL Western blotting assay kit is used to detect protein bands.

Statistical Analysis
All experiments were repeated at least three times, and data are shown as mean ± standard deviation or percentages. origin9.0 was used for plot analysis, and variance analysis between groups was compared using one-way ANOVA or ordinary two-way ANOVA, and variability with *P<0.05, **P<0.01 and #P<0.05, ##<0.01. was considered statistically signi cant.

Preparation and characterization of CMCT
The crude cordyceps militaris carotenoid was prepared according to the reported method with certain variations. The Cordyceps militaris carotenoids were intracellular pigments, destroy the cell wall and then extract CMCT by Cellulase and Pectinase. (the extraction ratio is 2:1, absorbance 3.322), Determine the acetone and 60% ethanol solution to dissolve the crude pigment extract and measure the absorbance at 445nm (Figure 1.A). Con gure standard curve as follows: y=0.1093x+0.0801, R2 =0.9992(Y was Absorbance and X was Pigment concentration). For subsequent cell experiments using higher purity carotenoids, CMCT was further fractionated by macroporous adsorbent resin as shown in the gure ( Figure 1.B) giving a puri ed carotenoids CMCT. Absorbance OD 455nm , the sample is colorless, determined by the color reaction, concentrated sulfuric acid is blue-green and the chloroform solution of antimony trichloride is green. The color characteristics were in line with the general features of ole n carotenoids. The ve prominent peaks were prepared by the UPLC method (Figure 1.C), the exact prime numbers were measured in the positive ion [M+H] + mode, Dry under vacuum at 60℃, UV spectrophotometers tested carotenoids dissolved in methanol the absorption maximums of the ve carotenoids had the same absorption spectrum and a same conjugate system of bond, According to its UV-MS and Infrared spectrograms, the ltered carotenoids CMCT ful ll the speci c characteristics when contrasting the retention period and peak region of the standard under the same conditions.

Effects of H 2 O 2 and CMCT in ARPE-19 Cells
The impact of CMCT on cell viability of ARPE-19 was rst investigated using the MTT system, and the cell survival rate is expressed as the control group's survival rate group (untreated cells). As shown in (Figure 2.A) (Figure 3 .A). The percentage of apoptotic cells was detected by V-FITC/PI staining on ow cytometry as shown in (Figure 3.B Many experiments have shown that the oxidative stress of the human retinal pigment epithelium (RPE) was due to imbalances between the excessive in vivo formation of free, reactive radicals and the premature reaction of oxidants to the stimulation of the RPE. Therefore, we further investigated the mechanism of cellular oxidative stress caused by H 2 O 2 and whether CMCT treatment of ARPE-19 in uences the activity of reactive species of oxygen as shown in ( Figure 5). The ARPE-19 cells were CMCT promotes H 2 O 2 -induced intracellular antioxidant activity down modulation in ARPE-19 cells.
Oxidative stress was necessary for normal cellular function; we investigated the expression of three antioxidant biomarkers (SOD, CAT, and GSH) activity in ARPE-19 cells treated during speci c treatment periods and assessed the levels of the lipid oxidation indicator (MDA). As showed in (Figure 6.A). We observed a substantial increase in MDA levels in ARPE-19 cells after H 2 O 2 induction and decreased MDA levels after CMCT treatment relative to the untreated cell group (p<0.05). Besides, CMCT treatment of cells alone did not affect SOD, CAT, and GSH. In contrast, the three oxidative parameters were substantially reduced in ARPE-19 cells after H 2 O 2 treatment and standardized after reduced SOD stimulation, CAT, and GSH levels up-regulated by CMCT treatment in culture. (Figure 6. B, C and D). Differences were observed compared to the control group. (p<0.05 or p<0.01)

Discussion
An increasing amount of studies have shown that oxidative stress was usually caused by excessive ROS induction, cell mitochondrial dysfunction, and damage to the antioxidant system bring about the degeneration of human retinal epithelial cells. However, In the pathogenesis of AMD, oxidative stress plays a signi cant function [25,26] . The disparity between the generation of active free radicals and the capacity of biological scavenging was the critical cause of oxidative stress. As the environment changes and age increases, fat accumulation, cleavage mutations of nucleic acid molecules, protein degeneration and inactivation, the repair and regeneration of photosensitive cells decrease [27]. Therefore, slowing the oxidative stress state of cells was essential to promote the development of potential vision loss treatment options.
It can be seen that, according to clinical and experimental research data, the intake of vitamin C, vitamin E, lutein, zeaxanthin, anthocyanins, it's possible that a regular dose of and other antioxidants is needed to keep the retina in good shape. And a potential strategy for the function of photoreceptor cells [22,[28][29][30][31].
There are indications that carotene may defend retinal epithelial cells against oxidative stress harm [32]. Cordyceps militaris has various natural biologically active ingredients, different pharmacological functions such as immunomodulation, antioxidant, anti-aging, anti-tumor and anti-bacterial [33]. For medicinal purposes and tonic health food, it has been used extensively in China and other Asian countries. Meanwhile, Cordyceps militaris various medicinal properties come from the unique chemical composition, mainly including polysaccharides, phenols, ascorbic acid, proteins, cordycepin, adenosine, ergosterol, carotenoids, etc. Especially, carotenoids have the natural properties of immunomodulation, antioxidant, antioxidant, aging, tumor and anti-bacterial [34,35]. In particular, carotenoids have natural antioxidant properties, and Cordyceps militaris has a high carotenoid content and is also a highly watersoluble carotenoid that the body can easily absorb. It can scavenge free radicals through single electron transfer, protect the retina from light and aging-induced oxidative stress by ltering phototoxic shortwavelength visible light [36] . Through removing single electron transmission, they may block and slow down AMD by ltering free radicals, ltering short-wavelength visible light with phototoxicity and function as antioxidants to defend the retina against light and aging oxidative stress, DPPH, FRAP and inhibitions of lipid peroxidation [37,38]. However, the biochemical and oxidative synthesis of eye carotenoids has not been completely known. Therefore, this research was intended to detect the possible protection impact on the death of ARPE as well as a decrease in apoptosis rate and improvement in oxidative stress levels. These ndings show a bene cial impact on retinal pigment epithelial cells of CMCT against oxidative stress.
Many experiments have shown the inextricable linkage of apoptosis caused by oxidative stress in retinal pigment epithelial cells to the production of certain reactive oxygen species, and antioxidant enzymes, as an antioxidant, can help the body to scavenge free radicals and peroxides to maintain relative stability of the organism [40]. So, Studies also con rmed antioxidant enzyme inhibition of reactive oxygen species and a greater degree of lower apoptosis [41]. In ARPE-19 cells, we studied ROS levels and the activity of