The alleviating effect of low-temperature plasma on bee stings

The alleviating effect of low-temperature plasma on bee stings | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article The alleviating effect of low-temperature plasma on bee stings Li Song, Shouguo Wang This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9016945/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract A great deal of research has been conducted on the surface sterilization and treatment of skin using low-temperature plasma. However, the effect of plasma on subcutaneous toxicity or inflammatory factors has not been experimentally verified to date. Bee stings can trigger a series of allergic reactions, with clinical manifestations mainly including redness, swelling and pain. In this paper, low-temperature plasma was used for the intervention treatment of swelling caused by bee stings, and its therapeutic effect was compared with that of fluticasone propionate cream (hereinafter referred to as unguent), a commonly used drug for mosquito bites. The inactivation effect of dielectric barrier discharge plasma directly applied to the swollen skin of mice on subcutaneous toxic substances was investigated. In the research method, three groups of mice were selected, and melittin was injected subcutaneously into the feet of mice to establish a bee sting model. Then, plasma treatment, drug treatment and no treatment were applied respectively for comparison, and the skin conditions were observed. In addition, the plasma of mice was collected to analyze the levels of inflammatory factors in the blood. It was found that the anti-inflammatory effect on the right paw of mice in the low-temperature plasma treatment group was satisfactory. Compared with the unguent group and the untreated group, low-temperature plasma acted more rapidly on such inflammatory responses. The results demonstrate that direct dielectric barrier plasma treatment on the skin can rapidly inactivate subcutaneous toxic substances and inhibit inflammatory responses, confirming that plasma can not only kill pathogens on the skin surface, but also quickly suppress subcutaneous inflammation and eliminate redness and swelling. In related applications, unlike traditional therapeutic methods based on thermal denaturation, the plasma pen changes the protein spatial structure of bee venom through free charge carriers generated by plasma combined with a high-voltage electric field, leading to the earliest resolution of paw swelling in the low-temperature plasma group. Health sciences/Diseases Biological sciences/Drug discovery Biological sciences/Immunology Health sciences/Medical research Bee sting Low-temperature plasma Therapeutic effect Modification Protein spatial structure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 INTRODUCTION With the rapid development of plasma physics, plasma technology has been widely applied in fields such as biomedicine, food safety, energy engineering, and agricultural production ( 1 – 3 ). Among them, the application of low-temperature plasma in the field of biomedicine has become a research hotspot, showing broad prospects in disinfection, dermatological treatment, wound healing, and tumor treatment. In addition, low-temperature plasma has degradation and inactivation effects on various biological toxins, such as bacterial endotoxins, mycotoxins, and staphylococcal enterotoxins ( 4 – 6 ). Sakudo et al. found through experiments that the α-helix content of bovine serum albumin (BSA) treated with nitrogen plasma increased and the β-turn content decreased; while heat treatment led to a decrease in α-helix content and an increase in β-sheet content. Based on this, they believed that the protein conformational changes induced by nitrogen plasma are mediated by a mechanism different from thermal denaturation ( 7 ). The above effects can reduce the pathological damages caused by toxins to humans and animals, such as fever, disseminated intravascular coagulation, endotoxemia, inflammation, and shock. Bee sting poisoning refers to a toxic disease caused by bees injecting venom into human skin through stinging, with clinical manifestations usually including local redness, swelling, and tingling, and may also present systemic symptoms such as dizziness and nausea ( 8 ). The pathogenic mechanism of bee sting injury is closely related to the toxic components in bee venom and the subsequent allergic reactions. However, there is still a lack of efficient treatment schemes for severe bee sting poisoning, and the mortality rate of related cases is relatively high ( 9 ). Based on the existing research conclusion that low-temperature plasma can reduce the content of toxins in the body, this study attempted to apply low-temperature plasma to the swollen area caused by bee stings, in order to remove the toxins produced during the bee sting process. For patients with severe bee sting poisoning, it is crucial to control the condition in a timely manner and carry out effective treatment. At present, the clinical treatment methods for severe cases are still relatively primitive and single, including traditional Chinese medicine intervention. Such methods generally have the problem of slow onset, which makes it difficult to achieve rapid cure and effectively relieve the pain of patients ( 10 ). In view of the wide application of low-temperature plasma in the degradation and inactivation of various biological toxins, this study compared low-temperature plasma treatment, unguent treatment, and no treatment for bee stings, and concluded that low-temperature plasma has a faster effect on reducing swelling of the swollen paws of mice, and simultaneously explored and explained its mechanism of action. MATERIALS AND METHODS Plasma Pen A plasma pen with output parameters of 5 V DC and 2 A can release plasma when its ceramic probe is close to a conductor. The ceramic probe was used for horizontal scanning treatment on the swollen area of the right foot pad to verify the feasibility of plasma therapy for the bee sting model. Discharge Waveform Discharge waveforms were detected using an oscilloscope, and precise detection could be achieved by combining partial discharge testing technology. The specific measurement process of the oscilloscope is as follows: Equipment Preparation First, connect the oscilloscope to the probe to ensure that the probe calibration is completed and the calibration result is accurate; then select the matching measurement range (such as current range or voltage range) according to the discharge type, and configure key parameters such as DC coupling and trigger point in turn. 1. Parameter Debugging Set the horizontal scanning range to 1 ms/div, adjust the vertical sensitivity to 25 A/div to ensure that the discharge pulse characteristics can be clearly observed; at the same time, adjust the screen display position to center the waveform, ensuring that the waveform is clear and unobstructed. 2. Waveform Analysis Focus on observing the phase characteristics of the discharge pulse and its synchronization relationship with the power frequency signal to determine the period when the discharge phenomenon occurs; adopt the elliptical scanning or horizontal scanning method to effectively distinguish the interference signal from the real discharge waveform, ensuring the accuracy of the analysis results. Reagents Melittin powder (purity ≥ 97%) was dissolved in 0.9% normal saline to prepare solutions with concentrations of 5 mg/mL, 2.5 mg/mL, and 1 mg/mL, respectively. Meanwhile, a 25% urethane solution was prepared. Fluticasone propionate cream was purchased from Hubei Heng'an Fuling Pharmaceutical Co., Ltd. SPF-grade Balb/c male mice (6 weeks old, weighing 20–22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Hubei Branch. During the experiment, the mice had free access to food and water and were reared in an environment-controlled facility. All experimental operations strictly followed the Principles of Laboratory Animal Care and the Guidelines for the Care and Use of Laboratory Animals issued by the State Science and Technology Commission of the People's Republic of China. The experimental protocol was approved by the Ethics Committee of Wuhan Servicebio Technology Co., Ltd., China (approval number: 2025376). All procedures were performed in accordance with the relevant guidelines and regulations. Construction of A Mouse Foot Blister Model and Determination of the Optimal Swelling-inducing Dose of Melittin The mice were randomly divided into 3 groups with 3 mice in each group. The hair on the tail, both hind limbs and feet of all mice was shaved. Subsequently, 10 µL of melittin normal saline solutions with concentrations of 5 mg/mL, 2.5 mg/mL, and 1 mg/mL were injected into the plantar space between the right hind toes of the three groups of mice, respectively. After injection, the mice were left to stand for 5–10 minutes to induce foot swelling, and the group with the most obvious swelling of the right foot was selected to establish the mouse foot swelling model. Groups and Treatments After acclimatization to the environment for 1 week, the mice were randomly divided into 3 groups with 15 mice in each group (n = 15): the plasma group, the unguent group, and the control group. According to the results of preliminary experiments, the optimal concentration of melittin normal saline with the most significant inhibitory effect on redness and swelling was determined to be 5 mg/mL. Subsequently, 10 µL of 5 mg/mL melittin normal saline was injected into the plantar space between the right hind toes of all mice to establish a foot swelling model, and the degree of foot swelling was monitored within 5 minutes after injection. Mice with redness and swelling of the right foot pad were selected and anesthetized with urethane. Plasma group: treated with low-temperature plasma for 1 minute and 20 seconds, with an interval of 5 minutes, for a total of 3 times; the ceramic discharge probe of the plasma pen was horizontally placed 0.5–0.8 cm above the mouse's foot to scan the swollen foot in the horizontal direction. Unguent group: an appropriate amount of fluticasone propionate cream was locally applied to the swollen area of the right hind foot. Control group: no intervention measures were taken ( 11 , 12 ). The redness and swelling of the feet of mice in each group were photographed and recorded at 0.5, 1, 2, 4, 24, 48, and 72 hours after treatment, respectively, and the foot thickness was measured. The degree of edema in the right foot of mice was calculated according to the formula: Edema degree = Thickness of the affected foot − Initial thickness of the affected foot The relative edema inhibition rate was calculated accordingly: Foot swelling inhibition rate = [(Edema degree of control group − Edema degree of intervention group) / Edema degree of control group] × 100% Indicators of Observation The therapeutic effects of the three groups were compared. The improvement of redness and swelling in the right plantar of mice was evaluated by histopathological section analysis and enzyme-linked immunosorbent assay (ELISA) for high-sensitivity C-reactive protein (hs-CRP) levels. High-sensitivity C-reactive protein (hs-CRP) is an important systemic inflammatory marker, whose level increases significantly under inflammatory stimuli such as infection and tissue damage, and can be used as a reliable indicator to evaluate the inflammatory state ( 13 ). ( 1 ) Histopathological Examination Fresh foot tissues were taken to make longitudinal paraffin sections. The paraffin sections were dewaxed to water, immersed in a high-definition constant staining pretreatment solution for 1 minute, stained with hematoxylin-eosin (HE), dehydrated, mounted, and then observed under a microscope to collect images for analysis. ( 2 ) ELISA Detection Mouse C-reactive protein (CRP) ELISA kit was used for detection, and the kit was purchased from Hangzhou Lianke Biotechnology Co., Ltd. 1. Sample Collection and Preservation Blood Collection by Enucleation Hold the skin on the back of the mouse's neck with the left hand, gently press and fix it on the experimental table, and take the lateral position. Press the skin around the mouse's eyes as far back to the neck as possible with the index finger of the left hand to make the eyeballs fully protrude. Quickly clamp and enucleate the eyeball with curved ophthalmic forceps, immediately invert the mouse, and collect the flowing blood with a pyrogen-free and endotoxin-free sterile container. Immediately press with gauze to stop bleeding after blood collection. The blood collection volume by this method is about 0.5-1 mL. The collected blood was left standing at room temperature for 30 minutes to coagulate naturally. 2. Centrifugal Separation Place the coagulated blood sample in a centrifuge and centrifuge at 3000 r/min for 10 minutes. After centrifugation, quickly and carefully separate the serum from the red blood cells to avoid serum contamination caused by red blood cell rupture. 3. Sample Preservation The separated serum can be detected immediately; if not detected temporarily, aliquot it according to a single dosage, store it frozen at -20 ℃, and repeated freezing and thawing is strictly prohibited. Thaw at room temperature before use and mix gently continuously to ensure complete and uniform thawing of the sample. Partial Materials to Be Prepared Standard microplate reader (450 nm), precision pipette and disposable tips, 37 ℃ incubator Result Determination Draw a standard curve: In Excel, draw a linear regression curve with the standard concentration as the abscissa and the corresponding OD value as the ordinate, and calculate the concentration of each sample according to the curve equation. RESULTS Plasma Pen Working Mode The plasma arc generator is composed of a ceramic probe, a pen body and a wire. A 5 V DC voltage and 2 A current were adopted. When the ceramic probe was used for horizontal scanning treatment on the swollen area of the right foot of the mouse, a voltage difference was formed between the probe and the sole of the mouse, inducing purple arc discharge. This discharge was used to treat the bee sting model, as shown in Fig. 1 . Discharge Waveform The discharge waveforms of the plasma pen under different working conditions are as follows: the waveform at room temperature (Fig. 2 A) and the waveform at body temperature (Fig. 2 B). It can be seen from the figures that there are differences in the discharge characteristics of the plasma pen between the animal body temperature condition and the room temperature discharge condition. The discharge at body temperature is smoother and more obvious, indicating that a relatively stable electromagnetic field is formed between the living body and the low-temperature plasma. In addition, various active substances are generated during plasma discharge, including reactive oxygen species (ROS) such as O, ·OH, H₂O₂, and O₃, as well as reactive nitrogen species (RNS) such as NO and NO₂ and related substances ( 14 , 15 ). These active substances can change and destroy the structure of toxins in organisms. If the active substances can enter the cells, they can change the structure of toxic substances in the body's tissue cells, thereby inhibiting symptoms such as inflammation caused by toxic substances and relieving pain. In fact, plasma generated under specific voltage conditions can produce certain biological effects on cells or organisms. Animal experiments conducted by Fathollah et al. showed that cold atmospheric plasma (CAP) generated under the conditions of 8 kV voltage and 6 kHz frequency can significantly promote the wound healing process of diabetic rats ( 16 ). Therefore, under the action of a uniform electric field with 5 V DC voltage and 2 A current, the ion channels on the animal cell membrane open, leading to the formation of pores in the cell membrane. Based on this, the active substances generated by plasma discharge can enter the cells, thereby affecting and changing the protein conformation of melittin ( 17 ). The Improvement of Redness and Swelling Symptoms The redness and swelling of the mouse foot pads were compared at 0.5, 1, 4, 24, and 48 h after treatment, respectively. The results showed that the redness and swelling of the right hind foot in both the control group and the unguent group were relatively severe, while the symptoms in the plasma group were the mildest (Fig. 3 A). At 1 h after treatment, the edema thickness of the plasma group was 1.18 mm, which was lower than that of the other two groups (Fig. 3 B); at the same time, the swelling inhibition rate of the plasma group reached 17.67%, which was higher than 13.72% of the unguent group (Fig. 3 C). This suggests that low-temperature plasma takes effect faster. At 4 h after treatment, the swelling of the plasma group subsided significantly and rapidly (Fig. 3 A). At this time, the morphology of the two feet in the plasma group was basically consistent, with a relative edema degree of 1.07 mm, which was lower than that of the unguent group (1.17 mm) and the control group (1.39 mm) (Fig. 3 B). The swelling inhibition rate of the plasma group reached 23.21%, which was significantly higher than 16.27% of the ointment group, and the inhibition rate of the control group was 0% (Fig. 3 C). At 48 h after treatment, the edema degree related to redness and swelling of the foot pads in the plasma group was still the lowest among the three groups (Fig. 3 B), and the inhibition rate was the highest among all observation time points (Fig. 3 C). In general, compared with the control group and the unguent group, the plasma group had a lower relative edema degree and a higher relative edema inhibition rate, indicating that the therapeutic effect of low-temperature plasma is superior to other commonly used intervention methods. Histopathology Histopathology is a medical technology that observes diseased tissue samples through a microscope, analyzes abnormalities in cell morphology and structure, and clarifies lesion characteristics, which is the core basis for disease diagnosis (11). At 0 h after treatment (Fig. 4 ), obvious tissue deformation, inflammatory cell infiltration, and significant plasma exudation were observed in the plantar tissue sections of the three groups of experimental animals. Plasma containing white blood cells and red blood cells infiltrated into the subcutaneous layer from peripheral blood vessels, which was particularly obvious in the unguent group and the control group. After 1 h, the myocyte morphology in the plasma group recovered to a long spindle shape, while the unguent group and the blank control group (untreated) had not fully recovered, and some cells were still round. At 4 h after treatment, the myocytes in the plasma group recovered well, and the tissue structure returned to a dense state; in contrast, the myocytes in the unguent group and the blank control group were still loose and prone to disintegration. Starting from 24 h, the muscle tissue in the plasma group had recovered to a normal and healthy morphology; the recovery rate of muscle tissue in the unguent group and the blank control group was significantly slower than that in the plasma group, and irreversible damage even occurred in some areas. The Change of hs-CRP Levels Existing studies have shown that high-sensitivity C-reactive protein (hs-CRP) is a sensitive protein secreted by the body during the inflammatory response, which can be detected even under mild inflammatory conditions, so it can be used as a sensitive indicator of the inflammatory response (18). After detection, when the swelling of the right hind foot of the mice reached the peak at 0 h after treatment, the serum hs-CRP level in the plasma group was 6.353 mg/L, 7.071 mg/L in the unguent group, and 7.157 mg/L in the control group (Fig. 5 ), indicating that the inflammatory level in the plasma group was relatively lower. At 24 h after plasma treatment, unguent application, and blank control treatment, the hs-CRP level in the plasma group decreased by 27.4%, which was the most significant decrease among the three groups; the control group only decreased by 1.58%; while the hs-CRP level in the unguent group increased instead of decreasing. At the same time, the differences in hs-CRP levels between the plasma group and the unguent group, and between the plasma group and the control group were statistically significant (p < 0.05), indicating that the effect of plasma in relieving redness and swelling was superior to that of unguent. At 48 h after the start of treatment in each group, the hs-CRP level in the plasma group decreased only slightly compared with 0 h and 24 h, but was still lower than that in the other two groups, and the difference was statistically significant (p < 0.05). At 72 h, although the hs-CRP levels in the unguent group and the control group continued to decrease, the plasma group still maintained the lowest level among the three groups. The 3-day treatment results showed that plasma was more effective than the commonly used unguent in the treatment of the bee sting model (i.e., melittin-induced redness and swelling). DISCUSSION Severe bee stings can lead to coma and even death. Melittin, the main toxic protein released by bee stings, can cause local tissue redness, swelling and pain. At present, there is no rapid and targeted treatment for this condition. After a bee sting, the level of interleukin-6 (IL-6) in the body rises rapidly, and high-sensitivity C-reactive protein (hs-CRP) fluctuates significantly, which are typical manifestations of the inflammatory response ( 19 ). Existing studies have shown that plasma has the ability to decompose biological toxins, which provides a research idea for using low-temperature plasma to degrade toxins produced by bee stings ( 20 ). In animal experiments, mice with swollen right foot pads were treated with low-temperature plasma. After 4 h of treatment, the morphology of the left and right foot pads of the mice was almost consistent, the relative swelling degree was the lowest compared with the unguent group and the untreated group, and the inflammation inhibition rate was the highest. In addition, after 0, 24, and 48 h of treatment, the serum hs-CRP level of the mice was low and gradually decreased. In contrast, the therapeutic effect of the unguent was relatively slow, indicating that low-temperature plasma has a significant regulatory effect on inflammation induced by melittin. Its mechanism of action may be as follows: low-temperature plasma generates charged particles, reactive oxygen species (ROS), reactive nitrogen species (RNS) and a small amount of heat during discharge, and forms a high-voltage electric field at the same time. It can induce changes in the conformational structure of toxin proteins through the oxidation process mediated by peroxynitrite (ONOO⁻) jointly generated by ROS and RNS. This mechanism is different from thermal degradation, but may be related to the modification of melittin by active chemical substances and other factors generated during the operation of the low-temperature plasma pen. Existing studies have confirmed that such active substances are the main factors leading to the degradation of biological toxins in mouse tissues ( 21 – 23 ). Nevertheless, it is still necessary to verify the therapeutic effect of low-temperature plasma through more inflammatory indicator detection, verify the correctness of the mechanism through more experiments, and carry out further clinical trials to confirm its clinical application potential. This study shows that low-temperature plasma can quickly reduce the redness and swelling symptoms related to the inflammatory response, and explores its mechanism of action, suggesting that low-temperature plasma has a rapid and effective therapeutic effect on the inflammatory response after bee stings, which can timely relieve the pain of patients and even save lives. Declarations Author Contributions Conceptualization, S.G.W. and L.S.; methodology, L.S.; formal analysis, L.S. and S.G.W.; investigation, L.S.; resources, S.G.W.; writing—original draft preparation, L.S.; writing—review and editing, L.S.; visualization, S.G.W.; supervision, S.G.W.; project administration, S.G.W.; funding acquisition, L.S.. All authors have read and agreed to the published version of the manuscript. Competing Interests The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. Funding Declaration This research was funded by Shandong Province Natural Science Foundation Project (NO.ZR2023QA010 ) Data availability The datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request. 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Primary hyperalgesia to mechanical and heat stimuli followingsubcutaneous bee venom injection into the plantar surface of hindpaw in the conscious rat: a comparative study with the formalin test. PAIN 83 , 67–76 (1999). Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 31 Mar, 2026 Reviews received at journal 30 Mar, 2026 Reviews received at journal 19 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers agreed at journal 18 Mar, 2026 Reviewers invited by journal 18 Mar, 2026 Editor assigned by journal 18 Mar, 2026 Editor invited by journal 17 Mar, 2026 Submission checks completed at journal 11 Mar, 2026 First submitted to journal 11 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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07:08:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9016945/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9016945/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105563109,"identity":"9c20f762-f13a-4c38-aca3-9dfd62179253","added_by":"auto","created_at":"2026-03-27 12:45:59","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":555028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePlasma pen treating bee stings model\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-9016945/v1/d4ff3c3cbc641e08f45378c9.png"},{"id":105073904,"identity":"a8a984dd-c8ac-4ecc-8cf2-13867aced8a2","added_by":"auto","created_at":"2026-03-20 15:49:46","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":570541,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe discharge waveforms of the plasma pen in different conditions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) discharge waveforms of the low-temperature plasma pen under High-voltage condition\u003c/p\u003e\n\u003cp\u003e(B) discharge waveforms of the low-temperature plasma pen under room temperature condition\u003c/p\u003e\n\u003cp\u003e(C) discharge waveforms of the low-temperature plasma pen under body temperature condition\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-9016945/v1/0852703e5e2d229a85e29883.png"},{"id":105073906,"identity":"24c9b4a4-c78e-4333-a7d5-788ad5666d4b","added_by":"auto","created_at":"2026-03-20 15:49:46","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":921941,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTreatment effect pictures of the mice's right foot swelling and redness.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Observation of the swelling degree of mouse paws for different time after treatments\u003c/p\u003e\n\u003cp\u003e(B) Use a line graph to represent the relative swelling degree of mouse paws at different time points after treatments\u003c/p\u003e\n\u003cp\u003e(C) Use a line graph to represent the paw edema inhibition rate at different time points after treatments\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-9016945/v1/543863c1d11b05d46f65c643.png"},{"id":105073908,"identity":"0142d017-67cb-4875-a699-df3841369554","added_by":"auto","created_at":"2026-03-20 15:49:46","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":709903,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHistopathological examination of the mice foot soles after treatments\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-9016945/v1/751bac563a5566625a08912d.png"},{"id":105073905,"identity":"dc0564c4-d971-4e9a-897f-7d2693205a5d","added_by":"auto","created_at":"2026-03-20 15:49:46","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":282939,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ehs-CRP levels in the serum of mice for 0h,24h,48h and 72h\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-9016945/v1/e43f0bc3a14d5e5ea222d54e.png"},{"id":105568963,"identity":"c4dd6ac0-267c-4307-b6df-81ac1952d3a7","added_by":"auto","created_at":"2026-03-27 13:10:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4183657,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9016945/v1/88566113-f4a7-435c-8ec6-9a6d1f3ea000.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The alleviating effect of low-temperature plasma on bee stings","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eWith the rapid development of plasma physics, plasma technology has been widely applied in fields such as biomedicine, food safety, energy engineering, and agricultural production (\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Among them, the application of low-temperature plasma in the field of biomedicine has become a research hotspot, showing broad prospects in disinfection, dermatological treatment, wound healing, and tumor treatment. In addition, low-temperature plasma has degradation and inactivation effects on various biological toxins, such as bacterial endotoxins, mycotoxins, and staphylococcal enterotoxins (\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Sakudo et al. found through experiments that the α-helix content of bovine serum albumin (BSA) treated with nitrogen plasma increased and the β-turn content decreased; while heat treatment led to a decrease in α-helix content and an increase in β-sheet content. Based on this, they believed that the protein conformational changes induced by nitrogen plasma are mediated by a mechanism different from thermal denaturation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The above effects can reduce the pathological damages caused by toxins to humans and animals, such as fever, disseminated intravascular coagulation, endotoxemia, inflammation, and shock.\u003c/p\u003e \u003cp\u003eBee sting poisoning refers to a toxic disease caused by bees injecting venom into human skin through stinging, with clinical manifestations usually including local redness, swelling, and tingling, and may also present systemic symptoms such as dizziness and nausea (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). The pathogenic mechanism of bee sting injury is closely related to the toxic components in bee venom and the subsequent allergic reactions. However, there is still a lack of efficient treatment schemes for severe bee sting poisoning, and the mortality rate of related cases is relatively high (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the existing research conclusion that low-temperature plasma can reduce the content of toxins in the body, this study attempted to apply low-temperature plasma to the swollen area caused by bee stings, in order to remove the toxins produced during the bee sting process.\u003c/p\u003e \u003cp\u003eFor patients with severe bee sting poisoning, it is crucial to control the condition in a timely manner and carry out effective treatment. At present, the clinical treatment methods for severe cases are still relatively primitive and single, including traditional Chinese medicine intervention. Such methods generally have the problem of slow onset, which makes it difficult to achieve rapid cure and effectively relieve the pain of patients (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). In view of the wide application of low-temperature plasma in the degradation and inactivation of various biological toxins, this study compared low-temperature plasma treatment, unguent treatment, and no treatment for bee stings, and concluded that low-temperature plasma has a faster effect on reducing swelling of the swollen paws of mice, and simultaneously explored and explained its mechanism of action.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlasma Pen\u003c/h2\u003e \u003cp\u003eA plasma pen with output parameters of 5 V DC and 2 A can release plasma when its ceramic probe is close to a conductor. The ceramic probe was used for horizontal scanning treatment on the swollen area of the right foot pad to verify the feasibility of plasma therapy for the bee sting model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eDischarge Waveform\u003c/b\u003eDischarge waveforms were detected using an oscilloscope, and precise detection could be achieved by combining partial discharge testing technology. The specific measurement process of the oscilloscope is as follows:\u003c/p\u003e \u003cp\u003eEquipment Preparation\u003c/p\u003e \u003cp\u003eFirst, connect the oscilloscope to the probe to ensure that the probe calibration is completed and the calibration result is accurate; then select the matching measurement range (such as current range or voltage range) according to the discharge type, and configure key parameters such as DC coupling and trigger point in turn.\u003c/p\u003e \u003cp\u003e1. Parameter Debugging\u003c/p\u003e \u003cp\u003eSet the horizontal scanning range to 1 ms/div, adjust the vertical sensitivity to 25 A/div to ensure that the discharge pulse characteristics can be clearly observed; at the same time, adjust the screen display position to center the waveform, ensuring that the waveform is clear and unobstructed.\u003c/p\u003e \u003cp\u003e2. Waveform Analysis\u003c/p\u003e \u003cp\u003eFocus on observing the phase characteristics of the discharge pulse and its synchronization relationship with the power frequency signal to determine the period when the discharge phenomenon occurs; adopt the elliptical scanning or horizontal scanning method to effectively distinguish the interference signal from the real discharge waveform, ensuring the accuracy of the analysis results.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eReagents\u003c/h3\u003e\n\u003cp\u003eMelittin powder (purity\u0026thinsp;\u0026ge;\u0026thinsp;97%) was dissolved in 0.9% normal saline to prepare solutions with concentrations of 5 mg/mL, 2.5 mg/mL, and 1 mg/mL, respectively. Meanwhile, a 25% urethane solution was prepared. Fluticasone propionate cream was purchased from Hubei Heng'an Fuling Pharmaceutical Co., Ltd. SPF-grade Balb/c male mice (6 weeks old, weighing 20\u0026ndash;22 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Hubei Branch. During the experiment, the mice had free access to food and water and were reared in an environment-controlled facility. All experimental operations strictly followed the Principles of Laboratory Animal Care and the Guidelines for the Care and Use of Laboratory Animals issued by the State Science and Technology Commission of the People's Republic of China. The experimental protocol was approved by the Ethics Committee of Wuhan Servicebio Technology Co., Ltd., China (approval number: 2025376). All procedures were performed in accordance with the relevant guidelines and regulations.\u003cb\u003eConstruction of A Mouse Foot Blister Model and Determination of the Optimal Swelling-inducing Dose of Melittin\u003c/b\u003eThe mice were randomly divided into 3 groups with 3 mice in each group. The hair on the tail, both hind limbs and feet of all mice was shaved. Subsequently, 10 \u0026micro;L of melittin normal saline solutions with concentrations of 5 mg/mL, 2.5 mg/mL, and 1 mg/mL were injected into the plantar space between the right hind toes of the three groups of mice, respectively. After injection, the mice were left to stand for 5\u0026ndash;10 minutes to induce foot swelling, and the group with the most obvious swelling of the right foot was selected to establish the mouse foot swelling model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eGroups and Treatments\u003c/b\u003eAfter acclimatization to the environment for 1 week, the mice were randomly divided into 3 groups with 15 mice in each group (n\u0026thinsp;=\u0026thinsp;15): the plasma group, the unguent group, and the control group.\u003c/p\u003e \u003cp\u003eAccording to the results of preliminary experiments, the optimal concentration of melittin normal saline with the most significant inhibitory effect on redness and swelling was determined to be 5 mg/mL. Subsequently, 10 \u0026micro;L of 5 mg/mL melittin normal saline was injected into the plantar space between the right hind toes of all mice to establish a foot swelling model, and the degree of foot swelling was monitored within 5 minutes after injection.\u003c/p\u003e \u003cp\u003eMice with redness and swelling of the right foot pad were selected and anesthetized with urethane.\u003c/p\u003e \u003cp\u003ePlasma group: treated with low-temperature plasma for 1 minute and 20 seconds, with an interval of 5 minutes, for a total of 3 times; the ceramic discharge probe of the plasma pen was horizontally placed 0.5\u0026ndash;0.8 cm above the mouse's foot to scan the swollen foot in the horizontal direction. Unguent group: an appropriate amount of fluticasone propionate cream was locally applied to the swollen area of the right hind foot. Control group: no intervention measures were taken (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe redness and swelling of the feet of mice in each group were photographed and recorded at 0.5, 1, 2, 4, 24, 48, and 72 hours after treatment, respectively, and the foot thickness was measured.\u003c/p\u003e \u003cp\u003eThe degree of edema in the right foot of mice was calculated according to the formula: Edema degree\u0026thinsp;=\u0026thinsp;Thickness of the affected foot\u0026thinsp;\u0026minus;\u0026thinsp;Initial thickness of the affected foot\u003c/p\u003e \u003cp\u003eThe relative edema inhibition rate was calculated accordingly:\u003c/p\u003e \u003cp\u003eFoot swelling inhibition rate = [(Edema degree of control group\u0026thinsp;\u0026minus;\u0026thinsp;Edema degree of intervention group) / Edema degree of control group] \u0026times; 100%\u003c/p\u003e\n\u003ch3\u003eIndicators of Observation\u003c/h3\u003e\n\u003cp\u003eThe therapeutic effects of the three groups were compared. The improvement of redness and swelling in the right plantar of mice was evaluated by histopathological section analysis and enzyme-linked immunosorbent assay (ELISA) for high-sensitivity C-reactive protein (hs-CRP) levels.\u003c/p\u003e \u003cp\u003eHigh-sensitivity C-reactive protein (hs-CRP) is an important systemic inflammatory marker, whose level increases significantly under inflammatory stimuli such as infection and tissue damage, and can be used as a reliable indicator to evaluate the inflammatory state (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Histopathological Examination\u003c/p\u003e \u003cp\u003eFresh foot tissues were taken to make longitudinal paraffin sections. The paraffin sections were dewaxed to water, immersed in a high-definition constant staining pretreatment solution for 1 minute, stained with hematoxylin-eosin (HE), dehydrated, mounted, and then observed under a microscope to collect images for analysis.\u003c/p\u003e \u003cp\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) ELISA Detection\u003c/p\u003e \u003cp\u003eMouse C-reactive protein (CRP) ELISA kit was used for detection, and the kit was purchased from Hangzhou Lianke Biotechnology Co., Ltd.\u003c/p\u003e \u003cp\u003e1. Sample Collection and Preservation\u003c/p\u003e \u003cp\u003eBlood Collection by Enucleation\u003c/p\u003e \u003cp\u003eHold the skin on the back of the mouse's neck with the left hand, gently press and fix it on the experimental table, and take the lateral position. Press the skin around the mouse's eyes as far back to the neck as possible with the index finger of the left hand to make the eyeballs fully protrude. Quickly clamp and enucleate the eyeball with curved ophthalmic forceps, immediately invert the mouse, and collect the flowing blood with a pyrogen-free and endotoxin-free sterile container. Immediately press with gauze to stop bleeding after blood collection. The blood collection volume by this method is about 0.5-1 mL.\u003c/p\u003e \u003cp\u003eThe collected blood was left standing at room temperature for 30 minutes to coagulate naturally.\u003c/p\u003e \u003cp\u003e2. Centrifugal Separation\u003c/p\u003e \u003cp\u003ePlace the coagulated blood sample in a centrifuge and centrifuge at 3000 r/min for 10 minutes. After centrifugation, quickly and carefully separate the serum from the red blood cells to avoid serum contamination caused by red blood cell rupture.\u003c/p\u003e \u003cp\u003e3. Sample Preservation\u003c/p\u003e \u003cp\u003eThe separated serum can be detected immediately; if not detected temporarily, aliquot it according to a single dosage, store it frozen at -20 ℃, and repeated freezing and thawing is strictly prohibited. Thaw at room temperature before use and mix gently continuously to ensure complete and uniform thawing of the sample.\u003c/p\u003e \u003cp\u003ePartial Materials to Be Prepared\u003c/p\u003e \u003cp\u003eStandard microplate reader (450 nm), precision pipette and disposable tips, 37 ℃ incubator\u003c/p\u003e \u003cp\u003eResult Determination\u003c/p\u003e \u003cp\u003eDraw a standard curve: In Excel, draw a linear regression curve with the standard concentration as the abscissa and the corresponding OD value as the ordinate, and calculate the concentration of each sample according to the curve equation.\u003c/p\u003e "},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003ePlasma Pen Working Mode\u003c/b\u003eThe plasma arc generator is composed of a ceramic probe, a pen body and a wire. A 5 V DC voltage and 2 A current were adopted. When the ceramic probe was used for horizontal scanning treatment on the swollen area of the right foot of the mouse, a voltage difference was formed between the probe and the sole of the mouse, inducing purple arc discharge. This discharge was used to treat the bee sting model, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eDischarge Waveform\u003c/b\u003eThe discharge waveforms of the plasma pen under different working conditions are as follows: the waveform at room temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and the waveform at body temperature (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). It can be seen from the figures that there are differences in the discharge characteristics of the plasma pen between the animal body temperature condition and the room temperature discharge condition. The discharge at body temperature is smoother and more obvious, indicating that a relatively stable electromagnetic field is formed between the living body and the low-temperature plasma.\u003c/p\u003e\u003cp\u003eIn addition, various active substances are generated during plasma discharge, including reactive oxygen species (ROS) such as O, \u0026middot;OH, H₂O₂, and O₃, as well as reactive nitrogen species (RNS) such as NO and NO₂ and related substances (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). These active substances can change and destroy the structure of toxins in organisms. If the active substances can enter the cells, they can change the structure of toxic substances in the body's tissue cells, thereby inhibiting symptoms such as inflammation caused by toxic substances and relieving pain.\u003c/p\u003e \u003cp\u003eIn fact, plasma generated under specific voltage conditions can produce certain biological effects on cells or organisms. Animal experiments conducted by Fathollah et al. showed that cold atmospheric plasma (CAP) generated under the conditions of 8 kV voltage and 6 kHz frequency can significantly promote the wound healing process of diabetic rats (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eTherefore, under the action of a uniform electric field with 5 V DC voltage and 2 A current, the ion channels on the animal cell membrane open, leading to the formation of pores in the cell membrane. Based on this, the active substances generated by plasma discharge can enter the cells, thereby affecting and changing the protein conformation of melittin (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Improvement of Redness and Swelling Symptoms\u003c/b\u003eThe redness and swelling of the mouse foot pads were compared at 0.5, 1, 4, 24, and 48 h after treatment, respectively. The results showed that the redness and swelling of the right hind foot in both the control group and the unguent group were relatively severe, while the symptoms in the plasma group were the mildest (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003eAt 1 h after treatment, the edema thickness of the plasma group was 1.18 mm, which was lower than that of the other two groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB); at the same time, the swelling inhibition rate of the plasma group reached 17.67%, which was higher than 13.72% of the unguent group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). This suggests that low-temperature plasma takes effect faster.\u003c/p\u003e \u003cp\u003eAt 4 h after treatment, the swelling of the plasma group subsided significantly and rapidly (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). At this time, the morphology of the two feet in the plasma group was basically consistent, with a relative edema degree of 1.07 mm, which was lower than that of the unguent group (1.17 mm) and the control group (1.39 mm) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). The swelling inhibition rate of the plasma group reached 23.21%, which was significantly higher than 16.27% of the ointment group, and the inhibition rate of the control group was 0% (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eAt 48 h after treatment, the edema degree related to redness and swelling of the foot pads in the plasma group was still the lowest among the three groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), and the inhibition rate was the highest among all observation time points (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). In general, compared with the control group and the unguent group, the plasma group had a lower relative edema degree and a higher relative edema inhibition rate, indicating that the therapeutic effect of low-temperature plasma is superior to other commonly used intervention methods.\u003c/p\u003e \u003cp\u003e \u003cb\u003eHistopathology\u003c/b\u003eHistopathology is a medical technology that observes diseased tissue samples through a microscope, analyzes abnormalities in cell morphology and structure, and clarifies lesion characteristics, which is the core basis for disease diagnosis (11).\u003c/p\u003e \u003cp\u003eAt 0 h after treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), obvious tissue deformation, inflammatory cell infiltration, and significant plasma exudation were observed in the plantar tissue sections of the three groups of experimental animals. Plasma containing white blood cells and red blood cells infiltrated into the subcutaneous layer from peripheral blood vessels, which was particularly obvious in the unguent group and the control group.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAfter 1 h, the myocyte morphology in the plasma group recovered to a long spindle shape, while the unguent group and the blank control group (untreated) had not fully recovered, and some cells were still round.\u003c/p\u003e \u003cp\u003eAt 4 h after treatment, the myocytes in the plasma group recovered well, and the tissue structure returned to a dense state; in contrast, the myocytes in the unguent group and the blank control group were still loose and prone to disintegration.\u003c/p\u003e \u003cp\u003eStarting from 24 h, the muscle tissue in the plasma group had recovered to a normal and healthy morphology; the recovery rate of muscle tissue in the unguent group and the blank control group was significantly slower than that in the plasma group, and irreversible damage even occurred in some areas.\u003c/p\u003e \u003cp\u003e \u003cb\u003eThe Change of hs-CRP Levels\u003c/b\u003e Existing studies have shown that high-sensitivity C-reactive protein (hs-CRP) is a sensitive protein secreted by the body during the inflammatory response, which can be detected even under mild inflammatory conditions, so it can be used as a sensitive indicator of the inflammatory response (18).\u003c/p\u003e \u003cp\u003eAfter detection, when the swelling of the right hind foot of the mice reached the peak at 0 h after treatment, the serum hs-CRP level in the plasma group was 6.353 mg/L, 7.071 mg/L in the unguent group, and 7.157 mg/L in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), indicating that the inflammatory level in the plasma group was relatively lower.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAt 24 h after plasma treatment, unguent application, and blank control treatment, the hs-CRP level in the plasma group decreased by 27.4%, which was the most significant decrease among the three groups; the control group only decreased by 1.58%; while the hs-CRP level in the unguent group increased instead of decreasing. At the same time, the differences in hs-CRP levels between the plasma group and the unguent group, and between the plasma group and the control group were statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that the effect of plasma in relieving redness and swelling was superior to that of unguent. At 48 h after the start of treatment in each group, the hs-CRP level in the plasma group decreased only slightly compared with 0 h and 24 h, but was still lower than that in the other two groups, and the difference was statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). At 72 h, although the hs-CRP levels in the unguent group and the control group continued to decrease, the plasma group still maintained the lowest level among the three groups. The 3-day treatment results showed that plasma was more effective than the commonly used unguent in the treatment of the bee sting model (i.e., melittin-induced redness and swelling).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eSevere bee stings can lead to coma and even death. Melittin, the main toxic protein released by bee stings, can cause local tissue redness, swelling and pain. At present, there is no rapid and targeted treatment for this condition. After a bee sting, the level of interleukin-6 (IL-6) in the body rises rapidly, and high-sensitivity C-reactive protein (hs-CRP) fluctuates significantly, which are typical manifestations of the inflammatory response (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eExisting studies have shown that plasma has the ability to decompose biological toxins, which provides a research idea for using low-temperature plasma to degrade toxins produced by bee stings (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In animal experiments, mice with swollen right foot pads were treated with low-temperature plasma. After 4 h of treatment, the morphology of the left and right foot pads of the mice was almost consistent, the relative swelling degree was the lowest compared with the unguent group and the untreated group, and the inflammation inhibition rate was the highest. In addition, after 0, 24, and 48 h of treatment, the serum hs-CRP level of the mice was low and gradually decreased. In contrast, the therapeutic effect of the unguent was relatively slow, indicating that low-temperature plasma has a significant regulatory effect on inflammation induced by melittin.\u003c/p\u003e \u003cp\u003eIts mechanism of action may be as follows: low-temperature plasma generates charged particles, reactive oxygen species (ROS), reactive nitrogen species (RNS) and a small amount of heat during discharge, and forms a high-voltage electric field at the same time. It can induce changes in the conformational structure of toxin proteins through the oxidation process mediated by peroxynitrite (ONOO⁻) jointly generated by ROS and RNS. This mechanism is different from thermal degradation, but may be related to the modification of melittin by active chemical substances and other factors generated during the operation of the low-temperature plasma pen. Existing studies have confirmed that such active substances are the main factors leading to the degradation of biological toxins in mouse tissues (\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNevertheless, it is still necessary to verify the therapeutic effect of low-temperature plasma through more inflammatory indicator detection, verify the correctness of the mechanism through more experiments, and carry out further clinical trials to confirm its clinical application potential.\u003c/p\u003e \u003cp\u003eThis study shows that low-temperature plasma can quickly reduce the redness and swelling symptoms related to the inflammatory response, and explores its mechanism of action, suggesting that low-temperature plasma has a rapid and effective therapeutic effect on the inflammatory response after bee stings, which can timely relieve the pain of patients and even save lives.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization, S.G.W. and L.S.; methodology, L.S.; formal analysis, L.S. and S.G.W.; investigation, L.S.; resources, S.G.W.; writing\u0026mdash;original draft preparation, L.S.; writing\u0026mdash;review and editing, L.S.; visualization, S.G.W.; supervision, S.G.W.; project administration, S.G.W.; funding acquisition, L.S.. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by Shandong Province Natural Science Foundation Project (NO.ZR2023QA010 )\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets generated and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Wuhan Servicebio Co., Ltd. for technical assistance.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWhittaker, A. G. et al. Plasma cleaning of dental instruments. \u003cem\u003eJ. 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Primary hyperalgesia to mechanical and heat stimuli followingsubcutaneous bee venom injection into the plantar surface of hindpaw in the conscious rat: a comparative study with the formalin test. \u003cem\u003ePAIN\u003c/em\u003e \u003cb\u003e83\u003c/b\u003e, 67\u0026ndash;76 (1999).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Bee sting, Low-temperature plasma, Therapeutic effect, Modification, Protein spatial structure","lastPublishedDoi":"10.21203/rs.3.rs-9016945/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9016945/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA great deal of research has been conducted on the surface sterilization and treatment of skin using low-temperature plasma. However, the effect of plasma on subcutaneous toxicity or inflammatory factors has not been experimentally verified to date. Bee stings can trigger a series of allergic reactions, with clinical manifestations mainly including redness, swelling and pain.\u003c/p\u003e \u003cp\u003eIn this paper, low-temperature plasma was used for the intervention treatment of swelling caused by bee stings, and its therapeutic effect was compared with that of fluticasone propionate cream (hereinafter referred to as unguent), a commonly used drug for mosquito bites. The inactivation effect of dielectric barrier discharge plasma directly applied to the swollen skin of mice on subcutaneous toxic substances was investigated.\u003c/p\u003e \u003cp\u003eIn the research method, three groups of mice were selected, and melittin was injected subcutaneously into the feet of mice to establish a bee sting model. Then, plasma treatment, drug treatment and no treatment were applied respectively for comparison, and the skin conditions were observed. In addition, the plasma of mice was collected to analyze the levels of inflammatory factors in the blood.\u003c/p\u003e \u003cp\u003eIt was found that the anti-inflammatory effect on the right paw of mice in the low-temperature plasma treatment group was satisfactory. Compared with the unguent group and the untreated group, low-temperature plasma acted more rapidly on such inflammatory responses. The results demonstrate that direct dielectric barrier plasma treatment on the skin can rapidly inactivate subcutaneous toxic substances and inhibit inflammatory responses, confirming that plasma can not only kill pathogens on the skin surface, but also quickly suppress subcutaneous inflammation and eliminate redness and swelling.\u003c/p\u003e \u003cp\u003eIn related applications, unlike traditional therapeutic methods based on thermal denaturation, the plasma pen changes the protein spatial structure of bee venom through free charge carriers generated by plasma combined with a high-voltage electric field, leading to the earliest resolution of paw swelling in the low-temperature plasma group.\u003c/p\u003e","manuscriptTitle":"The alleviating effect of low-temperature plasma on bee stings","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 15:49:41","doi":"10.21203/rs.3.rs-9016945/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-03-31T12:18:56+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-30T16:13:14+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-19T11:14:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157780827928147909954098219035073242355","date":"2026-03-18T13:06:35+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"16402101994770307783810486467487268527","date":"2026-03-18T07:58:35+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-18T07:04:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-18T04:43:51+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-17T16:35:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-11T11:27:01+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-11T07:53:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"cc75c2fe-469f-4e42-b062-a8c212fc307e","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":64787635,"name":"Health sciences/Diseases"},{"id":64787636,"name":"Biological sciences/Drug discovery"},{"id":64787637,"name":"Biological sciences/Immunology"},{"id":64787638,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2026-05-12T12:20:16+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 15:49:41","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9016945","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9016945","identity":"rs-9016945","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}