Topical bee venom nanoemulsion in collagen-induced arthritis model: effects on biochemical and hematological parameters

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This study aimed to assess the side effects of bee venom nanoemulsions (BV-NEs) in the collagen-induced arthritis (CIA) model by examining biochemical and hematological parameters. Methods: The BV-NEs were prepared, and the CIA model was induced in rats. After the seventh day, the groups were topically treated for two weeks as the following: blank (free treatment), negative control (NE-0), positive control (hydrocortisone acetate ointment 1%, 50 mg/day), BV control (37.5 µg/ml/day), and BV-NEs receiving 75, 37.5, 18.75, and 9.37 µg/ml/day. Three steps of blood sampling were done on days 0, 7, and 21 (healthy rats, before and at the end of treatment, respectively). Results: The results revealed that blood levels of glucose, cholesterol, urea, aspartate aminotransferase (AST), alkaline phosphatase (ALP), white blood cell (WBC), and %neutrophil significantly increased before the treatment. Nevertheless, most parameters declined at the end of the treatment compared to the blank and negative control groups about BV-NEs dose-dependently. The drastic changes in biochemical parameters in the CIA model indicated the effect of the immune system function on the metabolic system. Also, NE's impact of BV passed through the skin on these items. Conclusions: BV-NEs can reduce inflammation caused by arthritis without acute adverse effects on the routine biochemical and hematological parameters. Nanoemulsion Bee Venom Biochemical Hematological Arthritis Figures Figure 1 Figure 2 Highlights Topical bee venom nanoemulsion reduces inflammation caused by arthritis. This nanoemulsion passes bee venom through the skin. It has no acute adverse effects on the routine biochemical and hematological parameters. Introduction Nanoemulsions (NEs), as homogeneous systems in nanotechnology, consist of at least two immiscible liquids thoroughly dispersed by a surfactant(s) and have droplets with a mean size of less than 100 nm. They exist in water-in-oil (W/O) or oil-in-water (O/W) forms [ 1 , 2 ]. NEs as drug delivery systems have different advantages, including enhancing bioavailability and drug loading, controlled drug release, protection from enzymatic degradation, and carrying hydrophilic as well as hydrophobic components in a single form. Thus, they can be used as high-efficiency carriers for local and transdermal purposes [ 3 , 4 ]. Collagen-induced arthritis (CIA) is widely used as a model of human rheumatoid arthritis caused by injecting type 2 collagen that indicates both immunological and pathological features [ 5 ]. This model causes multi-joint inflammation, bone proliferation, and cartilage damage among the various models; it has also a more specific onset and prospers faster [ 6 ]. Although it is highly used in arthritis studies in rats, there is less evidence available for assessing their biochemical and hematological parameters. Bee Venom (BV) as a biotoxin is composed of at least 18 pharmacologically active components, including various enzymes (i.e., phospholipases and hyaluronidase), a variety of peptide components (i.e., melittin, apamin, adolapin, and mast-cell-degranulating (MCD)), non-peptide components (i.e., carbohydrates, lipids, and free amino acids), as well as active amines (i.e., histamine and epinephrine) [ 7 ]. It is reported that BV has various pharmaceutical properties, whose anti-inflammatory and anti-arthritis effects have been attributed to melittin [ 7 ], adolapin, mast cell degranulating peptides [ 8 ], and apamin [ 9 ]. BV suppresses leukocyte migration to the inflammation site by reducing cytokine production [ 10 ]. BV is used for treating rheumatoid arthritis (RA) as acupuncture in clinical trials [ 11 ]. It can be encapsulated by water-in-oil NEs to pass through the skin through topical/transdermal delivery without being stung by bees or the injection route [ 12 ]. In our previous study, topical BV nanoemulsions (BV-NEs) could pass BV through the skin and reduce the inflammation caused by the collagen-induced arthritis (CIA) model in rats [ 13 , 14 ]. On the other hand, studies have shown the side effects of BV on the body’s organs, such as the liver [ 15 ], kidney [ 16 ], pancreas [ 17 ], and adrenal glands [ 18 ], as well as its effect on glucose and lipids as parameters of biochemistry [ 19 ] or lytic impact on red blood cells [ 7 ]; however, there is no pharmacodynamic information about it in the local/transdermal delivery route by NEs. Accordingly, this study aimed to evaluate any changes in biochemical and hematological parameters in Wistar rats with the CIA model; also, CIA treated with BV-NEs. Materials And Methods Drugs and chemicals BV was prepared by Asghapoor Honey and Bee Products Co. (Iran). It was collected from healthy and approved hives, Apis mellifera strain, by electrical stimulation. Sorbitan monooleate (Span-80) and polyoxyethylene 20 sorbitan monooleate (Tween-80) as surfactants were purchased from Merck Chemicals (Germany), and olive oil was from Fadak Co. (Iran). Bovine type II collagen was from Xi'an Harmonious Natural Bio-Technology Co., Ltd (China), and incomplete Freund's adjuvant (IFA) was from Sigma-Aldrich (USA). Biochemistry kits were prepared by Pars Azmoon Co. (Iran). Sampling tubes containing EDTA (Ethylene diamine tetra acetic acid) as an anti-coagulant were prepared from FL Co. (Italy) for collecting whole blood as ready to use. Animal 88 male Wistar rats weighing ~ 200 g were used in this study, kept according to the Standard Laboratory Animal Guidelines. The Ethical Committee approved the experimental protocols of Kerman University of Medical Sciences (The Ethics approval code is IR.KMU.REC.1399.234) Preparation And Characterization Of NEs The nanoemulsion was prepared as a volumetric percentage formulation according to the previous study [ 14 ]. Briefly, 3% aqueous phase (containing different BV solutions as the following) was added to the container containing 30% surfactant (including 14% Span-80 and 16% Tween-80) and mixed using a magnetic stirrer (MS-300HS, Protraction Inter-trade co, Korea) for 10 min with the rotation speed of 1000 rpm at room temperature. Then, the oil phase (67% olive oil) was added and thoroughly mixed for 10 min. The mean size of droplets and polydispersity index were measured by dynamic light scattering at 25°C using Scatteroscope (K-one Ltd. Korea). Loading capacity was also calculated in terms of 3% aqueous phase and BV concentrations as 9.37, 18.75, 37.5, and 75 µg/ml. Induction Of CIA And Treatment Bovine type II collagen was dissolved (2 mg.ml − 1 ) in 0.05 M acetic acid by gently stirring overnight at 4°C. Next, an equal volume of IFA was added to the emulsified collagen solution, then mixed by a homogenizer (1000 RPM, 30 min, in an ice-water container) for the collagen-IFA emulsion to be used prepared [ 14 ]. 0.1 ml of this emulsion was injected subcutaneously into the plantar surface of the left hind paw as the inducer dose and 0.1 ml as a booster dose in an intradermal injection into the root of the tail on the same day to induce CIA in rats [ 10 , 20 ]. Maximum inflammation was observed on day 7; treatment was followed up from day 7 to day 21 for two weeks. Experimental Design Rats were assigned to 8 groups (n = 11). Each group was treated daily with one of the following treatments; blank (no treatment), negative control (NEs free BV, 0 µg/ml), positive control (hydrocortisone acetate ointment 1%, 50 mg/day), BV control (solution of bulk BV in NS, 37.5 µg/ml), and four groups of NE formulations (i.e., 9.37, 18.75, 37.5, and 75 µg/ml). All treatments were massaged for 5 min of the rat's paw with 1 ml of BV-solution or BV-NEs. Blood Sampling Deep anesthesia of rats was achieved using intraperitoneal injection of 87 mg ketamine/kg and 13 mg xylazine/kg of body weight [ 21 ], 4 ml of blood was taken directly from their hearts, 2.5 ml for biochemical parameters, and 1.5 ml for hematological parameters and then rats were sacrificed. Three steps of blood sampling were done, including 16 samples (2 samples per group) on day 0 as an indicator of healthy rats, 16 samples (2 samples per group) on day 7 as an indicator of maximum inflammatory rats, and 56 samples (7 samples per group) on day 21 at the end of treatment. After each sampling, rats were sacrificed due to the high volume of blood loss. Samples were collected from the EDTA anti-coagulant tubes (1.5 mg/ml whole blood) for hematological parameters and without anti-coagulant tubes for biochemical parameters (The serums were separated after coagulation by centrifuging at 3000 rpm, room temperature, 10 min). Measurement Of Biochemical And Hematological Parameters Serums were evaluated for biochemical parameters, including blood glucose (Glu), cholesterol (Ch), triglyceride (Tg), urea (Ur), creatinine (Cr), AST (aspartate aminotransferase or SGOT, serum glutamic-oxaloacetic transaminase), ALT (alanine aminotransferase or SGPT, serum glutamic pyruvic transaminase), ALP (alkaline phosphatase), calcium (Ca) and phosphorus (Ph) using an autoanalyzer (BT 1500, Biotechnica Co., Italy). Hematological parameters were counted as complete blood cells (CBC) using an automated cell counter (XP-300, Sysmex co., Japan), including white blood cells (WBCs), and the percentage of neutrophil (%Neu), red blood cells (RBCs), hemoglobin (Hb) concentration, hematocrit (Hct) percentage, and platelets (Plts) count. Also, RBC indices, including mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW), were evaluated. Statistical analysis One-way analysis of variance and Tukey comparison were performed to assess the statistical significance of differences among groups. Results with a p -value ≤ 0.05 were considered statistically significant. Statistical analyses were carried out using the SPSS software, v.19 (SPSS, Inc., USA). Results As mentioned in the previous study, droplets' mean sizes of NEs were 21.1, 16.9, 15.1, 14.2, and 12.7 nm, as well as PDIs were 0.147, 0.146, 0.143, 0.142, 0.142 for BV-NE-75, BV-NE-37.5, BV-NE-18.75, BV-NE-9.37, and BV-NE-0, respectively [ 14 ]. Also, the loading capacity (in terms of 3% aqueous phase) of BV-NEs were 75, 37.5, 18.75, 9.37, and 0, which are named accordingly. It states that in a certain amount of aqueous phase (3%), with increasing the concentration of BV from 0 to 75 µg/ml, the size increased from 12.7 to 21.1 nm, and the PDIs also increased from 0.142 to 0.147. The load capacities of NEs increased with the increase of BV concentrations from 9.37 to 75 µg/ml. Figure 1 (A to J) shows the values of biochemical parameters for healthy rats (day 0), before treatment CIA rats as maximum inflammation (day 7), and for treated groups (day 21), including blank, negative, positive, and BV controls, as well as BV-NEs including 75, 37.5, 18.75, and 9.37 µg/ml. Figure 1 . A shows the blood Glu level significantly increased in the CIA model from 188 to 227 mg/dl from day 0 to day 7. But, it was significantly returned to near the baseline level only for positive and BV controls, as well as BV-NE 18.75 and 9.37 µg/ml on day 21. In Fig. 1 . B, the Chol blood level was significantly increased in the CIA model from 63 to 70 mg/dl from day 0 to day 7. It states that Chol was significantly decreased for BV control on day 21. Figure 1 . C shows which Tg blood level has significantly decreased in the CIA model on day 7. however, it increased on day 21 in all groups. Changes in urea and creatinine in rat blood are shown in Fig. 1 . D, and E. These biochemical parameters were not changed on days 0, 7, and 21, except urea, which significantly increased from 33 to 41 mg/dl in the CIA model. Figure 1 . F, G, and H illustrate blood levels of AST, ALT, and ALP as a function of liver enzymes. Although all of them were increased in the CIA model on day 7, it was only significant for AST from 134 to 160 U/L. The AST and ALP liver enzymes were decreased in the positive and BV controls, as well as NE 9.37 µg/ml group on day 21 compared to day 7. Ca and Ph blood levels are given in Fig. 1 . I and J. for the treated groups, the levels of Ca and ph decreased on day 21 compared to the CIA model on day 7. Especially about Ca, there were significant differences for BV control and also BV-NEs groups. Figure 2 (L to T) illustrates the results of hematological parameters for understudy groups in the pattern of Fig. 1 . Figure 2 . L shows a significant increase in the counting of WBCs from 4.96 to 9.74 (×10 3 /mm 3 ) from day 0 to day 7 in the CIA model. However, it was significantly decreased for all treatment groups on day 21, although it was higher for blank and negative control groups. Hematological parameters of RBC, Hb, and HCT are shown in Fig. 1 . M, N, and O, respectively. Figure 2 . P, Q, R, and S also indicate indices of RBC (MCV, MCH, MCHC, and RDW). There was no significant difference between treated groups on days 0, 7, and 21. Plts count was increased on days 7 and 21 compared to day 0, but it was not significant (see Fig. 2 . T). Figure 2 . U shows %Neu for treated groups. It significantly increased from 19.9–54.4% from day 0 to day 7 in the CIA model and then was significantly decreased for all groups on day 21, although it was more significant for BV-NEs, BV control, and positive control groups compared to blank and negative control groups. Discussion Our previous studies found a significant effect of NEs containing BV on improving arthritis in an animal model [ 13 , 14 ]. We did not know how much BV could have adverse effects after entering the body nor did we know its benefits. Thus, this study aimed to identify the side effects of NEs containing BV on the body by measuring the biochemical and hematological parameters of arthritis rats. We also measured these changes in the CIA animal model on the full inflammation day that were not treated. The study showed that on day 7 of induction of the CIA model, as the maximum inflammation day, biochemical parameters, including Glu ( p ≤ 0.01), Ch ( p ≤ 0.001), Ur ( p ≤ 0.05), and AST ( p ≤ 0.05), had significantly increased, and only, TG ( p ≤ 0.05) had significantly decreased. Among the hematological parameters on day 7, only WBCs ( p ≤ 0.001) and %Neu ( p ≤ 0.001) had significantly increased. The other biochemical and hematological parameter changes in the CIA were insignificant from day 7 until the end of two weeks of the treatment (day 21, see the blank group). Nevertheless, the %Neu was still high compared to day 0 ( p ≤ 0.001). Zainab et al. reported that WBCs and Plts had increased in formaldehyde-induced arthritis, which agrees with our results in the CIA model. Also, she reported that RBCs, Hb, and HCT diminished, which was similar to the results of the present study [ 22 ]. Increasing WBCs, especially %Neu in the blood of CIA model rats on day 7, can be due to calling WBCs to the inflammation site in the rat's paw by some inflammatory cytokines after induction of arthritis [ 23 ]. In this regard, inflammatory mediators, immune cells, cytokines, and chemokines play a critical role in dyslipidemia, which hence cause metabolic effects in arthritis [ 24 ]. An increase in Chol and TG has been reported in formaldehyde-induced arthritis, while in the CIA model, only Chol increased and TG decreased [ 22 ]. Thus, there was a disorder of metabolism and dyslipidemia in arthritis and animal models of CIA. BV has several anti-inflammatory and anti-arthritic properties [ 11 ]. It has a variety of components with pharmacological and biochemical activities and can destroy location cells or enter the bloodstream and lyse other blood cells, especially RBCs, as it passes through the skin [ 7 ]. Studies have shown that BV can affect the body's metabolic systems to change their function, such as reducing blood glucose [ 25 ] and affecting the blood lipids [ 26 ]. Although some unfavorable effects of BV can be neutralized by albumin in the bloodstream [ 27 ], it can affect organs such as islands of Langerhans of the pancreas [ 17 ], liver [ 15 ], kidney [ 16 ], adrenal glands, and their hormones [ 18 ]. Imani et al. showed that BV injection decreased Glu, total Chol, and Tg levels in treated diabetic rats [ 28 ]. It has been shown that serum levels of Chol and TG decreased in formaldehyde-induced arthritis male rats receiving injection BV, which was similar to our results only about Chol [ 22 ]. The study indicated that the blood levels of Glu decreased at the end of treatment and were similar to the base level on day 0 for all groups, except for the BV control, which was lower than the base level. It suggests that topical BV can reduce the blood Glu, but this effect was not found for Chol and TG. It was reported that BV therapy is associated with multiple causes of kidney injury and hepatotoxicity [ 29 ]. Measuring Ur and Cr blood levels is a simple way to assess kidney injuries [ 30 ]. Ur and Cr increased in the CIA model on day 7. At the end of treatment on day 21, Ur and Cr dose-dependently dropped in all groups, compared to day 0 and even day 7. Although these increases were not significant, all groups were, especially the blank group. It increased over 21 days, which is more evident for the blank group. AST, ALT, and ALP are the most common tests used to diagnose liver disorders [ 31 , 32 ]. Our findings express which blood levels these enzymes have increased on day 7 compared to day 0, although only significant for AST ( p ≤ 0.05). They decreased in a dose-dependent manner for BV-NEs at the end of treatment. These results align with Nicodim et al.'s investigations about rats with RA induced by Freund complete adjuvant treated with BV [ 33 ]. Parameters of Ca and Ph were elevated in CIA rats on day 7, and their reduction in all treatment groups on day 21. The level of Ca significantly decreased dose-dependent manner for all BV-NEs and BV control at the end of treatment. Therefore, BV could reduce the level of Ca and Ph after possible entry into the bloodstream, which was recently reported by Kang [ 34 ]. Also, in another study, the reducing effect of melittin of BV on Ca of the equine skeletal muscle has been shown [ 35 ] due to the interaction of melittin as a 26-residue peptide with Ca [ 36 ]. BV has an anti-inflammatory effect on pro-inflammatory cytokines and is expected to lower WBC and %Neu [ 19 ]. A decrease in the total number of WBCs at the end of treatment in all groups compared to day 7 is related to reducing inflammation in the animal. There are more reductions in the positive and BV controls, as well as BV-NEs in comparison with blank and negative control groups, due to BV's immunosuppressive effects [ 22 ]. After an increase of %Neu on day 7, it significantly decreased on day 21. The highest %Neu depletion was in the BV-NE-9.37 group. However, this difference was insignificant, and the %Neu did not return to the base level of day 0. In CIA rats treated with BV control and BV-NEs, WBC and the %Neu had diminished, which agrees with the study of formaldehyde-induced arthritis in male rats [ 22 ]. On the maximum inflammation day in the CIA rats, anemia could be seen as decreased blood levels of RBCs, Hb, and Hct [ 19 ]. After two weeks of treatment, significant changes were not seen by topical BV-NEs, but a dose-dependent increase was observed for BV-NEs (specially BV-NE-9.37 and BV-NE-18.75) about parameters of RBC, Hb, Hct, which could be due to BV improvement of circulation of blood in the micro blood vessels, as well as its role in the stimulation of building erythrocytes [ 37 ]. Indices of RBCs, including MCV, MCH, MCHC, and RDW, help elucidate the etiology of anemias [ 38 ]. According to the study, there were no crucial changes in these indices on days 7 and 21 which seems that they were less affected by inflammation of CIA and treatment of BV-NEs. Plts have increased on days 7 and 21; however, these changes were insignificant. An increase of Plts on maximum inflammation day and at the end of treatment in the groups indicates inflammation in RA's early and secondary stages [ 33 ]. The critical role of Plts is known in inflammation and immune responses. They actively participate in leukocyte recruitment, especially neutrophils, and host defense regulation in response to exogenous pathogens. This increase in Plts can also be due to the significant effect of cytokines, especially IL-6, which affects the maturation of generated cells of Plts and causes increased production of Plts [ 39 ]. Conclusion Based on the findings, induced inflammation in the collagen-induced arthritis model in rats dramatically changed some routine biochemical and hematological parameters, including increased blood levels of Glu, Chol, Ur, and AST and an increase in the count of WBCs and %Neu. In collagen-induced arthritis model rats, after two weeks of maximum inflammation (day 21 in our study), blood Glu, Chol, Tg, Ur, ALT, Ca, and Ph, also MCV returned to baseline similar to day 0. Blood levels of Cr, AST, %Neu, MCH, MCHC, RDW, and Plts count increased. Nevertheless, blood levels of ALP, count of WBCs, Hb concentration, and Hct percentage decreased. Reduction in blood levels of Glu, liver enzymes, and Ph were higher for the bee venom control than bee venom-nanoemulsion. Overall, the use of bee venom in nanoemulsion formulation as topical routes has no adverse effects on the routine biochemical and hematological parameters and can reduce inflammation induced in this model in a dose-dependent manner, especially for 18.75 and 9.37 µg/ml. Declarations Acknowledgment We want to thank those who collaborated, Mr. Yaser Eskandari Torbaghan and Mrs. Hanieh kharratian from Khalil Abad Health center (Mashhad University of Medical Sciences). Ethics approval and consent to participate All methods and experimental protocols were performed following arrive guidelines, also the relevant guidelines and regulations by the Ethical Committee of Kerman University of Medical Sciences (The Ethics approval code is IR.KMU.REC.1399.234). Competing interests All authors declare no conflict of interest. Funding This project was supported by the Kerman University of Medical Sciences (grant No. 98001207). Authors’ Contributions Yaser Yousefpoor and Amir Amani conceived and designed the experiments and wrote the manuscript. Yaser Yousefpoor and Seyed Mohammad Hashemi performed the experiments. Ali Afgar and Seyed Mohammad Amini analyzed the data. Mohamad Javad Mirzaei-Parsa participated in the study's design and helped perform the analysis with constructive discussions. All authors read and approved the final manuscript. 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Beech, Bee venom melittin is a potent toxin for reducing the threshold for calcium-induced calcium release in human and equine skeletal muscle. Life Sci, 1992. 51 (22): p. 1731-8. Permyakov, E.A., et al., Calcium-regulated interactions of human alpha-lactalbumin with bee venom melittin. Biophys Chem, 1991. 39 (2): p. 111-7. Son, D.J., et al., Therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds. Pharmacology & therapeutics, 2007. 115 (2): p. 246-270. Walker, H.K., W.D. Hall, and J.W. Hurst, Peripheral Blood Smear--Clinical Methods: The History, Physical, and Laboratory Examinations . 1990: Butterworths. Pamuk, G.E., et al., Increased platelet activation markers in rheumatoid arthritis: are they related with subclinical atherosclerosis? Platelets, 2008. 19 (2): p. 146-154. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-2524028","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":172027310,"identity":"dfc1cbc1-0bd4-47c0-80f8-afda2b0c525d","order_by":0,"name":"Yaser Yousefpoor","email":"","orcid":"","institution":"Torbat Heydariyeh University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yaser","middleName":"","lastName":"Yousefpoor","suffix":""},{"id":172027313,"identity":"7de153a0-d758-44eb-a7ec-d97871beed49","order_by":1,"name":"Amir Amani","email":"","orcid":"","institution":"Natural Products and Medicinal Plants Research Center, North Khorasan University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Amir","middleName":"","lastName":"Amani","suffix":""},{"id":172027316,"identity":"9f68651e-d76d-424a-b9ca-924563a4d839","order_by":2,"name":"Seyed Mohammad Hashemi","email":"","orcid":"","institution":"Mashhad University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Mohammad","lastName":"Hashemi","suffix":""},{"id":172027318,"identity":"14b8a395-e8d6-4cc4-a31a-90ac169f586f","order_by":3,"name":"Ali Afgar","email":"","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Afgar","suffix":""},{"id":172027319,"identity":"ce99fa3a-0e33-45d3-88a3-0a9415fd7657","order_by":4,"name":"Seyed Mohammad Amini","email":"","orcid":"","institution":"Iran University of Medical Sciences (IUMS)","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Seyed","middleName":"Mohammad","lastName":"Amini","suffix":""},{"id":172027320,"identity":"4d1c89a7-a7f1-4fbb-b257-a06865e1934a","order_by":5,"name":"Mohamad Javad Mirzaei-Parsa","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzUlEQVRIiWNgGAWjYFCCAwwMCSCamfkwVISxgVgtbMnEaoEDHmPi1Ok2HmDd8HBPnTw/O89no5ttDPL8DcxtH/BpMTtwgO1GwjM2w5nNvJuTc9sYDGccYGyeQVjLAR7GDYd5Nx8GamHcwMDYjNdhUC0S9vsP8zwGabEnVotB4gZmHmaQwxKJ0HKwDaglIXnGYTZj45xzEkAGIS03Dh+7+eNAnW1//+HH0jllNrb97e2P8WphkDjYgMIFxil+DQwM/A2EVIyCUTAKRsGIBwB4C0sygPDRJgAAAABJRU5ErkJggg==","orcid":"","institution":"Kerman University of Medical Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Mohamad","middleName":"Javad","lastName":"Mirzaei-Parsa","suffix":""}],"badges":[],"createdAt":"2023-01-28 14:44:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-2524028/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-2524028/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":32389421,"identity":"be62fbc5-e91f-432c-9bc3-da0ef10f6949","added_by":"auto","created_at":"2023-02-02 16:04:21","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1045059,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe values of the rat's blood biochemical parameters\u003c/strong\u003e \u003cstrong\u003ewere\u003c/strong\u003e \u003cstrong\u003etreated with different groups.\u003c/strong\u003e Day 0: the base levels of biochemical parameters in the rat's blood on day 0. Day 7: biochemical parameters in the rat's blood of collagen-induced arthritis model on day 7 as maximum inflammation and start of treatment. Blank (no treatment); Neg Con: Negative control (nanoemulsion free BV 0 µg/ml); Pos Con: Positive control (hydrocortisone acetate ointment 1%, 50 mg/day); BV Control (BV solution, 37.5 µg/ml); BV-NE-75, BV-NE-37.5, BV-NE-18.75, BV-NE-9.37 (4 groups of nanoemulsions containing bee venom solutions with 75, 37.5, 18.75, and 9.37 µg/ml, respectively). All treatments were followed by 5 minutes of massage the rat's paw with 1 ml bee venom solution or nanoemulsions. \u003cstrong\u003eA\u003c/strong\u003e: Glucose, \u003cstrong\u003eB\u003c/strong\u003e: Cholesterol, \u003cstrong\u003eC\u003c/strong\u003e: Triglyceride, \u003cstrong\u003eD\u003c/strong\u003e: Urea, \u003cstrong\u003eE\u003c/strong\u003e: Creatinine, \u003cstrong\u003eF\u003c/strong\u003e: AST (aspartate aminotransferase), \u003cstrong\u003eG\u003c/strong\u003e: ALT (alanine aminotransferase), \u003cstrong\u003eH\u003c/strong\u003e: ALP (alkaline phosphatase), \u003cstrong\u003eI\u003c/strong\u003e: Calcium and, \u003cstrong\u003eJ\u003c/strong\u003e: Phosphorus. *, **, *** show \u003cem\u003ep\u003c/em\u003e-value≤ 0.05, 0.01, and 0.001, respectively\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2524028/v1/930f37528e5e8dc40d772549.jpg"},{"id":32390146,"identity":"df6e730b-14d2-468c-9b88-5936bfd61ad3","added_by":"auto","created_at":"2023-02-02 16:12:19","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1094936,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe values of the rat's hematological parameters were treated with different groups.\u003c/strong\u003e The details are similar to Fig.1.\u003cstrong\u003e L\u003c/strong\u003e: WBC (white blood cell), \u003cstrong\u003eM\u003c/strong\u003e: RBC (red blood cell), \u003cstrong\u003eN\u003c/strong\u003e: Hemoglobin, \u003cstrong\u003eO\u003c/strong\u003e: Hematocrit, \u003cstrong\u003eP\u003c/strong\u003e: MCV (mean cell volume), \u003cstrong\u003eQ\u003c/strong\u003e: MCH (mean corpuscular hemoglobin), \u003cstrong\u003eR\u003c/strong\u003e: MCHC (mean corpuscular hemoglobin concentration), \u003cstrong\u003eS\u003c/strong\u003e: RDW (red cell distribution width),\u003cstrong\u003e T\u003c/strong\u003e: Platelet, and, \u003cstrong\u003eU\u003c/strong\u003e: Neutrophil (percentage of neutrophil of white blood cell). *, **, *** show \u003cem\u003ep\u003c/em\u003e-value≤ 0.05, 0.01, and 0.001, respectively.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2524028/v1/75aa2e283dc3bde3a5ca55d7.jpg"},{"id":36333738,"identity":"cf8977cb-76b5-4909-b80b-b669b7f70097","added_by":"auto","created_at":"2023-04-26 15:14:44","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":615032,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-2524028/v1/f14049e2-e409-4d51-b25f-809ec28bd8c2.pdf"},{"id":32389420,"identity":"ea9dcca3-c959-49e9-8176-12ec2ec0b336","added_by":"auto","created_at":"2023-02-02 16:04:19","extension":"jpg","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":280107,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicalAbstract.jpg","url":"https://assets-eu.researchsquare.com/files/rs-2524028/v1/444429e63c10f88ceace045c.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Topical bee venom nanoemulsion in collagen-induced arthritis model: effects on biochemical and hematological parameters","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n\u003cli\u003e\n\u003cp\u003eTopical bee venom nanoemulsion\u0026nbsp;reduces inflammation caused by arthritis.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eThis nanoemulsion passes bee venom through the skin.\u003c/p\u003e\n\u003c/li\u003e\n\u003cli\u003e\n\u003cp\u003eIt has no acute adverse effects on the routine biochemical and hematological parameters.\u003c/p\u003e\n\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eNanoemulsions (NEs), as homogeneous systems in nanotechnology, consist of at least two immiscible liquids thoroughly dispersed by a surfactant(s) and have droplets with a mean size of less than 100 nm. They exist in water-in-oil (W/O) or oil-in-water (O/W) forms [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. NEs as drug delivery systems have different advantages, including enhancing bioavailability and drug loading, controlled drug release, protection from enzymatic degradation, and carrying hydrophilic as well as hydrophobic components in a single form. Thus, they can be used as high-efficiency carriers for local and transdermal purposes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCollagen-induced arthritis (CIA) is widely used as a model of human rheumatoid arthritis caused by injecting type 2 collagen that indicates both immunological and pathological features [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This model causes multi-joint inflammation, bone proliferation, and cartilage damage among the various models; it has also a more specific onset and prospers faster [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although it is highly used in arthritis studies in rats, there is less evidence available for assessing their biochemical and hematological parameters.\u003c/p\u003e \u003cp\u003eBee Venom (BV) as a biotoxin is composed of at least 18 pharmacologically active components, including various enzymes (i.e., phospholipases and hyaluronidase), a variety of peptide components (i.e., melittin, apamin, adolapin, and mast-cell-degranulating (MCD)), non-peptide components (i.e., carbohydrates, lipids, and free amino acids), as well as active amines (i.e., histamine and epinephrine) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. It is reported that BV has various pharmaceutical properties, whose anti-inflammatory and anti-arthritis effects have been attributed to melittin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], adolapin, mast cell degranulating peptides [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], and apamin [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. BV suppresses leukocyte migration to the inflammation site by reducing cytokine production [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. BV is used for treating rheumatoid arthritis (RA) as acupuncture in clinical trials [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It can be encapsulated by water-in-oil NEs to pass through the skin through topical/transdermal delivery without being stung by bees or the injection route [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn our previous study, topical BV nanoemulsions (BV-NEs) could pass BV through the skin and reduce the inflammation caused by the collagen-induced arthritis (CIA) model in rats [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. On the other hand, studies have shown the side effects of BV on the body\u0026rsquo;s organs, such as the liver [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], kidney [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], pancreas [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and adrenal glands [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], as well as its effect on glucose and lipids as parameters of biochemistry [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] or lytic impact on red blood cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]; however, there is no pharmacodynamic information about it in the local/transdermal delivery route by NEs. Accordingly, this study aimed to evaluate any changes in biochemical and hematological parameters in Wistar rats with the CIA model; also, CIA treated with BV-NEs.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch3\u003eDrugs and chemicals\u003c/h3\u003e\n\u003cp\u003eBV was prepared by Asghapoor Honey and Bee Products Co. (Iran). It was collected from healthy and approved hives, Apis mellifera strain, by electrical stimulation. Sorbitan monooleate (Span-80) and polyoxyethylene 20 sorbitan monooleate (Tween-80) as surfactants were purchased from Merck Chemicals (Germany), and olive oil was from Fadak Co. (Iran). Bovine type II collagen was from Xi'an Harmonious Natural Bio-Technology Co., Ltd (China), and incomplete Freund's adjuvant (IFA) was from Sigma-Aldrich (USA). Biochemistry kits were prepared by Pars Azmoon Co. (Iran). Sampling tubes containing EDTA (Ethylene diamine tetra acetic acid) as an anti-coagulant were prepared from FL Co. (Italy) for collecting whole blood as ready to use.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eAnimal\u003c/h3\u003e\n\u003cp\u003e88 male Wistar rats weighing\u0026thinsp;~\u0026thinsp;200 g were used in this study, kept according to the Standard Laboratory Animal Guidelines. The Ethical Committee approved the experimental protocols of Kerman University of Medical Sciences (The Ethics approval code is IR.KMU.REC.1399.234)\u003c/p\u003e\n\u003ch3\u003ePreparation And Characterization Of NEs\u003c/h3\u003e\n\u003cp\u003eThe nanoemulsion was prepared as a volumetric percentage formulation according to the previous study [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Briefly, 3% aqueous phase (containing different BV solutions as the following) was added to the container containing 30% surfactant (including 14% Span-80 and 16% Tween-80) and mixed using a magnetic stirrer (MS-300HS, Protraction Inter-trade co, Korea) for 10 min with the rotation speed of 1000 rpm at room temperature. Then, the oil phase (67% olive oil) was added and thoroughly mixed for 10 min.\u003c/p\u003e\n\u003cp\u003eThe mean size of droplets and polydispersity index were measured by dynamic light scattering at 25\u0026deg;C using Scatteroscope (K-one Ltd. Korea). Loading capacity was also calculated in terms of 3% aqueous phase and BV concentrations as 9.37, 18.75, 37.5, and 75 \u0026micro;g/ml.\u003c/p\u003e\n\u003ch3\u003eInduction Of CIA And Treatment\u003c/h3\u003e\n\u003cp\u003eBovine type II collagen was dissolved (2 mg.ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in 0.05 M acetic acid by gently stirring overnight at 4\u0026deg;C. Next, an equal volume of IFA was added to the emulsified collagen solution, then mixed by a homogenizer (1000 RPM, 30 min, in an ice-water container) for the collagen-IFA emulsion to be used prepared [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. 0.1 ml of this emulsion was injected subcutaneously into the plantar surface of the left hind paw as the inducer dose and 0.1 ml as a booster dose in an intradermal injection into the root of the tail on the same day to induce CIA in rats [\u003cspan class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e20\u003c/span\u003e]. Maximum inflammation was observed on day 7; treatment was followed up from day 7 to day 21 for two weeks.\u003c/p\u003e\n\u003ch3\u003eExperimental Design\u003c/h3\u003e\n\u003cp\u003eRats were assigned to 8 groups (n\u0026thinsp;=\u0026thinsp;11). Each group was treated daily with one of the following treatments; blank (no treatment), negative control (NEs free BV, 0 \u0026micro;g/ml), positive control (hydrocortisone acetate ointment 1%, 50 mg/day), BV control (solution of bulk BV in NS, 37.5 \u0026micro;g/ml), and four groups of NE formulations (i.e., 9.37, 18.75, 37.5, and 75 \u0026micro;g/ml). All treatments were massaged for 5 min of the rat's paw with 1 ml of BV-solution or BV-NEs.\u003c/p\u003e\n\u003ch3\u003eBlood Sampling\u003c/h3\u003e\n\u003cp\u003eDeep anesthesia of rats was achieved using intraperitoneal injection of 87 mg ketamine/kg and 13 mg xylazine/kg of body weight [\u003cspan class=\"CitationRef\"\u003e21\u003c/span\u003e], 4 ml of blood was taken directly from their hearts, 2.5 ml for biochemical parameters, and 1.5 ml for hematological parameters and then rats were sacrificed. Three steps of blood sampling were done, including 16 samples (2 samples per group) on day 0 as an indicator of healthy rats, 16 samples (2 samples per group) on day 7 as an indicator of maximum inflammatory rats, and 56 samples (7 samples per group) on day 21 at the end of treatment. After each sampling, rats were sacrificed due to the high volume of blood loss. Samples were collected from the EDTA anti-coagulant tubes (1.5 mg/ml whole blood) for hematological parameters and without anti-coagulant tubes for biochemical parameters (The serums were separated after coagulation by centrifuging at 3000 rpm, room temperature, 10 min).\u003c/p\u003e\n\u003ch3\u003eMeasurement Of Biochemical And Hematological Parameters\u003c/h3\u003e\n\u003cp\u003eSerums were evaluated for biochemical parameters, including blood glucose (Glu), cholesterol (Ch), triglyceride (Tg), urea (Ur), creatinine (Cr), AST (aspartate aminotransferase or SGOT, serum glutamic-oxaloacetic transaminase), ALT (alanine aminotransferase or SGPT, serum glutamic pyruvic transaminase), ALP (alkaline phosphatase), calcium (Ca) and phosphorus (Ph) using an autoanalyzer (BT 1500, Biotechnica Co., Italy).\u003c/p\u003e\n\u003cp\u003eHematological parameters were counted as complete blood cells (CBC) using an automated cell counter (XP-300, Sysmex co., Japan), including white blood cells (WBCs), and the percentage of neutrophil (%Neu), red blood cells (RBCs), hemoglobin (Hb) concentration, hematocrit (Hct) percentage, and platelets (Plts) count. Also, RBC indices, including mean cell volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and red cell distribution width (RDW), were evaluated.\u003c/p\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\n\u003ch3\u003eStatistical analysis\u003c/h3\u003e\n\u003cp\u003eOne-way analysis of variance and Tukey comparison were performed to assess the statistical significance of differences among groups. Results with a \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026le;\u0026thinsp;0.05 were considered statistically significant. Statistical analyses were carried out using the SPSS software, v.19 (SPSS, Inc., USA).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAs mentioned in the previous study, droplets' mean sizes of NEs were 21.1, 16.9, 15.1, 14.2, and 12.7 nm, as well as PDIs were 0.147, 0.146, 0.143, 0.142, 0.142 for BV-NE-75, BV-NE-37.5, BV-NE-18.75, BV-NE-9.37, and BV-NE-0, respectively [\u003cspan class=\"CitationRef\"\u003e14\u003c/span\u003e]. Also, the loading capacity (in terms of 3% aqueous phase) of BV-NEs were 75, 37.5, 18.75, 9.37, and 0, which are named accordingly. It states that in a certain amount of aqueous phase (3%), with increasing the concentration of BV from 0 to 75 \u0026micro;g/ml, the size increased from 12.7 to 21.1 nm, and the PDIs also increased from 0.142 to 0.147. The load capacities of NEs increased with the increase of BV concentrations from 9.37 to 75 \u0026micro;g/ml.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e (A to J) shows the values of biochemical parameters for healthy rats (day 0), before treatment CIA rats as maximum inflammation (day 7), and for treated groups (day 21), including blank, negative, positive, and BV controls, as well as BV-NEs including 75, 37.5, 18.75, and 9.37 \u0026micro;g/ml.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. A shows the blood Glu level significantly increased in the CIA model from 188 to 227 mg/dl from day 0 to day 7. But, it was significantly returned to near the baseline level only for positive and BV controls, as well as BV-NE 18.75 and 9.37 \u0026micro;g/ml on day 21.\u003c/p\u003e\n\u003cp\u003eIn Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. B, the Chol blood level was significantly increased in the CIA model from 63 to 70 mg/dl from day 0 to day 7. It states that Chol was significantly decreased for BV control on day 21. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. C shows which Tg blood level has significantly decreased in the CIA model on day 7. however, it increased on day 21 in all groups. Changes in urea and creatinine in rat blood are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. D, and E. These biochemical parameters were not changed on days 0, 7, and 21, except urea, which significantly increased from 33 to 41 mg/dl in the CIA model.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. F, G, and H illustrate blood levels of AST, ALT, and ALP as a function of liver enzymes. Although all of them were increased in the CIA model on day 7, it was only significant for AST from 134 to 160 U/L. The AST and ALP liver enzymes were decreased in the positive and BV controls, as well as NE 9.37 \u0026micro;g/ml group on day 21 compared to day 7.\u003c/p\u003e\n\u003cp\u003eCa and Ph blood levels are given in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. I and J. for the treated groups, the levels of Ca and ph decreased on day 21 compared to the CIA model on day 7. Especially about Ca, there were significant differences for BV control and also BV-NEs groups.\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e (L to T) illustrates the results of hematological parameters for understudy groups in the pattern of Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. L shows a significant increase in the counting of WBCs from 4.96 to 9.74 (\u0026times;10\u003csup\u003e3\u003c/sup\u003e/mm\u003csup\u003e3\u003c/sup\u003e) from day 0 to day 7 in the CIA model. However, it was significantly decreased for all treatment groups on day 21, although it was higher for blank and negative control groups.\u003c/p\u003e\n\u003cp\u003eHematological parameters of RBC, Hb, and HCT are shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. M, N, and O, respectively. Figure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. P, Q, R, and S also indicate indices of RBC (MCV, MCH, MCHC, and RDW). There was no significant difference between treated groups on days 0, 7, and 21. Plts count was increased on days 7 and 21 compared to day 0, but it was not significant (see Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. T).\u003c/p\u003e\n\u003cp\u003eFigure\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e. \u003cstrong\u003eU\u003c/strong\u003e shows %Neu for treated groups. It significantly increased from 19.9\u0026ndash;54.4% from day 0 to day 7 in the CIA model and then was significantly decreased for all groups on day 21, although it was more significant for BV-NEs, BV control, and positive control groups compared to blank and negative control groups.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur previous studies found a significant effect of NEs containing BV on improving arthritis in an animal model [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. We did not know how much BV could have adverse effects after entering the body nor did we know its benefits. Thus, this study aimed to identify the side effects of NEs containing BV on the body by measuring the biochemical and hematological parameters of arthritis rats. We also measured these changes in the CIA animal model on the full inflammation day that were not treated.\u003c/p\u003e \u003cp\u003eThe study showed that on day 7 of induction of the CIA model, as the maximum inflammation day, biochemical parameters, including Glu (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.01), Ch (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001), Ur (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05), and AST (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05), had significantly increased, and only, TG (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05) had significantly decreased. Among the hematological parameters on day 7, only WBCs (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001) and %Neu (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001) had significantly increased. The other biochemical and hematological parameter changes in the CIA were insignificant from day 7 until the end of two weeks of the treatment (day 21, see the blank group). Nevertheless, the %Neu was still high compared to day 0 (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.001).\u003c/p\u003e \u003cp\u003eZainab et al. reported that WBCs and Plts had increased in formaldehyde-induced arthritis, which agrees with our results in the CIA model. Also, she reported that RBCs, Hb, and HCT diminished, which was similar to the results of the present study [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Increasing WBCs, especially %Neu in the blood of CIA model rats on day 7, can be due to calling WBCs to the inflammation site in the rat's paw by some inflammatory cytokines after induction of arthritis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In this regard, inflammatory mediators, immune cells, cytokines, and chemokines play a critical role in dyslipidemia, which hence cause metabolic effects in arthritis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. An increase in Chol and TG has been reported in formaldehyde-induced arthritis, while in the CIA model, only Chol increased and TG decreased [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Thus, there was a disorder of metabolism and dyslipidemia in arthritis and animal models of CIA.\u003c/p\u003e \u003cp\u003eBV has several anti-inflammatory and anti-arthritic properties [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. It has a variety of components with pharmacological and biochemical activities and can destroy location cells or enter the bloodstream and lyse other blood cells, especially RBCs, as it passes through the skin [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Studies have shown that BV can affect the body's metabolic systems to change their function, such as reducing blood glucose [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and affecting the blood lipids [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Although some unfavorable effects of BV can be neutralized by albumin in the bloodstream [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], it can affect organs such as islands of Langerhans of the pancreas [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], liver [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], kidney [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], adrenal glands, and their hormones [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Imani et al. showed that BV injection decreased Glu, total Chol, and Tg levels in treated diabetic rats [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. It has been shown that serum levels of Chol and TG decreased in formaldehyde-induced arthritis male rats receiving injection BV, which was similar to our results only about Chol [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe study indicated that the blood levels of Glu decreased at the end of treatment and were similar to the base level on day 0 for all groups, except for the BV control, which was lower than the base level. It suggests that topical BV can reduce the blood Glu, but this effect was not found for Chol and TG. It was reported that BV therapy is associated with multiple causes of kidney injury and hepatotoxicity [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Measuring Ur and Cr blood levels is a simple way to assess kidney injuries [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Ur and Cr increased in the CIA model on day 7. At the end of treatment on day 21, Ur and Cr dose-dependently dropped in all groups, compared to day 0 and even day 7. Although these increases were not significant, all groups were, especially the blank group. It increased over 21 days, which is more evident for the blank group. AST, ALT, and ALP are the most common tests used to diagnose liver disorders [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Our findings express which blood levels these enzymes have increased on day 7 compared to day 0, although only significant for AST (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.05). They decreased in a dose-dependent manner for BV-NEs at the end of treatment. These results align with Nicodim et al.'s investigations about rats with RA induced by Freund complete adjuvant treated with BV [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eParameters of Ca and Ph were elevated in CIA rats on day 7, and their reduction in all treatment groups on day 21. The level of Ca significantly decreased dose-dependent manner for all BV-NEs and BV control at the end of treatment. Therefore, BV could reduce the level of Ca and Ph after possible entry into the bloodstream, which was recently reported by Kang [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Also, in another study, the reducing effect of melittin of BV on Ca of the equine skeletal muscle has been shown [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] due to the interaction of melittin as a 26-residue peptide with Ca [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBV has an anti-inflammatory effect on pro-inflammatory cytokines and is expected to lower WBC and %Neu [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. A decrease in the total number of WBCs at the end of treatment in all groups compared to day 7 is related to reducing inflammation in the animal. There are more reductions in the positive and BV controls, as well as BV-NEs in comparison with blank and negative control groups, due to BV's immunosuppressive effects [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. After an increase of %Neu on day 7, it significantly decreased on day 21. The highest %Neu depletion was in the BV-NE-9.37 group. However, this difference was insignificant, and the %Neu did not return to the base level of day 0. In CIA rats treated with BV control and BV-NEs, WBC and the %Neu had diminished, which agrees with the study of formaldehyde-induced arthritis in male rats [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOn the maximum inflammation day in the CIA rats, anemia could be seen as decreased blood levels of RBCs, Hb, and Hct [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. After two weeks of treatment, significant changes were not seen by topical BV-NEs, but a dose-dependent increase was observed for BV-NEs (specially BV-NE-9.37 and BV-NE-18.75) about parameters of RBC, Hb, Hct, which could be due to BV improvement of circulation of blood in the micro blood vessels, as well as its role in the stimulation of building erythrocytes [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Indices of RBCs, including MCV, MCH, MCHC, and RDW, help elucidate the etiology of anemias [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. According to the study, there were no crucial changes in these indices on days 7 and 21 which seems that they were less affected by inflammation of CIA and treatment of BV-NEs.\u003c/p\u003e \u003cp\u003ePlts have increased on days 7 and 21; however, these changes were insignificant. An increase of Plts on maximum inflammation day and at the end of treatment in the groups indicates inflammation in RA's early and secondary stages [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The critical role of Plts is known in inflammation and immune responses. They actively participate in leukocyte recruitment, especially neutrophils, and host defense regulation in response to exogenous pathogens. This increase in Plts can also be due to the significant effect of cytokines, especially IL-6, which affects the maturation of generated cells of Plts and causes increased production of Plts [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eBased on the findings, induced inflammation in the collagen-induced arthritis model in rats dramatically changed some routine biochemical and hematological parameters, including increased blood levels of Glu, Chol, Ur, and AST and an increase in the count of WBCs and %Neu. In collagen-induced arthritis model rats, after two weeks of maximum inflammation (day 21 in our study), blood Glu, Chol, Tg, Ur, ALT, Ca, and Ph, also MCV returned to baseline similar to day 0. Blood levels of Cr, AST, %Neu, MCH, MCHC, RDW, and Plts count increased. Nevertheless, blood levels of ALP, count of WBCs, Hb concentration, and Hct percentage decreased. Reduction in blood levels of Glu, liver enzymes, and Ph were higher for the bee venom control than bee venom-nanoemulsion. Overall, the use of bee venom in nanoemulsion formulation as topical routes has no adverse effects on the routine biochemical and hematological parameters and can reduce inflammation induced in this model in a dose-dependent manner, especially for 18.75 and 9.37 \u0026micro;g/ml.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe want to thank those who collaborated, Mr. Yaser Eskandari Torbaghan and Mrs. Hanieh kharratian from Khalil Abad Health center (Mashhad University of Medical Sciences).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll methods and experimental protocols were performed following arrive guidelines, also the relevant guidelines and regulations by the Ethical Committee of Kerman University of Medical Sciences (The Ethics approval code is IR.KMU.REC.1399.234).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was supported by the Kerman University of Medical Sciences (grant No. 98001207).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eYaser Yousefpoor and Amir Amani conceived and designed the experiments and wrote the manuscript. Yaser Yousefpoor and Seyed Mohammad Hashemi performed the experiments. Ali Afgar and Seyed Mohammad Amini analyzed the data. Mohamad Javad Mirzaei-Parsa participated in the study\u0026apos;s design and helped perform the analysis with constructive discussions. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOsanloo, M., et al., \u003cem\u003ePreparation and optimization nanoemulsion of Tarragon (Artemisia dracunculus) essential oil as effective herbal larvicide against Anopheles stephensi.\u003c/em\u003e Industrial crops and products, 2017. \u003cstrong\u003e109\u003c/strong\u003e: p. 214-219.\u003c/li\u003e\n\u003cli\u003eYousefpoor, Y., et al., \u003cem\u003eThe combined effects of Aloe vera gel and silver nanoparticles on wound healing in rats.\u003c/em\u003e Nanomedicine Journal, 2016. \u003cstrong\u003e3\u003c/strong\u003e(1): p. 57-64.\u003c/li\u003e\n\u003cli\u003eValizadeh, A., et al., \u003cem\u003eLevofloxacin nanoemulsion gel has a powerful healing effect on infected wound in streptozotocin-induced diabetic rats.\u003c/em\u003e Drug Delivery and Translational Research, 2021. \u003cstrong\u003e11\u003c/strong\u003e(1): p. 292-304.\u003c/li\u003e\n\u003cli\u003eRoozitalab, G., et al., \u003cem\u003eAntioxidative, anticancer, and antibacterial activities of a nanoemulsion-based gel containing Myrtus communis L. essential oil.\u003c/em\u003e Chemical Papers, 2022: p. 1-11.\u003c/li\u003e\n\u003cli\u003eBilliau, A. and P. 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Chimedragcha, \u003cem\u003eEffect of Honey Bee Venom (Apis mellifera) on Hyperglycemia and Hyperlipidemia in Alloxan Induced Diabetic Rabbits. J Diabetes Metab 6: 507. doi: 10.4172/2155-6156.1000 507 Page 2 of 4 Volume 6\u0026bull; Issue 3\u0026bull; 1000507 J Diabetes Metab ISSN: 2155-6156 JDM, an open access journal 2.71\u0026plusmn;0.24 mmol/l and continued dropping down to 2.24*\u0026plusmn;0.28 mmol/l by day 14, 14.2% lower than the diabetic group which was very close to the control values (P\u0026lt; 0.05).\u003c/em\u003e Figure, 2015. \u003cstrong\u003e5\u003c/strong\u003e: p. 1.66-0.07.\u003c/li\u003e\n\u003cli\u003eZarrinnahad, H., et al., \u003cem\u003eApoptotic effect of melittin purified from Iranian honey bee venom on human cervical cancer HeLa cell line.\u003c/em\u003e International journal of peptide research and therapeutics, 2018. \u003cstrong\u003e24\u003c/strong\u003e(4): p. 563-570.\u003c/li\u003e\n\u003cli\u003eMousavi, S.M., et al., \u003cem\u003eEffect of Iranian honey bee (Apis mellifera) venom on blood glucose and insulin in diabetic rats.\u003c/em\u003e Journal of arthropod-borne diseases, 2012. \u003cstrong\u003e6\u003c/strong\u003e(2): p. 136.\u003c/li\u003e\n\u003cli\u003eAlqutub, A.N., et al., \u003cem\u003eBee sting therapy-induced hepatotoxicity: A case report.\u003c/em\u003e World journal of hepatology, 2011. \u003cstrong\u003e3\u003c/strong\u003e(10): p. 268.\u003c/li\u003e\n\u003cli\u003eKamal, A., \u003cem\u003eEstimation of blood urea (BUN) and serum creatinine level in patients of renal disorder.\u003c/em\u003e Indian J Fundam Appl Life Sci, 2014. \u003cstrong\u003e4\u003c/strong\u003e(4): p. 199-202.\u003c/li\u003e\n\u003cli\u003ePu, N., et al., \u003cem\u003eAlkaline phosphatase-to-albumin ratio as a prognostic indicator in pancreatic ductal adenocarcinoma after curative resection.\u003c/em\u003e Journal of Cancer, 2017. \u003cstrong\u003e8\u003c/strong\u003e(16): p. 3362.\u003c/li\u003e\n\u003cli\u003eRej, R., \u003cem\u003eAminotransferases in disease.\u003c/em\u003e Clinics in laboratory medicine, 1989. \u003cstrong\u003e9\u003c/strong\u003e(4): p. 667-687.\u003c/li\u003e\n\u003cli\u003eFiț, N., et al., \u003cem\u003eHaematological and biochemical investigations in rats with rheumatoid arthritis induced by Freund Complete Adjuvant and treated with bee venom.\u003c/em\u003e Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca. Veterinary Medicine, 2011. \u003cstrong\u003e68\u003c/strong\u003e(1): p. 151-158.\u003c/li\u003e\n\u003cli\u003eKang, H.S., et al., \u003cem\u003eThe cardiovascular depression caused by bee venom in Sprague-Dawley rats associated with a decrease of developed pressure in the left ventricular and the ratio of ionized calcium/ionized magnesium.\u003c/em\u003e Am J Chin Med, 2008. \u003cstrong\u003e36\u003c/strong\u003e(3): p. 505-16.\u003c/li\u003e\n\u003cli\u003eFletcher, J.E., L. Tripolitis, and J. Beech, \u003cem\u003eBee venom melittin is a potent toxin for reducing the threshold for calcium-induced calcium release in human and equine skeletal muscle.\u003c/em\u003e Life Sci, 1992. \u003cstrong\u003e51\u003c/strong\u003e(22): p. 1731-8.\u003c/li\u003e\n\u003cli\u003ePermyakov, E.A., et al., \u003cem\u003eCalcium-regulated interactions of human alpha-lactalbumin with bee venom melittin.\u003c/em\u003e Biophys Chem, 1991. \u003cstrong\u003e39\u003c/strong\u003e(2): p. 111-7.\u003c/li\u003e\n\u003cli\u003eSon, D.J., et al., \u003cem\u003eTherapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds.\u003c/em\u003e Pharmacology \u0026amp; therapeutics, 2007. \u003cstrong\u003e115\u003c/strong\u003e(2): p. 246-270.\u003c/li\u003e\n\u003cli\u003eWalker, H.K., W.D. Hall, and J.W. Hurst, \u003cem\u003ePeripheral Blood Smear--Clinical Methods: The History, Physical, and Laboratory Examinations\u003c/em\u003e. 1990: Butterworths.\u003c/li\u003e\n\u003cli\u003ePamuk, G.E., et al., \u003cem\u003eIncreased platelet activation markers in rheumatoid arthritis: are they related with subclinical atherosclerosis?\u003c/em\u003e Platelets, 2008. \u003cstrong\u003e19\u003c/strong\u003e(2): p. 146-154.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"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":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Nanoemulsion, Bee Venom, Biochemical, Hematological, Arthritis","lastPublishedDoi":"10.21203/rs.3.rs-2524028/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-2524028/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eTraditionally, Bee venom (BV) is used through stinging or injection to treat rheumatoid arthritis (RA). This study aimed to assess the side effects of bee venom nanoemulsions (BV-NEs) in the \u0026nbsp;collagen-induced arthritis (CIA) model by examining biochemical and hematological parameters.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eThe BV-NEs were prepared, and the CIA model was induced in rats. After the seventh day, the groups were topically treated for two weeks as the following: blank (free treatment), negative control (NE-0), positive control (hydrocortisone acetate ointment 1%, 50 mg/day), BV control (37.5 µg/ml/day), and BV-NEs receiving 75, 37.5, 18.75, and 9.37 µg/ml/day. Three steps of blood sampling were done on days 0, 7, and 21 (healthy rats, before and at the end of treatment, respectively).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe results revealed that blood levels of glucose, cholesterol, urea, aspartate aminotransferase (AST), alkaline phosphatase (ALP), white blood cell (WBC), and %neutrophil significantly increased before the treatment. Nevertheless, most parameters declined at the end of the treatment compared to the blank and negative control groups about BV-NEs dose-dependently. The drastic changes in biochemical parameters in the CIA model indicated the effect of the immune system function on the metabolic system. Also, NE's impact of BV passed through the skin on these items.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eBV-NEs can reduce inflammation caused by arthritis without acute adverse effects on the routine biochemical and hematological parameters.\u003c/p\u003e","manuscriptTitle":"Topical bee venom nanoemulsion in collagen-induced arthritis model: effects on biochemical and hematological parameters","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-02-02 16:04:14","doi":"10.21203/rs.3.rs-2524028/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"4d1ac37e-4ba6-4c02-8b7a-c3bf78605aa4","owner":[],"postedDate":"February 2nd, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-04-26T15:14:32+00:00","versionOfRecord":[],"versionCreatedAt":"2023-02-02 16:04:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-2524028","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-2524028","identity":"rs-2524028","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}