Study on protein hydrolysate from Symplectoteuthis oualaniensis reconstructing fibroblast against oxidative stress by hydrogen peroxide

doi:10.21203/rs.3.rs-2803605/v1

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Study on protein hydrolysate from Symplectoteuthis oualaniensis reconstructing fibroblast against oxidative stress by hydrogen peroxide

https://doi.org/10.21203/rs.3.rs-2803605/v1

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The free radical hypothesis has been accepted in the researching of skin aging. As a small molecule oxidant, hydrogen peroxide (H₂O₂) is easily induce the apoptosis of HSF cells through biofilm system. Oxidative stress caused by H₂O₂ also leads to the synthesis of inflammatory cytokines, mainly including interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), which are involved in the regulation of skin inflammation and irritation. Protein protease hydrolysates were prepared from Iris cuttlefish (Symplectoteuthis oualaniensis) and then investigated for their antioxidant activities in vitro. The positive effect by PHCSO (protease hydrolysates from the carcass of Symplectoteuthis oualaniensis) on the reconstruction of HSF (human skin fibroblasts) cells against oxidative stress was investigated by the oxidative stress model via H₂O₂ inducement. PHCSO-1 or PHCSO-2 was a mixed peptide whose molecular weight is over 10 kDa or less 10 kDa from the ultrafiltration of PHCSO. The reconstructing effect of PHCSO-2 was superior to that of PHCSO on HSF cells according to the results from cell morphology of fluorescence staining, inflammatory factors and antioxidant activity analysis. 5 mg/mL PHCSO-2 showed the DPPH and hydroxyl radical scavenging activity as 57.96% and 56.86% respectively, with TNF-α reduced by 24.83%, which indicated the effective protection on skin from oxidative stress. The protein hydrolysate from Symplectoteuthis oualaniensis was proved to be the marine antioxidant peptide, which could be applied in cosmetics, pharmaceutical or food industries.

protein protease hydrolysate

fibroblast

oxidative stress

antioxidant activity

inflammatory cytokines

Natural products extracted from marine organisms beneficial to human health due to their specific living environment, which have been attracting continuous attentions. Proteins with antioxidant activity produced by marine organisms have been undergoing the screening for the potential antioxidants for years to be further applied in cosmetics, pharmaceutical or food industries.[1] Free radicals are the core factors of skin aging [2, 3]. While inducing HSF cells apoptosis through biofilm system, hydrogen peroxide (H₂O₂) is decomposed to produce OH free radicals that can trigger the degradation of hyaluronic acid probably with continuous generation of new H₂O₂ [4, 5]. In addition, oxidative stress induced by H₂O₂ leads to the synthesis of inflammatory cytokines such as IL-1, IL-6, and TNF-α, which are involved in the regulation of skin inflammation and irritation. The biological activity of protein peptides has been extensively studied since it was first introduced by Marcuse [6]. Antioxidant peptides commonly contain 5–16 amino acid residues [7].

Symplectoteuthis oualaniensis is one kind of deep-sea animal, which inhabits in the dark environment with high pressure, low temperature and low oxygen. It shows fine growth characteristics with short growth cycle and fast reproduction rate, contributing to its great potential in the production of new bioactive compounds, which could be a resource of marine protein worthwhile for the exploitation [8]. China owns rich Iris cuttlefish resources with high protein content, which mainly distributed in the Indian Ocean and the South China Sea. Much evidence has been given for the good biological activity of Iris cuttlefish. HMIDET et al. [9] have found that protease hydrolysates from the carcass of Symplectoteuthis oualaniensis had significant DPPH free radical scavenging ability, high chelating metal ion activity, and the ability to inhibit linoleic acid peroxidation. In the certain range, the antioxidant activity increased by the hydrolysis degree. Spontaneously hypertensive rats models suggected that the high ACE inhibitory peptides prepared from the enzymic hydrolysates of isolated and purified cuttlefish carcasses reduced blood pressure by BALTI et al. [10]. The protease hydrolysates isolated from Symplectoteuthis oualaniensis can be used to develop natural antioxidants and functional foods, which could show the promising effects on health [11, 12].

As the largest body part of the Symplectoteuthis oualaniensis, the carcass contains high protein and low fat (17.24% and 0.49% of fresh weight, respectively) [13], which can be a high-quality protein sources. The protein extracted from the carcass of cuttlefish was hydrolyzed by protease at a specific cleavage site to produce small molecular peptides and free amino acids, which were easily absorbed with high activity [14]. It has been confirmed that the protein has certain antioxidant activity [15, 16], hypoglycemic activity [17] and other biological activities, which has wide application in cosmetics, pharmaceutical or food industries. However, most of the previous studies focused on the protein extracted from Iris cuttlefish, instead of the antioxidant peptides from carcasses, which remained to be explored. Therefore, in this study, protein protease hydrolysates from the carcass of Iris cuttlefish (Symplectoteuthis oualaniensis) were prepared to be investigated for their antioxidant activities in vitro, providing theoretical basis for the further application in the industry.

2.1 Materials

Citric Acid (CA), sodium hydroxide (NaOH), hydrochloric acid (HCl), concentrated sulfuric acid (H₂SO₄), Potassium sulphate (K₂SO₄), Cupric sulfate (CuSO₄), hydrogen peroxide (H₂O₂) were purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). 1,1-diphenyl-2-picrylhydrazyl radical 2 was purchased from Tokyo Chemical Industry (Shanghai, China). 2,7-dichlorodihydrofluorescein diacetate was purchased from Sigma-Aldrich (St. Louis, USA). Pepsin and trypsin were purchased from Shanghai McLean Biochemical Technology Co., Ltd. (Shanghai, China). SDS-PAGE gel preparation kit was purchased from Beijing Solaibao Technology Co., Ltd. (Beijing, China). HSF cells and Fetal bovine serum (FBS) were purchased from Gibco, USA (California, USA). All reagents were used at an analytical grade unless otherwise specified.

2.2 Preparation of carcass protein from Symplectoteuthis oualaniensis

Iris cuttlefish (Symplectoteuthis oualaniensis) were fished in the northwest part of the Indian Ocean by the Fisheries College of Shanghai Ocean University. Thawed at room temperature at about 27 ° C and washed thoroughly with tap-water, then the head and foot were separated on the operating table, the carcass was retained, the skin was manually peeled and the fan-shaped bones were extracted. Cut the Symplectoteuthis oualaniensis into small pieces, add 0.1 mol/L citric acid solution to clean twice, remove impurities outside the carcass and weigh. The carcass was mixed with 0.1 mol/L NaOH at a sample-to-solution ratio of 1:3 (w/v) to remove water-soluble substances and the alkali solution was replaced 6 h later. The mixture was then centrifuged at 3,000× g for 10 min using a Himac CR 21G High-speed floor centrifuge (Hitachi, Tokyo, Japan), and 6 mol/L HCl was added to adjust the supernatant to pH 5.5. The precipitation was collected after centrifugation and the prepared protein was freeze-dried in a vacuum, then stored in plastic bags in a freezer at − 80◦C until further use.

2.3 Preparation of PHCSO、PHCSO-1 and PHCSO-2

Weigh 1.0 g from the carcass protein of Symplectoteuthis oualaniensis prepared in 2.2, adding deionized water to adjust the substrate concentration to 4%. Then pepsin with substrate ratio of 9 U/mg was added, after incubation at 37℃ for 4 h, PHCSO was obtained by enzymolysis. Half of the PHCSO was stored in plastic bags in a freezer at − 80◦C until further use, and the other half was ultrafilted at 4000g for 15 min with a test tube with a molecular weight of 10 kDa. The fraction with molecular weight > 10 kDa was recorded as PHCSO-1 and the remaining substance (< 10 kDa) was recorded as PHCSO-2.

2.4 Molecular Pattern of carcass protein from Symplectoteuthis oualaniensis

The molecular chromatogram of protein isolated from carcass of Symplectoteuthis oualaniensis was determined by using sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli’s method [18]. The samples were prepared with SDS to obtain 8 mg/mL. The samples were mixed with (3:1) 4 × Laemmli Sample Buffer (SDS, Tris-HCl, bromophenol blue, glycerol, and DTT) and oscillated slightly with a scroll oscillator (TianGen Biotech Co, Ltd., Beijing, China). The samples were boiled for 6 min and the boiled mixture was briefly centrifuged at 2,000 g. The test samples and standard protein standard marker (MW ranging from 37 kDa to 250 kDa) (Bio-Rad Laboratories Inc., Hercules, CA, USA) were loaded onto 5% stacking polyacrylamide gel with 8% separating gel (Cat#PG112, EpiZyme Biotechnology, Shanghai, China). The electrophoretic unit voltage is set to 80 V for the concentrated gel and 120 V for the separation gel. After the electrophoresis, the gel was stained with 0.25% Coomassie brilliant blue R-250 solution for 30 min and discolored with the mixture of 20% (v/v) ethanol and 10% (v/v) acetic acid twice, each for 1 h until clear protein bands were observed. The protein bands were then captured using the gel documentation system (Clinx GenoSens 2100(T), Shanghai, China).

2.5 Amino Acid Composition of carcass protein from Symplectoteuthis oualaniensis

The freeze-dried samples were completely hydrolyzed in 6 mol/L HCl at 110 ℃ for 24 h. The excess solvent is evaporated and the drying process is repeated until the dried sample is dissolved in distilled water. In the end, the dried sample was dissolved with a minimum amount of sodium citrate buffer solution (pH 2.2) and filtered through a 0.45 nm hydrophilic membrane. The amino acid content of samples was analyzed by using an amino acid analyzer (Hitachi LA-8080, Tokyo, Japan) [19].

2.6 Fourier Transform Infrared Spectroscopy (FTIR) Analysis

The 2 mg freeze-dried sample was mixed with dried KBr (100 mg) in order to make a 13 × 1 mm thin transparent disk by subjecting a pressure of approximately 5 × 10⁶ Pa. Subjected to FTIR analysis using an FT/IR-400 spectrometer (PerkinElmer, Waltham, MA, USA) 4000 − 600 cm^− 1 (mid-IR region) at 25 ℃.

2.7 Determination of antioxidant activity of protein protease hydrolysates from the carcass of Symplectoteuthis oualaniensis

a. DPPH radical scavenging activity

PHCSO, PHCSO-1 and PHCSO-2 were diluted by deionized water into 3 mg/L solution, respectively. 2 mL sample was thoroughly mixed with 2 mL 0.1mmol /L DPPH, which was denoted as sample group. 2 mL sample was thoroughly mixed with 2 mL ethanol and recorded as blank group. 2 mL deionized water was mixed with 2 mL 0.1mmol /L DPPH solution, which was recorded as the control group. Each group reacted at room temperature for 40 min in the dark, and then measured the absorbance at 517 nm with an ultraviolet spectrophotometer (UV-1800, Shimazu Earthquake Co., Kyoto, Japan). The free radical scavenging rate of sample DPPH was calculated as follow: DPPH radical scavenging activity (%) = [1-(A1-A2)/(A3-A2)] × 100 where A1, A2, and A3 represent the absorbances of the sample, blank, and control, respectively.

b. Hydroxyl radical scavenging activity

A 0.5 mL of FeSO4 solution (9 mmol), 0.5 mL of salicylic acid-ethanol solution (9 mmol), 5 mL distilled water was mixed with 1 mL of sample in sequnce. Then, the reaction was initiated by the addition of 0.5 mL of H₂O₂ (8.8 mmol) and incubated for 20 min at 37 ℃. Distilled water instead of the sample was used as control group, and distilled water instead of the H₂O₂ was used as blank group. The absorbance at 510 nm was measured by an ultraviolet spectrophotometer. The hydroxyl scavenging activity of sample was calculated as follow: Hydroxyl radical scavenging activity (%) = [1-(A1-A2)/(A3-A2)] × 100, where A1, A2, and A3 represent the absorbances of the sample, blank, and control, respectively.

2.8 HSF cells culture

HSF cells were removed from liquid nitrogen for resuscitation, maintained in DMEM (contain 10% FBS, 1.5% penicillin-streptomycin) in condition of 5% CO₂ and saturated humidity at 37 ℃, digested with trypsin solution, and passaged every 48 h. Cells in good growth condition and the log phase were subjected to follow-up experiments.

2.9 Effect of PHCSO and PHCSO-2 on cell viability of HSF cells

The CCK-8 kit was used to detect the effects of PHCSO and PHCSO-2 on the viability of HSF cells. HSF cells at logarithmic growth stage were cultured in 96-well plates, and about 1 × 10⁴ cells per well were cultured in cell incubator for 24 h. Different concentrations (1, 2, 3, 4 and 5 mg/mL) of PHCSO and PHCSO-2 were added to HSF cells, respectively, and DMEM was added as a blank group. Absorbance was measured at 450 nm using a microplate reader (SH-1000, Hitachi, Japan) after cell culture for 24 h.

2.10 Establishment of H₂O₂-induced oxidative stress model of HSF cells

The CCK-8 kit was used to detect the viability of HSF cells. HSF cells at logarithmic growth stage were cultured in 96-well plates, and about 1 × 10⁴ cells per well were cultured in cell incubator for 24 h. Different concentrations (10, 20, 30, 50, 100, 500 and 1000 µmol/L) of H₂O₂ was added to HSF cells, and DMEM was added as a blank group. Absorbance was measured at 450nm using a microplate reader (SH-1000, Hitachi, Japan) after cell culture for 1 h.

2.11 PHCSO and PHCSO-2 repairs HSF cells from oxidative stress induced by H₂O₂

The CCK-8 kit was used to detect the viability of HSF cells. HSF cells at logarithmic growth stage were cultured in 96-well plates, and about 1 × 10⁴ cells per well were cultured in cell incubator for 24 h. HSF cells were pretreated with different concentrations (1, 2, 3, 4, 5 mg/mL) of PHCSO and PHCSO-2, respectively, while only DMEM was added to the control group and the injured group. After 24 h, 20 µmol/L H₂O₂ was added to each well, and the cells were placed in the incubator for 1 h. Then, absorbance was measured at 450nm using a microplate reader (SH-1000, Hitachi, Japan).

2.12 ROS fluorescence staining

HSF cells at logarithmic growth stage were cultured in 24-well plates, and about 1 × 10⁵ cells per well were cultured in cell incubator for 24 h. Different concentrations of PHCSO-2 (1, 2, 3, 4, 5 mg/mL) were added to HSF cells, while only DMEM was added to control group and injury group. After 24 h, 20 µmol/L H₂O₂ was added to each well, and the cells were placed in an incubator for 1 h, and then washed twice with PBS buffer solution. At last, 10 µmol/L DCFH-DA was added to each well. After 30 min, the solution was removed and washed twice with PBS buffer solution, and then observed under an inverted fluorescence microscope (BDS200-FL, Chongqing Optec Instrument Co., Ltd, Chongqing, China).

2.13 Effect of PHCSO-2 on intracellular SOD activity, CAT activity, glutathione (GSH), malondialdehyde (MDA) level and cells secretes the inflammatory factors IL-1, IL-6 and TNF-α level

HSF cells at logarithmic growth stage were cultured in 6-well plates, and about 2 × 10⁵ cells per well were cultured in cell incubator for 24 h. Different concentrations of PHCSO-2 (1, 2, 3, 4, 5 mg/mL) were added to HSF cells, while only DMEM was added to control group and injury group. After 24 h, 20 µmol/L H₂O₂ was added to each well, and the cells were placed in an incubator for 1 h. (a) The supernatant was collected after centrifugation 2000× g for 30 min. The ELISA detection kit method was used to detect the contents of IL-1, IL-6 and TNF-α. (b) The cells were washed twice with PBS buffer, 300 µL RIPA lysis buffer was added, and the adherent cells were lysed on ice for 20 min, and centrifuged at 10,000 g for 5 min. SOD activity, CAT activity, GSH and MDA content in adherents were detected in strict accordance with the kit instructions.

2.14 Statistical Analysis

Each experiment was replicated three times. Data were expressed as mean ± standard deviation and mentioned in the figure legends. Statistical analysis was performed using GraphPad Prism 9 software (GraphPad Inc, San Diego, CA, USA) using two-way ANOVA with Fisher’s LSD test multiple comparison analysis. The values identified as outliers were excluded from statistical analysis. Results were considered statistically significant if the p-value < 0.05.

3.1 SDS-PAGE Protein Pattern of Carcass Protein from Symplectoteuthis oualaniensis

The electrophoretic protein pattern of carcass protein from Symplectoteuthis oualaniensis was presented in Figure 1. The protein consists of 7 bands with molecular weights of 240 ~ 200, 175, 60, 55, 44 and 36 kDa, respectively. 240 ~ 200 kDa protein is MHC, molecular weight of about 99 kDa protein is PM, molecular weight of about 44 kDa is A, and the relatively small molecular weight is TM, about 36 kDa. Among them, the band of MHC is wide and thickness, and this part of proteins has more mass and content of similar molecules, and the protein band is clear, without aggregation and degradation of macromolecular proteins caused by acid-base treatment. The molecular size and amino acid composition of protein isolates are closely related to their physicochemical and functional properties.

3.2 Amino Acid Composition of Carcass Protein from Symplectoteuthis oualaniensis

Automatic amino acid analysis was used to detect 17 amino acids of carcass protein from Symplectoteuthis oualaniensis, and the results were shown in Table 1. The contents of essential amino acids Leu, Lys, Val and Ile were higher in PHCSO, which were 5.59, 5.31, 3.04 and 3.27 g/100 g, respectively, accounting for 42.56% of the total amino acids. According to the ideal model of protein proposed by FAO/WHO, a protein is considered to be of good quality when its essential amino acids account for more than 40% of the total amino acids. The proportion of essential amino acids and non-essential amino acids in carcass protein was suitable, and the nutrient structure was reasonable. Amino acids commonly contained in antioxidant peptides include Met, Arg, Leu, Ala and Glu, which account for 42.19% of the total amino acids in carcass protein.

Table 1. Amino acid composition and content of carcass protein from Symplectoteuthis oualaniensis

	Amino acid	content（g/100g）	Percentage of total amino acids %
polar amino acid	Glycine (Gly)	2.46	4.12
	Tyrosine (Tyr)	2.43	4.06
	Serine (Ser)	2.73	4.58
	Threonine (Thr)	2.90	4.86
nonpolar amino acid	Alanine (Ala)	3.43	5.74
	Valine (Val)	3.04	5.10
	Leucine (Leu)	5.59	9.38
	Isoleucine (Ile)	3.27	5.48
	Phenylalanine (Phe)	2.69	4.51
	Proline (Pro)	0.93	1.56
	Tryptophan (Try)	0.61	1.02
	Methionine (Met)	1.97	3.30
Amino acids with negatively charged	Lysine (Lys)	5.31	8.91
	Arginine (Arg)	4.63	7.77
	Histidine (His)	1.77	2.97
Amino acids with positive charge	Aspartic (Asp)	6.34	10.63
Amino acids with positive charge	Glutamic (Glu)	9.54	16.00

3.3 Fourier Transform Infrared (FTIR) Spectra of Carcass Protein from Symplectoteuthis oualaniensis

FTIR transmission spectra of carcass protein from Symplectoteuthis oualaniensis was shown in Figure 2. Protein had general transmission patterns in major amide bands such as amide A, amide B, amide I, amide II and amide III, respectively. The maximum transmission wave numbers of amide A and amide B, which represent N-H stretch and CH₂ asymmetric stretch, respectively, were at 3290 and 2934 cm⁻¹, respectively. The amide I of PHCSO was observed at 1643 cm⁻¹, mainly related to the C=O stretching vibration on the polypeptide backbone. The presence of amide II in PHCSO at 1539 cm⁻¹is the result of the coupling of N-H bending vibration and CN tensile vibration. The absorption peak of amide III is located at 1235 cm⁻¹ and is related to intermolecular interactions, including N-C-N stretching and N-H deformation of amide bonds.

3.4 Analysis of Antioxidant Activity of PHCSO, PHCSO-1 and PHCSO-2

From the perspective of amino acid composition, carcass protein from Symplectoteuthis oualaniensis has the potential to prepare antioxidant peptides. The results of DPPH radical scavenging activity and hydroxyl radical scavenging activity are shown in Figure 3. The results showed that the antioxidant activity of PHCSO-1 was weak, and small molecule PHCSO-2 had higher DPPH and hydroxyl radical scavenging activity, but there was no significant difference compared with PHCSO (p< 0.05). PHCSO-2 of dosage 5 mg/mL improves the DPPH radical scavenging activity and hydroxyl radical scavenging activity with 57.96% and 56.86%, respectively. Therefore, PHCSO and PHCSO-2 will be selected to further explore the influence at the cellular level.

3.5 The effect of PHCSO and PHCSO-2 on the cell viability of HSF cells

Cytotoxicity assay is typical test for drug safety. To ensure the effect of PHCSO and PHCSO-2 on HSF cells, the CCK-8 kit was used to determine the cell viability of HSF cells with different concentrations of PHCSO and PHCSO-2. As can be seen from Figure 4 A and B, PHCSO and PHCSO-2 showed a dose-dependent proliferation effect on HSF cells viability, with significant differences compared with the blank group at 4 and 5 mg/mL (p<0.05). At 5 mg/mL, the proliferation effect of PHCSO and PHCSO-2 on HSF cells viability was the highest, which were 125.62% and 133.06%, respectively. However, the overall proliferation rate of HSF cells induced by PHCSO-2 was higher than that of PHCSO.

3.6 Repair effect of PHCSO and PHCSO-2 on H₂O₂-induced oxidative stress of HSF cells

The effects of seven H₂O₂ concentration gradients (10, 20, 30, 50, 100, 500, 1000 μmol/L) on the proliferation of HSF cells were studied to obtain the tolerance of HSF cells to H₂O₂ (Figure 5A). The results of CCK-8 cell proliferation showed that H₂O₂ inhibited HSF cells proliferation. Compared with blank control group, the cell viability was significantly decreased with the increase of H₂O₂ concentration (p < 0.05). The survival rate of cells treated with 20 μmol/L H₂O₂ was 47.74%, which was close to half cell survival rate, so the concentration was selected to establish the cell damage model. Cell survival was measured by CCK-8 kit to evaluate the repair effect of PHCSO and PHCSO-2 on H₂O₂-induced oxidative stress of HSF cells (Figure 5B and Figure 5C). Both PHCSO and PHCSO-2 had certain repair effects on HSF cells damage induced by 20 μmol/L H₂O₂. The repair effect was dose-dependent and significantly different from the injured control group except for the addition of 1mg/mL PHCSO (p＜0.05), when 5 mg/mL PHCSO-2 was added, the cell viability was as high as 96.33%. In addition, compared with PHCSO, PHCSO-2 has a higher repair effect on HSF cells, and considering the production cost, so PHCSO-2 was selected for the follow up study on the antioxidant mechanism.

3.7 Cell morphology observation and repair effects of PHCSO-2 against H₂O₂-induced intracellular ROS generation

Figure 6A shows the effect of different concentrations of PHCSO-2 on the morphology of HSF cells. In the blank control, the number of cells was small and adherent, and the cell morphology was spindle and fibrous. The addition of PHCSO-2 increased cell density in a dose-dependent manner. The highest cell density was obtained when the added concentration reached 5 mg/ml. Figure 6B shows the effect of different concentrations of PHCSO-2 on the morphology of HSF cells under oxidative stress. The cells in the injured control group were small, loosely arranged, and mostly floating dead cells. However, with the increase of PHCSO-2 concentration, the number of adherent cells increased in a dose-dependent manner. When 5 mg/ml PHCSO-2 was added, the cell density was the highest and the fibroblast morphology was obvious. The intracellular ROS production was evaluated by DCFH-DA to reflect the repair effect of PHCSO-2 on cells pretreated with H₂O₂, as shown in Figure 6C. ROS production was significantly increased in cells exposed to H₂O₂, and cells in the injured control group were small, loosely arranged, and had strong fluorescence intensity. When the concentration was 5 mg/mL, the fluorescence intensity was decreased, indicating that 5 mg/mL PHCSO-2 could effectively reduce the production of ROS.

3.8 Effect of PHCSO-2 on intracellular SOD, CAT, GSH and MDA level

The activity of antioxidant enzymes has a great influence on cell growth and senescence. SOD, CAT, GSH and MDA are necessary indexes to detect the level of intracellular free radicals. Compared with blank control, SOD (Figure 7A), CAT (Figure 7B) and GSH (Figure 7C) contents in injured control were significantly decreased, while MDA (Figure 7D) contents were significantly increased, indicating that the antioxidant capacity of cells after H₂O₂ oxidation was significantly decreased. Compared with the injured control, SOD, CAT and GSH contents increased and MDA contents decreased after addition of PHCSO-2, indicating that PHCSO-2 had a good repair effect on HSF cells damaged by H₂O₂ oxidation. When the concentration of PHCSO-2 increased in the range of 1-5 mg/mL, the antioxidant capacity of cells gradually increased. Therefore, PHCSO-2 may act as an antioxidant in a similar manner to GSH to prevent H₂O₂ induced oxidative stress in HSF cells.

3.9 Effect of PHCSO-2 on cells secretes the inflammatory factors IL-1, IL-6 and TNF-α level

The contents of IL-1 (Figure 8A), L-6 (Figure 8B) and TNF-α (Figure 8C) induced by oxidative stress were determined by ELISA to observe the occurrence of inflammation. As shown in Figure 9, compared with the blank control, the contents of IL-1, IL-6 and TNF-α in the injury control were significantly increased, indicating that H₂O₂ induced oxidative stress induced a significant increase in the levels of inflammatory factors and inflammation in HSF cells. After addition of PHCSO-2, inflammatory expression levels were reduced in a dose dependent manner in the range of 1 to 5 mg/mL. When 5 mg/mL PHCSO-2 was added, the contents of IL-1, IL-6 and TNF-α were significantly decreased (p＜0.05), however, the difference was not statistically significant compared with the blank control. Therefore, PHCSO-2 has a good inhibitory effect on inflammatory factors produced by oxidative stress of H₂O₂.

According to SDS-PAGE protein proﬁle, the carcass protein from Symplectoteuthis oualaniensis by alkali-solution and acid-isolation showed clear bands with no obvious aggregation or degradation, which indicated the compatibility of pepsin in the extraction of the carcass protein. The wide and thick band of MHC band was consistent with the results in previous studies [20]. The carcass protein contains over 40% essential amino acids including high contents of leucine and lysine. Similar to salmon, Symplectoteuthis oualaniensis produce high-quality aquatic protein with reasonable composition, which conforms to the ideal structural pattern of high-quality protein proposed by WHO [13, 21]. Antioxidant amino acid such as Met, Arg, Leu, Ala and Glu compose about 42.19% of the peptide from carcass protein, indicating its potentiality to produce antioxidant peptides. The intact molecular structure of the protein was shown by FTIR. Given by the fact that peptides of low molecular weight commonly have high antioxidant activity, PHCSO-2 has reasonable better DPPH and hydroxyl radical scavenging activity than PHCSO-1, which is also supported by the study on cottonseed protein hydrolysates (＜3 kDa) [22].

In this study, both PHCSO and PHCSO-2 improved the viability of HSF cells without cytotoxicity, between which PHCSO-2 owned better protectionthan PHCSO. Since the theory of replicative aging was proposed, H₂O₂, UV and many other methods have been applied to construct cell damage models [23], among which H₂O₂ is usually used to induce oxidative stress on cells for antioxidants screening in vitro [24]. DNA damage is caused by oxidative stress via activating P53/P21 signaling pathway, whose expressions are closely related to cell cycle and apoptosis. The increasing level of P21 gene inhibits the activity of CDK4/6, followed by the blocking on cell cycle, which consequently results in cell growth arrest and cell senescence delay [25, 26]. Therefore, PHCSO-2 was selected for further investigations including cell observation, fluorescence staining, antioxidant enzyme activity and inflammatory factor assay.

DCFH-DA was used to evaluate intracellular ROS production by producing 2 ', 7 '-dichlorofluorescein (DCF) after the hydrolyzation and oxidation in cells which can emit green fluorescence under the stimulation of H₂O₂[27]. Although reactive oxygen species (ROS) is a natural byproduct of normal oxygen metabolism in mitochondria, excessive amount of them are harmful to cell function and homeostasis [28]. By degrading extracellular matrix proteins (such as collagen) and changing connective tissue, ROS could accelerate skin aging [29]. ROS also can directly damage DNA, lipids and proteins in dermal cells [30], and alter transcription. Contrast to the result that more than half of HSF cells were apoptotic in the injured group, 5 mg/mL PHCSO-2 pretreated group showed vigorous growth of cells, with decreasing intracellular ROS level.

SOD, CAT, GSH and MDA are important indicators to detect the level of free radicals in cells. SOD and CAT are both important antioxidant enzymes for scavenging free radicals in cells [31, 32]. In general, endogenous antioxidant enzymes can effectively protect skin damage by ROS clearance [33]. Reduced GSH is a tripeptide that helps maintain the normal immune function of human body with detoxification and antioxidative effects [34]. MDA is one of the key substances of lipid peroxidation in cells [35]. To assess the antioxidant effect of PHCSO-2, key oxidation resistance gene including SOD, CAT, GSH, and the oxidation product MDA were investigated for their expression. According to the results, SOD, CAT and GSH transcripts in PHCSO-2 pretreated HSF cells were significantly up-regulated, while MDA transcripts were significantly down-regulated, compared to the injure group. In addition, it can also reduce the susceptibility of cells to the secondary oxidative stress.

The secretion of pro-inflammatory cytokines and the release of pro-inflammatory mediators induced by oxidative stress lead to the infiltration of by inflammatory cells into dermis , resulting in skin damage, accelerating skin aging and even skin cancer [36]. Therefore, inflammatory cytokines are the most intuitive factors to estimate the inflammation. Proven by previous study,low molecular peptides extracted from salmon and chicken inhibit the expressions of nuclear factor κ-B (NF-kB) and inducible nitric oxide synthase (iNOS) respectively [37, 38]. By the pretreatment of 5 mg/mL PHCSO-2, IL-1 almost keeps the similar transcription level with the blank group in HSF cells. Compared to the injured HSF cells, both IL-6 and TNF-α transcripts were significantly down-regulated in PHCSO-2 pretreated cells under oxidative stress induced by H₂O₂, which indicates the protective effect on skin against oxidative stress.

In this study, PHCSO-1 and PHCSO-2 as molecular weights over 10 kDa or less 10 kDa by ultrafiltration, were isolated from PHCSO of Symplectoteuthis oualaniensis followed by the investigation on their antioxidant activities. Small molecule PHCSO-2 showed higher antioxidant activity than PHCSO-1, supported by its higher DPPH and hydroxyl radical scavenging activity, which showed no significant difference compared with PHCSO (p< 0.05). By H₂O₂-induced HSF cells damage model, both cytotoxicity and protective effect on HSF cells of PHCSO and PHCSO-2 were evaluated, which indicated the superiority of the repairing effect by PHCSO-2 than PHCSO. Furthermore, experiments on cell morphology, fluorescence staining, antioxidant enzyme activity and inflammatory factor determination of PHCSO-2 were conducted. According to the results, PHCSO-2 showed significant antioxidant capacity on oxidation and inflammation caused by H₂O₂ injury on HSF cells, and it could effectively repair the skin damage caused by H₂O₂. The protection of PHCSO-2 against the damage by H₂O₂ indicated by this study contributes to its prospective application in cosmetics or pharmaceutical industries.

Author Contributions: Wenhui Wu., conceptualization, funding acquisition; Na Li, formal analysis, investigation, writing—original draft preparation; Xiaozhen Diao, Xinyi Pu and Jeevithan Elango, writing—review and editing; Pengjie Tang, methodology. All authors have read and agreed to the published version of the manuscript.

Funding: Project supported by the National Natural Science Foundation of China (Grant No.82173731); Project supported by the Research Fund for International Young Scientists (Grant No.81750110548); Project supported by Natural Science Foundation of Shanghai (No. 21ZR1427300).

Informed Consent Statement: No applicable.

Data Availability Statement: All data included in this study are available upon by contact with the corresponding author.

Conflicts of Interest: The authors declare no conﬂict of interest.

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No competing interests reported.

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Study on protein hydrolysate from Symplectoteuthis oualaniensis reconstructing fibroblast against oxidative stress by hydrogen peroxide

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Materials

2.2 Preparation of carcass protein from Symplectoteuthis oualaniensis

2.3 Preparation of PHCSO、PHCSO-1 and PHCSO-2

2.4 Molecular Pattern of carcass protein from Symplectoteuthis oualaniensis

2.5 Amino Acid Composition of carcass protein from Symplectoteuthis oualaniensis

2.6 Fourier Transform Infrared Spectroscopy (FTIR) Analysis

2.7 Determination of antioxidant activity of protein protease hydrolysates from the carcass of Symplectoteuthis oualaniensis

2.8 HSF cells culture

2.9 Effect of PHCSO and PHCSO-2 on cell viability of HSF cells

2.10 Establishment of H₂O₂-induced oxidative stress model of HSF cells

2.11 PHCSO and PHCSO-2 repairs HSF cells from oxidative stress induced by H₂O₂

2.12 ROS fluorescence staining

2.13 Effect of PHCSO-2 on intracellular SOD activity, CAT activity, glutathione (GSH), malondialdehyde (MDA) level and cells secretes the inflammatory factors IL-1, IL-6 and TNF-α level

2.14 Statistical Analysis

3. Results

4. Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

Study on protein hydrolysate from Symplectoteuthis oualaniensis reconstructing fibroblast against oxidative stress by hydrogen peroxide

Status:

Version 1

Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Materials

2.2 Preparation of carcass protein from Symplectoteuthis oualaniensis

2.3 Preparation of PHCSO、PHCSO-1 and PHCSO-2

2.4 Molecular Pattern of carcass protein from Symplectoteuthis oualaniensis

2.5 Amino Acid Composition of carcass protein from Symplectoteuthis oualaniensis

2.6 Fourier Transform Infrared Spectroscopy (FTIR) Analysis

2.7 Determination of antioxidant activity of protein protease hydrolysates from the carcass of Symplectoteuthis oualaniensis

2.8 HSF cells culture

2.9 Effect of PHCSO and PHCSO-2 on cell viability of HSF cells

2.10 Establishment of H2O2-induced oxidative stress model of HSF cells

2.11 PHCSO and PHCSO-2 repairs HSF cells from oxidative stress induced by H2O2

2.12 ROS fluorescence staining

2.13 Effect of PHCSO-2 on intracellular SOD activity, CAT activity, glutathione (GSH), malondialdehyde (MDA) level and cells secretes the inflammatory factors IL-1, IL-6 and TNF-α level

2.14 Statistical Analysis

3. Results

4. Discussion

Conclusion

Declarations

References

Additional Declarations

Status:

Version 1

2.10 Establishment of H₂O₂-induced oxidative stress model of HSF cells

2.11 PHCSO and PHCSO-2 repairs HSF cells from oxidative stress induced by H₂O₂