Bee venom reduces early inflammation and oxidative stress associated with lipopolysaccharide-induced alpha-synuclein in the substantia nigra-striatum axis

Bee venom reduces early inflammation and oxidative stress associated with lipopolysaccharide-induced alpha-synuclein in the substantia nigra-striatum axis | 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 Research Article Bee venom reduces early inflammation and oxidative stress associated with lipopolysaccharide-induced alpha-synuclein in the substantia nigra-striatum axis Alma Karen Lomeli-Lepe, José Luis Castañeda-Cabral, Mónica E. Ureña-Guerrero, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4551820/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Sep, 2024 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted 17 You are reading this latest preprint version Abstract Neuroinflammation and oxidative stress are important features in the pathogenesis and development of synucleinopathies, the glial activation and upregulation of pro-inflammatory and oxidative mediators induce alpha-synuclein (α-syn) accumulation. Recent studies have shown that bee venom (BV) has beneficial effects on symptoms of these neurodegenerative diseases. BV is known to exert anti-inflammatory and anti-oxidative effects. Here, we investigated the effects of BV over the different inflammatory and oxidative markers, and in the expression of α-syn and tyrosine hydroxylase (TH) in a lipopolysaccharide (LPS)-induced rat model of synucleinopathies. We examined whether BV (1.5 mg/kg by acupoint injection ST36 six times every 48 hours) could change the α-syn and TH expression measured by western blotting, also, observed the activation of microglia and astrocytes by immunofluorescence, quantify the proinflammatory cytokines levels (TNF-α and IL-1β) by ELISA, and estimated the lipid peroxidation and the activity of superoxide dismutase (SOD) and catalase (CAT) by colorimetric kits in LPS-treated rats (2.5 µg by a single dose intranigral injection) in substantia nigra (SN) and striatum (STR) brain areas. In the LPS-injected rat brain, BV treatment reduced α-syn levels and increased the TH levels. In addition, we observed lower microglia and astrocyte activation in SN and STR. Furthermore, BV decreases IL-1β and lipid peroxidation and increases the CAT activity in the STR. These results indicate that BV can restore the α-syn and TH levels possibly by the inhibition of LPS-induced neuroinflammation and oxidation, also, these results suggest that BV could be a promising treatment option for synucleinopathies. Bee venom alpha-synuclein monomers synucleinopathies- substantia nigra Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 INTRODUCTION Synucleinopathies are a group of neurodegenerative diseases characterized by exacerbated expression of alpha-synuclein (α-syn) and its dissemination in the brain. In Parkinson's disease, the best-characterized synucleinopathy, fibrils and tangles of α-syn underlie the disease’s pathological manifestations. However, α-syn aggregates are also observed in other clinical entities, such as Parkinson's-associated dementia, Lewy body dementia, multiple system atrophy, and pure autonomic failure (Savica et al. 2018 ; Simuni et al. 2024 ). The pathogenesis of these neurological disorders is complex, and the evolution of the disease adds side effects and collaterals due to the mainly symptomatic medication. The overexpression of α-syn in the dopaminergic axis substantia nigra-striatum seems to be favored by a neuroinflammatory state (Peixoto et al. 2023 ; Lomeli-Lepe et al. 2023 ; Eser et al. 2024 ). Neuroinflammation can be induced experimentally using lipopolysaccharide (LPS), a bacterial endotoxin that produces a potent glial activation and promotes the aggregation of α-syn (Sharma and Nehru 2015 ). Several natural compounds have therapeutic effects in human pathologies because they affect multiple molecular targets and signaling pathways through mechanisms not yet fully described (Rehman et al. 2019 ; Rahman et al. 2021 ). Bee venom (BV) has beneficial effects against cancer, neuroinflammatory processes, arthropathies (Aufschnaiter et al. 2020 ), and immune diseases (Wehbe et al. 2019 ). Several clinical trials have found evidence that BV could be beneficial in treating neurodegenerative diseases (Zhang et al. 2018 ). BV is a mixture of peptides, enzymes, amines, sugars, and minerals; melittin, phospholipase A2, apamin, adolapin, and mast cell degranulation peptide are the molecules found in high concentrations (Aufschnaiter et al. 2020 ; Shi et al. 2022 ). Given the tendency of α-syn to form oligomers in oxidative and inflammatory milieu, we hypothesize that BV components act synergistically to counteract the generation of mediators of this type in the brain and, as a consequence, reduce the overexpression of α-syn. To address this hypothesis, we applied a BV systemic treatment to animals with LPS-induced inflammation and oxidative stress in the SN and STR. We confirmed that BV treatment decreases the expression of monomeric α-syn and the astrocytic and microglial activation induced by LPS while maintaining a normal expression of TH in the rat SN and STR. We also observed that BV maintains lower levels of oxidative and inflammatory markers in the STR compared to animals that did not receive this compound. MATERIALS AND METHODS Animal treatment All animals used in this work were housed under standard animal care conditions, with free access to water and chow ad libitum . All procedures were carried out according to the ethical guidelines of the Animal Care and Use Committee of the University Campus of Biological and Agricultural Science (CUCBA) of the University of Guadalajara under the agreement CINV.104/12. Male Wistar rats between 200–250 g were anesthetized, and LPS (Escherichia coli 0111:B4; Sigma) (LPS group, n = 10) was stereotaxically injected into the right SN (2.5 µg in 2.5 µl of saline solution, SS) over 2.5 min using a motorized microinjection pump. The following coordinates were used for the injection: -5.8 mm posterior to the bregma, 1 mm lateral to the midline, and 7.8 mm ventral to the surface of the skull. The SHAM group (n = 10) received SS in the SN following the same procedure, and the Control group (n = 5) consisted of untreated animals. Five days after LPS injection, lyophilized BV (obtained from the Bee Research Center of the Southern University Center (CUSur), University of Guadalajara) was used to treat a subset of rats previously injected with LPS (LPS + BV group, n = 5), and animals randomly selected from the SHAM group (SHAM + BV group, n = 5). Each animal received a subcutaneous (s.c.) BV injection (1.5 mg per kg of body weight, dissolved in SS) at the ST36 acupuncture point (Oh and Kim 2022 ) every 48 hours for a total of 6 doses (Fig. 1 ). The animals were sacrificed 15 days after the end of the BV treatment (Fig. 1 ). Western Blot assay After sacrifice, the brain was removed, and the SN and STR were dissected, separating the ipsilateral and contralateral sides from the LPS injection. Protein concentration was determined according to the Lowry method (Lowry et al. 1951 ) with a DC Protein Assay Kit (Bio-Rad Laboratories, USA Cat. 500011) on a Multiskan Go spectrophotometer (Thermo Scientific, Finland), using bovine serum albumin (BSA) (Bio-Rad Laboratories, USA Cat. 500-0007) as the external standard. Briefly, 20 µg of denatured protein per lane were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Separated proteins were blotted to nitrocellulose membranes (Protean Premium 0.45 µm, Amersham, Germany), which were later placed in a 5% blocker solution (QuickBlocker, EMD Millipore, USA) and dissolved in 0.1 M PBS with 0.1% Tween-20 at 4°C for 1 hour. Then, the following primary antibodies and dilutions were used for detection of α-syn, TH, and GAPDH: Rabbit anti-alpha-synuclein (1:2000; Abcam, Cambridge, UK Cat. Ab212184), rabbit anti-TH (1:1000; Abcam, Cambridge, UK Cat. ab112), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5,000; Abcam, Cambridge, UK, Cat. Ab125247). Incubation with primary antibodies was carried out at 4°C overnight with gentle shaking. Horseradish peroxidase (HRP)-conjugated secondary antibodies anti-rabbit IgG (1:10,000; LICOR USA Cat.926-80011) and anti-mouse IgG (1:10,000; LICOR USA Cat.926-80010) were used to recognize the primary antibody. Finally, bands were revealed with a chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, USA). Band images were obtained using a C-DiGit Blot Scanner (LI-COR Bioscience, USA), and their density was estimated using Image Studio Lite 3.1.4 software (LI-COR Bioscience, USA). The results are presented as the expression ratio to constitutive protein (GAPDH). Immunofluorescence Animals were deeply anesthetized with pentobarbital until a negative toe-pinch response was achieved to proceed with intracardiac perfusion with 0.9% NaCl, followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4). After being carefully extracted from the skull, brains were transferred to a cryoprotectant solution (30% sucrose and 30% ethylene glycol in PBS) at 4°C for 5 days. Frozen tissues were embedded with Tissue Plus (O.C.T. compound, Fisher HealthCare, cat. 4585) (Sakura Finetek, Torrance, CA, USA) and transferred to a cryostat set to -20°C overnight. Brains were cut in the frontal plane into 30 µm-thick sections, which were immediately transferred to 24-well plates containing approximately 1 mL of PBS before proceeding with immunofluorescence (IF). Floating sections were permeabilized with 2% Triton X-100 in PBS overnight and blocked with 5% BSA for 30 min at room temperature under gentle shaking. The sections were incubated with mouse anti-IBA1 (1:1,000; Abcam, Cambridge, UK, Cat. AB209064) and mouse anti-GFAP (1:1000; Millipore, USA Cat. MAB360) at 4°C overnight. Slices were washed three times in PBS for 10 min and then incubated with the following secondary antibodies: anti-mouse Alexa Fluor 488 (1:1,000, Abcam, Cambridge, UK, cat. Ab209064) and anti-mouse Alexa Fluor 594 (1:1,000, Abcam, Cambridge, UK cat. Ab150077) for 2 h. After washing in PBS for 10 min thrice, sections were mounted on glass coverslips using PBS-Glycerol 1:1. Imaging and OD estimation To evaluate the astrocytic GFAP-positive and microglial IBA-1-positive immunoreactivity in SN and STR, we obtained fluorescence images of STR and SN sections. An inverted microscope (Nikon, model eclipse 55i with an integrated Nikon camera, model ds-fi1) and PLN 10X/0.1NA and 20X/0.5NA objective lens was used. Optical density (OD) was analyzed semiquantitatively in the software ImageJ by transforming the original images into grayscale images (assigning a value of 0 to white and a value of 255 to black) (Lai et al. 2021 ). In this way, the shades of gray are converted into OD using the following equation: OD = log (255/mean gray level) Six sections per animal per region (three ipsilateral and three contralateral; total area by side of 0.75 mm2) were used for quantification. The six values per region from each rat were averaged to calculate a total value for glial and astrocytic activation. Enzyme-linked immunosorbent assay (ELISA) To assess the presence of neuroinflammatory agents in response to LPS stimulation, we measured TNF-α and IL-1β concentration by ELISA in STR homogenates. Tissue samples from all groups of STR ipsilateral and contralateral (100 mg/mL of protein) to LPS injection were prepared for commercial TNF-α (ab100785, Abcam) and IL-1β (ab100768, Abcam) kits, according to the manufacturer’s instructions. Standards and samples were assessed in duplicate, and the absorbance was measured at 450 nm in a spectrophotometer (Multiskan GO, Thermo Scientific, Finland). Cytokine concentrations were normalized to the total protein content and reported as mean ± standard deviation (SD) in pg/mg of total protein. Oxidative markers To evaluate the oxidative state in response to the treatments, we quantified the levels of some oxidative stress markers. For this, 100 mg/mL of homogenate of STR (ipsilateral and contralateral to LPS injection) was applied to the following commercial kits, as per the manufacturer’s instructions: Lipid peroxidation (MDA) colorimetric assay kit (Cat. ab233471, Abcam), Superoxide dismutase (SOD) activity colorimetric assay kit (Cat. ab65354, Abcam), and Catalase (CAT) activity colorimetric assay kit (Cat. ab83464, Abcam). In all cases, standards and samples were measured in duplicates. Absorbance was measured using a spectrophotometer (Multiskan GO, Thermo Scientific, Finland). Lipid peroxidation and CAT activity were expressed as nanomoles per gram of tissue protein (nmol/g), and superoxide dismutase activity was expressed as the percentage of inhibition (%). Statistical data analysis The results of inflammatory, oxidative stress markers, and immunolabeled cells are expressed as the mean ± SD. For WB, results are expressed as a ratio to constitutive protein (GAPDH). Statistical differences between groups were analyzed by one-way ANOVA followed by Tukey's post hoc test, with p < 0.05. The results were analyzed using the Prism Graph Pad v 8.0 software (GraphPad Prism Software Inc., USA). RESULTS Monomeric α-syn expression In the SN ipsilateral to LPS injection, no significant difference was found between the Control, SHAM, and SHAM + BV groups (p = 0.9769 Control vs SHAM; p = 0.9943 Control vs SHAM + BV; p = 0.9997 SHAM vs SHAM + BV), suggesting that the mechanical lesion combined with the series of BV doses does not alter the expression of monomeric α-syn in this region. In contrast, the LPS group showed a significant increase in monomeric α-syn expression compared to the Control (p = 0.0211 Control vs LPS), SHAM (p = 0.0059 SHAM vs LPS), and SHAM + BV (p = 0.0088 SHAM + BV vs LPS) groups. The group that received BV after LPS injection maintained the expression of monomeric α-syn at a level comparable to the Control, SHAM, and SHAM + BV groups (p = 0.9984 Control vs LPS + BV; p = 0.9983 SHAM vs LPS + BV; p = 0.9999 SHAM + BV vs LPS + BV), and significantly lower than the LPS group (p = 0.0113 LPS vs LPS + BV). On the contralateral side of the SN, no significant changes were observed between the groups (Fig. 2 a). In STR ipsilateral to LPS lesion, there were also no significant differences between the Control, SHAM, and SHAM + BV group (p = 0.3054 Control vs SHAM; p = 0.5637 Control vs SHAM + BV; p = 0.2563 SHAM vs SHAM + BV). In contrast, the LPS group shows an elevated expression of monomeric α-syn (p = 0.0222 Control vs LPS), while the LPS + BV combination and SHAM + BV maintains this expression at the level of the Control group (p = 0.0478 LPS vs LPS + BV; p = 0.9471 Control vs LPS + BV; p = 0.0007 SHAM + BV vs LPS; p = 0.5637 SHAM + BV vs Control) (Fig. 2 c). These data confirm our previous results indicating that LPS increases the expression of monomeric α-syn, and evidence that BV has a favorable outcome preventing the effect of LPS and maintaining protein expression at a physiological level. TH expression On the SN ipsilateral to LPS injection, TH was significantly reduced in the LPS group compared to the Control group (p = 0.0057 Control vs LPS); and the combination of LPS + BV maintained the TH expression level comparable to the Control (p = 0.3684, Control vs LPS + BV) (Fig. 3 a). In STR, TH expression also had no significant changes between control groups (p = 0.9360 Control vs SHAM; p = 0.1594 Control vs SHAM + BV; p = 0.9999 SHAM vs SHAM + BV), whereas the animals with LPS show significantly decreased TH compared to the Control group (p = 0.0346 LPS vs Control). In contrast, animals in the LPS + BV group maintain the TH expression close to the control level (p = 0.6813 Control vs LPS + BV) but significantly higher than the group with only LPS (p = 0.0499 LPS vs LPS + BV) (Fig. 3 c). Taken together, these results support that BV may be useful for the neuroprotection of TH-positive cells that are susceptible to α-syn deregulation. Reactivity of astrocytes and microglia in response to LPS As expected, LPS increased reactive astrocytes and microglia on the ipsilateral and contralateral (to LPS injection) sides of the SN (Fig. 4 ) and STR (Fig. 5 ). The OD of the IF signal showed a pronounced rise in GFAP + and IBA-1 + cells in both the SN and the STR compared to the controls (p < 0.0001). The treatment with BV greatly prevents this glial activation, evidenced by a significant decrease of both markers in the LPS + BV group (Fig. 4 – 5 ), suggesting that BV controls the proinflammatory molecules released by astrocytes and microglia. Proinflammatory cytokines We did not find statistically significant changes in TNF-α expression, although there was an upward trend in the LPS group and a downward trend in the BV group on the STR ipsi- (p = 0.4220 Control vs LPS; p = 0.7186, LPS vs LPS + BV; p = 0.9998, Control vs LPS + BV) and contralateral (p = 0.1419, Control vs LPS; p = 0.3823, LPS vs LPS + BV; p = 0.9885, Control vs LPS + BV) sides to the LPS injection (Fig. 6 a). We found no notable differences in IL-1β in animals with LPS compared to the control group in the ipsi- (p = 0.4924, Control vs LPS) and contralateral STR (p = 0.3336, Control vs LPS). Interestingly, the control group differed from the BV-treated groups treated in the ipsi- (p = 0.0766, Control vs LPS + BV; p = 0.0197, Control vs SHAM + BV) and contralateral STR (p = 0.0051, Control vs LPS + BV; p = 0.0690, Control vs SHAM + BV). Furthermore, IL-1β expression was higher in the untreated LPS group than in the LPS + BV and SHAM + BV groups in the ipsilateral STR (p = 0.0485, LPS vs LPS + BV; p = 0.0716, LPS vs SHAM + BV), and higher than the LPS + BV group in the contralateral STR (p = 0.0406, LPS vs LPS + BV). The untreated Control and SHAM groups show no differences in both sides of the STR (p = 0.8913, Control vs SHAM ipsilateral; p = 0.9234, Control vs SHAM contralateral; p = 0.8916, SHAM vs SHAM + BV ipsilateral, p = 0.3573, SHAM vs SHAM + BV contralateral). These results suggest that BV treatment alone is sufficient to decrease the expression of IL-1β, even in the absence of an inflammatory response in ipsilateral STR (Fig. 6 b). Oxidative markers Lipid peroxidation was estimated indirectly through the quantification of MDA. MDA concentrations were not different between the Control and LPS groups in the ipsilateral STR (p = 0.4918 Control vs LPS); however, the contralateral side showed a significant change (p = 0.0981, Control vs LPS). No differences were found in MDA concentration between Control, SHAM, and SHAM + BV groups, in the ipsi- (p = 0.9997, Control vs SHAM; p = 0.9993, SHAM vs SHAM + BV; p = 0.9931, Control vs SHAM + BV), and contralateral sides of STR (p = 0.6610, Control vs SHAM; p = 0.9523, SHAM vs SHAM + BV; p = 0.9580, Control vs SHAM + BV) (Fig. 7 ). The combination of LPS + BV significantly decreased MDA concentrations compared to the untreated LPS group in the ipsi- (p = 0.0094, LPS vs LPS + BV) and contralateral (p = 0.0141, LPS vs LPS + BV) STR, and even below the Control group in the ipsilateral STR (p = 0.0637, Control vs LPS + BV). Interestingly, MDA levels significantly increased in the LPS group compared to the SHAM and SHAM + BV groups (p = 0.0136, SHAM vs LPS; p = 0.0369, SHAM + BV vs LPS) only in the contralateral STR (Fig. 7 ). Regarding the inhibition of SOD activity, no significant changes were found between groups in this enzymatic marker (Fig. 8 a). Similarly, no significant difference in CAT activity was found between Control and LPS groups. Surprisingly, the LPS + BV combination increased CAT activity compared to all groups in the ipsilateral (p = < 0.0001, SHAM vs LPS + BV, SHAM + BV vs LPS + BV, LPS vs LPS + BV and Control vs LPS + BV) and except the Control in contralateral STR (p = 0.0212, SHAM vs LPS + BV; p = 0.0351, SHAM + BV vs LPS + BV; p = 0.0166, LPS vs LPS + BV; p = 0.2285, Control vs LPS + BV), (Fig. 8 b). Therefore, we conclude that there is an inverse relationship between the expression of α-syn and CAT activity, which can be attributed to the effect of BV. Taken together, our results suggest that BV is a promising molecule for effectively controlling oxidative markers and modulating IL-1β in the context of an inflammatory response. DISCUSSION This work challenged BV's neuroprotective capacity, its potential to control α-syn expression in the rat brain, and the self-sustaining inflammatory and oxidative environment generated around this process. Our results show that LPS stimulation led to the following temporally and spatially coinciding changes: an increase in monomeric α-synuclein expression, an increase in the density of GFAP + and IBA-1 + cells, and a decrease in TH. Conversely, LPS increases IL-1β and lipid peroxidation and reduces catalase activity, promoting oxidative stress and inflammation. The treatment with BV effectively countered this effect, showing a potent anti-inflammatory and antioxidant activity under these experimental conditions. Previous studies have shown that BV decreases the degeneration of nigral dopaminergic cells and microglia in an MPTP + Parkinson's disease model in mice (Kim et al. 2011 ; Doo et al. 2012 ). Applying BV to cultures of LPS-activated microglia decreases the production of NO, iNOS, and TNF-α, and decreases the presence or expression of NF-kB, IL-1β, IL-6, and prostaglandins (Park et al. 2004 ; Han et al. 2007 ; Moon et al. 2007 ). Our immunofluorescence results confirm and support these observations by demonstrating a decrease in the OD of IBA-1 + cells after BV treatment in the SN and STR. Notably, the results presented here extend BV’s effects to astrocytes, since we reported for the first time a decrease in GFAP + cells in SN and STR in BV-treated animals. Astrocytes represent approximately 30% of brain cells, and their activation (in this case, by LPS) triggers the release of inflammatory modulators, chemokines, cytokines, and neurotrophic factors, either neuroprotective or neurotoxic (Trujillo-Estrada et al. 2019 ). These molecules, along with microglia activation, enhance and sustain neuroinflammation. Thus, BV was beneficial to control the pro-inflammatory cellular response induced by LPS. We also found that BV diminished IL-1β, a pleiotropic cytokine that leads to the synthesis of proinflammatory and chemotactic mediators (Lee et al. 2010 ) and contributes to the pathogenesis of neurodegenerative diseases (Mendiola and Cardona 2018 ). Together, these results suggest that controlling microglial and astrocytic activation by BV decreases IL-1β production, resulting in a neuroprotective effect towards TH + cells, which maintained their immunoreactivity in animals treated with BV. However, we cannot rule out that IL-1β is involved in the initial activation of microglia, since this molecule is rapidly induced in the brain after acute brain injury (Mendiola and Cardona 2018 ). In addition, oligomeric α-syn released by neurons has been shown to induce inflammatory responses of microglia through activation of the toll-like receptors 2 (TLR2) (Kim et al. 2013 ). Interestingly, the work of Kim (2022) showed that the pro-inflammatory cytokines TNF-α and IL-1β stimulate the cell-to-cell transmission of α-syn in vitro . Therefore, although we found no significant changes in TNF-α concentration (which may have been due to an insufficient LPS stimulus), our results demonstrate that BV can reduce IL-1β in conditions of neuroinflammation, likely preventing the spread of α-syn in vivo (Kim et al. 2022 ). This observation should be further studied in future research. Although the molecular mechanisms explaining BV’s effects are still unknown, it has been proposed that BV could exert an anti-inflammatory effect by regulating microglia activity, the transcription of nuclear factor kappa B (NF-kB), the mitogen-activated protein kinase (MAPK) pathway and protein kinase B pathway (Akt) (Zhang et al. 2018 ). In addition, melittin, the major component of the venom, suppresses the signaling pathways of TLR2 and TLR4, cluster of differentiation (CD14), the essential modulator of nuclear factor kappa-B (NEMO) and platelet-derived growth factor receptor beta (PDGFRβ) (Park et al. 2004 ; Son et al. 2006 ; Moon et al. 2007 ; Park et al. 2008 ; Lee et al. 2014 ). By inhibiting these pathways, melittin decreases the activation of p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), Akt, PLCγ1, and the translocation of NF-κB to the nucleus, resulting in reduced inflammation. The anti-inflammatory effect of bvPLA2 was previously established (Lee and Bae 2016 ), and both BV components (melittin and bvPLA2) synergistically enhance its effects (Damianoglou et al. 2010 ). This explains why the use of complete BV has better effects than its individual components. Likely, the inhibition of these pro-inflammatory pathways by BV is significantly participating in maintaining adequate levels of α-syn, TH, and IL-1β and reducing the activation of microglia and astrocytes, which highlights its anti-neuroinflammatory potential. However, more studies are still needed to elucidate the mechanisms by which BV could influence the expression of these proteins. So far, oxidative stress is considered a primary cause of α-syn neurotoxicity (Luk 2019 ; Li et al. 2024 ) because the presence of aggregates alters neuronal intracellular redox balance. Therefore, it is assumed that reducing oxidative stress can effectively avoid aggregation of this protein. The activity of catalase and glutathione peroxidase, two enzymes responsible for ROS removal and thus markers of oxidative stress, is reduced in PD brains (Silva et al. 2022 ) and in animal models of the disease (Anjum et al. 2024 ; Kumar et al. 2023 ), Furthermore these markers are increased along with lipid oxidation in brains with synucleinopathies (Fu et al. 2022 ). These results are consistent with those of the current study, where we found an imbalance in the oxidative status represented by various alterations in CAT activity and lipid peroxidation. An interesting relationship between CAT activity and α-syn arises from the work done by Yakunin (2014), who observed that PPARγ (a member of the nuclear receptor family) inhibition by α-syn overexpression also inhibits CAT activity. These results match the LPS-induced α-syn overexpression and CAT reduction observed in our results. Limiting cellular antioxidant mechanisms favors the production of ROS and lipid peroxidation. Our results showed that BV reduces lipid peroxidation and significantly increases CAT activity, indicating a strengthening of oxidant removal systems associated with decreased α-syn expression. Although the mechanism by which BV mediates this effect is still unclear, previous work has shown that BV upregulates brain-derived neurotrophic factor (BDNF) expression through ERK activation (Cai et al. 2017 ). This suggests a positive effect of BV on the BDNF/tyrosine kinase B (TrkB)/cAMP-response element-binding protein (CREB) signaling pathway, which is key to strengthening the cellular intrinsic antioxidant defense (Jin 2020 ). BV is also known to upregulate the activation of the Nrf2/HO-1 signaling pathway (Jin 2020 ), which is responsible for producing the intrinsic antioxidant enzyme HO-1. This enzyme is considered the first line of cellular defense against oxidative damage (Chen et al. 2020 ). Although it is generally believed that these interactions are mediated by melittin or PLA2 present in BV, it is more likely that the interaction of several components enhances the effect of BV. CONCLUSIONS The pathologies associated with α-syn represent a challenge to human health. Generating effective therapies is still pending since multiple environmental and genetic factors contribute to its etiology. Our results allow us to conclude that BV reduces the expression of α-syn, increases TH, and has an important anti-inflammatory and antioxidant effect in the SN-STR circuit after LPS-induced neuroinflammation. These results suggest that BV could be a viable element for the design of pharmacological therapies against synucleinopathies. Declarations Author contributions The study conception and design were performed by S.J.L.P. and A.K.L.L., material preparation, data collection, and analysis were performed by A.K.L.L. and J.L.C.C.; results analysis and discussión were performed by all authors. The first draft of the manuscript was written by A.K.L.L, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Data statements Data is available from the authors on request. Declaration of competing interests None. Acknowledgments This study received partial support from the National Council of Humanities, Sciences, and Technologies of Mexico (CONAHCYT) through scholarship No. 1028543 awarded to A.K.L.L. and from the University of Guadalajara through the P3E-UDG-2022-23 program to S.J.L.P. 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Supplementary Files Graphicalabstract.tif Cite Share Download PDF Status: Published Journal Publication published 29 Sep, 2024 Read the published version in Cell Biochemistry and Biophysics → Version 1 posted Editorial decision: Revision requested 18 Jun, 2024 Reviews received at journal 17 Jun, 2024 Reviewers agreed at journal 13 Jun, 2024 Reviews received at journal 12 Jun, 2024 Reviewers agreed at journal 11 Jun, 2024 Reviewers agreed at journal 11 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers agreed at journal 10 Jun, 2024 Reviewers invited by journal 10 Jun, 2024 Editor assigned by journal 10 Jun, 2024 Submission checks completed at journal 10 Jun, 2024 First submitted to journal 08 Jun, 2024 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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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-4551820","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":315989460,"identity":"6535ac95-9c13-4601-9275-fad179063605","order_by":0,"name":"Alma Karen Lomeli-Lepe","email":"","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":false,"prefix":"","firstName":"Alma","middleName":"Karen","lastName":"Lomeli-Lepe","suffix":""},{"id":315989462,"identity":"afe77f4d-f4ae-4876-88a5-e0d25cc38e0a","order_by":1,"name":"José Luis Castañeda-Cabral","email":"","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":false,"prefix":"","firstName":"José","middleName":"Luis","lastName":"Castañeda-Cabral","suffix":""},{"id":315989464,"identity":"c2a4f41d-2c94-4921-9728-c7757bc6d9f9","order_by":2,"name":"Mónica E. Ureña-Guerrero","email":"","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":false,"prefix":"","firstName":"Mónica","middleName":"E.","lastName":"Ureña-Guerrero","suffix":""},{"id":315989465,"identity":"e15e17ae-d072-40dd-aa04-8d46ec4faf06","order_by":3,"name":"Graciela Gudiño Cabrera","email":"","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":false,"prefix":"","firstName":"Graciela","middleName":"Gudiño","lastName":"Cabrera","suffix":""},{"id":315989466,"identity":"7bca8afc-11f0-4367-b4b6-2606113b635a","order_by":4,"name":"Silvia Josefina López-Pérez","email":"data:image/png;base64,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","orcid":"","institution":"Universidad de Guadalajara","correspondingAuthor":true,"prefix":"","firstName":"Silvia","middleName":"Josefina","lastName":"López-Pérez","suffix":""}],"badges":[],"createdAt":"2024-06-08 20:44:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4551820/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4551820/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s12013-024-01552-x","type":"published","date":"2024-09-29T15:57:12+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":58969308,"identity":"fde47080-c306-4629-8bb3-94f4c9ce73f7","added_by":"auto","created_at":"2024-06-24 20:04:28","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":220556,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTimeline of BV treatment\u003c/strong\u003e The time course is represented from the intranigral injection with LPS or FSS, until the sacrifice of the animals for the subsequent extraction of proteins from brain areas of interest. The treatment with BV (1.5 mg/kg) consisted of 6 injections, with 48 hours of rest between each one, subcutaneously (SC) at the ST36 acupuncture point, 15 days after the end of the treatment, the animals were sacrificed. Created with BioRender.com\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/e7144e6dfa7b559c59c2842e.jpg"},{"id":58969317,"identity":"f9ca3a39-c233-4638-9d1d-119d2f4d36e5","added_by":"auto","created_at":"2024-06-24 20:04:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":256342,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExpression of α-syn after BV in SN and STR\u003c/strong\u003eThe expression levels of α-syn protein were analyzed by WB using 12% SDS-PAGE gels for the Control, SHAM, SHAM+BV, LPS and LPS+BV groups 30 days after stereotaxic surgery. a,c: Relative expression of monomeric α-syn in SN and STR respectively in ipsi and contralateral side of injection; b,d: Representative images of the WB for α-syn (15 kDa) and GAPDH (37 kDa). The results are expressed as the mean ± SD with *p\u0026lt;0.05, in the indicated pairs, by one-way ANOVA with Tukey's post-hoc.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/509359f2ea9abae9cdc4fbac.jpg"},{"id":58969311,"identity":"2f1a1a60-a1cb-45d5-a3dd-b665570ae437","added_by":"auto","created_at":"2024-06-24 20:04:28","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":246586,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTH expression in SN and STR after BV treatment\u003c/strong\u003e The expression of the protein tyrosine hydroxylase (TH), a marker of dopaminergic neurons, was analyzed by WB using 12% SDS-PAGE gels for the Control, SHAM, SHAM+BV, LPS and LPS+BV 30 days groups, after stereotaxic surgery, on the ipsi and contralateral sides to the injection in the SN and STR. a, c: The relative expression index of TH is shown based on the expression of the constitutive protein GAPDH; c, d: Representative images of the WB of the proteins of interest TH (65 kDa) and GAPDH (37 kDa). The results are expressed as the mean ± SD with *p\u0026lt;0.05 in the indicated pairs, by one-way ANOVA with Tukey's post-hoc.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/4a1672c78dda2efa2ec6aeef.jpg"},{"id":58970013,"identity":"e7bacfc2-47fd-42dc-8aad-9e8fb379778a","added_by":"auto","created_at":"2024-06-24 20:12:29","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4420547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunoreactivity of astrocytes and microglia in SN after treatment with BV\u003c/strong\u003e Representative images of microglia and astrocytes in the SN. Images show the immunoreactivity of GFAP (taken with the 10x objective) in green (a) and IBA-1 (taken with the 20x objective) in red (b), for all groups on the side ipsilateral and contralateral to the injection with LPS or FSS. c, d: Mean optical density of GFAP and IBA-1. The results are expressed as the mean ± SD with ***p \u0026lt; 0.001 in the indicated pairs, by one-way ANOVA with Tukey's post-hoc. Scale bar, 100 and 200 μm.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/0a17d706c3a3fa2b6dc63671.jpg"},{"id":58969309,"identity":"df31c0c9-a4b3-47c1-8032-5240c4e6dee7","added_by":"auto","created_at":"2024-06-24 20:04:28","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4397569,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunoreactivity of astrocytes and microglia in the STR after treatment with BV\u003c/strong\u003e Representative images of microglia and astrocytes in the STR. The images show the immunoreactivity of GFAP (taken with the 10x objective) in green (a) and IBA-1 (taken with the 20x objective) in red (b), for all groups in ipsilateral and contralateral side to the injection with LPS or FSS. c, d: average optical density of GFAP and IBA-1. The results are expressed as the mean ± SD with ***p \u0026lt; 0.001 in the indicated pairs, by one-way ANOVA with Tukey's post-hoc. Scale bar, 100 and 200 μm.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/e4fa5930e858c5f00eba026c.jpg"},{"id":58969315,"identity":"d785f4c9-e518-4e16-9fd1-a016edf9af1e","added_by":"auto","created_at":"2024-06-24 20:04:29","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":171665,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLevels of proinflammatory cytokines after BV treatment\u003c/strong\u003e The expression of TNF-α (a) and IL-1β (b) was measured in tissue lysates by sandwich ELISA for the Control, SHAM, SHAM+VA, LPS and LPS+VA groups 30 days after stereotaxic surgery on the ipsilateral and contralateral side to the injection of LPS or FSS in the STR. The results are expressed as the mean ± SD with *p\u0026lt;0.10 in the indicated pairs, by one-way ANOVA with Tukey's post-hoc.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/e079b66d4cd0e87df1d40eec.jpg"},{"id":58969314,"identity":"fe7b4d96-a235-4304-9a6a-b890906ab8d8","added_by":"auto","created_at":"2024-06-24 20:04:29","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":166204,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLipid peroxidation after BV treatment \u003c/strong\u003eLipid peroxidation was measured indirectly by the formation of malondialdehyde (MDA) in tissue lysates of Control, SHAM, SHAM+BV, LPS and LPS+BV groups 30 days after stereotaxic surgery on the ipsilateral and contralateral side to injection of LPS or FSS into the STR. The results are expressed as the mean ± SD with *p\u0026lt;0.10 and **p\u0026lt;0.01 in the indicated pairs, by one-way ANOVA with Tukey's post-hoc.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/fe619740757ff048b8bb2cab.jpg"},{"id":58970012,"identity":"83de5bfb-5726-4452-910e-6306c6539d79","added_by":"auto","created_at":"2024-06-24 20:12:28","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":172783,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSOD and CAT levels after BV treatment \u003c/strong\u003eSuperoxide dismutase and catalase activity were measured in tissue lysates of Control, SHAM, SHAM+BV, LPS and LPS+BV groups 30 days after stereotaxic surgery on the ipsilateral and contralateral side to injection of LPS or FSS into the STR. The results are expressed as the mean ± SD with ***p\u0026lt;0.001 in the indicated pairs, by one-way ANOVA with Tukey's post-hoc.\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/a550fded69ea03e4fd1308fc.jpg"},{"id":65627168,"identity":"39138956-f371-4a33-aebb-6b4375934511","added_by":"auto","created_at":"2024-09-30 16:12:46","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10610283,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/8b603f1d-de55-4320-b629-449319194185.pdf"},{"id":58969313,"identity":"b6cc2c76-32c3-4694-8eeb-da2bd5edfee1","added_by":"auto","created_at":"2024-06-24 20:04:29","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":4048936,"visible":true,"origin":"","legend":"","description":"","filename":"Graphicalabstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-4551820/v1/4afa97fffb0d42e16ac281f6.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Bee venom reduces early inflammation and oxidative stress associated with lipopolysaccharide-induced alpha-synuclein in the substantia nigra-striatum axis","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSynucleinopathies are a group of neurodegenerative diseases characterized by exacerbated expression of alpha-synuclein (α-syn) and its dissemination in the brain. In Parkinson's disease, the best-characterized synucleinopathy, fibrils and tangles of α-syn underlie the disease\u0026rsquo;s pathological manifestations. However, α-syn aggregates are also observed in other clinical entities, such as Parkinson's-associated dementia, Lewy body dementia, multiple system atrophy, and pure autonomic failure (Savica et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Simuni et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The pathogenesis of these neurological disorders is complex, and the evolution of the disease adds side effects and collaterals due to the mainly symptomatic medication. The overexpression of α-syn in the dopaminergic axis substantia nigra-striatum seems to be favored by a neuroinflammatory state (Peixoto et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Lomeli-Lepe et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Eser et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Neuroinflammation can be induced experimentally using lipopolysaccharide (LPS), a bacterial endotoxin that produces a potent glial activation and promotes the aggregation of α-syn (Sharma and Nehru \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSeveral natural compounds have therapeutic effects in human pathologies because they affect multiple molecular targets and signaling pathways through mechanisms not yet fully described (Rehman et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Rahman et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Bee venom (BV) has beneficial effects against cancer, neuroinflammatory processes, arthropathies (Aufschnaiter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), and immune diseases (Wehbe et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Several clinical trials have found evidence that BV could be beneficial in treating neurodegenerative diseases (Zhang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). BV is a mixture of peptides, enzymes, amines, sugars, and minerals; melittin, phospholipase A2, apamin, adolapin, and mast cell degranulation peptide are the molecules found in high concentrations (Aufschnaiter et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Shi et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eGiven the tendency of α-syn to form oligomers in oxidative and inflammatory milieu, we hypothesize that BV components act synergistically to counteract the generation of mediators of this type in the brain and, as a consequence, reduce the overexpression of α-syn. To address this hypothesis, we applied a BV systemic treatment to animals with LPS-induced inflammation and oxidative stress in the SN and STR. We confirmed that BV treatment decreases the expression of monomeric α-syn and the astrocytic and microglial activation induced by LPS while maintaining a normal expression of TH in the rat SN and STR. We also observed that BV maintains lower levels of oxidative and inflammatory markers in the STR compared to animals that did not receive this compound.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimal treatment\u003c/h2\u003e \u003cp\u003eAll animals used in this work were housed under standard animal care conditions, with free access to water and chow \u003cem\u003ead libitum\u003c/em\u003e. All procedures were carried out according to the ethical guidelines of the Animal Care and Use Committee of the University Campus of Biological and Agricultural Science (CUCBA) of the University of Guadalajara under the agreement CINV.104/12.\u003c/p\u003e \u003cp\u003eMale Wistar rats between 200\u0026ndash;250 g were anesthetized, and LPS (Escherichia coli 0111:B4; Sigma) (LPS group, n\u0026thinsp;=\u0026thinsp;10) was stereotaxically injected into the right SN (2.5 \u0026micro;g in 2.5 \u0026micro;l of saline solution, SS) over 2.5 min using a motorized microinjection pump. The following coordinates were used for the injection: -5.8 mm posterior to the bregma, 1 mm lateral to the midline, and 7.8 mm ventral to the surface of the skull. The SHAM group (n\u0026thinsp;=\u0026thinsp;10) received SS in the SN following the same procedure, and the Control group (n\u0026thinsp;=\u0026thinsp;5) consisted of untreated animals.\u003c/p\u003e \u003cp\u003eFive days after LPS injection, lyophilized BV (obtained from the Bee Research Center of the Southern University Center (CUSur), University of Guadalajara) was used to treat a subset of rats previously injected with LPS (LPS\u0026thinsp;+\u0026thinsp;BV group, n\u0026thinsp;=\u0026thinsp;5), and animals randomly selected from the SHAM group (SHAM\u0026thinsp;+\u0026thinsp;BV group, n\u0026thinsp;=\u0026thinsp;5). Each animal received a subcutaneous (s.c.) BV injection (1.5 mg per kg of body weight, dissolved in SS) at the ST36 acupuncture point (Oh and Kim \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) every 48 hours for a total of 6 doses (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The animals were sacrificed 15 days after the end of the BV treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot assay\u003c/h2\u003e \u003cp\u003eAfter sacrifice, the brain was removed, and the SN and STR were dissected, separating the ipsilateral and contralateral sides from the LPS injection. Protein concentration was determined according to the Lowry method (Lowry et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1951\u003c/span\u003e) with a DC Protein Assay Kit (Bio-Rad Laboratories, USA Cat. 500011) on a Multiskan Go spectrophotometer (Thermo Scientific, Finland), using bovine serum albumin (BSA) (Bio-Rad Laboratories, USA Cat. 500-0007) as the external standard. Briefly, 20 \u0026micro;g of denatured protein per lane were separated by electrophoresis on 12% sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE). Separated proteins were blotted to nitrocellulose membranes (Protean Premium 0.45 \u0026micro;m, Amersham, Germany), which were later placed in a 5% blocker solution (QuickBlocker, EMD Millipore, USA) and dissolved in 0.1 M PBS with 0.1% Tween-20 at 4\u0026deg;C for 1 hour. Then, the following primary antibodies and dilutions were used for detection of α-syn, TH, and GAPDH: Rabbit anti-alpha-synuclein (1:2000; Abcam, Cambridge, UK Cat. Ab212184), rabbit anti-TH (1:1000; Abcam, Cambridge, UK Cat. ab112), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:5,000; Abcam, Cambridge, UK, Cat. Ab125247). Incubation with primary antibodies was carried out at 4\u0026deg;C overnight with gentle shaking. Horseradish peroxidase (HRP)-conjugated secondary antibodies anti-rabbit IgG (1:10,000; LICOR USA Cat.926-80011) and anti-mouse IgG (1:10,000; LICOR USA Cat.926-80010) were used to recognize the primary antibody. Finally, bands were revealed with a chemiluminescent substrate (SuperSignal West Femto Maximum Sensitivity Substrate, Thermo Scientific, USA). Band images were obtained using a C-DiGit Blot Scanner (LI-COR Bioscience, USA), and their density was estimated using Image Studio Lite 3.1.4 software (LI-COR Bioscience, USA). The results are presented as the expression ratio to constitutive protein (GAPDH).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence\u003c/h2\u003e \u003cp\u003eAnimals were deeply anesthetized with pentobarbital until a negative toe-pinch response was achieved to proceed with intracardiac perfusion with 0.9% NaCl, followed by 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS, pH 7.4). After being carefully extracted from the skull, brains were transferred to a cryoprotectant solution (30% sucrose and 30% ethylene glycol in PBS) at 4\u0026deg;C for 5 days. Frozen tissues were embedded with Tissue Plus (O.C.T. compound, Fisher HealthCare, cat. 4585) (Sakura Finetek, Torrance, CA, USA) and transferred to a cryostat set to -20\u0026deg;C overnight. Brains were cut in the frontal plane into 30 \u0026micro;m-thick sections, which were immediately transferred to 24-well plates containing approximately 1 mL of PBS before proceeding with immunofluorescence (IF).\u003c/p\u003e \u003cp\u003eFloating sections were permeabilized with 2% Triton X-100 in PBS overnight and blocked with 5% BSA for 30 min at room temperature under gentle shaking. The sections were incubated with mouse anti-IBA1 (1:1,000; Abcam, Cambridge, UK, Cat. AB209064) and mouse anti-GFAP (1:1000; Millipore, USA Cat. MAB360) at 4\u0026deg;C overnight. Slices were washed three times in PBS for 10 min and then incubated with the following secondary antibodies: anti-mouse Alexa Fluor 488 (1:1,000, Abcam, Cambridge, UK, cat. Ab209064) and anti-mouse Alexa Fluor 594 (1:1,000, Abcam, Cambridge, UK cat. Ab150077) for 2 h. After washing in PBS for 10 min thrice, sections were mounted on glass coverslips using PBS-Glycerol 1:1.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eImaging and OD estimation\u003c/h2\u003e \u003cp\u003eTo evaluate the astrocytic GFAP-positive and microglial IBA-1-positive immunoreactivity in SN and STR, we obtained fluorescence images of STR and SN sections. An inverted microscope (Nikon, model eclipse 55i with an integrated Nikon camera, model ds-fi1) and PLN 10X/0.1NA and 20X/0.5NA objective lens was used.\u003c/p\u003e \u003cp\u003eOptical density (OD) was analyzed semiquantitatively in the software ImageJ by transforming the original images into grayscale images (assigning a value of 0 to white and a value of 255 to black) (Lai et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this way, the shades of gray are converted into OD using the following equation:\u003c/p\u003e \u003cp\u003eOD\u0026thinsp;=\u0026thinsp;log (255/mean gray level)\u003c/p\u003e \u003cp\u003eSix sections per animal per region (three ipsilateral and three contralateral; total area by side of 0.75 mm2) were used for quantification. The six values per region from each rat were averaged to calculate a total value for glial and astrocytic activation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eEnzyme-linked immunosorbent assay (ELISA)\u003c/h2\u003e \u003cp\u003eTo assess the presence of neuroinflammatory agents in response to LPS stimulation, we measured TNF-α and IL-1β concentration by ELISA in STR homogenates. Tissue samples from all groups of STR ipsilateral and contralateral (100 mg/mL of protein) to LPS injection were prepared for commercial TNF-α (ab100785, Abcam) and IL-1β (ab100768, Abcam) kits, according to the manufacturer\u0026rsquo;s instructions. Standards and samples were assessed in duplicate, and the absorbance was measured at 450 nm in a spectrophotometer (Multiskan GO, Thermo Scientific, Finland). Cytokine concentrations were normalized to the total protein content and reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD) in pg/mg of total protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eOxidative markers\u003c/h2\u003e \u003cp\u003eTo evaluate the oxidative state in response to the treatments, we quantified the levels of some oxidative stress markers. For this, 100 mg/mL of homogenate of STR (ipsilateral and contralateral to LPS injection) was applied to the following commercial kits, as per the manufacturer\u0026rsquo;s instructions: Lipid peroxidation (MDA) colorimetric assay kit (Cat. ab233471, Abcam), Superoxide dismutase (SOD) activity colorimetric assay kit (Cat. ab65354, Abcam), and Catalase (CAT) activity colorimetric assay kit (Cat. ab83464, Abcam). In all cases, standards and samples were measured in duplicates. Absorbance was measured using a spectrophotometer (Multiskan GO, Thermo Scientific, Finland). Lipid peroxidation and CAT activity were expressed as nanomoles per gram of tissue protein (nmol/g), and superoxide dismutase activity was expressed as the percentage of inhibition (%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical data analysis\u003c/h2\u003e \u003cp\u003eThe results of inflammatory, oxidative stress markers, and immunolabeled cells are expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. For WB, results are expressed as a ratio to constitutive protein (GAPDH). Statistical differences between groups were analyzed by one-way ANOVA followed by Tukey's post hoc test, with p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. The results were analyzed using the Prism Graph Pad v 8.0 software (GraphPad Prism Software Inc., USA).\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eMonomeric α-syn expression\u003c/h2\u003e \u003cp\u003eIn the SN ipsilateral to LPS injection, no significant difference was found between the Control, SHAM, and SHAM\u0026thinsp;+\u0026thinsp;BV groups (p\u0026thinsp;=\u0026thinsp;0.9769 Control vs SHAM; p\u0026thinsp;=\u0026thinsp;0.9943 Control vs SHAM\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9997 SHAM vs SHAM\u0026thinsp;+\u0026thinsp;BV), suggesting that the mechanical lesion combined with the series of BV doses does not alter the expression of monomeric α-syn in this region. In contrast, the LPS group showed a significant increase in monomeric α-syn expression compared to the Control (p\u0026thinsp;=\u0026thinsp;0.0211 Control vs LPS), SHAM (p\u0026thinsp;=\u0026thinsp;0.0059 SHAM vs LPS), and SHAM\u0026thinsp;+\u0026thinsp;BV (p\u0026thinsp;=\u0026thinsp;0.0088 SHAM\u0026thinsp;+\u0026thinsp;BV vs LPS) groups. The group that received BV after LPS injection maintained the expression of monomeric α-syn at a level comparable to the Control, SHAM, and SHAM\u0026thinsp;+\u0026thinsp;BV groups (p\u0026thinsp;=\u0026thinsp;0.9984 Control vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9983 SHAM vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9999 SHAM\u0026thinsp;+\u0026thinsp;BV vs LPS\u0026thinsp;+\u0026thinsp;BV), and significantly lower than the LPS group (p\u0026thinsp;=\u0026thinsp;0.0113 LPS vs LPS\u0026thinsp;+\u0026thinsp;BV). On the contralateral side of the SN, no significant changes were observed between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eIn STR ipsilateral to LPS lesion, there were also no significant differences between the Control, SHAM, and SHAM\u0026thinsp;+\u0026thinsp;BV group (p\u0026thinsp;=\u0026thinsp;0.3054 Control vs SHAM; p\u0026thinsp;=\u0026thinsp;0.5637 Control vs SHAM\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.2563 SHAM vs SHAM\u0026thinsp;+\u0026thinsp;BV). In contrast, the LPS group shows an elevated expression of monomeric α-syn (p\u0026thinsp;=\u0026thinsp;0.0222 Control vs LPS), while the LPS\u0026thinsp;+\u0026thinsp;BV combination and SHAM\u0026thinsp;+\u0026thinsp;BV maintains this expression at the level of the Control group (p\u0026thinsp;=\u0026thinsp;0.0478 LPS vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9471 Control vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.0007 SHAM\u0026thinsp;+\u0026thinsp;BV vs LPS; p\u0026thinsp;=\u0026thinsp;0.5637 SHAM\u0026thinsp;+\u0026thinsp;BV vs Control) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). These data confirm our previous results indicating that LPS increases the expression of monomeric α-syn, and evidence that BV has a favorable outcome preventing the effect of LPS and maintaining protein expression at a physiological level.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eTH expression\u003c/h2\u003e \u003cp\u003eOn the SN ipsilateral to LPS injection, TH was significantly reduced in the LPS group compared to the Control group (p\u0026thinsp;=\u0026thinsp;0.0057 Control vs LPS); and the combination of LPS\u0026thinsp;+\u0026thinsp;BV maintained the TH expression level comparable to the Control (p\u0026thinsp;=\u0026thinsp;0.3684, Control vs LPS\u0026thinsp;+\u0026thinsp;BV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). In STR, TH expression also had no significant changes between control groups (p\u0026thinsp;=\u0026thinsp;0.9360 Control vs SHAM; p\u0026thinsp;=\u0026thinsp;0.1594 Control vs SHAM\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9999 SHAM vs SHAM\u0026thinsp;+\u0026thinsp;BV), whereas the animals with LPS show significantly decreased TH compared to the Control group (p\u0026thinsp;=\u0026thinsp;0.0346 LPS vs Control). In contrast, animals in the LPS\u0026thinsp;+\u0026thinsp;BV group maintain the TH expression close to the control level (p\u0026thinsp;=\u0026thinsp;0.6813 Control vs LPS\u0026thinsp;+\u0026thinsp;BV) but significantly higher than the group with only LPS (p\u0026thinsp;=\u0026thinsp;0.0499 LPS vs LPS\u0026thinsp;+\u0026thinsp;BV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Taken together, these results support that BV may be useful for the neuroprotection of TH-positive cells that are susceptible to α-syn deregulation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eReactivity of astrocytes and microglia in response to LPS\u003c/h2\u003e \u003cp\u003eAs expected, LPS increased reactive astrocytes and microglia on the ipsilateral and contralateral (to LPS injection) sides of the SN (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e) and STR (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The OD of the IF signal showed a pronounced rise in GFAP\u0026thinsp;+\u0026thinsp;and IBA-1\u0026thinsp;+\u0026thinsp;cells in both the SN and the STR compared to the controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The treatment with BV greatly prevents this glial activation, evidenced by a significant decrease of both markers in the LPS\u0026thinsp;+\u0026thinsp;BV group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), suggesting that BV controls the proinflammatory molecules released by astrocytes and microglia.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eProinflammatory cytokines\u003c/h2\u003e \u003cp\u003eWe did not find statistically significant changes in TNF-α expression, although there was an upward trend in the LPS group and a downward trend in the BV group on the STR ipsi- (p\u0026thinsp;=\u0026thinsp;0.4220 Control vs LPS; p\u0026thinsp;=\u0026thinsp;0.7186, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9998, Control vs LPS\u0026thinsp;+\u0026thinsp;BV) and contralateral (p\u0026thinsp;=\u0026thinsp;0.1419, Control vs LPS; p\u0026thinsp;=\u0026thinsp;0.3823, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9885, Control vs LPS\u0026thinsp;+\u0026thinsp;BV) sides to the LPS injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eWe found no notable differences in IL-1β in animals with LPS compared to the control group in the ipsi- (p\u0026thinsp;=\u0026thinsp;0.4924, Control vs LPS) and contralateral STR (p\u0026thinsp;=\u0026thinsp;0.3336, Control vs LPS). Interestingly, the control group differed from the BV-treated groups treated in the ipsi- (p\u0026thinsp;=\u0026thinsp;0.0766, Control vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.0197, Control vs SHAM\u0026thinsp;+\u0026thinsp;BV) and contralateral STR (p\u0026thinsp;=\u0026thinsp;0.0051, Control vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.0690, Control vs SHAM\u0026thinsp;+\u0026thinsp;BV). Furthermore, IL-1β expression was higher in the untreated LPS group than in the LPS\u0026thinsp;+\u0026thinsp;BV and SHAM\u0026thinsp;+\u0026thinsp;BV groups in the ipsilateral STR (p\u0026thinsp;=\u0026thinsp;0.0485, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.0716, LPS vs SHAM\u0026thinsp;+\u0026thinsp;BV), and higher than the LPS\u0026thinsp;+\u0026thinsp;BV group in the contralateral STR (p\u0026thinsp;=\u0026thinsp;0.0406, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV). The untreated Control and SHAM groups show no differences in both sides of the STR (p\u0026thinsp;=\u0026thinsp;0.8913, Control vs SHAM ipsilateral; p\u0026thinsp;=\u0026thinsp;0.9234, Control vs SHAM contralateral; p\u0026thinsp;=\u0026thinsp;0.8916, SHAM vs SHAM\u0026thinsp;+\u0026thinsp;BV ipsilateral, p\u0026thinsp;=\u0026thinsp;0.3573, SHAM vs SHAM\u0026thinsp;+\u0026thinsp;BV contralateral). These results suggest that BV treatment alone is sufficient to decrease the expression of IL-1β, even in the absence of an inflammatory response in ipsilateral STR (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eOxidative markers\u003c/h2\u003e \u003cp\u003eLipid peroxidation was estimated indirectly through the quantification of MDA. MDA concentrations were not different between the Control and LPS groups in the ipsilateral STR (p\u0026thinsp;=\u0026thinsp;0.4918 Control vs LPS); however, the contralateral side showed a significant change (p\u0026thinsp;=\u0026thinsp;0.0981, Control vs LPS). No differences were found in MDA concentration between Control, SHAM, and SHAM\u0026thinsp;+\u0026thinsp;BV groups, in the ipsi- (p\u0026thinsp;=\u0026thinsp;0.9997, Control vs SHAM; p\u0026thinsp;=\u0026thinsp;0.9993, SHAM vs SHAM\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9931, Control vs SHAM\u0026thinsp;+\u0026thinsp;BV), and contralateral sides of STR (p\u0026thinsp;=\u0026thinsp;0.6610, Control vs SHAM; p\u0026thinsp;=\u0026thinsp;0.9523, SHAM vs SHAM\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.9580, Control vs SHAM\u0026thinsp;+\u0026thinsp;BV) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). The combination of LPS\u0026thinsp;+\u0026thinsp;BV significantly decreased MDA concentrations compared to the untreated LPS group in the ipsi- (p\u0026thinsp;=\u0026thinsp;0.0094, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV) and contralateral (p\u0026thinsp;=\u0026thinsp;0.0141, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV) STR, and even below the Control group in the ipsilateral STR (p\u0026thinsp;=\u0026thinsp;0.0637, Control vs LPS\u0026thinsp;+\u0026thinsp;BV). Interestingly, MDA levels significantly increased in the LPS group compared to the SHAM and SHAM\u0026thinsp;+\u0026thinsp;BV groups (p\u0026thinsp;=\u0026thinsp;0.0136, SHAM vs LPS; p\u0026thinsp;=\u0026thinsp;0.0369, SHAM\u0026thinsp;+\u0026thinsp;BV vs LPS) only in the contralateral STR (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eRegarding the inhibition of SOD activity, no significant changes were found between groups in this enzymatic marker (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea). Similarly, no significant difference in CAT activity was found between Control and LPS groups. Surprisingly, the LPS\u0026thinsp;+\u0026thinsp;BV combination increased CAT activity compared to all groups in the ipsilateral (p\u0026thinsp;=\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, SHAM vs LPS\u0026thinsp;+\u0026thinsp;BV, SHAM\u0026thinsp;+\u0026thinsp;BV vs LPS\u0026thinsp;+\u0026thinsp;BV, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV and Control vs LPS\u0026thinsp;+\u0026thinsp;BV) and except the Control in contralateral STR (p\u0026thinsp;=\u0026thinsp;0.0212, SHAM vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.0351, SHAM\u0026thinsp;+\u0026thinsp;BV vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.0166, LPS vs LPS\u0026thinsp;+\u0026thinsp;BV; p\u0026thinsp;=\u0026thinsp;0.2285, Control vs LPS\u0026thinsp;+\u0026thinsp;BV), (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eb). Therefore, we conclude that there is an inverse relationship between the expression of α-syn and CAT activity, which can be attributed to the effect of BV.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTaken together, our results suggest that BV is a promising molecule for effectively controlling oxidative markers and modulating IL-1β in the context of an inflammatory response.\u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThis work challenged BV's neuroprotective capacity, its potential to control α-syn expression in the rat brain, and the self-sustaining inflammatory and oxidative environment generated around this process. Our results show that LPS stimulation led to the following temporally and spatially coinciding changes: an increase in monomeric α-synuclein expression, an increase in the density of GFAP\u0026thinsp;+\u0026thinsp;and IBA-1\u0026thinsp;+\u0026thinsp;cells, and a decrease in TH. Conversely, LPS increases IL-1β and lipid peroxidation and reduces catalase activity, promoting oxidative stress and inflammation. The treatment with BV effectively countered this effect, showing a potent anti-inflammatory and antioxidant activity under these experimental conditions.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that BV decreases the degeneration of nigral dopaminergic cells and microglia in an MPTP\u0026thinsp;+\u0026thinsp;Parkinson's disease model in mice (Kim et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Doo et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Applying BV to cultures of LPS-activated microglia decreases the production of NO, iNOS, and TNF-α, and decreases the presence or expression of NF-kB, IL-1β, IL-6, and prostaglandins (Park et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Moon et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). Our immunofluorescence results confirm and support these observations by demonstrating a decrease in the OD of IBA-1\u0026thinsp;+\u0026thinsp;cells after BV treatment in the SN and STR. Notably, the results presented here extend BV\u0026rsquo;s effects to astrocytes, since we reported for the first time a decrease in GFAP\u0026thinsp;+\u0026thinsp;cells in SN and STR in BV-treated animals. Astrocytes represent approximately 30% of brain cells, and their activation (in this case, by LPS) triggers the release of inflammatory modulators, chemokines, cytokines, and neurotrophic factors, either neuroprotective or neurotoxic (Trujillo-Estrada et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). These molecules, along with microglia activation, enhance and sustain neuroinflammation. Thus, BV was beneficial to control the pro-inflammatory cellular response induced by LPS. We also found that BV diminished IL-1β, a pleiotropic cytokine that leads to the synthesis of proinflammatory and chemotactic mediators (Lee et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2010\u003c/span\u003e) and contributes to the pathogenesis of neurodegenerative diseases (Mendiola and Cardona \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Together, these results suggest that controlling microglial and astrocytic activation by BV decreases IL-1β production, resulting in a neuroprotective effect towards TH\u0026thinsp;+\u0026thinsp;cells, which maintained their immunoreactivity in animals treated with BV. However, we cannot rule out that IL-1β is involved in the initial activation of microglia, since this molecule is rapidly induced in the brain after acute brain injury (Mendiola and Cardona \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn addition, oligomeric α-syn released by neurons has been shown to induce inflammatory responses of microglia through activation of the toll-like receptors 2 (TLR2) (Kim et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). Interestingly, the work of Kim (2022) showed that the pro-inflammatory cytokines TNF-α and IL-1β stimulate the cell-to-cell transmission of α-syn \u003cem\u003ein vitro\u003c/em\u003e. Therefore, although we found no significant changes in TNF-α concentration (which may have been due to an insufficient LPS stimulus), our results demonstrate that BV can reduce IL-1β in conditions of neuroinflammation, likely preventing the spread of α-syn \u003cem\u003ein vivo\u003c/em\u003e (Kim et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). This observation should be further studied in future research.\u003c/p\u003e \u003cp\u003eAlthough the molecular mechanisms explaining BV\u0026rsquo;s effects are still unknown, it has been proposed that BV could exert an anti-inflammatory effect by regulating microglia activity, the transcription of nuclear factor kappa B (NF-kB), the mitogen-activated protein kinase (MAPK) pathway and protein kinase B pathway (Akt) (Zhang et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). In addition, melittin, the major component of the venom, suppresses the signaling pathways of TLR2 and TLR4, cluster of differentiation (CD14), the essential modulator of nuclear factor kappa-B (NEMO) and platelet-derived growth factor receptor beta (PDGFRβ) (Park et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Son et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Moon et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2007\u003c/span\u003e; Park et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). By inhibiting these pathways, melittin decreases the activation of p38, extracellular signal-regulated kinases 1 and 2 (ERK1/2), Akt, PLCγ1, and the translocation of NF-κB to the nucleus, resulting in reduced inflammation. The anti-inflammatory effect of bvPLA2 was previously established (Lee and Bae \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and both BV components (melittin and bvPLA2) synergistically enhance its effects (Damianoglou et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). This explains why the use of complete BV has better effects than its individual components. Likely, the inhibition of these pro-inflammatory pathways by BV is significantly participating in maintaining adequate levels of α-syn, TH, and IL-1β and reducing the activation of microglia and astrocytes, which highlights its anti-neuroinflammatory potential. However, more studies are still needed to elucidate the mechanisms by which BV could influence the expression of these proteins.\u003c/p\u003e \u003cp\u003eSo far, oxidative stress is considered a primary cause of α-syn neurotoxicity (Luk \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) because the presence of aggregates alters neuronal intracellular redox balance. Therefore, it is assumed that reducing oxidative stress can effectively avoid aggregation of this protein. The activity of catalase and glutathione peroxidase, two enzymes responsible for ROS removal and thus markers of oxidative stress, is reduced in PD brains (Silva et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) and in animal models of the disease (Anjum et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Kumar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), Furthermore these markers are increased along with lipid oxidation in brains with synucleinopathies (Fu et al. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These results are consistent with those of the current study, where we found an imbalance in the oxidative status represented by various alterations in CAT activity and lipid peroxidation.\u003c/p\u003e \u003cp\u003eAn interesting relationship between CAT activity and α-syn arises from the work done by Yakunin (2014), who observed that PPARγ (a member of the nuclear receptor family) inhibition by α-syn overexpression also inhibits CAT activity. These results match the LPS-induced α-syn overexpression and CAT reduction observed in our results.\u003c/p\u003e \u003cp\u003eLimiting cellular antioxidant mechanisms favors the production of ROS and lipid peroxidation. Our results showed that BV reduces lipid peroxidation and significantly increases CAT activity, indicating a strengthening of oxidant removal systems associated with decreased α-syn expression. Although the mechanism by which BV mediates this effect is still unclear, previous work has shown that BV upregulates brain-derived neurotrophic factor (BDNF) expression through ERK activation (Cai et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). This suggests a positive effect of BV on the BDNF/tyrosine kinase B (TrkB)/cAMP-response element-binding protein (CREB) signaling pathway, which is key to strengthening the cellular intrinsic antioxidant defense (Jin \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). BV is also known to upregulate the activation of the Nrf2/HO-1 signaling pathway (Jin \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which is responsible for producing the intrinsic antioxidant enzyme HO-1. This enzyme is considered the first line of cellular defense against oxidative damage (Chen et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Although it is generally believed that these interactions are mediated by melittin or PLA2 present in BV, it is more likely that the interaction of several components enhances the effect of BV.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eThe pathologies associated with α-syn represent a challenge to human health. Generating effective therapies is still pending since multiple environmental and genetic factors contribute to its etiology. Our results allow us to conclude that BV reduces the expression of α-syn, increases TH, and has an important anti-inflammatory and antioxidant effect in the SN-STR circuit after LPS-induced neuroinflammation. These results suggest that BV could be a viable element for the design of pharmacological therapies against synucleinopathies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study conception and design were performed by S.J.L.P. and A.K.L.L., material preparation, data collection, and analysis were performed by A.K.L.L. and J.L.C.C.; results analysis and discussi\u0026oacute;n were performed by all authors. The first draft of the manuscript was written by A.K.L.L, and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData statements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData is available from the authors on request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNone.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study received partial support from the National Council of Humanities, Sciences, and Technologies of Mexico (CONAHCYT) through scholarship No. 1028543 awarded to A.K.L.L. and from the University of Guadalajara through the P3E-UDG-2022-23 program to S.J.L.P.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAnjum R, Raza C, Faheem M, Ullah A, Chaudhry M (2024) Neuroprotective Potential of Mentha Piperita Extract Prevents Motor Dysfunctions in Mouse Model of Parkinson\u0026rsquo;s Disease through Anti-Oxidant Capacities. 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Molecules 24(16):2997. https://doi.org/10.3390/molecules24162997.\u003c/li\u003e\n\u003cli\u003eYakunin E, Kisos H, Kulik W, Grigoletto J, Wanders RJ, Sharon R (2014) The Regulation of Catalase Activity by PPAR \u0026gamma; Is Affected by \u0026alpha;-Synuclein. Ann Clin Transl Neurol 1(3):145\u0026ndash;59. https://doi.org/10.1002/acn3.38.\u003c/li\u003e\n\u003cli\u003eZhang S, Liu Y, Ye Y, Wang XR, Lin LT, Xiao LY, Zhou P, Shi GX, Liu CZ (2018) Bee Venom Therapy: Potential Mechanisms and Therapeutic Applications. Toxicon 148:64\u0026ndash;73. https://doi.org/10.1016/j.toxicon.2018.04.012.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Bee venom, alpha-synuclein, monomers, synucleinopathies- substantia nigra","lastPublishedDoi":"10.21203/rs.3.rs-4551820/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4551820/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeuroinflammation and oxidative stress are important features in the pathogenesis and development of synucleinopathies, the glial activation and upregulation of pro-inflammatory and oxidative mediators induce alpha-synuclein (α-syn) accumulation. Recent studies have shown that bee venom (BV) has beneficial effects on symptoms of these neurodegenerative diseases. BV is known to exert anti-inflammatory and anti-oxidative effects. Here, we investigated the effects of BV over the different inflammatory and oxidative markers, and in the expression of α-syn and tyrosine hydroxylase (TH) in a lipopolysaccharide (LPS)-induced rat model of synucleinopathies. We examined whether BV (1.5 mg/kg by acupoint injection ST36 six times every 48 hours) could change the α-syn and TH expression measured by western blotting, also, observed the activation of microglia and astrocytes by immunofluorescence, quantify the proinflammatory cytokines levels (TNF-α and IL-1β) by ELISA, and estimated the lipid peroxidation and the activity of superoxide dismutase (SOD) and catalase (CAT) by colorimetric kits in LPS-treated rats (2.5 \u0026micro;g by a single dose intranigral injection) in substantia nigra (SN) and striatum (STR) brain areas. In the LPS-injected rat brain, BV treatment reduced α-syn levels and increased the TH levels. In addition, we observed lower microglia and astrocyte activation in SN and STR. Furthermore, BV decreases IL-1β and lipid peroxidation and increases the CAT activity in the STR. These results indicate that BV can restore the α-syn and TH levels possibly by the inhibition of LPS-induced neuroinflammation and oxidation, also, these results suggest that BV could be a promising treatment option for synucleinopathies.\u003c/p\u003e","manuscriptTitle":"Bee venom reduces early inflammation and oxidative stress associated with lipopolysaccharide-induced alpha-synuclein in the substantia nigra-striatum axis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-24 20:04:23","doi":"10.21203/rs.3.rs-4551820/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-06-18T15:02:26+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-17T05:00:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101249114498133107509339749000979236975","date":"2024-06-13T17:28:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-12T17:41:50+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"135946933885555838955149076883369580955","date":"2024-06-11T16:40:24+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"282797046962080085098088153480388477312","date":"2024-06-11T16:21:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101486275446698932074605309752593322016","date":"2024-06-11T03:52:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"268806775815607241850668756980134635087","date":"2024-06-10T21:24:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"240842040615846460547783997450450113631","date":"2024-06-10T19:54:22+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"195968960843261961751350943100515541185","date":"2024-06-10T19:12:00+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151044217422158272668589750692641516841","date":"2024-06-10T19:08:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"31315569931286542275710472238966340734","date":"2024-06-10T19:01:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"94772846591713949809804598240113766526","date":"2024-06-10T18:57:25+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-10T18:54:16+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-10T07:32:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-10T07:32:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Biochemistry and Biophysics","date":"2024-06-08T20:43:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-biochemistry-and-biophysics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cbbi","sideBox":"Learn more about [Cell Biochemistry and Biophysics](http://link.springer.com/journal/12013)","snPcode":"12013","submissionUrl":"https://submission.nature.com/new-submission/12013/3","title":"Cell Biochemistry and Biophysics","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f9f683d3-47d0-480b-8220-ff09f8399275","owner":[],"postedDate":"June 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-09-30T16:01:18+00:00","versionOfRecord":{"articleIdentity":"rs-4551820","link":"https://doi.org/10.1007/s12013-024-01552-x","journal":{"identity":"cell-biochemistry-and-biophysics","isVorOnly":false,"title":"Cell Biochemistry and Biophysics"},"publishedOn":"2024-09-29 15:57:12","publishedOnDateReadable":"September 29th, 2024"},"versionCreatedAt":"2024-06-24 20:04:23","video":"","vorDoi":"10.1007/s12013-024-01552-x","vorDoiUrl":"https://doi.org/10.1007/s12013-024-01552-x","workflowStages":[]},"version":"v1","identity":"rs-4551820","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4551820","identity":"rs-4551820","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}