Protective effect of phosphorylated Athyrium multidentatum (Doll.) Ching polysaccharide on vascular endothelial cells in vitro and in vivo

The purpose of this study was to prepare phosphorylated Athyrium multidentatum (Doll.) Ching polysaccharide (PPS) and investigate its protective effect on vascular endothelial cells (VECs) in vitro and in vivo and the underlying mechanisms. Sodium tripolyphosphate (STPP) and sodium trimetaphosphate (STMP) were used as phosphorylation reagents and PPS was characterized by Fourier transform infrared (FT‐IR), 13C nuclear magnetic resonance (13C NMR) and 31P nuclear magnetic resonance (31P NMR) spectra. Chemical analysis demonstrated that PPS was composed of mannose, glucosamine, rhamnose, glucuronic acid, galacturonic acid, galactosamine, glucose, galactose, xylose, arabinose, and fucose with a molar ratio of 11.36:0.42:4.03:1.12:1.81:0.26:33.25:24.12:6.85:14.46:2.32 and a molecular weight of 28,837 Da. Results from in vitro and in vivo assays revealed that PPS protected human umbilical vein endothelial cells (HUVECs) against H2O2‐induced oxidative injury and attenuated D‐galactose‐induced VECs damage in mice. RNA sequencing (RNA‐seq) analysis identified 18 differentially expressed genes (DEGs) between D‐galactose‐treated and PPS‐pretreated mice abdominal aorta. A deep analysis of these DEGs disclosed that PPS regulated the expression of genes involved in the functions of vascular endothelium repairment, cell growth and proliferation, cell survival and apoptosis, inflammation, angiogenesis and antioxidant, indicating that these biological processes might play crucial roles in the protective actions of PPS on VECs.

cells may trigger a significant decrease in blood flow and subsequent tissue damages in certain organs.Therefore, protecting VECs from damage is regarded as an essential strategy for CVD prevention.
Athyrium multidentatum (Doll.)Ching (AMC), a delicious and nutritious potherb in Changbai Mountain area of China, has been used for curing hypertensive, rheumatisms and flu for thousands of years (Liu et al., 2016).Polysaccharides from AMC were reported to possess excellent antioxidant, antiaging, cytoprotective and immuneenhancing activities, denoting broad application prospects in the field of medicine, foods, and health products (Jing et al., 2019;Jing et al., 2020;Wang et al., 2022).In recent years, polysaccharides have aroused extensive attention of researchers because of their diverse biological activities and unique physical and chemical properties.Biological activities of polysaccharides are intimately related to several structure parameters such as molecular weight distributions, monosaccharide compositions and linkage types (Zhang et al., 2008).Structural modifications including methylation, sulfation, acetylation and phosphorylation can change the physicochemical and biological properties of polysaccharides and thus acquire better or unique activities (Ge et al., 2008).Phosphorylation is considered as an effective covalent modification method that introduces the phosphate groups to the hydroxy groups of polysaccharides.Evidence had proved that the introduction of the phosphate groups may lead to better biological activities due to their high nucleophilic characteristic and remarkable hydrogen donation capacity.Deng et al. (2015) reported that phosphorylated polysaccharides from Dictyophora indusiata displayed more powerful effects on the growth of MCF-7/B16 tumor cells than unmodified Dictyophora indusiata polysaccharides.Ye et al. (2013) reported that polysaccharides from Lachnum YM120 had more excellent antitumor activity after phosphorylation.Our previous study suggested that phosphorylated AMC polysaccharide exhibited a potential protective effect on HUVECs.However, to the best of our knowledge, the phosphorylated polysaccharide of AMC was never investigated.Therefore, the protective effect of phosphorylated AMC polysaccharide on VECs in vitro and in vivo were explored in this study.
To achieve this objective, phosphorylated AMC polysaccharide was prepared by sodium tripolyphosphate (STPP) and sodium trimetaphosphate (STMP) method, and characterized by determination of the monosaccharide composition, molecular weight and phosphorus content, FT-IR, 13 C and 31 P NMR spectra.Protective effect on VECs was evaluated employing H 2 O 2 -injured HUVECs and D-galactose-induced aging mice.RNA-seq analysis was applied to explore the underlying mechanisms and quantitative real-time reverse-transcription PCR (RT-qPCR) was performed to testify its reliability.Current study aimed to provide an experimental basis for the development and utilization of phosphorylated AMC polysaccharide as a novel vasoprotective agent.

| Preparation of phosphorylated polysaccharides
Polysaccharides from AMC were prepared according to the method described previously (Wang et al., 2022).Briefly, 2.5 kg of AMC rhizomes were defatted with a 5fold volume of methanol and then extracted with 18 L of distilled water at 100°C for 1.5 h twice.Afterwards, the water extract was concentrated and precipitated with a 3-fold volume of anhydrous ethanol.After filtering, the resultant precipitate was collected and extracted repeatly with a mixture of n-butanol and chloroform (1:4, v/v).The protein-free supernatant was dialyzed against tap water for 48 h and against distilled water for 24 h using 3500 Da Mw cutoff dialysis membranes to acquire AMC polysaccharides (PS).To prepare the phosphorylated polysaccharides, 5 g of polysaccharides was dissolved in a 500 mL mixture of 5% STPP and 2% STMP, and then stirred at room temperature.After the solids were entirely dissolved, the pH value of the solution was adjusted to 9 with saturated NaHCO 3 solution and the mixture was allowed to react at 88°C for 5 h.At the end of reaction, the mixture was cooled to room temperature and precipitated with three times the volume of anhydrous ethanol.The resultant precipitant was redissolved in distilled water and dialyzed against tap water for 48 h and against distilled water for 48 h using 3500 Da Mw cutoff dialysis membranes.After lyophilization, phosphorylated AMC polysaccharide (PPS) was synthesized.

| Structure characterization
2.3.1 | Determination of phosphorus content 0.1091 g of polysaccharide sample was digested with a mixture of HNO 3 (5 mL), HCl (1 mL), and H 2 O 2 (0.5 mL) at 25°C for 15 min and 200°C for 60 min.The obtained solution was subsequently diluted to 10 mL with distilled water.Measurement of phosphorus (P) content was carried out on an inductively coupled plasma mass spectrometer (ICP-MS, Aglient 7700, USA) under the conditions of carrier gas 1.10 L/min, omega bias −105 V, omega lens 10.1 V, cell entrance −45 V, deflect 3.8 V, cell exit −62 V, plate bias −60 V, RF power 1.50 KW and RF matching 1.80 V.A calibration curve was established by plotting the absorption intensities against the phosphorus concentrations (0, 5, 10, 25, 50, and 100 mg/L).Degree of substitution (DS) represented the average number of phosphate groups on each monosaccharide residue and was calculated according to the following formula, where P% stranded for the phosphate group content.DS = (162 × P%)/(3100-84 × P%).

| Measurement of molecular weight
The molecular weights were achieved on a Shimadzu LC-20A HPLC system (Shimadzu Co.) equipped with a TSK-GEL G3000 PWxl column (300 × 7.8 mm, 5 μm) and a refractive index detector.20 μL sample solution was injected in each perform and eluted with 0.1 mol/L Na 2 SO 4 buffer at a flow rate of 0.5 mL/min.The molecular weight was estimated by reference to a calibration curve prepared from the T-series Dextran standards of known molecular weights (Mw 13.05,36.80,64.65,135.35,and 300.60 kDa).

| Spectral analysis
The ultraviolet absorption spectra were measured on a UV-Vis spectrophotometer (UV-2700, Shimadzu, Japan) in the wavelength range 200-400 nm.Fourier transform infrared spectrophotometer (Shimadzu FTIR-8300) was used for IR spectrum analysis within the scanning area from 4000 to 400 cm −1 with a resolution of 4 cm −1 .The NMR spectra were recorded on a 500 MHz Bruker Avance III spectrometer equipped with a 5 mm multinuclear inverse detection probe, operating at 126 and 202 MHz for 13 C and 31 P nuclei, respectively.Here, samples were analyzed in deuterated dimethylsulfoxide (DMSO-d 6 ) reagent and the chemical shifts were given in parts per million (ppm).Human umbilical vein endothelial cells were donated by Institute of Plastic Surgery, Weifang Medical University (Weifang, China).Cells were propagated in sterile RPMI 1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin mixture, and incubated in a humidified atmosphere containing 5% CO 2 at 37°C.After reaching 90%-95% confluence, cells were passaged at a ratio of 1:2.

| Measurement of the cell proliferative rate
Cell proliferative rate was detected by CCK-8 method and all operations were performed according to the manufacturer's instructions.Briefly, cells were seeded at a density of 5 × 10 3 cells/well in a 96-well microplate and allowed to adhere for 12 h.Afterwards, cells were cocultured with 25, 50, 100, and 150 μg/mL polysaccharide samples or 10 μM VE for 24 or 48 h.At the end of treatment, 300 μM of H 2 O 2 was added and incubated with the cells for another 50 min.Finally, 20 μL of CCK-8 was added and incubated with the cells for 4 h.The absorbance was measured at 450 nm on a SpectraMax M5 microplate reader (Molecular Devices Corporation).The cell proliferative rate was calculated according to the following equation.
2.4.3 | SOD/CAT/GSH-Px activities and MDA/NO/ROS levels Cells were seeded at a density of 1 × 10 5 cells/well in a 6well plate.12 h later, the cells were pretreated with 25, 50, 100 and 150 μg/mL polysaccharide samples or 10 μM VE for 48 h, and then exposed to 300 μM of H 2 O 2 for 50 min.At the end of treatment, antioxidant enzymes (SOD, CAT, and GSH-Px) activities and nitric oxide/malondialdehyde (NO/MDA) contents in the supernatants were respectively measured using a SpectraMax M5 microplate reader followed the manufacturer's protocols.2′, 7'-Dichlorofluorescein diacetate (DCFH-DA) was used as a ROS capturing reagent.In brief, cells were seeded into a 96-well black plate at a density of 1 × 10 4 cells/well and cultured at 37°C for 12 h.Afterwards, 100 μL of different concentrations of polysaccharide solution (25, 50, 100, and 150 μg/mL) or VE (10 μM) was added and incubated at 37°C for 48 h.At the end of treatment, cell supernatant was abandoned and cells were exposed to DCFH-DA for 20 min.After rinsing with FBS-free 1640 medium twice, cells were exposed to 300 μM H 2 O 2 for 50 min.The fluorescence intensity of DCF in cells was detected at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a SpectraMax M5 microplate reader.The intracellular ROS level was directly recorded as the fluorescent intensity.

| Apoptosis analysis
Annexin V-FITC/PI double staining was performed to label apoptotic cells and the percentage of the apoptotic cells was analyzed by flow cytometry.Briefly, cells were cultured at a density of 1 × 10 5 cells/well in a 6-well plate and allowed to attach for 12 h at 37°C.After cultivation with 150 μg/mL of polysaccharide sample or 10 μM of VE for 48 h, cells were treated with 300 μM of H 2 O 2 for 50 min.Afterwards, cells were respectively stained with Annexin V-FITC and PI according to the manufacturer's instruction.Finally, the cells was analyzed by a Becton Dickinson FACS Calibur flow cytometer (BD Biosciences).

| Determination of the mitochondrial membrane potential
Assessment of the mitochondrial membrane potential (MMP) was performed using JC-1 reagent.Specifically, cells were seeded at a density of 1 × 10 5 cells/well in a 6well plate and cultured at 37°C for 12 h.Afterwards, cells were pretreated with the polysaccharide sample (150 μg/ mL) or VE (10 μM) for 48 h and then exposed to 300 μM H 2 O 2 for 50 min.At the end of treatment, cells were collected and stained with JC-1 following the manufacturer's protocols.Determination was performed on a Becton Dickinson FACS Caliber flow cytometer.
2.5 | Protective effect of phosphorylated polysaccharide on VECs in D-galactose-induced aging mice

| Experimental protocols
To implement in vivo assays, male Kunming mice weighing 26 ± 2 g (12-week-old) were purchased from Experimental Animal Center of Shandong Province, Jinan, China (Certificate No. SCXK 20140007).All mice were housed at 25 ± 2°C under a standard 12 h light/12 h dark cycle with ad libitum usage of food and water.After 3 days of acclimatization, the D-galactose-induced (100 mg/kg/day, ip) aging mice were randomly divided into five groups (16 mice per group), including: D-galactose model control group, positive control group (using VE as the positive drug, 200 mg/kg/day, ig), PPS low-dose group (50 mg/ kg/day, ig), PPS medium-dose group (100 mg/kg/day, ig) and PPS high-dose group (200 mg/kg/day, ig).Another 16 healthy mice were selected as the normal control group.After 60 consecutive days of administration, mice were anesthetized and euthanized by cervical dislocation.The blood was collected by eyeball extraction and the abdominal aorta was meticulously excised and fixed in 4% paraformaldehyde for hematoxylin-eosin (HE) and TdTmediated dUTP nick-end labeling (TUNEL) staining or kept in liquid nitrogen for RNA-seq and RT-qPCR assays.All experiments were approved by the ethics committee of Weifang Medical University.

| HE and TUNEL staining
After fixing overnight, HE and TUNEL staining of the abdominal aorta was carried out.In brief, abdominal aorta was embedded in paraffin wax, sectioned at 5 μm thickness and stained with hematoxylin and eosin for microscopic observation under an Olympus BX51 light microscope (Olympus).TUNEL staining was conducted using an in situ cell death detection kit according to the manufacturer's recommendation.Five areas were selected randomly in each sample and the percentage of TUNEL-positive cells was calculated by Image J software.
2.5.4 | RNA-seq analysis RNA-seq was performed to seek for target genes related to the endothelial cell protective effect of PPS.Briefly, the abdominal aorta tissues from the model group and PPS highdose group were respectively treated with Trizol reagent to isolate total RNAs.The RNA purity and concentration were checked by a NanoPhotometer® spectrophotometer (IMPLEN), and RNA integrity was measured using the Agilent RNA 6000 Nano Kit and a 2100 Bioanalyzer (Agilent Technologies). 1 μg of RNA was used to construct the sequencing libraries by employing the Illumina's TruSeq Small RNA Sample Prep Kit.After library profile analysis, the libraries were sequenced on an Illumina HiSeq × 10 platform (Annoroad Genomics).The gene levels were quantified as fragments per kilobase of transcript per million mapped reads (FPKM) and normalized across all samples by the cuffdiff program.Gene Ontology (GO) function and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses of the DEGs were performed with clusterProfiler package in R language.The |log 2 FC| ≥1 and adjusted p value of <.05 compared with the model group were adopted as standards to screen the DEGs.
2.5.5 | RT-qPCR assay RT-qPCR assay was implemented to testify RNA-seq results.The total RNAs were extracted from the abdominal aorta of the mice by Trizol method.The concentration and purity of the isolated RNA were assessed according to the absorbance ratio at 260 and 280 nm with a NanoDrop one microvolume UV-Vis spectrophotometer from Thermo Fisher Scientific (USA).Total RNA was reversetranscribed into cDNA according to the manufacturer's instructions.RT-qPCR was conducted on a 7500 Fast Dx real-time PCR instrument (Shanghai, China) adopting the procedures of 95°C for 30 s, 40 cycles at 95°C for 5 s, 55°C for 20 s and 72°C for 30 s. Sequences of the primers used in this study were listed in Table 1.

| Statistical analysis
All experiments were performed in triplicate, and the values were presented as means ± standard deviation and analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's test using SPSS 17.0 software.A p value of less than .05was considered statistically significant.
According to the regression equation y = 718.5677x-442.3883(R 2 = 0.9998), the phosphorus contents in PPS and polysaccharide were respectively calculated to be 7.71% and 0.06%, implying that the phosphorylated derivative of polysaccharide with a DS of 0.51 was prepared successfully.DS is believed to have a great influence on the bioactivity of phosphorylated polysaccharides.However, the phosphorylated modification was very difficult to accomplish owing to the tough esterification process between -PO 4 and -OH and the complicated structures of polysaccharides.Multifarious phosphorylation reagents, including phosphoric acid, phosphorous acid, phosphonopropionic acid and tripolyphosphate, have been employed to prepare the phosphorylated polysaccharides.It is believed that phosphorus content and DS are closely associated with the types and ratios of phosphorylation reagents and/or the reaction conditions.Wang et al. (2018) synthesized phosphorylated Artemisia sphaerocephala polysaccharides with a DS of 0.34-0.54using POCl 3 /pyridine.Here, STPP and STMP were proved to be convenient and efficient phosphorylation reagents owing to the desirable DS and mild reaction conditions.

| Spectroscopic characteristics
The nucleic acid and protein were monitored by UV absorption at 260 and 280 nm.As depicted in Figure 1a, scanned UV spectra showed no absorption at 260 or 280 nm, manifesting that polysaccharide and PPS were nucleic acid and protein free.FT-IR spectra of PS and PPS were displayed in Figure 1b.It could be seen from the FT-IR spectra that there were significant differences between polysaccharide and PPS.To be specific, the broad peaks at around 3254.31 cm −1 and 2932.38 cm −1 represented the characteristic absorption bands of O-H and C-H stretching vibration.The bands at around 1722.49, 1602.89 and 1371.52 cm −1 were separately due to the asymmetric and symmetric vibration of -COOH, indicating that polysaccharide and PPS were acidic polysaccharides, which was consistent with the monosaccharide composition analysis.The peaks near 1145.58cm −1 and 926.91 cm −1 were ascribed to the characteristic absorptions of carbohydrate.Compared with the FT-IR spectrum of PS, a new characteristic absorption peak at 895.39 cm −1 appeared in the FT-IR spectrum of PPS, which should be attributed to the symmetrical C-O-P vibration of C-O-PO 3 group (Wang et al., 2018).Beyond that, the increment of the signal at 1240.14 cm −1 could be ascribed to the stretching vibration of P=O (Kim et al., 2015;Ye et al., 2013).FT-IR analysis showed that the phosphorylated modification of polysaccharide occurred successfully.
It is generally recognized that the carbon signal will shift towards lower field position if an electron-withdrawing phosphate group directly attaches to the carbon, while the signal will shift to higher field position if the phosphate group is indirectly connected (Chen et al., 2011;Chen et al., 2014).However, compared the carbon signals of PPS with that of PS, significant induction did not occur.We speculated that the hydroxyl groups of polysaccharide were not completely substituted.Encouragingly, three signals at −17.28, −17.69 and − 19.07 ppm emerged in the 31 P NMR spectrum of PPS (Figure 1d), proclaiming the existence of phosphorus atoms at different phosphorylated positions of the glucosidic units.Taken together, phosphorylated AMC polysaccharides with high DS were prepared successfully.
PPS displayed the most strongest cytoproliferative activity at the concentration of 150 μg/mL and the treatment time of 48 h.These results also suggested that PPS exhibited no harmful effects on HUVECs within the concentration ranges of 25-150 μg/mL.Therefore, the following in vitro assays were performed employing PPS at this optimum concentration and treatment time. 3.3.2| Effects of PPS on SOD, CAT, GSH-Px, MDA, NO, and ROS Seen from Table 3, PPS had positive impacts on SOD, CAT and GSH-Px activities, MDA and NO contents, and ROS level.Compared with the normal group, the activities of SOD, CAT and GSH-Px and the content of NO in the HUVEC supernatant of the model group were decreased, while the levels of MDA and intracellular ROS were increased dramatically.In contrast, PPS pretreatment enhanced SOD, CAT and GSH-Px activities, raised NO content, and reduced MDA and ROS levels.PPS regulated the levels of SOD, CAT and ROS in concentration-dependent manners.These results demonstrated that PPS possessed significant antioxidant activity in vitro.In previous study, we found that polysaccharide failed to increase the activity of GSH-Px in the HUVEC supernatant (Jing et al., 2020).
Comprehensive consideration of these results, PPS displayed more powerful effects on these biomarkers than polysaccharide and VE at 100 and 150 μg/mL.Membrane-permeant JC-1 dye was used to estimate the mitochondrial membranes permeability of HUVECs in this study.From Figure 3b, it could be seen that the MMP level was significantly reduced in H 2 O 2 -alone treated group (1.90 ± 0.28%, p < .01)compared to the normal group (9.65 ± 0.18%).After 48 h treatment with 150 μg/mL PPS, the positive staining rate of the cells was enhanced to 6.00 ± 0.56% (p < .05,p < .01),which was higher than the VE-treated group (4.8 ± 0.01%, p < .05).In previous study, we reported that the MMP level in polysaccharide treatment group was 4.10 ± 0.46% under the same conditions (Jing et al., 2020), which was lower than that of PPS treatment group, indicating that PPS exhibited more powerful effects on MMP levels than PS.The artery wall has a three-layered structure (intima, media, and adventitia) governing the mechanical properties of blood vessels.Seen from the images of the abdominal aorta depicted in Figure 4a, the artery wall of the normal group displayed a compact structure.The intima was smooth and covered with intact endothelial cells.The smooth muscle cells were in fusiform shape and regularly arranged in the media.The elastic membranes were intact, flabby, clear and continuous.After the mice were treated with D-galactose, the smooth muscle cells shrinked apparently and some cells appeared apoptotic characteristics.The elastic membranes turned straight and even ruptured.The media of the artery was loosely organized and many endothelial cells seemed to peel off the intima.A comparative analysis showed that PPS pretreatment presented significant ameliorations in Dgalactose-induced mice abdominal aorta injuries, including increased smooth muscle and endothelial cells, flappy and intact elastic membranes, and tight artery wall structure, which could be clearly observed in the HE staining images.Meanwhile, PPS exhibited the most powerful protective effect on the abdominal aorta at 100 mg/kg, which was stronger than VE.TUNEL staining enables researchers to visualize and quantify cells undergoing apoptosis.In this assay, the apoptotic cells were stained brown and the living cells were stained blue.As displayed in Figure 4b, the cell apoptosis rates in the normal group and the Dgalactose-treated group were respectively 4.8 ± 0.12% and 78.1 ± 5.46%, indicating that D-galactose induced cell apoptosis in the artery wall, which was consistent with the HE staining results.The apoptosis rate was decreased to 3.2 ± 0.31% after treatment with 50 mg/kg PPS daily, but the apoptotic cells were not observed in 100 and 150 mg/kg PPS treatment groups, which was similar to the VE treatment group.All things considered, PPS exhibited marked anti-apoptosis capacity, which agreed with our cellular apoptosis results in vitro.We concluded that the decrease in cellular apoptosis might be one of the potential protective mechanisms of PPS on VECs.
3.4.2| Effects of PPS on SOD, CAT, GSH-Px, ROS, NO, eNOS, and ET-1 Similar to the in vitro experimental results, PPS showed powerful effects on the activities of SOD, CAT and GSH-Px as well as the levels of NO, eNOS, and ROS in vivo (Table 4).Compared with the normal group, the activities of SOD, CAT, and GSH-Px in the model group were attenuated.Reversely, PPS markedly enhanced the antioxidant enzyme activities in mice serum.Except for CAT activity in the PPS1 group, the antioxidant enzymes activities in the PPS treatment groups were stronger than the normal group, and the CAT and GSH-Px activities were increased in dose-dependent manners.In comparison with VE, PPS had a more robust influence on the antioxidant enzyme activities.Meanwhile, the ROS levels in the PPS-treated groups were decreased in a dose-dependent manner, all were lower than the model group.
NO, the strongest diastolic vascular factor, has multiple functions including platelet aggregation prevention, neutrophil activation inhibition, anti-inflammatory, and anti-apoptotic effects, and plays a crucial role in maintaining the balance of circulation and modulating the vascular tone (Yao et al., 2013).The generation of NO mainly depends on the activity of eNOS, which functions as a pivotal enzyme in the synthesis of NO.In this assay, NO and eNOS contents in the serum were minimized after the mice were treated with D-galactose.After intervention with PPS, the NO and eNOS levels were elevated, indicating its powerful protective effects on VECs.Oddly, a reversed dose-response relationship was presented in the NO levels.We concluded that NO might be scavenged by PPS due to its increased antioxidant capacity at high concentrations since NO belongs to the ROS.ET-1, a strong vasoconstrictor secreted by endothelial cells, acts as the natural counterpart of NO.The dynamic balance between NO and ET-1 plays an important role in instructing the normal tension of blood vessels (Zhu et al., 2020).Our results showed that the ratios of NO/ET-1 in the NG, MG, VE, PPS1, PPS2, and PPS3 groups were respectively 0.45, 0.23, 0.24, 0.60, 0.52, and 0.34, suggesting that PPS could effectively ameliorate vasomotor function.Therefore, the antioxidative activity and the regulating capacity of PPS on NO/ET-1 balance could probably contribute to its protective effect on VECs in vivo. 3.4.3| Effects of PPS on TNF-α, IFN-γ, IL-2, ICAM-1, VCAM-1, and MCP-1 In this assay, the biomarkers including TNF-α, IFN-γ, IL-2, ICAM-1, VCAM-1, and MCP-1 in the mice serum were monitored.In the model group, IFN-γ, ICAM-1, VCAM-1, and MCP-1 levels were increased, while IL-2 levels were decreased (Table 5), implying that D-galactose induced inflammatory damage in mice.In contrast, PPS ameliorated D-galactose-induced damage through elevating IL-2 or by lessening TNF-α, IFN-γ, ICAM-1, VCAM-1, and MCP-1 levels, and the IFN-γ, IL-2, and ICAM-1 levels were in dose-dependent manners.
3.5 | Effects of phosphorylated polysaccharide on genes in mouse abdominal aorta 3.5.1 | RNA-seq analysis Genes associated with the protective actions of PPS were identified by RNA-seq analysis.As displayed in the volcano plots (Figure 5a), a total of 175 DEGs (105 upregulated and 70 downregulated DEGs, Figure S3) were identified in the PPS-treated abdominal aorta.Among these DEGs, 18 DEGs might be contributed to the protective effect of PPS owing to their involvement in the functions of anti-oxidant, anti-apoptosis, anti-inflammation, promoting proliferation, and stimulating angiogenesis and repair (Table 6).GO analysis is a useful method for annotating genes and gene sets with biological characteristics for high-throughput genome or transcriptome data.The upregulated and downregulated genes correlated with the protective actions of PPS were respectively involved in the biological processes of immune system process, cell proliferation, cellular process, reproductive process, biological adhesion, developmental process, growth, response to stimulus and biological regulation in the GO terms (Figure 5b).The KEGG pathway is a knowledge base for systematic analysis of gene functions (Kanehisa & Goto, 2000).As shown in the KEGG barplot (Figure 5c), MAPK (mitogen-activated protein kinase) signaling pathway might be responsible for the protective capacity of PPS, in which Nr4a1 (nuclear receptor subfamily 4, group a, member 1), Hspa1a (heat shock protein family a [Hsp70] member 1a), Dusp1 (dual specificity phosphatase 1) and Jun were involved.However, other KEGG pathways or signaling pathways seemed not to be intimately associated with the protective actions of PPS.
3.5.2| RT-qPCR analysis Lastly, RT-qPCR was conducted to validate the DEGs identified by the RNA-seq analysis.The mRNA expression levels of Klf6 (Krüppel-like factor 6), Nr4a1, Nfkbiz (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, zeta), Hspa1a, H2-Q10, and Btc (betacellulin) in the abdominal aorta were respectively examined in this assay.As displayed in Table 7, the mRNA expression levels of Klf6, Nr4a1, Nfkbiz, Hspa1a, and H2-Q10 in the model group were significantly downregulated, whereas the expression of Btc mRNA was similar to the normal group.PPS upregulated the expression levels of these genes, which was entirely consistent with the RNAseq results.Apart from the expression levels of Nr4a1 in PPS1 group and Btc in PPS3 group, the mRNA levels of the genes in PPS-treated groups were higher than that in the VE-treated group.The transcription levels of Nr4a1 were found to be in a dose-dependent manner.At a dosage of 100 mg/kg, PPS exhibited marked effects on Klf6, Hspa1a, H2-Q10, and Btc genes.All things considered, PPS exerted anti-oxidative, anti-inflammatory, and anti-apoptotic activities by regulating the expression of the genes associated with cell apoptosis, cell proliferation, inflammation, anti-oxidant and so forth.
Oxidative damages are associated with the pathogenesis of many chronic diseases, including atherosclerosis, stroke, chronic inflammatory diseases, cancers, and degenerative diseases (Halliwell & Grootveld, 1987).ROSinduced oxidative damage has distinct physiological and pathophysiological impacts on vascular cells.NO is an important cellular signaling molecule involved in many physiological and pathological processes.It can act directly on VECs and smooth muscle cells to activate guanylyl cyclase, which relaxes blood vessels through elevated cGMP (Archer et al., 1994).In this assay, PPS increased antioxidative enzyme activities, promoted NO production and attenuated MDA and ROS levels, exerting strong antioxidative and cytoprotective activities.Phosphate groups | 1227 YIN et al. enable polysaccharides to become excellent hydrogen atom donors through activating the hydrogen atom of the anomeric carbon, thus enhancing the antioxidant activities of polysaccharides (Hu et al., 2019).Song et al. (2015) reported that phosphorylated pumpkin polysaccharide exhibited relevant higher antioxidant activities and cytoprotective effects both in vitro and in a cell system than unmodified polysaccharide.Moreover, the introduced phosphate groups can trigger electrostatic effect and change the chain conformation of polysaccharides, leading to improved antioxidant activities.Phosphorylated garlic polysaccharide exhibited more powerful hydroxyl radical scavenging effect than unmodified garlic polysaccharide owing to the change in the molecular structure (Chen & Huang, 2019).Hence, we conjectured that the introduction of phosphate groups contributed to the enhanced antioxidant activities of PPS, which might play a key role in the protective actions of PPS towards VECs.
Apoptosis is an accurately controlled, energydependent process of cell death, normally occuring during the development and aging.It is considered as an essential homeostatic mechanism that maintains cell populations in tissues (Norbury & Hickson, 2001).However, excessive apoptosis may gradually lead to the loss of the target-cell population and its effector functions (Brunner & Mueller, 2003).Accumulating evidence has revealed that ROS-mediated oxidative stress is a key inducer of cell apoptosis.Our results suggested that PPS attenuated the apoptosis of vascular endothelial cells induced by H 2 O 2 and D-galactose via suppressing ROS overproduction.Atherosclerosis is closely related to the development of CVD and the endothelial cell apoptosis is the first step of atherosclerosis (Duan et al., 2021).Therefore, PPS might have great potential to ameliorate atherosclerosis due to its capacity of minimizing endothelial cell apoptosis.MMP is a key indicator of mitochondrial activity because it reflects the process of electron transport and oxidative phosphorylation, and the driving force behind ATP production (Lee et al., 2019).The dissipation of MMP constitutes an early irreversible step in the cascade of events leading to apoptosis (Zamzami et al., 1996).Mitochondria disfunction may lead to decreased MMP and excessive ROS, and thus induce apoptosis (Suski et al., 2012).Therefore, chemical constituents that contribute to enhance the level of MMP may lessen ROS generation and protect VECs from oxidative damage or apoptosis.PPS treatment led to the increase of SOD, CAT, and GSH-Px activities and the decrease of ROS levels in the HUVECs and the mice serum, which might contribute to the augmentation of MMP levels and the ensuing reduction in apoptosis.Das et al. (2012) found that nitrones protected endothelial cells against oxidative stress by modulating phase II antioxidant enzymes and subsequently inhibiting mitochondria-dependent apoptotic cascade, which was similar to our results.
Endothelial cell injury is a critical event in acute inflammatory processes, during which inflammatory cytokines and reactive oxygen metabolites may be involved (Yamaoka et al., 2002).TNF-α, a potent inflammatory cytokine, can induce the expression and release of various   (Ding et al., 2022).It was reported that TNF-α and IFN-γ treatment promoted distinct expression of a range of endothelial surface makers (selectins, ICAM-1, and VCAM-1) (Jaczewska et al., 2014).Here, PPS exerted beneficial effects on endothelial cells by abating the secretion of inflammatory cytokines and cell adhesion molecules susceptible to damage the endothelial cells.Endothelial dysfunction, an important pathological mechanism for the development of atherosclerosis, can be triggered and deteriorated by a cluster of cardiometabolic risk factors and then provokes oxidative stress, inflammation, cell cycle arrest or apoptosis in the endothelial cells (Sun et al., 2019).Anti-oxidation and antiinflammation interventions are considered to be one of the most effective strategies to inhibit endothelial cell damage and accelerate the recovery of the endothelium functions so as to prevent the occurrence and development of atherosclerosis.In this experiment, RNA-seq analysis revealed that PPS regulated the expression of cell proliferation-related genes Kit, Btc, and Btg2 (B-cell translocation gene 2), antioxidant-related genes H2-Q10 and Hspa1a, cell apoptosis and survival-related genes Nr4a1, Nr4a3 (nuclear receptor subfamily 4, group a, member 3), Nfkbiz, Ucp2 (uncoupling protein 2), Hapa1b (heat shock protein family a (Hsp70) member 1b), Tnfrsf10b (tumor necrosis factor receptor superfamily, member 10b) and Sfrp5 (secreted frizzled-related protein 5), inflammation-related genes C5ar1 (complement component 5a receptor 1), Dusp1 and Errfi1 (ERBB receptor feedback inhibitor 1), and angiogenesis, repairment and NO production-related genes Jun, Klf6 and Ddah1 (dimethylarginine dimethylaminohydrolase 1).However, the most enriched KEGG pathway analysis found no network or crosstalk between these genes.We conjectured that PPS might exert its cytoprotective activity by directly regulating the expression of these genes.

DEGs
H2-Q10, a fully reduced form of coenzyme Q10 (ubiquinol), has robust anti-oxidant and free radical scavenger effects and is widely used in clinic for cardiovascular and cerebrovascular diseases, nervous system diseases, hypertension, diabetes and so on (Grammenandi et al., 2016).In current study, the increased endogenous enzymes including SOD, CAT, GSH-Px, and H2-Q10 contributed to the antioxidative effect of PPS.Dusp1, a negative regulator of the MAPK signaling pathway, is involved in various cellular responses including inflammation, cell proliferation, differentiation, apoptosis, stress responses, and immune defense (Peng et al., 2017).It was reported that Dusp1 participated in the regulation of hypoxia-induced RAW264.7 macrophage inflammatory and immune response by modulating the expression and the secretion of TNF-α, IL-10, and IL-1β (Dong et al., 2020).Likewise, Errfi1 plays an important role in lessening inflammation, apoptosis and proliferation.It was reported that overexpression of Errfi1 could prevent the proliferation of nucleus pulposus cells and pro-inflammatory cytokine (IL-6, TNF-α, and IL-1β) production (Guo et al., 2019).We suggested that the decreased TNF-α levels in mice serum might be related to the overexpression of Dusp1 and Errfi1 in response to PPS treatment.The anti-apoptosis effect of PPS on VECs might also be mediated by the up-regulated genes Nr4a1, Hspa1a, Hapa1b, Nfkbiz, Sfrp5 and downregulated gene Ucp2.To our surprise, the up-regulation of pro-apoptosis genes Tnfrsf10b and Nr4a3 occurred concurrently, and the upregulation of pro-proliferative genes Kit/Btc and anti-proliferative gene Btg2 also happened simultaneously.We conjectured that the upregulation of Tnfrsf10b, Nr4a3, and Btg2 might help prevent T A B L E 7 Effect of PPS on the expression levels of genes in mice abdominal aorta.NG, normal group; MG, model group, mice treated with D-galactose alone (100 mg/kg/day, ip) for 60 days; VE, positive control group, mice treated with VE (200 mg/kg/day, ig) and D-galactose (100 mg/kg/day, ip) for 60 days; PPS1, PPS2 and PPS3, mice respectively treated with 50, 100 or 200 mg/kg/day PPS (ig) and D-galactose (100 mg/kg/day, ip) for 60 days.Compared with the normal group, *p < 0.05, **p < 0.01; compared with the model group, ∆ p < 0.05, ∆∆ p < 0.01.VECs from excessive proliferation.In addition, genes such as Jun, Klf6 and Ddah1 might also have important impacts on the protective activity of PPS.

| CONCLUSIONS
Current results showed that PPS had significant antioxidation, anti-inflammation, and anti-apoptosis activities in vitro and in vivo, implying its powerful protective capacity on VECs.The DEGs, including Klf6, Kit, Nr4a1, Nr4a3, Nfkbiz, C5ar1, Jun, H2-Q10, Btc, Dusp1, Hspa1a, Hapa1b, Errfi1, Btg2, Ucp2, Ddah1, Tnfrsf10b and Sfrp5, contributed to the protective effect of PPS.In light of the involvement of these genes in the functions of damaged vascular endothelium repairment, cell growth and proliferation, cell survival and apoptosis, inflammation, antioxidant, NO production inducement and angiogenesis, the molecular mechanisms underlying the protective actions of PPS might be characterized by synergistic effects of multiple targets.The phosphorylated AMC polysaccharides might provide us a novel type of polysaccharide derivative with more potentials for prevention and treatment of CVD.

F I G U R E 1
The UV (a), FT-IR (b),13 C NMR (c) and 31 P NMR (d) spectra of PPS and polysaccharide.

3. 4 |
Protective effect of phosphorylated polysaccharide on mouse VECs 3.4.1 |HE and TUNEL staining

F
I G U R E 3 Apoptosis (a) and MMP (b) analyses of HUVECs by flow cytometry.NG, normal group; MG, model group, cells treated with 300 μM H 2 O 2 alone for 50 min; VE, positive control group, cells treated with 10 μM VE for 24 or 48 h and 300 μM H 2 O 2 for 50 min; PPS, cells treated with 150 μg/mL PPS for 24 or 48 h and 300 μM H 2 O 2 for 50 min.Compared with the normal group, *p < .05,**p < .01;compared with the model group, ∆ p <.05, ∆∆ p < .01.

F
Effect of PPS on genes in mice abdominal aorta, compared with the model group (MG).(a), the volcano plot for DEGs (|log 2 FC| ≥1 and adjusted p value of <0.05), the yellow and blue circles indicate up-and downregulated genes, respectively; (b), GO analysis for the biological attributes of DEGs; (c), KEGG pathway enrichment analysis of DEGs.The figures were representative of three independent experiments.T A B L E 6 Differentially expressed genes associated with the protective effect of PPS on vascular endothelial cells.
Effect of PPS on SOD, CAT, GSH-Px, ROS, NO, eNOS and ET-1 in mice serum.NG, normal group; MG, model group, mice treated with D-galactose alone (100 mg/kg/day, ip) for 60 days; VE, positive control group, mice treated with VE (200 mg/kg/day, ig) and D-galactose (100 mg/kg/day, ip) for 60 days; PPS1, PPS2 and PPS3, mice respectively treated with 50, 100 or 200 mg/kg/day PPS (ig) and D-galactose (100 mg/kg/day, ip) for 60 days.Compared with the normal group, *p T A B L E 4