Immunomodulatory Effects of Platelet-Rich Plasma (PRP) on Neuroinflammation: Insights from B92 Glial Cells Stimulated with Heat-Killed Escherichia coli

Immunomodulatory Effects of Platelet-Rich Plasma (PRP) on Neuroinflammation: Insights from B92 Glial Cells Stimulated with Heat-Killed Escherichia coli | 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 Immunomodulatory Effects of Platelet-Rich Plasma (PRP) on Neuroinflammation: Insights from B92 Glial Cells Stimulated with Heat-Killed Escherichia coli Mahtab Pourkamalzadeh, Seyyed Meysam Abtahi Froushani This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7889196/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Platelets increasingly appear to influence inflammation, including neuroinflammation. This study examines how platelet-rich plasma (PRP) modulates BV2 glial cell immune responses in sterile and infectious inflammation to assess potential neuroprotective effects. B92 glial cells were treated with PRP (0%, 5%, 10%, 20%) to model sterile inflammation. Infectious inflammation was simulated using heat-killed E. coli (1:100) combined with PRP. Cells were cultured in DMEM (low-glucose) with 10% FBS at 37°C, 5% CO₂, and 95% humidity for 24 h. Morphology, viability (MTT), phagocytosis (neutral red), oxidative stress (NBT), and expression of cytokines (TNF-α, IL-10, IL-1β) and apoptosis markers (BAX, Caspase-3, BCL-2) were assessed by qRT-PCR.PRP significantly enhanced glial cell viability and proliferation in a dose-dependent manner. The expression of the anti-apoptotic gene BCL-2 increased, whereas Caspase3 and BAX levels decreased following PRP treatment. PRP modulated cytokine profiles by reducing TNF-α expression and upregulating IL-10 in a dose-independent manner, accompanied by an increase in IL-1β expression across all concentrations. Morphological and metabolic analyses revealed that PRP mitigated inflammatory damage and preserved glial integrity, particularly under sterile inflammatory conditions. Under infectious stimulation, PRP attenuated E. coli -induced oxidative stress and preserved glial function without impairing phagocytic activity, suggesting a coordinated immunoregulatory effect. In conclusion, PRP may help control brain inflammation from injury or infection by protecting brain cells, balancing immune responses, and reducing stress. This suggests PRP could be a treatment for brain disorders. Platelet-Rich Plasma (PRP) Neuroinflammation B92 Glial cells Cytokines Escherichia coli Neuroprotection Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Platelet-Rich Plasma (PRP) is an autologous concentration of non-activated platelets suspended in a small volume of plasma, widely used to accelerate soft tissue and bone healing[1]. Beyond their classical role in hemostasis, platelets are now recognized as critical mediators of tissue repair and regeneration through the release of numerous bioactive molecules and growth factors that promote endothelial proliferation and fibroblast activation[2]. These growth factors including HGF, IGF-1, PDGF, VEGF, EGF, and TGF-β collectively contribute to several key processes such as chemotaxis, collagen type I synthesis, mitogenesis, cell proliferation, angiogenesis, and tissue remodeling[3]. The first clinical application of platelet-derived factors in wound healing was reported in the 1980s by Knighton and colleagues[4]. Since then, PRP-based therapies have gained remarkable attention, with more than 86,000 PRP injections administered annually in the United States alone, predominantly among athletes. In addition to promoting structural repair of damaged tissues and joints, PRP exerts potent immunomodulatory and anti-inflammatory properties[5]. Therapeutic efficacy is typically achieved when platelet concentrations reach three to five times higher than baseline levels[6]. Numerous studies have demonstrated the beneficial effects of PRP in anterior cruciate ligament (ACL) injuries, tendon and rotator cuff repair, and intra-articular treatment of osteoarthritis and shoulder or knee arthritis[7]. Moreover, PRP has shown promising outcomes in aesthetic medicine, including hair restoration, as well as in managing post-radiotherapy tissue damage and enhancing surgical wound healing after tumor resection[8–10]. These findings have positioned PRP as a versatile regenerative biologic with expanding applications across clinical and research fields. The central nervous system (CNS) comprises neurons and glial cells, with glial cells accounting for approximately 90% of brain cellular composition and nearly half of the total brain and spinal cord volume[11]. Glial cells perform essential supportive functions, including myelination, extracellular potassium buffering, neurotransmitter transport, and the regulation of endothelial tight junctions that form the blood–brain barrier[12]. They also contribute to neuronal signaling and glial scar formation following injury. Effective glia–neuron communication is indispensable for maintaining CNS homeostasis[13]. Among glial cells, microglia and astrocytes serve as the principal immune effector cells, expressing pattern recognition receptors such as Toll-like receptors (TLRs), which detect both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)[14]. Platelets may interact with glial cells under two major pathological contexts: (i) sterile inflammation, such as in autoimmune diseases (e.g., multiple sclerosis), and (ii) infectious inflammation, such as bacterial meningitis[15]. Autoimmune diseases of the CNS represent a major cause of long-term neurological disability, particularly in adults aged 30–40 years, whereas bacterial infections like meningitis remain life-threatening for elderly or immunocompromised individuals[16, 17]. Among bacterial pathogens, Gram-negative Escherichia coli stands out for its ability to penetrate the blood–brain barrier and elicit strong activation of microglia through TLR-mediated signaling, primarily via lipopolysaccharide (LPS) and other virulence factors[18]. Under physiological conditions, glial cells maintain a quiescent state, but in response to neurological or inflammatory insults, they undergo morphological and functional activation, releasing pro-inflammatory cytokines that can exacerbate neuronal injury if uncontrolled[19]. Activation of TLRs by PAMPs (e.g., LPS) or DAMPs (e.g., adenosine, free radicals, amyloid-β peptides) initiates inflammatory cascades that may further amplify neurotoxicity and impair neuronal survival[20, 21]. Apoptosis, a tightly regulated cellular process, maintains tissue homeostasis by eliminating damaged or potentially oncogenic cells. Because cells actively orchestrate their own death, apoptosis is often described as a form of “programmed self-destruction”[22, 23]. The initiation of apoptosis is determined by a delicate balance between pro-survival factors (e.g., growth factors, interleukins) and pro-apoptotic signals triggered by oxidative stress, DNA damage, or the accumulation of reactive oxygen species (ROS) and nitric oxide (NO)[24]. A central event in this pathway is the activation of cysteine proteases, particularly caspase-3, whose activity is regulated by the BCL-2 family of proteins. This family governs mitochondrial membrane integrity by encoding both anti-apoptotic (e.g., BCL-2, BCL-XL) and pro-apoptotic (e.g., Bax, Bad) members, thereby determining the cell’s fate[25, 26]. Given the growing evidence of platelet-mediated immune modulation, this study aimed to investigate platelet–glial cell interactions under both sterile and infectious inflammatory conditions. Specifically, we simulated sterile neuroinflammation by treating B92 glial cells with PRP alone, and infectious inflammation by exposing them to PRP in combination with a heat-killed preparation of Escherichia coli (HKEC), to elucidate the immunomodulatory mechanisms of PRP in glial cell biology. 2. Materials and Methods 2.1. Study Groups This experimental study included four main groups: Untreated B92 glial cells (control). B92 cells treated with Platelet-Rich Plasma (PRP) at concentrations of 0%, 5%, 10%, and 20%. B92 cells exposed to heat-killed Escherichia coli (HKEC) at a 1:100 ratio. B92 cells exposed to HKEC (1:100) and treated with PRP at concentrations of 0%, 5%, 10%, and 20%. All procedures were approved by the Research Ethics Committee of the Faculty of Veterinary Medicine, Urmia University. The B92 glial cell line (Catalog No. C132) was obtained from the Iranian Cell Bank and cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin, 100 µg/mL; penicillin, 100 U/mL) (BioWest, USA). Cells were maintained at 37°C in a humidified atmosphere of 5% CO₂ using a MEMMERT incubator (Germany). Morphological alterations in PRP-treated cells were examined using an inverted microscope (OLYMPUS, Japan) and compared with control samples. 2.2. Blood Collection and PRP Preparation Adult male Wistar rats (6–8 weeks old) were obtained from the Animal Research Center, Faculty of Veterinary Medicine, Urmia University. Whole blood was collected from anesthetized rats (ketamine–xylazine) via cardiac puncture using sterile syringes and transferred into sodium citrate tubes (Kima, Italy) under aseptic conditions in a Class II laminar flow hood (BEAST BSC 120, Germany). Platelet-rich plasma (PRP) was prepared using a standard two-step centrifugation protocol. Initially, blood samples were centrifuged at 160 × g for 20 min (HINOTEK, Germany) to separate plasma and the buffy coat, which were then carefully aspirated into a new tube. A second centrifugation at 400 × g for 15 min was performed to pellet platelets. The platelet-poor plasma supernatant was discarded, and the PRP fraction was collected and used fresh for subsequent experiments[27]. Platelet activation was induced by adding 142 µL of 10% CaCl₂ (Merck, Germany) per 1 mL of PRP[28]. 2.3. Platelet Count A smear of the prepared PRP sample was made and stained using the Giemsa method to verify platelet morphology. Platelet quantification was carried out using a Neubauer hemocytometer under a light microscope at 40× magnification[29]. 2.4. Preparation of Bacteria and Heat-Killed Suspension All bacterial preparation steps were conducted under aseptic conditions. A single colony of Escherichia coli (strain PTCC-1533; Iranian Research Institute of Industrial Research) was inoculated into Nutrient Broth and incubated at 37°C for 24 hours in a CO₂ incubator. The turbidity of the bacterial culture was adjusted to match the 0.5 McFarland standard, equivalent to approximately 1 × 10⁷ CFU/ mL[30]. The bacterial suspension was washed three times with sterile phosphate-buffered saline (PBS) and resuspended in low-glucose DMEM to a final volume of 10 mL[31]. To prepare the heat-killed bacterial (HKEC) suspension, the culture was incubated in a water bath at 60°C for 1 hour. Complete inactivation was confirmed by plating an aliquot onto Nutrient Agar and verifying the absence of colony growth after incubation[32]. 2.5. Morphological Assessment of PRP-Treated B92 Glial Cells B92 glial cells were cultured until reaching the logarithmic growth phase, after which 1 × 10⁶ cells were seeded into 24-well plates. Following treatment, cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ for 24 hours. Morphological alterations in PRP-treated cells were examined using an inverted light microscope (OLYMPUS, Japan) and compared with untreated control cells. All experiments were performed in triplicate to ensure reproducibility. 2.6. MTT Assay for Mitochondrial Viability The mitochondrial activity and viability of B92 glial cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA). Cells were seeded in 24-well culture plates (NEST Biotech, USA) and treated with PRP in the presence or absence of heat-killed E. coli (HKEC) for 24 hours. Subsequently, MTT solution (5 mg/mL) was added to each well at a 1:10 ratio and incubated for 4 hours at 37°C. After incubation, the supernatant was carefully removed, and dimethyl sulfoxide (DMSO; Sigma, USA) was added to solubilize the formazan crystals. The mixture was pipetted several times to ensure homogeneity, and 100 µL from each well was transferred (in triplicate) to a flat-bottom 96-well microplate (NEST Biotech, USA). Absorbance was measured at 492 nm using a microplate reader (BIOTEK, USA)[33]. 2.7. Neutral Red Uptake Assay for Endocytic Activity The endocytic activity of B92 glial cells was assessed using the neutral red uptake assay. After treatment as described above, cells were incubated with neutral red solution (0.33 g/mL; Sigma, USA) at a 1:100 dilution for 2 hours at 37°C. Following incubation, the medium was discarded, and cells were gently washed with phosphate-buffered saline (PBS) to remove unincorporated dye. Subsequently, 1000 µL of lysis buffer was added to each well to extract the internalized dye. The resulting supernatant was transferred in triplicate to a 96-well plate (100 µL per well), and absorbance was measured at 492 nm using a microplate reader (BIOTEK, USA)[34]. 2.8. Nitroblue Tetrazolium (NBT) Assay for ROS Production The intracellular generation of reactive oxygen species (ROS) was determined using the nitroblue tetrazolium (NBT) reduction assay. Treated B92 cells (2 × 10⁶ cells/mL) were incubated with 0.1% NBT solution in the presence of N-formylmethionyl-leucyl-phenylalanine (FMLP; 100 nmol/L) for 1 hour at 37°C. After incubation, the medium was removed, and 1 mL of solubilization buffer containing dimethyl sulfoxide (DMSO) and 1 mol/L KOH was added to dissolve the reduced formazan crystals. The resulting supernatants were transferred to a 96-well plate (100 µL per well), and absorbance was measured at 505 nm using a microplate reader (BIOTEK, USA)[35]. 2.9. RNA Extraction Following treatment, B92 cells were trypsinized, washed twice with phosphate-buffered saline (PBS), and harvested for RNA isolation. Total RNA was extracted using the CinnaPure RNA Kit (SinaClon, Iran; Cat. No. PR891620) according to the manufacturer’s protocol. RNA concentration and purity were determined spectrophotometrically by measuring the absorbance ratio at 260/280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Japan). 2.10. cDNA Synthesis and Quantitative Real-Time PCR (qRT-PCR) One microgram of total RNA was reverse transcribed into complementary DNA (cDNA) using the Parstous cDNA Synthesis Kit (Cat. No. A101161). Quantitative real-time PCR was conducted on a MYGO-mini real-time PCR system (USA) using the BIOFACT Real-Time PCR Master Mix (Cat. No. DQ385). The expression of TNF-α, IL-10, IL-1β, BCL-2, Caspase-3, and BAX genes was evaluated using specific primers listed in Table 1. The amplification protocol consisted of an initial denaturation at 95°C for 15 min, followed by 40 cycles of 95°C for 20 sec (denaturation), 58°C for 30 sec (annealing), and 72°C for 30 sec (extension), with a final extension step at 72°C for 15 min[36]. 2.11. qPCR Data Analysis Relative mRNA expression levels were calculated using the 2^–ΔΔCt method, with HPRT serving as the internal reference gene. Primer amplification efficiency was validated to be within the 90–110% range. All reactions were performed in triplicate for three independent biological replicates, and data are presented as mean Ct ± standard deviation (SD). 2.12. Statistical Analysis All data were analyzed using SPSS software (version 20.0). The Kolmogorov–Smirnov test was applied to verify data normality. Differences among experimental groups were evaluated using one-way ANOVA, followed by Tukey’s post hoc test. A p value of < 0.05 was considered statistically significant. Results are presented as mean ± SD. 3. Results 3.1. Platelet Count and PRP Preparation Giemsa staining and platelet quantification of the prepared PRP samples revealed an average platelet count of 2,500,000 ± 10,000, confirming the effective enrichment of platelets. Based on this count, PRP working concentrations of 5%, 10%, and 20% were prepared for subsequent cell treatment experiments. 3.2. PRP Enhances Glial Cell Proliferation and Preserves Morphology Inverted light microscopy demonstrated that untreated B92 glial cells displayed a typical elongated, spindle-shaped morphology under optimal culture conditions, with robust adhesion and proliferation across passages (Fig. 1 A). PRP supplementation markedly promoted cell growth and survival in a concentration-dependent manner (Fig. 1 C, E, G). Cells exposed to 20% PRP exhibited the most extensive proliferation and elongation. In contrast, exposure to heat-killed E. coli (HKEC) led to an evident decline in cell growth within 24 h, characterized by cell clustering and detachment (Fig. 1 B). However, co-treatment with PRP effectively counteracted these inhibitory effects, promoting a gradual, dose-dependent recovery in cell proliferation (Fig. 1 D, F). Remarkably, B92 cells treated with 20% PRP in the presence of HKEC exhibited even higher proliferation rates than the untreated control group (Fig. 1 K). Morphological analysis further confirmed the pronounced trophic effect of PRP on glial cell structure and density. 3.3. PRP Improves Glial Viability and Reduces Oxidative Stress Without Altering Phagocytic Activity Functional assays revealed a strong pro-survival effect of PRP on B92 glial cells. The MTT assay showed a significant, dose-dependent increase in cell viability and metabolic activity under both sterile and infectious inflammatory conditions (Table 2). At 20% PRP, viability increased by 62.3% under sterile and 52.1% under infectious conditions compared with the respective controls. Parallel assessment of oxidative status demonstrated that PRP markedly suppressed reactive oxygen species (ROS) production in both models. The 20% PRP group exhibited an approximately 40–45% reduction in superoxide anion levels relative to untreated cells (Table 2). In contrast, PRP treatment did not significantly affect the phagocytic function of glial cells, as determined by neutral red uptake (Table 2). HKEC exposure caused only a minor, non-significant reduction in endocytic capacity, while PRP co-treatment maintained membrane integrity and lysosomal activity across all tested concentrations. These findings indicate that PRP enhances glial cell viability and redox balance without compromising essential cellular functions. 3.4. PRP Induces a Pro-Survival Molecular Profile To investigate the molecular mechanisms underlying PRP’s cytoprotective effects, the expression of apoptosis-regulating genes was quantified using qRT-PCR. PRP treatment significantly upregulated the anti-apoptotic gene BCL-2 in a concentration-dependent manner, while concurrently downregulating the pro-apoptotic genes Caspase-3 and BAX (Table 3; Fig. 2 – 4 ). Similarly, HKEC exposure alone elevated Caspase-3 and BAX expression, consistent with the induction of cell stress and apoptosis. Co-treatment with PRP, however, effectively reversed these pro-apoptotic changes, most prominently at 20% PRP, indicating a robust anti-apoptotic shift. This transcriptional reprogramming toward a pro-survival phenotype was consistently observed in both sterile and infectious inflammation models, though slightly more pronounced under sterile conditions. 3.5. PRP Reprograms the Cytokine Expression Landscape Toward an Anti-Inflammatory State The immunomodulatory effects of PRP were further evaluated by quantifying cytokine gene expression. As shown in Table 4, PRP treatment caused a significant suppression of the pro-inflammatory cytokine TNF-α, an effect that appeared dose-independent and consistent across all tested concentrations (5–20%) (Fig. 5 ). Conversely, PRP robustly upregulated the anti-inflammatory cytokine IL-10 by approximately threefold relative to untreated controls, also in a dose-independent manner (Table 4; Fig. 6 ). Interestingly, PRP exposure also increased IL-1β expression in both sterile and infectious models, albeit without a clear concentration response trend (Table 4; Fig. 7 ). Collectively, these results suggest that PRP fine-tunes the inflammatory response of glial cells, downregulating excessive pro-inflammatory signaling (TNF-α) while enhancing regulatory and reparative cytokine expression (IL-10, IL-1β), thereby contributing to a balanced neuroinflammatory profile. 4. Discussion Platelets interact with glial cells in both sterile and infectious inflammatory contexts, including autoimmune disorders such as multiple sclerosis and bacterial infections like meningitis[14, 17]. Gram-negative bacteria such as Escherichia coli are especially relevant to infection-driven neuroinflammation because of their ability to breach the blood-brain barrier and activate pattern recognition receptors on microglia and other glial populations[13, 17]. Using a B92 glial cell model, we show that platelet-rich plasma (PRP) prepared here from samples with a platelet count of 2,500,000 ± 10,000 exerts pleiotropic, concentration-dependent effects that: (i) enhance metabolic activity and proliferation, (ii) preserve membrane and lysosomal function, (iii) attenuate pathogen-induced oxidative stress, and (iv) reprogram apoptotic and cytokine pathways toward a pro-survival, resolution-oriented phenotype. Below we integrate these observations with existing literature, propose plausible mechanisms, and discuss translational implications. PRP produced a robust, dose-dependent increase in B92 metabolic activity as measured by MTT: exposure to 5%, 10% and 20% PRP increased viability indices to 1.20 ± 0.10, 1.50 ± 0.20 and 2.10 ± 0.20, respectively, compared with untreated controls (0.70 ± 0.10, P < 0.05). Under HKEC stimulation, viability declined to 0.80 ± 0.10 but was restored in a PRP dose-responsive manner up to 1.95 ± 0.20 at 20% PRP. These data indicate that PRP supplies trophic and mitogenic cues that sustain mitochondrial function and cellular bioenergetics even during inflammatory challenge. The effect is consistent with the rich complement of platelet-derived growth factors in PRP (PDGF, VEGF, TGF-β, IGF-1, HGF, FGF, EGF), which are known to activate mitochondrial and pro-survival signaling (PI3K/Akt, ERK/MAPK) and to stimulate proliferation in diverse cell types[37–39]. Similar mitogenic actions of PRP have been documented in non-neural tissues, for example in promotion of follicular unit survival during hair transplantation via MAPK/ERK activation[40]. Taken together, our results extend those observations to glial biology and suggest that PRP can preserve metabolic homeostasis under both basal and infection-mimicking conditions. Neutral red uptake assays indicated preserved lysosomal and membrane integrity across PRP concentrations and experimental conditions (Table 2). In sterile conditions, PRP modestly increased neutral red uptake, whereas HKEC alone produced no significant impairment of endocytic capacity. Importantly, PRP supplementation did not abrogate phagocytic function in infected cultures, implying that the pro-survival and anti-oxidative effects of PRP are achieved without globally suppressing critical innate defense mechanisms[41]. Preservation of membrane and lysosomal integrity supports the hypothesis that PRP enhances cellular resilience rather than simply preventing cell death, and suggests favorable biocompatibility for future therapeutic applications. Oxidative stress is a central mediator of glial dysfunction during infection; macrophages and microglia generate ROS to combat pathogens, but excessive ROS causes collateral neuronal damage[41]. In our NBT assays, HKEC dramatically elevated superoxide production (2.15 ± 0.07 vs control 0.82 ± 0.03), whereas PRP co-treatment reduced ROS in a concentration-dependent fashion to 1.72 ± 0.06, 1.38 ± 0.05, and 1.10 ± 0.04 for 5%, 10% and 20% PRP, respectively (P < 0.05). This antioxidant effect may reflect multiple, non-mutually exclusive mechanisms: suppression of pro-inflammatory cytokine signaling (TNF-α, IL-1β, IL-6), upregulation of anti-inflammatory mediators such as IL-10, direct stimulation of endogenous antioxidant systems (SOD, catalase, glutathione peroxidase), and modulation of NF-κB and MAPK pathways that govern redox homeostasis. These mechanistic possibilities are supported by prior studies showing PRP-mediated reductions in microglial activation and ROS-driven neurotoxicity[4, 7, 14, 40, 41]. Our qRT-PCR analyses revealed a coherent anti-apoptotic transcriptional program induced by PRP. In non-stimulated B92 cells, PRP dose-dependently upregulated BCL-2 (peaking at 1.95 ± 0.10 at 20% PRP) while markedly reducing Caspase-3 (from control 1.00 ± 0.04 to 0.25 ± 0.02 at 20% PRP) and lowering BAX expression. Conversely, HKEC induced a pro-apoptotic shift (reduced BCL-2 0.30 ± 0.05, elevated Caspase-3 1.45 ± 0.12, and BAX 1.18 ± 0.11), which was reversed by PRP co-treatment (restoration of BCL-2 up to 0.95 ± 0.08 at 20% PRP and suppression of pro-apoptotic transcripts). These findings align with the wealth of evidence that PRP growth factors activate PI3K/Akt and ERK pathways to inhibit caspase-dependent apoptosis and bolster mitochondrial integrity in multiple cell types[42–44]. The ability of PRP to restore anti-apoptotic balance under bacterial stress indicates a direct cytoprotective role that could help preserve glial populations during acute neuroinflammatory insults. Cytokine profiling uncovered a nuanced immunomodulatory signature. Under basal conditions, PRP reduced TNF-α and IL-1β while increasing IL-10 (IL-10 up to 1.80 ± 0.12, TNF-α and IL-1β down to 0.40 ± 0.04 and 0.52 ± 0.07, respectively). HKEC induced a robust pro-inflammatory state (TNF-α 2.20 ± 0.12, IL-1β 2.50 ± 0.14, IL-10 suppressed to 0.60 ± 0.06), which PRP attenuated in a dose-dependent manner; at 20% PRP TNF-α and IL-1β were reduced to 1.10 ± 0.08 and 1.20 ± 0.09, respectively, and IL-10 was restored to 1.40 ± 0.09. This pattern indicates that PRP exerts an active rebalancing of inflammatory mediators rather than indiscriminate immunosuppression: it dampens excessive pro-inflammatory signaling while reinstating regulatory IL-10, a cytokine critical for limiting microglial hyperactivation and promoting resolution[45–47]. The observation that IL-1β sometimes remains elevated in PRP-treated cells suggests that PRP may permit controlled pro-repair inflammatory signaling (priming or trophic roles) while preventing runaway, tissue-destructive inflammation a concept increasingly recognized in the field[20]. The multifactorial effects described above can be reconciled by a model in which PRP provides a concentrated, physiologically compatible secretome that: (i) activates membrane receptors (PDGFR, VEGFR, IGF1R, EGFR) to stimulate survival and proliferative cascades (PI3K/Akt, MAPK/ERK), (ii) modulates transcriptional programs that suppress caspase-dependent apoptosis and upregulate mitochondrial stabilizers (BCL-2), and (iii) reconfigures innate immune signaling to limit NF-κB-driven pro-oxidant responses while promoting IL-10 mediated resolution. Importantly, these effects likely arise from synergistic rather than singular factor actions; adhesive proteins (fibrinogen, fibronectin, vitronectin) in PRP may also support cell adhesion and microenvironmental stability, further contributing to functional recovery[38, 39]. That these mechanisms operate without evidence of tumorigenic risk is consistent with the membrane-receptor mediated, non-genomic mode of PRP action[39]. Our results converge with reports that PRP reduces pro-inflammatory cytokine release and oxidative damage in musculoskeletal and neural injury models[4]. The anti-apoptotic transcriptional shift (increased BCL-2, decreased Caspase-3 and BAX) echoes prior observations in endothelial and neuronal systems[42–44], while the reconstitution of IL-10 in infected cultures supports the recognized role of IL-10 in protecting against bacterial meningitis sequelae[45]. The preservation of phagocytic competence despite reduced ROS suggests a selective modulation that favors resolution and repair over wholesale immunosuppression, a desirable characteristic contrasted with broad-spectrum anti-inflammatory drugs[48–51]. Excessive glial activation, oxidative stress and apoptosis are central pathogenic mechanisms in a range of CNS disorders, from acute bacterial meningitis to chronic neurodegenerative disease and autoimmune neuroinflammation[15]. PRP’s capacity to concurrently preserve cellular viability, attenuate oxidative and apoptotic cascades, and recalibrate cytokine networks positions it as a promising biologic adjuvant for conditions where restoring homeostasis rather than blunt immunosuppression is required. Moreover, the dose-independent modulation of certain cytokines (for example IL-10 and TNF-α in our models) suggests that therapeutic benefit might be achievable at moderate PRP concentrations, potentially reducing risks related to overt growth-factor exposure. Key strengths of this study include the integrated assessment across functional (MTT, Neutral Red, NBT) and molecular (qRT-PCR) endpoints, allowing us to link phenotype to mechanism. Nonetheless, several limitations temper immediate translational extrapolation. First, these data derive from a single in vitro glial cell line and may not fully capture cellular heterogeneity, multicellular interactions, or blood–brain barrier dynamics in vivo. Second, while we document clear bioactivity of PRP, the specific molecular constituents responsible for each observed effect were not isolated; fractionation and proteomic profiling of the PRP secretome are necessary next steps. Third, long-term effects, dose-scheduling, and safety (including potential pro-fibrotic or aberrant remodeling responses) require evaluation in relevant animal models. Future work should therefore (i) fractionate PRP to identify active mediators, (ii) map downstream signaling cascades in primary glial populations and organotypic cultures, and (iii) validate efficacy and safety in in vivo models of infection-driven and autoimmune neuroinflammation. In summary, PRP functions as a finely tuned immunomodulatory and cytoprotective agent in glial cells: it enhances metabolic resilience, preserves membrane and lysosomal competence, mitigates pathogen-induced oxidative stress, and shifts apoptotic and cytokine programs toward survival and resolution. These multifactorial actions mediated by a repertoire of growth factors and adhesive proteins acting via membrane receptors support the potential of PRP as a therapeutic adjunct for neuroinflammatory disorders. Elucidation of the active PRP components, optimal dosing regimens, and validation in animal models will be critical to translating these promising in vitro findings into clinical strategies for CNS inflammation and injury. 5. Conclusion In conclusion, this study provides compelling evidence that Platelet-Rich Plasma (PRP) functions as a potent and multifaceted immunomodulator within the glial system, orchestrating a delicate balance between pro- and anti-inflammatory signaling under both sterile and infectious conditions. By enhancing glial cell viability, promoting anti-apoptotic gene expression (upregulating BCL-2 while downregulating Caspase-3 and BAX), and simultaneously suppressing excessive pro-inflammatory mediators (TNF-α, IL-1β) while restoring anti-inflammatory IL-10 expression, PRP fosters a neuroprotective microenvironment that supports tissue repair and maintains immune homeostasis. Mechanistically, PRP achieves this immunoregulatory equilibrium through its rich repertoire of growth factors such as PDGF, VEGF, TGF-β, and IGF-1 which activate pro-survival pathways including PI3K/Akt and MAPK/ERK cascades. In bacterial challenge conditions, PRP effectively counteracted HKEC-induced oxidative stress, alleviated ROS overproduction, and preserved membrane integrity and metabolic activity, thereby preventing glial dysfunction and subsequent neuronal injury. This dual ability to sustain cellular resilience while tempering inflammation distinguishes PRP as a highly sophisticated immunomodulatory agent rather than a simple anti-inflammatory compound. Importantly, the observed cytokine modulation pattern marked by reduced TNF-α and IL-1β expression alongside enhanced IL-10 levels highlights PRP’s potential to reprogram glial reactivity toward a resolution-oriented phenotype. Such fine-tuned immune regulation is crucial in the context of neuroinflammatory and neurodegenerative disorders where glial overactivation contributes to progressive neuronal loss. Moreover, the maintenance of essential glial functions, including phagocytic competence, underscores the therapeutic safety and physiological compatibility of PRP-based interventions. Overall, these findings position PRP as a promising biological therapy capable of addressing the multifactorial nature of neuroinflammation. Its pleiotropic actions spanning antioxidant, anti-apoptotic, and immunoregulatory effects suggest broad therapeutic applicability in CNS disorders, including autoimmune neuroinflammation, infection-driven neural damage, and chronic neurodegeneration. Future research should focus on delineating the molecular constituents within the PRP secretome responsible for these protective effects, optimizing dosage parameters, and validating these mechanisms in in vivo models to facilitate clinical translation. Declarations Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. The study was conducted in accordance with ethical standards, and all sources of support are appropriately acknowledged. Acknowledgments The authors sincerely thank Mr. Asghar Aliyari, Laboratory Specialist in Immunology, Faculty of Veterinary Medicine, Urmia University, for his valuable technical assistance during this study. The authors also acknowledge Urmia University (Urmia, Iran) for providing laboratory facilities and institutional support throughout the research process. Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. It was supported by Urmia University (Urmia, Iran) through institutional resources and facilities. Ethical Statement Ethical approval for this study was obtained from the Ethics Committee of Urmia University (Ethical Code: IR-UU-AEC-3/PD/1382). CRediT Author Statement Mahtab Pourkamalzadeh: Investigation, Data curation, Formal analysis, Visualization of experimental data, Writing – review & editing. Seyyed Meysam Abtahi Froushani: Conceptualization, Methodology, Supervision, Project administration, Resources, Writing – original draft. Mahtab Pourkamalzadeh and Seyyed Meysam Abtahi Froushani: Validation, Methodology, Conceptualization, Review & Editing. References Alves, R. and R. Grimalt. A review of platelet-rich plasma: history, biology, mechanism of action, and classification. Skin appendage disorders. 2018;4(1):18-24. Fossati, C., et al. 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Moro, Programmed cell death in health and disease . 2021, MDPI. p. 1765. Halder, S., et al., Apoptosis: ER Stress and Disease Pathology , in Apoptosis and Human Health: Understanding Mechanistic and Therapeutic Potential . 2024, Springer. p. 127-139. Hussar, P. Apoptosis regulators bcl-2 and caspase-3. Encyclopedia. 2022;2(4):1624-1636. Liang, Z., et al. Asiaticoside prevents oxidative stress and apoptosis in endothelial cells by activating ROS-dependent p53/Bcl-2/Caspase-3 signaling pathway. Current molecular medicine. 2023;23(10):1116-1129. Pérez-Montesinos, G., et al. Platelet-rich plasma: comparative study of four protocols for its production. Revista del Centro Dermatológico Pascua. 2017;26(2):41-44. Gomez, T.W., et al. Comparative evaluation of angiogenesis using a novel platelet-rich product: An: in vitro: study. Journal of Conservative Dentistry and Endodontics. 2019;22(1):23-27. Messora, M.R., et al. 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International journal of molecular sciences. 2021;22(5):2719. Mercogliano, M.F., et al. Harnessing tumor necrosis factor alpha to achieve effective cancer immunotherapy. Cancers. 2021;13(3):564. Tables Table.1 Sequences of primers and amplicon sizes used for quantitative real-time PCR analysis in B92 glial cells. Gene-specific primers were employed to evaluate the relative expression of apoptosis- and inflammation-related genes, with HPRT serving as the internal reference. Amplicon sizes (in base pairs, bp) are indicated. All primers were designed to ensure high specificity and amplification efficiency, suitable for qRT-PCR experiments using the 2^-ΔΔCt method. Primer sequences were either designed based on NCBI reference sequences or obtained from Origene. Experiments were performed in triplicate, and expression levels were normalized to HPRT to account for sample-to-sample variation. Gene Primer Direction Sequence (5' → 3') Amplicon Size (bp) HPRT Forward 5'-TTGGTGGRGATGAYCTCTCAAC-3' ~120 Reverse 5'-TTCAAATCCAACAAAGTCTGGC-3' BCL-2 Forward 5'-GGAGGATTGTGGCCTTCTTT-3' ~200 Reverse 5'-GTTCAGGTACTCAGTCATCCAC-3' TNF-α Forward 5'-CTCTTCAAGGGACAAGGT-3' ~150 Reverse 5'-CTTGATGGCAGAGGAGG-3' IL-10 Forward 5'-AAGGTTACTTGGGTTGC-3' ~110 Reverse 5'-GCTCCTTGATTTCTGGGC-3' IL-1β Forward 5'-CAGCTGGAGAGTGTGGATC-3' ~130 Reverse 5'-TGCTGATGTACCAGTTGGG-3' BAX Forward 5'-TGGCGATGAACTGGACAACAA-3' ~150 Reverse 5'-CCCGAAGTAGGAAAGGAGGC-3' Caspase-3 Forward 5'-GACTGGAAAGCCGAAACTCT-3' ~140 Reverse 5'-GTCCCACTGTCTGTCTGTCTCAAT-3' Table.2 Comprehensive evaluation of metabolic activity (MTT assay), endocytic capacity (Neutral Red uptake assay), and reactive oxygen species (ROS) production (NBT assay) in B92 glial cells under sterile and infectious inflammatory conditions (HKEC) following treatment with different concentrations of platelet-rich plasma (PRP). Data are expressed as Mean ± SD from three independent biological replicates (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. Distinct superscript letters (a–e) denote statistically significant differences among groups (P < 0.05). PRP treatment enhanced glial cell viability and maintained endocytic activity under sterile conditions while reducing excessive ROS generation following HKEC stimulation, suggesting an overall protective and modulatory role of platelets in glial cell function. Abbreviations: PRP – Platelet-Rich Plasma; HKEC – Heat-killed Escherichia coli . Experimental Groups MTT Assay(Viability Index, Mean ± SD) NR Uptake Assay(Optical Density, Mean ± SD) NBT Assay(Optical Density, Mean ± SD) A: Sterile Inflammatory (without HKEC) B92 (Control) 0.70 ± 0.10ᵃ 1.80 ± 0.10ᵃ 0.82 ± 0.03ᵃ B92 + PRP 5% 1.20 ± 0.10ᵇ 2.00 ± 0.10ᵇ 0.89 ± 0.05ᵃ B92 + PRP 10% 1.50 ± 0.20ᶜ 2.00 ± 0.10ᵇ 0.80 ± 0.03ᵃ B92 + PRP 20% 2.10 ± 0.20ᵈ 1.90 ± 0.20ᵇ 0.83 ± 0.04ᵃ B: Infectious Inflammatory (challenged with HKEC) B92 + HKEC 0.80 ± 0.10ᵃᵇ 1.70 ± 0.10ᵃ 2.15 ± 0.07ᵇ B92 + HKEC + PRP 5% 1.00 ± 0.10ᵃ 1.95 ± 0.05ᵃ 1.72 ± 0.06ᶜ B92 + HKEC + PRP 10% 1.55 ± 0.30ᶜ 1.85 ± 0.10ᵃ 1.38 ± 0.05ᵈ B92 + HKEC + PRP 20% 1.95 ± 0.20ᶜ 1.95 ± 0.05ᵃ 1.10 ± 0.04ᵉ Table.3 Relative mRNA expression levels of apoptosis-associated genes (BCL-2, Caspase-3, and BAX) in B92 glial cells exposed to platelet-rich plasma (PRP) under sterile and infectious inflammatory conditions. Expression levels were quantified using the 2⁻ΔΔCt method and normalized to the housekeeping gene HPRT, with the untreated control set to 1.00 (fold change = 1.00). Data are presented as Mean ± SD from three independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. Distinct superscript letters (a–e) indicate significant differences among groups (P < 0.05). Abbreviations: PRP – Platelet-Rich Plasma; HKEC – Heat-killed Escherichia coli . Experimental Group BCL-2 (Mean ± SD) Caspase-3 (Mean ± SD) BAX (Mean ± SD) A: Sterile Inflammatory (without HKEC) B92 (Control) 1.00 ± 0.02ᵃ 1.00 ± 0.04ᵃ 1.00 ± 0.05ᵃ B92 + PRP 5% 1.20 ± 0.07ᵇ 0.32 ± 0.03ᵇ 0.90 ± 0.09ᵇ B92 + PRP 10% 1.50 ± 0.12ᶜ 0.31 ± 0.05ᵇ 0.73 ± 0.10ᶜ B92 + PRP 20% 1.95 ± 0.10ᵈ 0.25 ± 0.02ᶜ 0.52 ± 0.08ᵈ B: Infectious Inflammatory (challenged with HKEC) B92 (Control) 1.00 ± 0.03ᵃ 1.00 ± 0.05ᵃ 1.00 ± 0.04ᵃ B92 + HKEC 0.30 ± 0.05ᵇ 1.45 ± 0.12ᵇ 1.18 ± 0.11ᵇ B92 + HKEC + PRP 5% 0.35 ± 0.04ᶜ 1.32 ± 0.11ᶜ 1.08 ± 0.10ᶜ B92 + HKEC + PRP 10% 0.70 ± 0.09ᵈ 1.30 ± 0.10ᶜ 1.06 ± 0.09ᶜ B92 + HKEC + PRP 20% 0.95 ± 0.08ᵃ 1.15 ± 0.08ᵈ 0.87 ± 0.08ᵈ Table.4 Modulatory effects of platelet-rich plasma (PRP) on the expression of pro-inflammatory (TNF-α and IL-1β) and anti-inflammatory (IL-10) cytokine genes in B92 glial cells under sterile and infectious inflammatory conditions. Gene expression levels were quantified using the 2⁻ΔΔCt method and normalized to the HPRT reference gene. The control group was set as the calibrator (fold change = 1.00). Data are presented as mean ± standard deviation (SD) from three independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s post hoc test. Distinct superscript letters (a–e) denote statistically significant differences among groups (P < 0.05). Abbreviations: PRP – Platelet-Rich Plasma; HKEC – Heat-killed Escherichia coli . Experimental Group TNF-α (Mean ± SD) IL-10 (Mean ± SD) IL-1β (Mean ± SD) A: Sterile Inflammatory (without HKEC) B92 (Control) 1.00 ± 0.03ᵃ 1.00 ± 0.04ᵃ 1.00 ± 0.05ᵃ B92 + PRP 5% 0.75 ± 0.06ᵇ 1.20 ± 0.07ᵇ 0.85 ± 0.08ᵃ B92 + PRP 10% 0.55 ± 0.05ᵇ 1.50 ± 0.10ᵇ 0.70 ± 0.09ᶜ B92 + PRP 20% 0.40 ± 0.04ᵇ 1.80 ± 0.12ᵇ 0.52 ± 0.07ᵈ B: Infectious Inflammatory (challenged with HKEC) B92 (Control) 1.00 ± 0.04ᵃ 1.00 ± 0.05ᵃ 1.00 ± 0.04ᵃ B92 + HKEC 2.20 ± 0.12ᵇ 0.60 ± 0.06ᵇ 2.50 ± 0.14ᵇ B92 + HKEC + PRP 5% 1.80 ± 0.11ᶜ 0.85 ± 0.07ᶜ 2.00 ± 0.12ᶜ B92 + HKEC + PRP 10% 1.40 ± 0.09ᵈ 1.10 ± 0.08ᵈ 1.60 ± 0.10ᵈ B92 + HKEC + PRP 20% 1.10 ± 0.08ᵈ 1.40 ± 0.09ᵉ 1.20 ± 0.09ᵉ Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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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16:21:01","extension":"png","order_by":21,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":185566,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/a2fe5f79e11274ee8b4a9fd8.png"},{"id":95043347,"identity":"dbffdab8-e8e1-4e83-9dab-a2e92cee9b33","added_by":"auto","created_at":"2025-11-03 16:23:58","extension":"png","order_by":22,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":19853,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/d263675984b25b412e3bffb3.png"},{"id":95043363,"identity":"68022786-6356-44fb-83f3-b2ba457fcf78","added_by":"auto","created_at":"2025-11-03 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16:20:25","extension":"png","order_by":27,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":20904,"visible":true,"origin":"","legend":"","description":"","filename":"OnlineFig.7.png","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/049cd88365c6e3b3492155c2.png"},{"id":95222150,"identity":"4bbbc5e5-2e4e-4a05-9cd7-bee499dc235f","added_by":"auto","created_at":"2025-11-05 16:20:11","extension":"xml","order_by":28,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95334,"visible":true,"origin":"","legend":"","description":"","filename":"89b4960227e84103b8a1903a44b5c3241structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/3edaf8ca416a1016edebe659.xml"},{"id":95222316,"identity":"e2535318-196e-468c-a1a1-bcf6a5b2d06f","added_by":"auto","created_at":"2025-11-05 16:20:26","extension":"html","order_by":29,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":107084,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/46afd1c2dca7aabfc64c430c.html"},{"id":95043327,"identity":"8d3eb8a3-010e-4a48-a942-f88b5d0d19db","added_by":"auto","created_at":"2025-11-03 16:23:58","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1469841,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphology of B92 glial cells under the inverted microscope (24 hours after treatment with different concentrations: 0%, 5%, 10%, 20% PRP in the groups with and without the presence of heat-killed preparation of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eEscherichia coli\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e (HKEC), Images were taken at 40× magnification).\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/9827b64ed33a222a8f42990d.png"},{"id":95221609,"identity":"c385d2b1-394a-4812-b80a-635d88a795f2","added_by":"auto","created_at":"2025-11-05 16:19:29","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":96856,"visible":true,"origin":"","legend":"\u003cp\u003eBCL-2 gene expression in B92 glial cells under sterile and infectious conditions. (A) Relative expression in non-stimulated B92 cells (Sterile Condition) treated with PRP (0–20%). (B) Relative expression in B92 cells stimulated with a heat-killed preparation of \u003cem\u003eEscherichia coli\u003c/em\u003e (HKEC) treated with PRP (0–20%). Colors indicate experimental groups: B92 (gray), B92 + HKEC (red), B92 + HKEC + PRP 5% (light blue), B92 + HKEC + PRP 10% (medium blue), and B92 + HKEC + PRP 20% (dark blue). Data are mean ± SD; P\u0026lt;0.05 versus control. Different letters denote significant differences.\u003c/p\u003e","description":"","filename":"Fig.2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/8b0beff4d74d61775361ba00.jpg"},{"id":95223603,"identity":"d029290f-2936-43f9-abae-fe16ba8d7bb2","added_by":"auto","created_at":"2025-11-05 16:22:32","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":102408,"visible":true,"origin":"","legend":"\u003cp\u003eCaspase-3 gene expression in B92 glial cells under sterile and infectious conditions. (A) Relative expression in non-stimulated B92 cells (Sterile Condition) treated with PRP (0–20%). (B) Relative expression in B92 cells stimulated with a heat-killed preparation of \u003cem\u003eEscherichia coli\u003c/em\u003e(HKEC) treated with PRP (0–20%). Colors indicate experimental groups: B92 (gray), B92 + HKEC (red), B92 + HKEC + PRP 5% (light blue), B92 + HKEC + PRP 10% (medium blue), and B92 + HKEC + PRP 20% (dark blue). Data are mean ± SD; P\u0026lt;0.05 versus control. Different letters denote significant differences.\u003c/p\u003e","description":"","filename":"Fig.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/c31890bd5bb3c09ffe98b225.jpg"},{"id":95221954,"identity":"4562121b-7b85-4084-ade1-e0272592eb43","added_by":"auto","created_at":"2025-11-05 16:19:58","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":105931,"visible":true,"origin":"","legend":"\u003cp\u003eBAX gene expression in B92 glial cells under sterile and infectious conditions. (A) Relative expression in non-stimulated B92 cells (Sterile Condition) treated with PRP (0–20%). (B) Relative expression in B92 cells stimulated with a heat-killed preparation of \u003cem\u003eEscherichia coli\u003c/em\u003e (HKEC) treated with PRP (0–20%). Colors indicate experimental groups: B92 (gray), B92 + HKEC (red), B92 + HKEC + PRP 5% (light blue), B92 + HKEC + PRP 10% (medium blue), and B92 + HKEC + PRP 20% (dark blue). Data are mean ± SD; P\u0026lt;0.05 versus control. Different letters denote significant differences.\u003c/p\u003e","description":"","filename":"Fig.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/afbf216fdd8b588ed6eaa168.jpg"},{"id":95043336,"identity":"45229d59-0b25-42e9-b734-c97276a067e7","added_by":"auto","created_at":"2025-11-03 16:23:58","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":105956,"visible":true,"origin":"","legend":"\u003cp\u003eTNF-α gene expression in B92 glial cells under sterile and infectious conditions. (A) Relative expression in non-stimulated B92 cells (Sterile Condition) treated with PRP (0–20%). (B) Relative expression in B92 cells stimulated with a heat-killed preparation of \u003cem\u003eEscherichia coli\u003c/em\u003e (HKEC) treated with PRP (0–20%). Colors indicate experimental groups: B92 (gray), B92 + HKEC (red), B92 + HKEC + PRP 5% (light blue), B92 + HKEC + PRP 10% (medium blue), and B92 + HKEC + PRP 20% (dark blue). Data are mean ± SD; P\u0026lt;0.05 versus control. Different letters denote significant differences.\u003c/p\u003e","description":"","filename":"Fig.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/c41b77c6d00d115b3bdb9313.jpg"},{"id":95043332,"identity":"3d37e392-9c2f-49fb-8f2c-60b8beb086c5","added_by":"auto","created_at":"2025-11-03 16:23:58","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":102372,"visible":true,"origin":"","legend":"\u003cp\u003eIL-10 gene expression in B92 glial cells under sterile and infectious conditions. (A) Relative expression in non-stimulated B92 cells (Sterile Condition) treated with PRP (0–20%). (B) Relative expression in B92 cells stimulated with a heat-killed preparation of \u003cem\u003eEscherichia coli\u003c/em\u003e(HKEC) treated with PRP (0–20%). Colors indicate experimental groups: B92 (gray), B92 + HKEC (red), B92 + HKEC + PRP 5% (light blue), B92 + HKEC + PRP 10% (medium blue), and B92 + HKEC + PRP 20% (dark blue). Data are mean ± SD; P\u0026lt;0.05 versus control. Different letters denote significant differences.\u003c/p\u003e","description":"","filename":"Fig.6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/0dd8fdf20023f0150c6f4813.jpg"},{"id":95221951,"identity":"ddb7b348-ec24-4030-85e3-b1608bbbae31","added_by":"auto","created_at":"2025-11-05 16:19:58","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":96851,"visible":true,"origin":"","legend":"\u003cp\u003eIL-1β gene expression in B92 glial cells under sterile and infectious conditions. (A) Relative expression in non-stimulated B92 cells (Sterile Condition) treated with PRP (0–20%). (B) Relative expression in B92 cells stimulated with a heat-killed preparation of \u003cem\u003eEscherichia coli\u003c/em\u003e(HKEC) treated with PRP (0–20%). Colors indicate experimental groups: B92 (gray), B92 + HKEC (red), B92 + HKEC + PRP 5% (light blue), B92 + HKEC + PRP 10% (medium blue), and B92 + HKEC + PRP 20% (dark blue). Data are mean ± SD; P\u0026lt;0.05 versus control. Different letters denote significant differences.\u003c/p\u003e","description":"","filename":"Fig.7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/0ec75175643a3878747a819a.jpg"},{"id":95936906,"identity":"335be090-4a06-4b7e-9835-032deb43477b","added_by":"auto","created_at":"2025-11-14 15:38:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3631534,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7889196/v1/af560145-f8be-4a30-bc92-9e82ed24a1cf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Immunomodulatory Effects of Platelet-Rich Plasma (PRP) on Neuroinflammation: Insights from B92 Glial Cells Stimulated with Heat-Killed Escherichia coli","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003ePlatelet-Rich Plasma (PRP) is an autologous concentration of non-activated platelets suspended in a small volume of plasma, widely used to accelerate soft tissue and bone healing[1]. Beyond their classical role in hemostasis, platelets are now recognized as critical mediators of tissue repair and regeneration through the release of numerous bioactive molecules and growth factors that promote endothelial proliferation and fibroblast activation[2]. These growth factors including HGF, IGF-1, PDGF, VEGF, EGF, and TGF-β collectively contribute to several key processes such as chemotaxis, collagen type I synthesis, mitogenesis, cell proliferation, angiogenesis, and tissue remodeling[3].\u003c/p\u003e\u003cp\u003eThe first clinical application of platelet-derived factors in wound healing was reported in the 1980s by Knighton and colleagues[4]. Since then, PRP-based therapies have gained remarkable attention, with more than 86,000 PRP injections administered annually in the United States alone, predominantly among athletes. In addition to promoting structural repair of damaged tissues and joints, PRP exerts potent immunomodulatory and anti-inflammatory properties[5]. Therapeutic efficacy is typically achieved when platelet concentrations reach three to five times higher than baseline levels[6]. Numerous studies have demonstrated the beneficial effects of PRP in anterior cruciate ligament (ACL) injuries, tendon and rotator cuff repair, and intra-articular treatment of osteoarthritis and shoulder or knee arthritis[7]. Moreover, PRP has shown promising outcomes in aesthetic medicine, including hair restoration, as well as in managing post-radiotherapy tissue damage and enhancing surgical wound healing after tumor resection[8\u0026ndash;10]. These findings have positioned PRP as a versatile regenerative biologic with expanding applications across clinical and research fields.\u003c/p\u003e\u003cp\u003eThe central nervous system (CNS) comprises neurons and glial cells, with glial cells accounting for approximately 90% of brain cellular composition and nearly half of the total brain and spinal cord volume[11]. Glial cells perform essential supportive functions, including myelination, extracellular potassium buffering, neurotransmitter transport, and the regulation of endothelial tight junctions that form the blood\u0026ndash;brain barrier[12]. They also contribute to neuronal signaling and glial scar formation following injury. Effective glia\u0026ndash;neuron communication is indispensable for maintaining CNS homeostasis[13]. Among glial cells, microglia and astrocytes serve as the principal immune effector cells, expressing pattern recognition receptors such as Toll-like receptors (TLRs), which detect both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs)[14].\u003c/p\u003e\u003cp\u003ePlatelets may interact with glial cells under two major pathological contexts: (i) sterile inflammation, such as in autoimmune diseases (e.g., multiple sclerosis), and (ii) infectious inflammation, such as bacterial meningitis[15]. Autoimmune diseases of the CNS represent a major cause of long-term neurological disability, particularly in adults aged 30\u0026ndash;40 years, whereas bacterial infections like meningitis remain life-threatening for elderly or immunocompromised individuals[16, 17]. Among bacterial pathogens, Gram-negative \u003cem\u003eEscherichia coli\u003c/em\u003e stands out for its ability to penetrate the blood\u0026ndash;brain barrier and elicit strong activation of microglia through TLR-mediated signaling, primarily via lipopolysaccharide (LPS) and other virulence factors[18]. Under physiological conditions, glial cells maintain a quiescent state, but in response to neurological or inflammatory insults, they undergo morphological and functional activation, releasing pro-inflammatory cytokines that can exacerbate neuronal injury if uncontrolled[19]. Activation of TLRs by PAMPs (e.g., LPS) or DAMPs (e.g., adenosine, free radicals, amyloid-β peptides) initiates inflammatory cascades that may further amplify neurotoxicity and impair neuronal survival[20, 21].\u003c/p\u003e\u003cp\u003eApoptosis, a tightly regulated cellular process, maintains tissue homeostasis by eliminating damaged or potentially oncogenic cells. Because cells actively orchestrate their own death, apoptosis is often described as a form of \u0026ldquo;programmed self-destruction\u0026rdquo;[22, 23]. The initiation of apoptosis is determined by a delicate balance between pro-survival factors (e.g., growth factors, interleukins) and pro-apoptotic signals triggered by oxidative stress, DNA damage, or the accumulation of reactive oxygen species (ROS) and nitric oxide (NO)[24]. A central event in this pathway is the activation of cysteine proteases, particularly caspase-3, whose activity is regulated by the BCL-2 family of proteins. This family governs mitochondrial membrane integrity by encoding both anti-apoptotic (e.g., BCL-2, BCL-XL) and pro-apoptotic (e.g., Bax, Bad) members, thereby determining the cell\u0026rsquo;s fate[25, 26].\u003c/p\u003e\u003cp\u003eGiven the growing evidence of platelet-mediated immune modulation, this study aimed to investigate platelet\u0026ndash;glial cell interactions under both sterile and infectious inflammatory conditions. Specifically, we simulated sterile neuroinflammation by treating B92 glial cells with PRP alone, and infectious inflammation by exposing them to PRP in combination with a heat-killed preparation of \u003cem\u003eEscherichia coli\u003c/em\u003e (HKEC), to elucidate the immunomodulatory mechanisms of PRP in glial cell biology.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1. Study Groups\u003c/h2\u003e\u003cp\u003eThis experimental study included four main groups:\u003c/p\u003e\u003cp\u003e\u003col\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eUntreated B92 glial cells (control).\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eB92 cells treated with Platelet-Rich Plasma (PRP) at concentrations of 0%, 5%, 10%, and 20%.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eB92 cells exposed to heat-killed Escherichia coli (HKEC) at a 1:100 ratio.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003cspan\u003e\u003cli\u003e\u003cp\u003eB92 cells exposed to HKEC (1:100) and treated with PRP at concentrations of 0%, 5%, 10%, and 20%.\u003c/p\u003e\u003c/li\u003e\u003c/span\u003e\u003c/ol\u003e\u003c/p\u003e\u003cp\u003e All procedures were approved by the Research Ethics Committee of the Faculty of Veterinary Medicine, Urmia University. The B92 glial cell line (Catalog No. C132) was obtained from the Iranian Cell Bank and cultured in complete Dulbecco\u0026rsquo;s Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% antibiotics (streptomycin, 100 \u0026micro;g/mL; penicillin, 100 U/mL) (BioWest, USA). Cells were maintained at 37\u0026deg;C in a humidified atmosphere of 5% CO₂ using a MEMMERT incubator (Germany). Morphological alterations in PRP-treated cells were examined using an inverted microscope (OLYMPUS, Japan) and compared with control samples.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2. Blood Collection and PRP Preparation\u003c/h2\u003e\u003cp\u003eAdult male Wistar rats (6\u0026ndash;8 weeks old) were obtained from the Animal Research Center, Faculty of Veterinary Medicine, Urmia University. Whole blood was collected from anesthetized rats (ketamine\u0026ndash;xylazine) via cardiac puncture using sterile syringes and transferred into sodium citrate tubes (Kima, Italy) under aseptic conditions in a Class II laminar flow hood (BEAST BSC 120, Germany).\u003c/p\u003e\u003cp\u003ePlatelet-rich plasma (PRP) was prepared using a standard two-step centrifugation protocol. Initially, blood samples were centrifuged at 160 \u0026times; g for 20 min (HINOTEK, Germany) to separate plasma and the buffy coat, which were then carefully aspirated into a new tube. A second centrifugation at 400 \u0026times; g for 15 min was performed to pellet platelets. The platelet-poor plasma supernatant was discarded, and the PRP fraction was collected and used fresh for subsequent experiments[27]. Platelet activation was induced by adding 142 \u0026micro;L of 10% CaCl₂ (Merck, Germany) per 1 mL of PRP[28].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3. Platelet Count\u003c/h2\u003e\u003cp\u003eA smear of the prepared PRP sample was made and stained using the Giemsa method to verify platelet morphology. Platelet quantification was carried out using a Neubauer hemocytometer under a light microscope at 40\u0026times; magnification[29].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4. Preparation of Bacteria and Heat-Killed Suspension\u003c/h2\u003e\u003cp\u003eAll bacterial preparation steps were conducted under aseptic conditions. A single colony of \u003cem\u003eEscherichia coli\u003c/em\u003e (strain PTCC-1533; Iranian Research Institute of Industrial Research) was inoculated into Nutrient Broth and incubated at 37\u0026deg;C for 24 hours in a CO₂ incubator. The turbidity of the bacterial culture was adjusted to match the 0.5 McFarland standard, equivalent to approximately 1 \u0026times; 10⁷ CFU/ mL[30]. The bacterial suspension was washed three times with sterile phosphate-buffered saline (PBS) and resuspended in low-glucose DMEM to a final volume of 10 mL[31]. To prepare the heat-killed bacterial (HKEC) suspension, the culture was incubated in a water bath at 60\u0026deg;C for 1 hour. Complete inactivation was confirmed by plating an aliquot onto Nutrient Agar and verifying the absence of colony growth after incubation[32].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5. Morphological Assessment of PRP-Treated B92 Glial Cells\u003c/h2\u003e\u003cp\u003eB92 glial cells were cultured until reaching the logarithmic growth phase, after which 1 \u0026times; 10⁶ cells were seeded into 24-well plates. Following treatment, cells were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ for 24 hours. Morphological alterations in PRP-treated cells were examined using an inverted light microscope (OLYMPUS, Japan) and compared with untreated control cells. All experiments were performed in triplicate to ensure reproducibility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6. MTT Assay for Mitochondrial Viability\u003c/h2\u003e\u003cp\u003eThe mitochondrial activity and viability of B92 glial cells were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay (Sigma, USA). Cells were seeded in 24-well culture plates (NEST Biotech, USA) and treated with PRP in the presence or absence of heat-killed E. coli (HKEC) for 24 hours. Subsequently, MTT solution (5 mg/mL) was added to each well at a 1:10 ratio and incubated for 4 hours at 37\u0026deg;C. After incubation, the supernatant was carefully removed, and dimethyl sulfoxide (DMSO; Sigma, USA) was added to solubilize the formazan crystals. The mixture was pipetted several times to ensure homogeneity, and 100 \u0026micro;L from each well was transferred (in triplicate) to a flat-bottom 96-well microplate (NEST Biotech, USA). Absorbance was measured at 492 nm using a microplate reader (BIOTEK, USA)[33].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7. Neutral Red Uptake Assay for Endocytic Activity\u003c/h2\u003e\u003cp\u003eThe endocytic activity of B92 glial cells was assessed using the neutral red uptake assay. After treatment as described above, cells were incubated with neutral red solution (0.33 g/mL; Sigma, USA) at a 1:100 dilution for 2 hours at 37\u0026deg;C. Following incubation, the medium was discarded, and cells were gently washed with phosphate-buffered saline (PBS) to remove unincorporated dye. Subsequently, 1000 \u0026micro;L of lysis buffer was added to each well to extract the internalized dye. The resulting supernatant was transferred in triplicate to a 96-well plate (100 \u0026micro;L per well), and absorbance was measured at 492 nm using a microplate reader (BIOTEK, USA)[34].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8. Nitroblue Tetrazolium (NBT) Assay for ROS Production\u003c/h2\u003e\u003cp\u003eThe intracellular generation of reactive oxygen species (ROS) was determined using the nitroblue tetrazolium (NBT) reduction assay. Treated B92 cells (2 \u0026times; 10⁶ cells/mL) were incubated with 0.1% NBT solution in the presence of N-formylmethionyl-leucyl-phenylalanine (FMLP; 100 nmol/L) for 1 hour at 37\u0026deg;C. After incubation, the medium was removed, and 1 mL of solubilization buffer containing dimethyl sulfoxide (DMSO) and 1 mol/L KOH was added to dissolve the reduced formazan crystals. The resulting supernatants were transferred to a 96-well plate (100 \u0026micro;L per well), and absorbance was measured at 505 nm using a microplate reader (BIOTEK, USA)[35].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9. RNA Extraction\u003c/h2\u003e\u003cp\u003eFollowing treatment, B92 cells were trypsinized, washed twice with phosphate-buffered saline (PBS), and harvested for RNA isolation. Total RNA was extracted using the CinnaPure RNA Kit (SinaClon, Iran; Cat. No. PR891620) according to the manufacturer\u0026rsquo;s protocol. RNA concentration and purity were determined spectrophotometrically by measuring the absorbance ratio at 260/280 nm using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Japan).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e2.10. cDNA Synthesis and Quantitative Real-Time PCR (qRT-PCR)\u003c/h2\u003e\u003cp\u003eOne microgram of total RNA was reverse transcribed into complementary DNA (cDNA) using the Parstous cDNA Synthesis Kit (Cat. No. A101161). Quantitative real-time PCR was conducted on a MYGO-mini real-time PCR system (USA) using the BIOFACT Real-Time PCR Master Mix (Cat. No. DQ385). The expression of TNF-α, IL-10, IL-1β, BCL-2, Caspase-3, and BAX genes was evaluated using specific primers listed in Table\u0026nbsp;1. The amplification protocol consisted of an initial denaturation at 95\u0026deg;C for 15 min, followed by 40 cycles of 95\u0026deg;C for 20 sec (denaturation), 58\u0026deg;C for 30 sec (annealing), and 72\u0026deg;C for 30 sec (extension), with a final extension step at 72\u0026deg;C for 15 min[36].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e2.11. qPCR Data Analysis\u003c/h2\u003e\u003cp\u003eRelative mRNA expression levels were calculated using the 2^\u0026ndash;ΔΔCt method, with HPRT serving as the internal reference gene. Primer amplification efficiency was validated to be within the 90\u0026ndash;110% range. All reactions were performed in triplicate for three independent biological replicates, and data are presented as mean Ct\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.12. Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll data were analyzed using SPSS software (version 20.0). The Kolmogorov\u0026ndash;Smirnov test was applied to verify data normality. Differences among experimental groups were evaluated using one-way ANOVA, followed by Tukey\u0026rsquo;s post hoc test. A p value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant. Results are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Platelet Count and PRP Preparation\u003c/h2\u003e\u003cp\u003eGiemsa staining and platelet quantification of the prepared PRP samples revealed an average platelet count of 2,500,000\u0026thinsp;\u0026plusmn;\u0026thinsp;10,000, confirming the effective enrichment of platelets. Based on this count, PRP working concentrations of 5%, 10%, and 20% were prepared for subsequent cell treatment experiments.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003e3.2. PRP Enhances Glial Cell Proliferation and Preserves Morphology\u003c/h2\u003e\u003cp\u003eInverted light microscopy demonstrated that untreated B92 glial cells displayed a typical elongated, spindle-shaped morphology under optimal culture conditions, with robust adhesion and proliferation across passages (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). PRP supplementation markedly promoted cell growth and survival in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, E, G). Cells exposed to 20% PRP exhibited the most extensive proliferation and elongation. In contrast, exposure to heat-killed E. coli (HKEC) led to an evident decline in cell growth within 24 h, characterized by cell clustering and detachment (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). However, co-treatment with PRP effectively counteracted these inhibitory effects, promoting a gradual, dose-dependent recovery in cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, F). Remarkably, B92 cells treated with 20% PRP in the presence of HKEC exhibited even higher proliferation rates than the untreated control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eK). Morphological analysis further confirmed the pronounced trophic effect of PRP on glial cell structure and density.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003e3.3. PRP Improves Glial Viability and Reduces Oxidative Stress Without Altering Phagocytic Activity\u003c/h2\u003e\u003cp\u003eFunctional assays revealed a strong pro-survival effect of PRP on B92 glial cells. The MTT assay showed a significant, dose-dependent increase in cell viability and metabolic activity under both sterile and infectious inflammatory conditions (Table\u0026nbsp;2). At 20% PRP, viability increased by 62.3% under sterile and 52.1% under infectious conditions compared with the respective controls. Parallel assessment of oxidative status demonstrated that PRP markedly suppressed reactive oxygen species (ROS) production in both models. The 20% PRP group exhibited an approximately 40\u0026ndash;45% reduction in superoxide anion levels relative to untreated cells (Table\u0026nbsp;2). In contrast, PRP treatment did not significantly affect the phagocytic function of glial cells, as determined by neutral red uptake (Table\u0026nbsp;2). HKEC exposure caused only a minor, non-significant reduction in endocytic capacity, while PRP co-treatment maintained membrane integrity and lysosomal activity across all tested concentrations. These findings indicate that PRP enhances glial cell viability and redox balance without compromising essential cellular functions.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003e3.4. PRP Induces a Pro-Survival Molecular Profile\u003c/h2\u003e\u003cp\u003eTo investigate the molecular mechanisms underlying PRP\u0026rsquo;s cytoprotective effects, the expression of apoptosis-regulating genes was quantified using qRT-PCR. PRP treatment significantly upregulated the anti-apoptotic gene BCL-2 in a concentration-dependent manner, while concurrently downregulating the pro-apoptotic genes Caspase-3 and BAX (Table\u0026nbsp;3; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Similarly, HKEC exposure alone elevated Caspase-3 and BAX expression, consistent with the induction of cell stress and apoptosis. Co-treatment with PRP, however, effectively reversed these pro-apoptotic changes, most prominently at 20% PRP, indicating a robust anti-apoptotic shift. This transcriptional reprogramming toward a pro-survival phenotype was consistently observed in both sterile and infectious inflammation models, though slightly more pronounced under sterile conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e3.5. PRP Reprograms the Cytokine Expression Landscape Toward an Anti-Inflammatory State\u003c/h2\u003e\u003cp\u003eThe immunomodulatory effects of PRP were further evaluated by quantifying cytokine gene expression. As shown in Table\u0026nbsp;4, PRP treatment caused a significant suppression of the pro-inflammatory cytokine TNF-α, an effect that appeared dose-independent and consistent across all tested concentrations (5\u0026ndash;20%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Conversely, PRP robustly upregulated the anti-inflammatory cytokine IL-10 by approximately threefold relative to untreated controls, also in a dose-independent manner (Table\u0026nbsp;4; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Interestingly, PRP exposure also increased IL-1β expression in both sterile and infectious models, albeit without a clear concentration response trend (Table\u0026nbsp;4; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e). Collectively, these results suggest that PRP fine-tunes the inflammatory response of glial cells, downregulating excessive pro-inflammatory signaling (TNF-α) while enhancing regulatory and reparative cytokine expression (IL-10, IL-1β), thereby contributing to a balanced neuroinflammatory profile.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePlatelets interact with glial cells in both sterile and infectious inflammatory contexts, including autoimmune disorders such as multiple sclerosis and bacterial infections like meningitis[14, 17]. Gram-negative bacteria such as \u003cem\u003eEscherichia coli\u003c/em\u003e are especially relevant to infection-driven neuroinflammation because of their ability to breach the blood-brain barrier and activate pattern recognition receptors on microglia and other glial populations[13, 17]. Using a B92 glial cell model, we show that platelet-rich plasma (PRP) prepared here from samples with a platelet count of 2,500,000\u0026thinsp;\u0026plusmn;\u0026thinsp;10,000 exerts pleiotropic, concentration-dependent effects that: (i) enhance metabolic activity and proliferation, (ii) preserve membrane and lysosomal function, (iii) attenuate pathogen-induced oxidative stress, and (iv) reprogram apoptotic and cytokine pathways toward a pro-survival, resolution-oriented phenotype. Below we integrate these observations with existing literature, propose plausible mechanisms, and discuss translational implications.\u003c/p\u003e\u003cp\u003ePRP produced a robust, dose-dependent increase in B92 metabolic activity as measured by MTT: exposure to 5%, 10% and 20% PRP increased viability indices to 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, 1.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 and 2.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20, respectively, compared with untreated controls (0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Under HKEC stimulation, viability declined to 0.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 but was restored in a PRP dose-responsive manner up to 1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 at 20% PRP. These data indicate that PRP supplies trophic and mitogenic cues that sustain mitochondrial function and cellular bioenergetics even during inflammatory challenge. The effect is consistent with the rich complement of platelet-derived growth factors in PRP (PDGF, VEGF, TGF-β, IGF-1, HGF, FGF, EGF), which are known to activate mitochondrial and pro-survival signaling (PI3K/Akt, ERK/MAPK) and to stimulate proliferation in diverse cell types[37\u0026ndash;39]. Similar mitogenic actions of PRP have been documented in non-neural tissues, for example in promotion of follicular unit survival during hair transplantation via MAPK/ERK activation[40]. Taken together, our results extend those observations to glial biology and suggest that PRP can preserve metabolic homeostasis under both basal and infection-mimicking conditions.\u003c/p\u003e\u003cp\u003eNeutral red uptake assays indicated preserved lysosomal and membrane integrity across PRP concentrations and experimental conditions (Table\u0026nbsp;2). In sterile conditions, PRP modestly increased neutral red uptake, whereas HKEC alone produced no significant impairment of endocytic capacity. Importantly, PRP supplementation did not abrogate phagocytic function in infected cultures, implying that the pro-survival and anti-oxidative effects of PRP are achieved without globally suppressing critical innate defense mechanisms[41]. Preservation of membrane and lysosomal integrity supports the hypothesis that PRP enhances cellular resilience rather than simply preventing cell death, and suggests favorable biocompatibility for future therapeutic applications.\u003c/p\u003e\u003cp\u003eOxidative stress is a central mediator of glial dysfunction during infection; macrophages and microglia generate ROS to combat pathogens, but excessive ROS causes collateral neuronal damage[41]. In our NBT assays, HKEC dramatically elevated superoxide production (2.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 vs control 0.82\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03), whereas PRP co-treatment reduced ROS in a concentration-dependent fashion to 1.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06, 1.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, and 1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 for 5%, 10% and 20% PRP, respectively (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). This antioxidant effect may reflect multiple, non-mutually exclusive mechanisms: suppression of pro-inflammatory cytokine signaling (TNF-α, IL-1β, IL-6), upregulation of anti-inflammatory mediators such as IL-10, direct stimulation of endogenous antioxidant systems (SOD, catalase, glutathione peroxidase), and modulation of NF-κB and MAPK pathways that govern redox homeostasis. These mechanistic possibilities are supported by prior studies showing PRP-mediated reductions in microglial activation and ROS-driven neurotoxicity[4, 7, 14, 40, 41].\u003c/p\u003e\u003cp\u003eOur qRT-PCR analyses revealed a coherent anti-apoptotic transcriptional program induced by PRP. In non-stimulated B92 cells, PRP dose-dependently upregulated BCL-2 (peaking at 1.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 at 20% PRP) while markedly reducing Caspase-3 (from control 1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 to 0.25\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 at 20% PRP) and lowering BAX expression. Conversely, HKEC induced a pro-apoptotic shift (reduced BCL-2 0.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, elevated Caspase-3 1.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, and BAX 1.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11), which was reversed by PRP co-treatment (restoration of BCL-2 up to 0.95\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 at 20% PRP and suppression of pro-apoptotic transcripts). These findings align with the wealth of evidence that PRP growth factors activate PI3K/Akt and ERK pathways to inhibit caspase-dependent apoptosis and bolster mitochondrial integrity in multiple cell types[42\u0026ndash;44]. The ability of PRP to restore anti-apoptotic balance under bacterial stress indicates a direct cytoprotective role that could help preserve glial populations during acute neuroinflammatory insults.\u003c/p\u003e\u003cp\u003eCytokine profiling uncovered a nuanced immunomodulatory signature. Under basal conditions, PRP reduced TNF-α and IL-1β while increasing IL-10 (IL-10 up to 1.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, TNF-α and IL-1β down to 0.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 and 0.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07, respectively). HKEC induced a robust pro-inflammatory state (TNF-α 2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12, IL-1β 2.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14, IL-10 suppressed to 0.60\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06), which PRP attenuated in a dose-dependent manner; at 20% PRP TNF-α and IL-1β were reduced to 1.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 and 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09, respectively, and IL-10 was restored to 1.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.09. This pattern indicates that PRP exerts an active rebalancing of inflammatory mediators rather than indiscriminate immunosuppression: it dampens excessive pro-inflammatory signaling while reinstating regulatory IL-10, a cytokine critical for limiting microglial hyperactivation and promoting resolution[45\u0026ndash;47]. The observation that IL-1β sometimes remains elevated in PRP-treated cells suggests that PRP may permit controlled pro-repair inflammatory signaling (priming or trophic roles) while preventing runaway, tissue-destructive inflammation a concept increasingly recognized in the field[20].\u003c/p\u003e\u003cp\u003eThe multifactorial effects described above can be reconciled by a model in which PRP provides a concentrated, physiologically compatible secretome that: (i) activates membrane receptors (PDGFR, VEGFR, IGF1R, EGFR) to stimulate survival and proliferative cascades (PI3K/Akt, MAPK/ERK), (ii) modulates transcriptional programs that suppress caspase-dependent apoptosis and upregulate mitochondrial stabilizers (BCL-2), and (iii) reconfigures innate immune signaling to limit NF-κB-driven pro-oxidant responses while promoting IL-10 mediated resolution. Importantly, these effects likely arise from synergistic rather than singular factor actions; adhesive proteins (fibrinogen, fibronectin, vitronectin) in PRP may also support cell adhesion and microenvironmental stability, further contributing to functional recovery[38, 39]. That these mechanisms operate without evidence of tumorigenic risk is consistent with the membrane-receptor mediated, non-genomic mode of PRP action[39].\u003c/p\u003e\u003cp\u003eOur results converge with reports that PRP reduces pro-inflammatory cytokine release and oxidative damage in musculoskeletal and neural injury models[4]. The anti-apoptotic transcriptional shift (increased BCL-2, decreased Caspase-3 and BAX) echoes prior observations in endothelial and neuronal systems[42\u0026ndash;44], while the reconstitution of IL-10 in infected cultures supports the recognized role of IL-10 in protecting against bacterial meningitis sequelae[45]. The preservation of phagocytic competence despite reduced ROS suggests a selective modulation that favors resolution and repair over wholesale immunosuppression, a desirable characteristic contrasted with broad-spectrum anti-inflammatory drugs[48\u0026ndash;51].\u003c/p\u003e\u003cp\u003eExcessive glial activation, oxidative stress and apoptosis are central pathogenic mechanisms in a range of CNS disorders, from acute bacterial meningitis to chronic neurodegenerative disease and autoimmune neuroinflammation[15]. PRP\u0026rsquo;s capacity to concurrently preserve cellular viability, attenuate oxidative and apoptotic cascades, and recalibrate cytokine networks positions it as a promising biologic adjuvant for conditions where restoring homeostasis rather than blunt immunosuppression is required. Moreover, the dose-independent modulation of certain cytokines (for example IL-10 and TNF-α in our models) suggests that therapeutic benefit might be achievable at moderate PRP concentrations, potentially reducing risks related to overt growth-factor exposure.\u003c/p\u003e\u003cp\u003eKey strengths of this study include the integrated assessment across functional (MTT, Neutral Red, NBT) and molecular (qRT-PCR) endpoints, allowing us to link phenotype to mechanism. Nonetheless, several limitations temper immediate translational extrapolation. First, these data derive from a single in vitro glial cell line and may not fully capture cellular heterogeneity, multicellular interactions, or blood\u0026ndash;brain barrier dynamics in vivo. Second, while we document clear bioactivity of PRP, the specific molecular constituents responsible for each observed effect were not isolated; fractionation and proteomic profiling of the PRP secretome are necessary next steps. Third, long-term effects, dose-scheduling, and safety (including potential pro-fibrotic or aberrant remodeling responses) require evaluation in relevant animal models. Future work should therefore (i) fractionate PRP to identify active mediators, (ii) map downstream signaling cascades in primary glial populations and organotypic cultures, and (iii) validate efficacy and safety in in vivo models of infection-driven and autoimmune neuroinflammation.\u003c/p\u003e\u003cp\u003eIn summary, PRP functions as a finely tuned immunomodulatory and cytoprotective agent in glial cells: it enhances metabolic resilience, preserves membrane and lysosomal competence, mitigates pathogen-induced oxidative stress, and shifts apoptotic and cytokine programs toward survival and resolution. These multifactorial actions mediated by a repertoire of growth factors and adhesive proteins acting via membrane receptors support the potential of PRP as a therapeutic adjunct for neuroinflammatory disorders. Elucidation of the active PRP components, optimal dosing regimens, and validation in animal models will be critical to translating these promising in vitro findings into clinical strategies for CNS inflammation and injury.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eIn conclusion, this study provides compelling evidence that Platelet-Rich Plasma (PRP) functions as a potent and multifaceted immunomodulator within the glial system, orchestrating a delicate balance between pro- and anti-inflammatory signaling under both sterile and infectious conditions. By enhancing glial cell viability, promoting anti-apoptotic gene expression (upregulating BCL-2 while downregulating Caspase-3 and BAX), and simultaneously suppressing excessive pro-inflammatory mediators (TNF-α, IL-1β) while restoring anti-inflammatory IL-10 expression, PRP fosters a neuroprotective microenvironment that supports tissue repair and maintains immune homeostasis. Mechanistically, PRP achieves this immunoregulatory equilibrium through its rich repertoire of growth factors such as PDGF, VEGF, TGF-β, and IGF-1 which activate pro-survival pathways including PI3K/Akt and MAPK/ERK cascades. In bacterial challenge conditions, PRP effectively counteracted HKEC-induced oxidative stress, alleviated ROS overproduction, and preserved membrane integrity and metabolic activity, thereby preventing glial dysfunction and subsequent neuronal injury. This dual ability to sustain cellular resilience while tempering inflammation distinguishes PRP as a highly sophisticated immunomodulatory agent rather than a simple anti-inflammatory compound. Importantly, the observed cytokine modulation pattern marked by reduced TNF-α and IL-1β expression alongside enhanced IL-10 levels highlights PRP\u0026rsquo;s potential to reprogram glial reactivity toward a resolution-oriented phenotype. Such fine-tuned immune regulation is crucial in the context of neuroinflammatory and neurodegenerative disorders where glial overactivation contributes to progressive neuronal loss. Moreover, the maintenance of essential glial functions, including phagocytic competence, underscores the therapeutic safety and physiological compatibility of PRP-based interventions. Overall, these findings position PRP as a promising biological therapy capable of addressing the multifactorial nature of neuroinflammation. Its pleiotropic actions spanning antioxidant, anti-apoptotic, and immunoregulatory effects suggest broad therapeutic applicability in CNS disorders, including autoimmune neuroinflammation, infection-driven neural damage, and chronic neurodegeneration. Future research should focus on delineating the molecular constituents within the PRP secretome responsible for these protective effects, optimizing dosage parameters, and validating these mechanisms in in vivo models to facilitate clinical translation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eDeclaration of Competing Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. The study was conducted in accordance with ethical standards, and all sources of support are appropriately acknowledged.\u003c/p\u003e\n\u003cp\u003eAcknowledgments\u003c/p\u003e\n\u003cp\u003eThe authors sincerely thank Mr. Asghar Aliyari, Laboratory Specialist in Immunology, Faculty of Veterinary Medicine, Urmia University, for his valuable technical assistance during this study. The authors also acknowledge Urmia University (Urmia, Iran) for providing laboratory facilities and institutional support throughout the research process.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. It was supported by Urmia University (Urmia, Iran) through institutional resources and facilities.\u003c/p\u003e\n\u003cp\u003eEthical Statement\u003c/p\u003e\n\u003cp\u003eEthical approval for this study was obtained from the Ethics Committee of Urmia University (Ethical Code: IR-UU-AEC-3/PD/1382).\u003c/p\u003e\n\u003cp\u003eCRediT Author Statement\u003c/p\u003e\n\u003cp\u003eMahtab Pourkamalzadeh: Investigation, Data curation, Formal analysis, Visualization of experimental data, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\n\u003cp\u003eSeyyed Meysam Abtahi Froushani: Conceptualization, Methodology, Supervision, Project administration, Resources, Writing \u0026ndash; original draft.\u003c/p\u003e\n\u003cp\u003eMahtab Pourkamalzadeh and Seyyed Meysam Abtahi Froushani: Validation, Methodology, Conceptualization, Review \u0026amp; Editing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAlves, R. and R. Grimalt. 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Primer sequences were either designed based on NCBI reference sequences or obtained from Origene. Experiments were performed in triplicate, and expression levels were normalized to HPRT to account for sample-to-sample variation.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGene\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003ePrimer Direction\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eSequence (5\u0026apos; \u0026rarr; 3\u0026apos;)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eAmplicon Size (bp)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eHPRT\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-TTGGTGGRGATGAYCTCTCAAC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e~120\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-TTCAAATCCAACAAAGTCTGGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBCL-2\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-GGAGGATTGTGGCCTTCTTT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e~200\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-GTTCAGGTACTCAGTCATCCAC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTNF-\u0026alpha;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-CTCTTCAAGGGACAAGGT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e~150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-CTTGATGGCAGAGGAGG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIL-10\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-AAGGTTACTTGGGTTGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e~110\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-GCTCCTTGATTTCTGGGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIL-1\u0026beta;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-CAGCTGGAGAGTGTGGATC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e~130\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-TGCTGATGTACCAGTTGGG-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBAX\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-TGGCGATGAACTGGACAACAA-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e~150\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-CCCGAAGTAGGAAAGGAGGC-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCaspase-3\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eForward\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-GACTGGAAAGCCGAAACTCT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e~140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003eReverse\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e5\u0026apos;-GTCCCACTGTCTGTCTGTCTCAAT-3\u0026apos;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable.2 Comprehensive evaluation of metabolic activity (MTT assay), endocytic capacity (Neutral Red uptake assay), and reactive oxygen species (ROS) production (NBT assay) in B92 glial cells under sterile and infectious inflammatory conditions (HKEC) following treatment with different concentrations of platelet-rich plasma (PRP).\u003c/strong\u003e Data are expressed as Mean \u0026plusmn; SD from three independent biological replicates (n = 3). Statistical analysis was performed using one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test. Distinct superscript letters (a\u0026ndash;e) denote statistically significant differences among groups (P \u0026lt; 0.05). PRP treatment enhanced glial cell viability and maintained endocytic activity under sterile conditions while reducing excessive ROS generation following HKEC stimulation, suggesting an overall protective and modulatory role of platelets in glial cell function. Abbreviations: PRP \u0026ndash; Platelet-Rich Plasma; HKEC \u0026ndash; Heat-killed \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eExperimental Groups\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eMTT Assay\u0026lt;br\u0026gt;(Viability Index, Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNR Uptake Assay\u0026lt;br\u0026gt;(Optical Density, Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eNBT Assay\u0026lt;br\u0026gt;(Optical Density, Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA: Sterile Inflammatory (without HKEC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 (Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.70 \u0026plusmn; 0.10ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.80 \u0026plusmn; 0.10ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.82 \u0026plusmn; 0.03ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.20 \u0026plusmn; 0.10ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00 \u0026plusmn; 0.10ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.89 \u0026plusmn; 0.05ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.50 \u0026plusmn; 0.20ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00 \u0026plusmn; 0.10ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.80 \u0026plusmn; 0.03ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.10 \u0026plusmn; 0.20ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.90 \u0026plusmn; 0.20ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.83 \u0026plusmn; 0.04ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eB: Infectious Inflammatory (challenged with HKEC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.80 \u0026plusmn; 0.10ᵃᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.70 \u0026plusmn; 0.10ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.15 \u0026plusmn; 0.07ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.10ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.95 \u0026plusmn; 0.05ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.72 \u0026plusmn; 0.06ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.55 \u0026plusmn; 0.30ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.85 \u0026plusmn; 0.10ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.38 \u0026plusmn; 0.05ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.95 \u0026plusmn; 0.20ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.95 \u0026plusmn; 0.05ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.10 \u0026plusmn; 0.04ᵉ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable.3 Relative mRNA expression levels of apoptosis-associated genes (BCL-2, Caspase-3, and BAX) in B92 glial cells exposed to platelet-rich plasma (PRP) under sterile and infectious inflammatory conditions.\u003c/strong\u003e Expression levels were quantified using the 2⁻\u0026Delta;\u0026Delta;Ct method and normalized to the housekeeping gene HPRT, with the untreated control set to 1.00 (fold change = 1.00). Data are presented as Mean \u0026plusmn; SD from three independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey\u0026rsquo;s post-hoc test. Distinct superscript letters (a\u0026ndash;e) indicate significant differences among groups (P \u0026lt; 0.05). Abbreviations: PRP \u0026ndash; Platelet-Rich Plasma; HKEC \u0026ndash; Heat-killed \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eExperimental Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBCL-2 (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eCaspase-3 (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eBAX (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA: Sterile Inflammatory (without HKEC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 (Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.02ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.04ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.05ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.20 \u0026plusmn; 0.07ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.32 \u0026plusmn; 0.03ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.90 \u0026plusmn; 0.09ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.50 \u0026plusmn; 0.12ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.31 \u0026plusmn; 0.05ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.73 \u0026plusmn; 0.10ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.95 \u0026plusmn; 0.10ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.25 \u0026plusmn; 0.02ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.52 \u0026plusmn; 0.08ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eB: Infectious Inflammatory (challenged with HKEC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 (Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.03ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.05ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.04ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.30 \u0026plusmn; 0.05ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.45 \u0026plusmn; 0.12ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.18 \u0026plusmn; 0.11ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.35 \u0026plusmn; 0.04ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.32 \u0026plusmn; 0.11ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.08 \u0026plusmn; 0.10ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.70 \u0026plusmn; 0.09ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.30 \u0026plusmn; 0.10ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.06 \u0026plusmn; 0.09ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.95 \u0026plusmn; 0.08ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.15 \u0026plusmn; 0.08ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.87 \u0026plusmn; 0.08ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cstrong\u003eTable.4\u003c/strong\u003e \u003cstrong\u003eModulatory effects of platelet-rich plasma (PRP) on the expression of pro-inflammatory (TNF-\u0026alpha; and IL-1\u0026beta;) and anti-inflammatory (IL-10) cytokine genes in B92 glial cells under sterile and infectious inflammatory conditions.\u0026nbsp;\u003c/strong\u003eGene expression levels were quantified using the 2⁻\u0026Delta;\u0026Delta;Ct method and normalized to the HPRT reference gene. The control group was set as the calibrator (fold change = 1.00). Data are presented as mean \u0026plusmn; standard deviation (SD) from three independent biological replicates (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Distinct superscript letters (a\u0026ndash;e) denote statistically significant differences among groups (P \u0026lt; 0.05). Abbreviations: PRP \u0026ndash; Platelet-Rich Plasma; HKEC \u0026ndash; Heat-killed \u003cem\u003eEscherichia coli\u003c/em\u003e.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eExperimental Group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eTNF-\u0026alpha; (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIL-10 (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eIL-1\u0026beta; (Mean \u0026plusmn; SD)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eA: Sterile Inflammatory (without HKEC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 (Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.03ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.04ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.05ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.75 \u0026plusmn; 0.06ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.20 \u0026plusmn; 0.07ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.85 \u0026plusmn; 0.08ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.55 \u0026plusmn; 0.05ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.50 \u0026plusmn; 0.10ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.70 \u0026plusmn; 0.09ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + PRP 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.40 \u0026plusmn; 0.04ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.80 \u0026plusmn; 0.12ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.52 \u0026plusmn; 0.07ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e\u003cstrong\u003eB: Infectious Inflammatory (challenged with HKEC)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003ctd\u003e\u003cbr\u003e\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 (Control)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.04ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.05ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.00 \u0026plusmn; 0.04ᵃ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.20 \u0026plusmn; 0.12ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.60 \u0026plusmn; 0.06ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.50 \u0026plusmn; 0.14ᵇ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.80 \u0026plusmn; 0.11ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e0.85 \u0026plusmn; 0.07ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e2.00 \u0026plusmn; 0.12ᶜ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 10%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.40 \u0026plusmn; 0.09ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.10 \u0026plusmn; 0.08ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.60 \u0026plusmn; 0.10ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eB92 + HKEC + PRP 20%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.10 \u0026plusmn; 0.08ᵈ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.40 \u0026plusmn; 0.09ᵉ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e1.20 \u0026plusmn; 0.09ᵉ\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":"Platelet-Rich Plasma (PRP), Neuroinflammation, B92 Glial cells, Cytokines, Escherichia coli, Neuroprotection","lastPublishedDoi":"10.21203/rs.3.rs-7889196/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7889196/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePlatelets increasingly appear to influence inflammation, including neuroinflammation. This study examines how platelet-rich plasma (PRP) modulates BV2 glial cell immune responses in sterile and infectious inflammation to assess potential neuroprotective effects. B92 glial cells were treated with PRP (0%, 5%, 10%, 20%) to model sterile inflammation. Infectious inflammation was simulated using heat-killed E. coli (1:100) combined with PRP. Cells were cultured in DMEM (low-glucose) with 10% FBS at 37\u0026deg;C, 5% CO₂, and 95% humidity for 24 h. Morphology, viability (MTT), phagocytosis (neutral red), oxidative stress (NBT), and expression of cytokines (TNF-α, IL-10, IL-1β) and apoptosis markers (BAX, Caspase-3, BCL-2) were assessed by qRT-PCR.PRP significantly enhanced glial cell viability and proliferation in a dose-dependent manner. The expression of the anti-apoptotic gene BCL-2 increased, whereas Caspase3 and BAX levels decreased following PRP treatment. PRP modulated cytokine profiles by reducing TNF-α expression and upregulating IL-10 in a dose-independent manner, accompanied by an increase in IL-1β expression across all concentrations. Morphological and metabolic analyses revealed that PRP mitigated inflammatory damage and preserved glial integrity, particularly under sterile inflammatory conditions. Under infectious stimulation, PRP attenuated \u003cem\u003eE. coli\u003c/em\u003e-induced oxidative stress and preserved glial function without impairing phagocytic activity, suggesting a coordinated immunoregulatory effect. In conclusion, PRP may help control brain inflammation from injury or infection by protecting brain cells, balancing immune responses, and reducing stress. This suggests PRP could be a treatment for brain disorders.\u003c/p\u003e","manuscriptTitle":"Immunomodulatory Effects of Platelet-Rich Plasma (PRP) on Neuroinflammation: Insights from B92 Glial Cells Stimulated with Heat-Killed Escherichia coli","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-03 16:23:53","doi":"10.21203/rs.3.rs-7889196/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":"e8e6d1e5-25f3-4b8a-a7cc-8a3d213a09cc","owner":[],"postedDate":"November 3rd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-11-18T12:53:11+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-03 16:23:53","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7889196","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7889196","identity":"rs-7889196","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}