Introduction

Blood is a crucial component in human body, comprising approximately 7%-8% of the body weight of an adult. In the circulatory system, blood functions not only as a transport medium but also plays a pivotal role in maintaining redox homeostasis through its intrinsic antioxidant defense network. Oxidative stress arises when there is an imbalance between oxidant generation and antioxidant capacity, leading to the accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), ultimately resulting in cellular injury and dysfunction1. The serum contains several key antioxidant enzymes, including glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), collectively forming an essential enzymatic defense against oxidative injury2. Additionally, glutathione reductase (GR) supports this antioxidant system by sustaining the glutathione (GSH) cycle and regeneration, thus enhancing the serum’s capacity for ROS detoxification3. This collective enzymatic activity effectively mitigates oxidative stress-induced inflammation, cellular damage, and apoptosis4. Additionally, the ratio of reduced to oxidized glutathione (GSH/GSSG) is widely regarded as a sensitive indicator of the oxidative state in blood, reflecting dynamic shifts in antioxidant capacity5. Recent advances in proteomic technologies have enabled systematic analysis of the expression patterns and regulatory mechanisms of these antioxidant enzymes and markers, providing valuable insights into the complex redox regulation within blood6.

Conventional “biological plasma” refers to the cell-free extracellular fluid obtained from blood after the removal of cellular components; whereas “plasma” here is referred to the “physical plasma”, which is the ionized gas also known as the fourth state of matter, and represents the macroscopic system in the unbound state with quasi-electrical neutrality7. Notably, plasma emissions mainly consist of free electrons, free radicals, positively- or negatively-charged particles, and neutral reactive oxygen/nitrogen species (ROS/RNS)8. Typically, ROS and RNS are related to various biological processes and have been identified to be the major effectors of plasma on biological tissues and cells9,10. As a presently widely adopted source of plasma for biomedical applications, atmospheric pressure plasma (APP) jet can confine the plasma into a smaller space and keep a gas flow temperature close to room temperature, allowing the direct treatment of biological target. Therefore, studies have reported the applications of APP in many biomedical fields including stomatology, disinfection and sterilization, coagulation, and cancer treatment11,12.

In recent years, extensive research on the interactions between APP and liquids has garnered significant attention. In response to this emerging interdisciplinary domain, Patrick first coined the term “plasma physics of liquids”13. There are numerous studies elucidating the mechanisms of APP interactions with various liquids, including water, diverse solutions, oils, buffer solutions, and culture media, as well as their applications in the environmental, material processing, agricultural, and medical fields14,15. However, research on the interaction between plasma and the blood system, particularly serum, remains relatively limited. Some studies on the effects of APP treatment on blood have primarily focused on coagulation16. Meanwhile, it is also indicated that plasma treatment of blood accelerates the coagulation process by promoting the aggregation of platelets and the formation of platelet-like membrane structures. Further analysis using mass spectrometry of plasma-treated platelet supernatant reveals the up-regulation of 16 proteins, which may be crucial for the coagulation-promoting process17.

Currently, most studies concentrate on the impact of APP on the intracellular redox state, including the APP generated reactive species on cellular oxidative stress, leading to lipid peroxidation, DNA damage, mitochondrial dysfunction, and apoptosis18. In contrast, relatively few studies are performed to explore the antioxidant capacity of APP. Meanwhile, there is no direct evidence regarding the interaction of APP treatment with serum. Consequently, this work focused on elucidating the APP-serum interactions and the underlying mechanisms. Here, we prepared a needle-to-ring dielectric barrier helium (He) discharge plasma jet device and used it for rat serum treatment. The reactive species in the serum were detected and compared with those in the plasma-treated saline. Then, the serum glutathione levels and proteomics were analyzed to clarify the mechanisms of plasma-serum interactions.

Methods and materials

Ethical approval

All animal experiments were conducted following the regulatory standards approved by the Animal Ethics Committee of Beijing Neurosurgical Institute (approval no. BNI202406001). The study was conducted in compliance with the ARRIVE guidelines (https://arriveguidelines.org). All methods were carried out in accordance with relevant guidelines and regulations.

Animals and serum collection

Specific pathogen-free (SPF) male adult Sprague–Dawley rats (weight 250–300 g, n = 10) were obtained from the Beijing Vital River Laboratory Animal Technology Co., Ltd. Animals were raised at 24 ± 2 °C, 50%–60% humidity, and 12-h/12-h light–dark cycle conditions, with four rats per cage. Animals were allowed to take water and food freely.

At 7 days post-adaptation, rats received intraperitoneal injection of the mixture comprising 10 mg/kg xylazine and 100 mg/kg ketamine for anesthesia. Then, blood was sampled in the abdominal aorta and centrifuged for 15 min at 2000 g to obtain serum, which was subsequently retained and preserved at − 80 °C for subsequent analyses.

APP device

Figure 1a shows a sketch map for our experimental setup. To be specific, APP was generated with the needle-to-ring dielectric barrier discharge (DBD) structure. In this setup, one tungsten needle (radius of curvature, 15 μm) was utilized to be the HV electrode connected with the sinusoidal high-voltage AC power supply, while one aluminum ring encircled the quartz tube (inner and outer diameters, 1 and 3 mm separately), functioning as the ground electrode. More details regarding the plasma source as well as the discharge conditions can be found in our previous report19. A sinusoidal voltage at the 5 kHz frequency was applied to the needle electrode, whereas the low voltage probe (Tektronix TPP1000) was adopted for collecting the current flowing through the sampling resistance (R = 1 kΩ). In the experiment, the voltage applied in the needle had a peak-to-peak value of 5.6 kV, as measured through the oscilloscope (Tektronix DPO 4104B) through the high-voltage probe (Tektronix P6015A). At the same time, high-purity helium (volume fraction 99.999%) was injected in this tube at the 1.4 slm continuous flow rate. The fiber optic spectrometer (AvaSpec‐ULS3648) was used to monitor the APP emission spectrum, which simultaneously obtained information in different wavelength ranges (200–900 nm). The gas temperature of APP was predicted through fitting the transition line of OH radical \(\left( {{\text{A2}}\sum + ({\upnu }^{\prime } = 0) \to {\text{X2}}\prod \left( {{\upnu }^{\prime \prime } = 0} \right)} \right)\), 306–312 nm, where A and X stand for high and low electron energy states, separately) according to the emission spectrum of APP. The best fitting results between the experimental spectra and the simulated spectra were obtained by using the LIFBASE Spectroscopy Tool (SRI International).

Fig. 1
figure 1

(a) APP experimental platform and diagnostic system diagram. (b) APP voltage-current waveform diagram, at the 5 kHz frequency, 5 kV voltage, and 1.4 slm gas flow rate. (c) The APP emission spectrum at the quartz nozzle. (d) The diagram showing the typical gas temperature of APP, Tgas ≈ Trot = 310 K. APP: atmospheric pressure plasma.

Full size image

Serum treatment

Serum from one rat was randomly divided into the control group and different APP treatment groups. The Eppendorfs containing the 200 μL serum were placed right below the nozzle for APP treatment, with the nozzle-serum surface distance being fixed at 10 mm. Three replicates were set for each group. The serum samples without APP treatment served as controls, while serum samples in the treatment group received APP treatment for the indicated durations.

Detection of nitric oxide (NO) production

The serum NO concentrations in different groups were measured with the NO assay kit (Beyotime, Haimen, China) on the basis of Griess reaction following specific instructions. NO concentrations were calculated by determining NO2− level at 540 nm and the NaNO2 standard curve (M200 Pro, Tecan, Switzerland). Meanwhile, the content of total NO was analyzed through determining nitrate and nitrite with the Total Nitric Oxide assay kit (Beyotime, Haimen, China) according to the manufacturer’s protocols. The nitrate was firstly converted to nitrite, and the nitrite concentrations were later assayed with the Greiss reaction.

ROS measurement

The serum ROS levels were determined using hydrogen peroxide Assay kit (Beyotime, Haimen, China). Serum samples in different groups were incubated with detection reagent for 30 min at ambient temperature, while absorbance values were measured at 560 nm (M200 Pro, Tecan, Switzerland). The H2O2 level (μM) was measured based on the standard curve.

Reduced glutathione (GSH) and oxidized glutathione (GSSG) measurements

The serum GSH levels were determined using GSH and GSSG Assay Kits (Beyotime, Haimen, China). The absorbance values were recorded at 412 nm (M200 Pro, Tecan, Switzerland) to ascertain the total glutathione (GSH + GSSG) concentration based on a standard curve. Subsequently, GSH was cleared with suitable reagents for measuring the GSSG level. Later,the GSH level was obtained through taking away GSSG level from the total glutathione level.

Proteomics profiling of serum

This study also conducted proteomics analysis for clarifying how APP treatment affected serum composition. To this end, 200 μL serum was treated with APP for 240 s and supplemented with 1% protease inhibitor (LabLead, China). The proteomics experiments were performed at Jingjie PTM BioLab (Hangzhou), Co, Ltd, Hangzhou, China. In brief, 5 mM dithiothreitol was added to reduce proteins at 56 °C for a 30-min duration. Then, 11 mM iodoacetamide was employed for 15 min of alkylation at room temperature, whereas 100 mM TEAB was added to dilute the mixture to a urea concentration < 2 M. Thereafter, trypsin at 1:50 was poured for overnight digestion and that at 1:100 was introduced for additional 4 h of digestion, followed by peptide desalting using a C18 SPE column. By utilizing the home-made reversed-phase column, we then conducted Liquid Chromatograph Mass Spectrometry (LC–MS/MS) analysis was later conducted with a gradient of solvents A (2% acetonitrile, 0.1% formic acid within water) and B (90% acetonitrile, 0.1% formic acid within water) using the EASY-nLC 1200 UPLC system. Orbitrap Exploris 480 was used for MS analysis with specific settings for full-MS and MS/MS scans. Further, the Data-Independent Acquisition (DIA) data were processed using the DIA-NN search engine. Tandem mass spectra were searched based on Rattus_norvegicus_10116_PR_20230103.fasta (47,945 entries) concatenated with reverse decoy database. The threshold of differential expression was set at 1.5-fold change. False discovery rate (FDR) was set at < 1%.

Bioinformatics analysis

Gene Ontology (GO) analysis involved extracting GO IDs from the identified proteins using eggnog-mapper based on the EggNOG database, and classifying proteins into cellular component, molecular function, and biological process categories. Domain annotation was later conducted to identify the conserved protein regions using the Pfam database. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation involved identifying protein-enriched pathways through BLAST comparisons. Besides, subcellular localization annotation predicted the protein locations in eukaryotic and prokaryotic cells. The biological states were summarized by the HallMark’s Signature Gene Sets. Through transcription factor annotation, proteins were identified as transcription factors. Functional enrichment was assessed using Fisher’s exact test on differentially expressed proteins (DEPs). Enrichment-based clustering was performed to group proteins based on functional classifications. Protein–protein interaction (PPI) networks were constructed with STRING database (https://stringdb.org/; v.10.5) and visualized with the visNetwork R package.

Target proteins quantified through parallel reaction monitoring (PRM)

For further confirming the proteomics data, fifty DEPs were selected and measured through PRM-MS analysis at Jingjie PTM BioLab (Hangzhou), Co, Ltd, Hangzhou, China. After defining signature peptides for target proteins, just unique peptide sequences were applied in PRM analysis. Proteins were prepared and trypsinized according to the descriptions in proteomics experiments. Following LC–MS/MS analysis, the PRM acquisition approach was adopted to collect data from every sample to measure target protein levels. There were three biological replicates being set in every group. Skyline (v.3.6) was applied in processing those resulting MS data, under specific settings for peptide and transition parameters.

Superoxide dismutase (SOD), total antioxidant capacity (T-AOC) and catalase (CAT) activity assay

Total SOD Assay Kit with WST‐8 (Cat#S0058), T-AOC Assay Kit with FRAP approach (Cat#S0119) and CAT Assay Kit (Cat#S0051), which were provided by Beyotime Institute of Biotechnology (Haimen, China), were utilized to test SOD, lipid peroxidation, T-AOC and CAT activities. Every experiment was carried out in line with specific instructions. Data were recorded with the microplate reader (M200 Pro, Tecan, Switzerland).

Glutathione peroxidase (GPX) activity measurement

The serum GPX activity was further confirmed based on the proteomics and PRM results in line with the GPX Assay Kit instructions (S0058, Beyotime, Haimen, China). The serum in each group was blended into working solution and supplemented with peroxide reagent. Then, absorbance values were measured at 340 nm. The GPX activity was calculated by the relevant formula based on nicotinamide adenine dinucleotide phosphate (NADPH) reduction.

Determination of glutathione reductase (GR) activity

The glutathione-mediated NADPH oxidation was monitored to analyze GR activity at OD412 nm using the GR Assay Kit (Cat#S0055, Beyotime, Haimen, China) according to the manufacturer’ instructions. Briefly, 5 μL serum (after tenfold dilution with saline) was added to the total reaction system containing GSSG, NADPH and DTNB (5,5ʹ-dithiobis-(2-nitrobenzoic acid). Then, absorbance values at OD 412 nm were continuously recorded every 2 min for 10 min at 25 °C. Results were denoted as U/mg protein.

Statistical analysis

Results were represented by mean ± standard deviation (SD). Prism software (version 8.2.1) was employed for data analysis. Multiple groups were compared by one-way ANOVA followed by Tukey’s multiple comparisons, whereas two groups were compared by Student’s t-test. P < 0.05 stood for significant differences.

Results

APP characteristics

Figure 1b shows the current–voltage waveform diagram at the 5 kHz frequency, 5 kV voltage and 1.4 slm gas flow rate. The applied voltage had a current pulse every half cycle. The discharge current cycle was identical to the applied sinusoidal voltage. The positive current pulse amplitude was 2.88 mA, the voltage was 920 V, the negative current pulse amplitude was 1.92 mA, the voltage was -212 V, and the discharge current appeared in a single cycle. There were two asymmetric current pulses (positive and negative). Figure 1c shows the emission spectra of APP at the quartz nozzle. Multiple excited He atomic lines in the emission spectrum were observed, including He 587.6, He 667.8, He 706.5 and He 728.1 nm. Among them, the excited state He 706.5 nm spectral line was the strongest, corresponding to the transition from He (33S1) to He (23P0) state. Based on diffusion, N2 and O2 in the atmospheric air participate in a series of ionization reactions. There were also nitrogen spectra (mainly concentrated in 300–500 nm) and oxygen spectra (mainly concentrated in 700–900 nm) in the spectral lines. Figure 1d shows the typical fitting results under the same discharge condition, where Tgas and Trot represent the gas temperature and rotational temperature, respectively. APP had a gas temperature of 309 K, approaching room temperature and suitable for serum treatment.

APP treatment slightly increases serum ROS levels compared with saline

First of all, the APP treatment-induced changes in reactive species in serum and saline were compared. As shown in Fig. 2, certain basal ROS, NO and total NO concentrations were detected in serum, while gradual increases in ROS and NO contents with the increasing treatment time were also detected after APP treatment. When the treatment time reached 240 s, the concentrations of ROS, NO and total NO reached 26.93 ± 7.291 µM (Fig. 2a), 6.435 ± 1.138 µM (Fig. 2b) and 17.55 ± 2.006 µM (Fig. 2c), separately, which were remarkably higher than those in the control groups. In saline, APP treatment also induced significant changes in NO, total NO, and ROS contents in a treatment time-dependent manner, with their concentrations reaching 90.55 ± 15.90 µM (Fig. 2d), 1.301 ± 0.220 µM (Fig. 2e) and 3.080 ± 1.049 µM (Fig. 2f) after 240 s of treatment, respectively. It should be noticed that, the APP treatment-induced changes in ROS levels in saline were much greater than those induced by plasma in serum, implying that plasma may have altered the antioxidant properties and inhibited ROS production in serum. Meanwhile, the serum itself has a certain concentration of nitrite and nitrate, resulting in higher basal NO values than saline. Therefore, the NO concentration in serum increases compared to saline with increasing APP treatment.

Fig. 2
figure 2

Impacts of APP treatment on the reactive species production in serum and saline. The H2O2 (a), NO (b) and total NO (c) concentrations in serum were measured. The H2O2 (d), NO (e) and total NO (f) concentrations in serum were detected. Results were represented by mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with the untreated control group (n = 3). APP: atmospheric pressure plasma; H2O2: Hydrogen peroxide; NO: Nitric Oxide.

Full size image

APP treatment alters serum proteome expression

Next, we conducted proteomics analysis for examining changes in protein expression in serum after APP treatment. Relative to control group, altogether 59 DEPs could be detected from the treatment group (Fig. 3a), including 29 up-regulated whereas 30 with down-regulated ones. The volcano plot of DEPs displaying down-regulated proteins and up-regulated proteins, respectively, is shown. The names of the top 5 up- and down-regulated DEPs are labeled in the figure (Fig. 3b). Then, this study carried out GO and KEGG analysis to obtain an overview of the functions of these significant proteins. The most significant enrichment terms for GO were COP9 signalosome, protein deneddylation and azurophil granule lumen (Fig. 3c). Moreover, based on KEGG analysis, DEPs exerted an important effect on the glutathione metabolism and the leukocyte migration signaling pathway (Fig. 3d). Then, the expression of Glutathione Peroxidase 1 (GPX1), an enzymatic antioxidant, was further analyzed in the subsequent PRM validation. The fragment ion area distribution of the selected peptide in the samples quantified using the peptide peak area is shown in Fig. 3e. Clearly, the APP-treated group showed an elevated peak area compared with the untreated group. Based on these findings, GPX protein content significantly elevated after treatment. Then, the “visNetwork” tool of R package was employed to visualize the DEP interaction network, as shown in Fig. 3f. For showing PPIs, those top 50 proteins with the closest interactions were selected and the PPI network was mapped (Fig. 3f).

Fig. 3
figure 3

Bioinformatics analysis of the DEPs in APP-treated serum. (a) Heatmap displaying hierarchical clustering of DEPs in rats. Red and blue stand for high and low expression, separately, whereas gray indicates not significant. (b) Volcano plot showing the DEPs in rats and mice against the control samples. Red and blue indicate up-regulated and down-regulated proteins, respectively. GO (c) and KEGG (d) pathway analyses were performed among 59 DEPs. (e) PRM validation of GPX1. (f) The constructed PPI networks. Confidence score > 0.7. APP: atmospheric pressure plasma; DEPs: differentially expressed proteins; GO: Gene Ontology. KEGG: Kyoto Encyclopedia of Genes and Genomes; PRM: Parallel Reaction Monitoring. PPI: Protein–protein interaction; GPX1: Glutathione Peroxidase 1.

Full size image

APP treatment increases the GSH level in serum

Therefore, the antioxidant capacity of serum in each group was further examined by measuring the oxidized GSSG and reduced GSH levels. As shown in Fig. 4a, APP treatment slightly elevated GSSG level in the APP treatment groups. The serum GSH level, which serves as an antioxidant agent excessively consumed in oxidative stress injury, was significantly enhanced by APP treatment time-dependently (Fig. 4b). The GSH/GSSG ratio, which is usually adopted to indicate oxidative stress and damage in toxicological research, was calculated. From Fig. 4c, APP treatment modestly restored the serum GSH/GSSG ratio, which significantly increased after 240 s of APP treatment (P < 0.05), suggesting the enhanced antioxidant capacity of APP-treated serum.

Fig. 4
figure 4

Effects of APP treatment on GSSG (a), GSH (b) levels and GSH/GSSG ratio (c). Results are represented by mean ± SD. *P < 0.05 versus control group (n = 3). APP: atmospheric pressure plasma; GSSG: Glutathione disulfide. GSH: Glutathione.

Full size image

Impact of APP treatment on antioxidant enzymes

The antioxidant enzymes in APP-treated serum were further examined. Compared with the untreated control group, GPX, GR, and SOD enzyme activities increased remarkably after 240 s of APP treatment, whereas CAT activity decreased. Then, the T-AOC of the APP-treated serum was analyzed. According to Fig. 6, the T-AOC was significantly enhanced after 240 s of APP treatment compared with the control group, explaining why APP-treated serum could scavenged ROS.

APP-treated serum scavenges ROS in saline

To further verify the antioxidant property of the APP-treated serum, 200 µL saline or serum was treated by APP separately for another 240 s. It was observed from Fig. 5 that, the concentrations of ROS increased dramatically in saline after APP treatment. Although the ROS contents in serum after APP treatment also increased significantly, the multiplicity of change was significantly smaller than that of APP-treated saline. Furthermore, 20 µL untreated or APP-treated (240 s) serum was added to 180 µL APP-treated (240 s) saline, respectively. The results showed that the APP-treated serum scavenged ROS in the saline, which verified the antioxidant property of the APP-treated serum (Fig. 6).

Fig. 5
figure 5

Impacts of APP treatment on the antioxidant enzymes. Serum GPX (a), GR (b), SOD (c) and CAT (d) activities of diverse groups were measured. (e) The T-AOC of serum in different groups was calculated. Results were represented by mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 relative to untreated control group (n = 3). GPX: Glutathione peroxidase; GR: Glutathione reductase; SOD: Superoxide dismutase; CAT: Catalase; T-AOC: Total antioxidant capacity.

Full size image
Fig. 6
figure 6

The ROS scavenging capacity of APP-treated serum. (a) Flowchart of this experiment. (b) The H2O2 concentrations in each group were measured. Results were represented by mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001 versus control group (n = 3). ROS: reactive oxygen species; APP: atmospheric pressure plasma; H2O2: Hydrogen peroxide.

Full size image

Discussion

In our prior research, transnasal APP inhalation protects the brain from ischemic injuries, but it remains unknown how the plasma acts on the area of cerebral ischemic injury. The nasal mucosa has an adequate blood supply and is distributed with abundant capillaries, which is an important route for transnasal drug absorption20. It is hypothesized that transnasal absorption into the bloodstream may be an important way for APP inhalation to act on the ischemic brain injury area. Therefore, this work first detected how APP treatment affected serum. It was found that the ROS contents in serum after APP treatment did not increase as much as those in the APP-treated saline, while the NO content increased to about the same extent as those in APP-treated serum and saline. To explore the underlying mechanisms, the proteomics change in APP-treated serum was investigated through LC–MS/MS and bioinformatics analyses. There were 59 DEPs identified in APP-treated serum in comparison with control group. As revealed by KEGG enrichment, the above DEPs were predominantly associated with the glutathione metabolism pathway. Therefore, the GSH and GSSG levels were further evaluated. Based on our results, APP treatment significantly increased the serum GSH level and GSH/GSSG ratio, suggesting the enhanced antioxidant capacity of APP-treated serum. Furthermore, the antioxidant enzymes in APP-treated serum were examined. As a result, the GPX protein level and enzyme activity both increased according to the PRM analysis and GPX enzyme activity analysis. Meanwhile, the GR and SOD enzyme activities were also enhanced after APP treatment, whereas the CAT activity decreased. The T-AOC was also improved, as evidenced by the better ROS scavenging capacity of the APP-treated serum.

The interaction between non-thermal plasma and the liquids has been fully investigated. Based on the reactive species (such as ·OH, O·, ·OH, HO2·, O2-·, NO·, NO2·) produced when plasma interacts with liquids, the efficacy in eliminating toxic and environmentally persistent substances, as well as organic compounds in polluted water has been extensively confirmed21,22. The plasma-activated liquids show significant promise in the realm of biomedicine based on the reactive species and the oxidative/nitrative stresses. The utilization of plasma-activated solutions (PAS), like water, saline, Ringer’s lactate, culture medium, and phosphate-buffered saline (PBS), as an important complementary application way to direct plasma treatment, has demonstrated favorable treatment effects and application prospects in sterilization, tumor treatment and skin disease management23,24,25. Furthermore, recent studies have reported the wound healing effects and anti-cancer properties of plasma-activated oil (PAO), which are achieved by the plasma-generated reactive agents26,27. Different from most previous studies, the present work showed that the APP treatment-induced changes in the serum ROS levels were much smaller than those induced by plasma in saline, and the extent of plasma-induced change in the NO level differed slightly between saline and serum, implying the potential antioxidant properties of the plasma-treated serum.

We further analyzed how APP treatment affected serum antioxidant activities. Previously, plasma treatment is found to produce varying amounts of reactive species in diverse liquid media28. Under the identical plasma exposure conditions, ROS accumulation is higher in PBS than in saline solution29, indicating that the chemical properties of the medium have a substantial impact on both the generation and consumption of ROS/RNS30. This effect is particularly evident in complex organic-containing solutions, where both the ROS/RNS production rates and levels vary significantly. The efficacy of plasma treatment differs depending on the liquid composition31. For instance, the components of culture media, especially fetal bovine serum (FBS), significantly influence ROS concentration and their impact on cells32. Plasma treatment of serum-containing culture media results in significantly lower ROS levels than that those of the serum-free media, likely due to the components in FBS scavenging-specific ROS/RNS, thereby mitigating the influence of plasma treatment on tumor cells33. Abraham et al. demonstrated that plasma-generated ROS/RNS were stable within PBS, yet they were scavenged by the processed whole blood and blood plasma (BP). Following primary scavenging, RNS was still stable within BP, but they were totally reduced within processed whole blood, emphasizing a crucial role of serum in antioxidative defense34. In this study, APP-treated serum exhibited superior ROS scavenging capacity to untreated serum, underscoring a substantial enhancement of antioxidative potential induced by plasma treatment.

Furthermore, the mechanism of APP in serum antioxidant properties was explored.The serum contains a certain amount of basal nitrite /nitrate and ROS, as well as enzymatic and non-enzymatic antioxidant systems to maintain a state of redox homeostasis35,36. Notably, NO and its derivatives (e.g., nitrite and nitrate) are more stable and less susceptible to rapid degradation under the antioxidant systems of serum37.The enzymatic antioxidants, including GPX, CAT, SOD and thioredoxin reductase (TrxR), play key roles in mitigating oxidative stress induced by ROS and free radicals38. SOD is primarily responsible for converting superoxide anion radicals into hydrogen H2O239. Subsequently, GPX and CAT act in concert to further convert H2O2 into water and oxygen, preventing its accumulation and maintaining the redox homeostasis40. Collectively, these enzymatic systems function synergistically to counteract oxidative stress41. On the other hand, non-enzymatic antioxidants, including vitamin C, uric acid42, bilirubin39, vitamin E43, and glutathione44, directly neutralize free radicals or reactive species, thus protecting biomolecules in serum from oxidative damage. These non-enzymatic antioxidants work in synergy with enzymatic systems to preserve the systemic redox homeostasis45. Noteworthily, most antioxidant enzymes are predominantly localized intracellularly, and play essential roles in maintaining cellular redox homeostasis. Nevertheless, these enzymes can also be released into extracellular compartments, including serum, under physiological or pathological conditions46. For example, under the pathological conditions of cerebral ischemia, the antioxidant enzyme system in serum exerts a critical effect on maintaining the redox homeostasis and mitigating the oxidative stress-induced damage47. In the current study, the decrease in CAT activity was observed, despite the overall increase in antioxidant markers , which may be attributed to the predominantly intracellular localization of CAT. Previous studies have well-documented the direct interaction between APP-generated reactive species and isolated proteins, along with their significant impacts on protein functionality48. Building upon this evidence, we propose that limited extracellular CAT pool in serum is particularly vulnerable to oxidative modification through direct exposure to APP-derived reactive species, which may account for its diminished activity. This hypothesis, while mechanistically plausible, requires further experimental validation.

In this study, the APP-treated serum was analyzed through proteomics profiling and 59 DEPs were identified compared with control group. According to further KEGG enrichment analysis, the altered proteins were mostly associated with the glutathione metabolic pathway, an important antioxidant pathway in the body49. GPX, the most prominent family of enzymes within the glutathione metabolic pathway, can catalyze the reduction of hydrogen peroxide and organic peroxides and convert these harmful oxidants into harmless water or alcohols, thereby mitigating oxidative damage to cells and maintaining the redox balance40. Our results revealed that APP treatment not only increased GPX4 level but also significantly enhanced GPX activity in comparison with control group. Additionally, APP treatment increased GSH level and GSH/GSSG ratio, thereby augmenting the antioxidant capacity of the plasma-treated serum.

Moreover, the activities of other serum antioxidant enzymes in serum were assessed. Previous studies have identified three common SOD isoforms, namely, cytosolic SOD1, mitochondrial SOD2, and extracellular SOD3, which are present within blood, lymph, and other tissues50. SOD1 and SOD2 are responsible for converting superoxide radicals (O2−) into H2O2, thereby preventing cellular damage caused by free radicals51. Serum SOD3, which is a secreted extracellular Cu/Zn-containing SOD, plays a role in scavenging ROS generated by metabolic processes, thus alleviating oxidative damage from both endogenous and exogenous sources52. GR is another critical enzyme involved in antioxidant defense and responsible for reducing GSSG back to its reduced form GSH, which is pivotal for the cellular antioxidant protection53. By sustaining the GSH level, GR can enhance the cellular antioxidant capacity and strengthen the resilience against oxidative stress54. GR activity is also significant in serum, where it is often used as a prognostic marker and treatment evaluation indicator under conditions such as hepatocellular carcinoma44. Experimental results demonstrated that APP treatment significantly enhanced SOD and GR activities in serum. Although CAT activity was inhibited following APP treatment, the T-AOC of serum was markedly enhanced.

Notably, the observed downregulation of Metallothionein-2 (Mt2) in the serum proteome results, a well-established inducible antioxidant and metal-chelating protein in serum, strongly supports the interpretation of reduced oxidative stress. Mt2 is typically upregulated in response to elevated ROS and oxidative damage55. Therefore, its decreased expression after plasma treatment indicates the alleviation of oxidative burden and the enhanced redox stability. Similarly, the concomitant downregulation of Adam19, a metalloproteinase closely associated with inflammation and oxidative stress-induced tissue remodeling56, further reinforces the hypothesis that APP treatment actively promotes endogenous antioxidant defenses and improves redox homeostasis rather than provoking nonspecific oxidative damage. However, it also should aslo be noticed that Small Nuclear Ribonucleoprotein D1 (SNRPD1), a protein associated with tumor proliferation57, was found to be upregulated. SNRPD1, a core component of the spliceosome, has been found to be highly expressed in various cancers and is closely associated with tumor initiation and progression. Studies have indicated that the overexpression of SNRPD1 may promote cancer cell proliferation and survival by regulating key biological processes such as RNA splicing, cell cycle progression, and DNA replication. With the advancement of liquid biopsy technologies, the detection of SNRPD1 in blood has attracted increasing attention, highlighting its potential utility in cancer diagnosis and prognostic assessment58. In this study, although SNRPD1 was not enriched in any specific pathway, the increase in SNRPD1 expression suggests potential risks related to tumor proliferation that deserve future monitoring during APP treatment. Meanwhile, it may serve as a useful reference for future investigations into the potential adverse effects of APP treatment. Future studies are required to define the optimal treatment parameters and ensure the safety and efficacy of APP- treated serum for biomedical applications.

Similar to the APP treatment method, the ozone autohemotherapy has demonstrated potential applications in improving the blood antioxidant properties, resisting inflammation, improving microcirculation, enhancing immune function, and promoting tissue repair59,60. During ozone autohemotherapy, blood drawn from the patient’s vein is treated with medical-grade ozone and then slowly reinfused intravenously to the patients61. Our results demonstrate that APP treatment represents a novel blood processing technique for enhancing antioxidant properties. These findings suggest that re-infusion of autologous APP-treated serum may be developed as a novel antioxidant therapy, with potential applications in age-related cardio/cerebrovascular protection. Our future studies will prioritize this therapeutic potential. Meanwhile, establishing comprehensive in vivo efficacy and safety profiles is essential to optimize treatment parameters and address potential biosafety concerns, which will be critical for translating APP-based therapies into clinical practice.

Several limitations merit consideration. First, the prolonged APP treatment time, as well as the potential long-term biological effects after APP treatment, whether beneficial or detrimental, should be explored in our future studies. Second, our current analysis primarily examined the serum biochemical markers without directly investigating molecular mechanisms or intracellular antioxidant pathways, future studies will more focus on the sites and mechanisms of APP interaction with serum proteins and the impact on the intracellular functions. Third, detailed baseline characterizations of serum composition would further enhance the reproducibility and allow for deeper mechanistic insights into APP-induced biochemical alterations.

In summary, the present study highlights the impact of APP treatment on serum in vitro. The APP generated by the needle-to-ring helium DBD is applied in the serum treatment in vitro, which is further analyzed by proteomics profiling by LC–MS/MS and bioinformatics analyses. The serum antioxidant ability is remarkably enhanced after APP treatment, which is closely related to the up-regulated serum GSH level, GSH/GSSG ratio and antioxidant enzyme activities after APP treatment. Our results shed new lights and provide a new blood treatment method to enhance its antioxidant properties, which may be potentially used as a novel health care and rehabilitation method for organisms. Meanwhile, validation of these findings in vivo will be an essential next step towards translating our results into clinical applications.