Analgesic effects of human placental hydrolysate on capsaicin-induced hyperalgesia in rats

Analgesic effects of human placental hydrolysate on capsaicin-induced hyperalgesia in rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Analgesic effects of human placental hydrolysate on capsaicin-induced hyperalgesia in rats Baoji Lu, Tae Woo Kim, Eun-ee Jung, Guanghai Nan, Jae-Won Kim, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9079119/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Aim To explore the analgesic potential of HPH, we examined whether HPH could reduce primary and secondary hyperalgesia in a rat model of capsaicin-induced pain and whether co-administration with dexamethasone could provide enhanced analgesia with fewer side effects. Method HPH was administered in capsaicin-induced pain model. Primary and secondary hyperalgesia were assessed using von Frey filaments. Voltage-sensitive dye imaging (VSDI) was conducted in spinal cord slices to examine neuronal excitability. Immunohistochemistry was performed to evaluate c-Fos and phosphorylated PKCε (p-PKCε) expression. Body weight was monitored as an indicator of corticosteroid-associated toxicity in rats treated with dexamethasone alone or in combination with HPH. Result HPH dose-dependently attenuated capsaicin-induced both primary and secondary hyperalgesia, suppressed spinal dorsal horn hyperactivity, and reduced expression of c-Fos and p-PKCε. Combination of HPH with low-dose dexamethasone produced analgesia comparable to high-dose dexamethasone while reducing corticosteroid-related systemic toxicity, as reflected by body weight loss. Conclusion HPH effectively alleviated capsaicin-induced neurogenic inflammatory pain and enhances the therapeutic profile of dexamethasone. Co-treatment with low-dose dexamethasone may provide synergistic analgesic benefits while minimizing systemic toxicity, supporting HPH as a promising adjunctive therapy for corticosteroid-based pain management. Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Medical research Biological sciences/Neuroscience Human placental hydrolysate capsaicin neurogenic inflammatory pain analgesia PKCε dexamethasone toxicity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Pain, especially inflammatory pain, represents a major clinical challenge worldwide, affecting up to 20% of the global population and imposing substantial personal and socioeconomic burdens ( 1 , 2 ). Such pain conditions often lead to disability, reduced quality of life, and high healthcare costs ( 3 , 4 ). Corticosteroids are widely prescribed for their potent anti-inflammatory and analgesic properties and remain a mainstay in the management of pain associated with inflammation ( 5 , 6 ). However, their long-term use is restricted by serious side effects, including metabolic disturbances ( 7 ), osteoporosis ( 8 ), and immune suppression ( 9 ), which limit their clinical utility. These limitations underscore the need for adjunctive strategies that preserve therapeutic efficacy while reducing systemic toxicity. Human placental hydrolysate (HPH) contains a complex mixture of biologically active substances, including amino acids, peptides, nucleotides ( 10 ) that contribute to pharmacological effects. The antioxidant and anti-inflammatory effects of HPH are relatively well known in inflammatory conditions, especially in liver diseases, based on findings from both experimental and clinical studies ( 11 – 15 ). In the patients with osteoarthritis, HPH has also been clinically applied to alleviate pain symptoms ( 16 , 17 ). While some clinical studies have shown promise in the treatment of pain conditions like osteoarthritis, shoulder impingement syndrome and chronic back pain ( 16 – 18 ), the mechanism underlying analgesic effect of HPH remains unclear. Capsaicin, the pungent compound in chili peppers, induces pain by activating transient receptor potential vanilloid 1 (TRPV1) channel on nociceptive fibers, producing a burning sensation and local inflammation. Intradermal injection of capsaicin results in primary hyperalgesia at the site of application, characterized by increased sensitivity to thermal and mechanical stimuli due to peripheral sensitization of nociceptors. Secondary hyperalgesia also develops in surrounding uninjured areas and reflects central sensitization, leading primarily to enhanced mechanical sensitivity. This model is particularly useful for determining whether the analgesic effects of compound are mediated through peripheral or central site of action ( 19 ). In this context, previous in vitro and animal studies provide mechanistic evidence supporting the potential of HPH in alleviating pain ( 14 , 20 – 22 ). Specifically, HPH significantly attenuated inflammatory pain behaviors and reduced pro-inflammatory cytokine expression (TNF-α, IL-1β, IL-6) in a complete Freund’s adjuvant (CFA)-induced inflammatory pain model in mice ( 21 ), implicating its ability to modulate pathways involved in nociceptive sensitization. Additionally, HPH suppressed inflammatory responses in a dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model and reduced cytokine levels, further supporting its anti-inflammatory and neuro-modulatory potentials ( 22 ). Collectively, these preclinical findings provide a mechanistic rationale for investigating whether HPH can attenuate pain underlying capsaicin-induced hyperalgesia, which is driven by peripheral inflammatory sensitization. The present study investigated whether HPH could alleviate capsaicin-induced primary hyperalgesia and secondary hyperalgesia and compared its efficacy with dexamethasone, a standard corticosteroid used for pain relief ( 23 ) but limited by systemic side effects ( 24 , 25 ). Furthermore, we examined whether combining HPH with a low dose of dexamethasone could provide a synergistic analgesic effect while reducing corticosteroid-associated adverse outcomes such as body weight loss. Behavioral assessments and molecular analyses were performed to explore potential mechanisms underlying the analgesic effects of HPH. Materials and methods Chemicals Capsaicin (M2028; Sigma-Aldrich, St. Louis, MO, USA), olive oil (O1514; Sigma-Aldrich, St. Louis, MO, USA), dexamethasone disodium phosphate (08806505003025; Jeil Pharmaceutical Co., Ltd., Daegu, Korea), and human placental hydrolysate (HPH; Laennec®, GC Wellbeing, Seoul, Korea) were used. Experimental animals Adult male Sprague–Dawley rats (8 weeks; 250–300 g; Orient Bio, Sungnam, Gyonggi, Korea) were used. The rats were housed in groups of three in plastic cages with soft bedding under a 12-h light/dark cycle with free access to food and water. All experimental procedures involving animals were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System (permit no.: 2024-0001). Capsaicin-induced pain model The procedure for capsaicin injection in rats was performed as described previously ( 26 ). Briefly, rats were lightly anesthetized with isoflurane (Kyongbo Pharmaceutical CO., LTD, Asan, Republic of Korea), and a 30-gauge needle (with a Hamilton syringe) was inserted through the skin at the heel (designated as site X in Fig. 1 A) and advanced to the central plantar area of the left hind paw (site I), which was used consistently across all experiments. At this location, capsaicin (20 µg in 20 µl of olive oil) was administered via intradermal injection slowly. After the procedure, rats were returned to their home cage and closely observed until they recovered fully from anesthesia (typically within 5 minutes). Drug administration Two different doses of HPH were chosen and administered intraperitoneally: 3.6 mL/kg as the reference therapeutic dose and 1.8 mL/kg as a half-dose to evaluate dose dependency, based on previous studies ( 27 , 28 ). Dexamethasone was administered intraperitoneally at 1 mg/kg (low dose) or 5 mg/kg (high dose). Saline was used as the control. Behavioral assessment of capsaicin-induced pain As previously described ( 26 ), mechanical hypersensitivity was assessed using the up-down method with a series of von Frey filaments (Stoelting, Chicago, IL, USA) applied to the injection site to evaluate primary hyperalgesia and to an adjacent area to evaluate secondary hyperalgesia. Baseline assessment was performed at 0.5 h before HPH application (-0.5h). Following capsaicin injection (0h), behavioral test was conducted at 0.5, 1, 1.5, 2, and 24 h post-injection. The 50% mechanical withdrawal threshold (MWT) was calculated, and all behavioral test was conducted in a blinded manner. Voltage sensitive dye imaging (VSDI) in spinal dorsal horns Voltage sensitive dye imaging (VSDI) was carried out as performed in our laboratory ( 29 ). In brief, rats were deeply anesthetized with urethan (1.25 g/kg, intraperitoneally) after 30 minutes of capsaicin injection and transcardially perfused with ice-cold solution containing 213 mM sucrose, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 10 mM MgSO₄, 0.5 mM CaCl₂, 26 mM NaHCO₃, and 11 mM glucose. The L4-L5 spinal cord segments were surgically extracted and immediately immersed in ice-cold artificial cerebrospinal fluid (aCSF) solution for 5 minutes. The spinal cord was mounted on an agarose block and sectioned into 400-µm thick transverse slices using a vibratome (Leica Biosystems Inc., Buffalo Grove, IL, USA). These sections were immediately placed in interface chambers perfused with oxygenated aCSF containing: 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mM glucose, continuously bubbled with 95% O₂/5% CO₂ to maintain pH 7.2. After a 1-hour recovery period at room temperature under continuous aCSF perfusion, sections were incubated for 1 h with voltage-sensitive dye (di-2-ANEPEQ, 50 µg/mL in aCSF; Molecular Probes, Eugene, OR, USA). For optical recordings, a concentric bipolar microelectrode (30213, FHC, Bowdoin, Maine, USA) was positioned within the region of dorsal horn (laminae I–II) using an optical microscope (Olympus Optical Co. Ltd., Tokyo, Japan) equipped with a 10× objective and 0.35× projection lens. Electrical stimulation consisted of square pulses (2 ms duration, 5-second interstimulus interval) delivered through a stimulus isolation unit (World Precision Instruments, Sarasota, FL, USA), with intensity adjusted to evoke reliable responses. Neuronal activity was captured using a high-resolution CCD camera-based optical imaging system (Brainvision Inc., Tokyo, Japan) configured with a dichroic mirror, 510–555 nm excitation filter, and 590 nm emission filter. A 150 W tungsten-halogen lamp provided fluorescence excitation. The imaging field encompassed 184 × 124 pixels. Fluorescence measurements were acquired over 943.5 ms periods using the MiCAM02 optical imaging device (Brainvision Inc.) at a temporal resolution of 3.7 ms per frame. Signals were averaged over 20 trials to improve signal-to-noise ratio. Fluorescence changes were normalized by calculating the fractional change (ΔF/F) relative to baseline fluorescence for each pixel. Signal amplitude and spatial extent of activation were analyzed using spatial filtering (9 × 9 pixels) and cubic filtering (3 × 3 pixels). After the baseline responses were recorded, the slice was either maintained in aCSF (vehicle) or treated with HPH. For HPH treatment in vitro, lyophilized HPH was dissolved in aCSF and transferred into the chamber. The slice was incubated with HPH for 30 minutes. Following incubation, the same electrical stimulation protocol (0.3, 0.6, 0.9 mA) was applied, and the post-treatment VSD responses were recorded. All data acquisition and analysis were performed using BV Analyzer software (Brainvision Inc.). Optical signals were quantified as percentage fluorescence change (%ΔF/F) within circular areas (radius = 5 pixels) positioned specifically in the dorsal horn region. Both temporal dynamics and spatial patterns of activation were systematically analyzed. Immunohistochemistry for c-Fos or PKC expression in spinal dorsal horns As performed in our laboratory ( 29 ), rats were deeply anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) and perfused with 150 mL of 0.01M phosphate-buffered saline (PBS, pH 7.4), followed by 500 mL of 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The L5 spinal cord segment was dissected out and post-fixed in 4% paraformaldehyde for 2 hours at 4°C, then cryoprotected in 30% sucrose solution for 12 hours at 4°C. Tissue sections (40 µm thickness) were prepared using a freezing microtome (Cryostat 1720; Leitz, Mannheim, Germany). Free-floating sections were washed three times in 0.01 M PBS before blocking for 1 hour at room temperature with 10% normal goat serum (ab7481; Abcam, Cambridge, MA) or donkey serum (ab7475; Abcam) in PBS containing 0.3% Triton X-100. For c-Fos labeling, primary antibody incubation was performed at room temperature using rabbit anti-Fos antibody (1:1000, ab190289; Abcam). Following three-time PBS washes, sections were incubated for 2 hours at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:200; AB_2576217; Invitrogen, Thermo Fisher Scientific, Waltham, MA) in 0.3% Triton X-100 PBS solution. For phosphorylated protein kinase C (P-PKC) staining, a similar protocol was followed on separate sections: a goat monoclonal anti-phospho-PKC (p-PKCε (Ser 729): sc-12355, 1:500, Santa Cruz Biotechnology, Dallas, TX) was used as primary, and an Alexa Fluor 647–conjugated donkey anti-goat IgG (AB_2535864; Invitrogen) as secondary. In all cases, nuclei were counterstained with DAPI (4′,6-diamidino-2-phenylindole, blue fluorescence, Sigma-Aldrich, St. Louis, MO 63103 USA) before mounting the sections. Finally, sections were mounted onto glass slides and cover slipped with an anti-fade mounting medium. Immunofluorescence imaging was performed using a Zeiss LSM 710 confocal laser scanning microscope (Axio Examiner.Z1, Jena, Germany). For each rat, multiple sections of the L5 dorsal horn were imaged, and the number of c-Fos–positive nuclei was counted in a standardized region of the superficial dorsal horn (laminae I–III) on the side of capsaicin injection. P-PKCε immunoreactivity was quantified by calculating the percentage of neurons (identified by DAPI-stained nuclei) that showed clear PKC phosphorylation (red cytoplasmic staining) in the dorsal horn. Quantification was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA). Statistical Analysis All statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). Behavioral and body weight data were analyzed by two-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test. Optical signal intensities were compared by two-way ANOVA, followed by Bonferroni’s post hoc test. Immunohistochemistry data were analyzed using one-way ANOVA followed by Tukey’s post hoc test. Data are presented as means ± standard error of the mean (SEM). P values less than 0.05 were considered statistically significant. Result HPH alleviates capsaicin-induced hyperalgesia in a dose-dependent manner To evaluate the analgesic effect of HPH in the capsaicin-induced pain model, capsaicin was injected into X site and primary hyperalgesia at the injection site (site P) and secondary hyperalgesia at the base of the third-fourth toes (site S) were measured (Fig. 1 A). Rats were randomly assigned to three groups: ( 1 ) CAP-Veh (capsaicin + saline), ( 2 ) CAP-HPH 1.8 mL/kg (capsaicin + HPH 1.8 mL/kg), ( 3 ) CAP-HPH 3.6 mL/kg (capsaicin + HPH 3.6 mL/kg). HPH was administered 0.5 h before capsaicin injection. Behavioral tests were performed at 0.5 h before capsaicin injection and 0.5, 1, 1.5, 2 and 24 h after capsaicin injection (Fig. 1 B). Thirty minutes after intraplantar injection of capsaicin, the mean paw withdrawal threshold (MWT) rapidly dropped from a pre-injection value (log value = 5.1) of approximately 12 g to around 1 g (CAP-Veh) (p < 0.01; for time: F 5, 84 = 17.85, p < 0.0001; for group: F 2, 84 = 14.01, p < 0.0001; for time × group: F 10, 84 = 0.7767, p = 0.6508, Fig. 1 C), indicating development of primary hyperalgesia by intradermal capsaicin. This reduction remained statistically significant for about 2 hours (p < 0.0001), followed by a gradual recovery over 24 hours. Intraperitoneal administration of HPH attenuated capsaicin-induced changes of MWTs at both primary and secondary sites in a dose-dependent manner, compared to the CAP-Veh group. CAP-HPH (3.6 mL/kg) group significantly reduced primary hyperalgesia at 1 and 2 h after capsaicin injection (p < 0.01, Fig. 1 C) and suppressed secondary hyperalgesia up to 24 hr (Fig. 1 D) compared with the vehicle (p < 0.05). It showed that HPH, particularly at 3.6 mL/kg, effectively attenuated both primary and secondary hyperalgesia induced by capsaicin, with significant analgesic effects evident from 1 h and persisting up to 24 h. HPH reduces neuronal activity in the spinal cord dorsal horn of the capsaicin-induced pain model To investigate the effect of HPH on the neuronal excitability of spinal dorsal horns, VSDI was performed on spinal cord slices from capsaicin-injected rats. Animals were divided into Veh and HPH groups. Neuronal activity was recorded before and during vehicle or HPH treatment. Based on the previous study ( 30 ), the electrical stimulation intensities of 0.3, 0.6, 0.9 mA were applied. Neuronal signals and the activated areas increased in a stimulation intensity-dependent manner (Fig. 2 A). While no significant differences were observed between the pre- and vehicle treatment phases at any stimulation intensity in the Veh group (Fig. 2 B). On the other hand, HPH treatment reduced stimulation-induced peak amplitudes at 0.9 mA compared with the values before HPH treatment (p < 0.01; for phase: F 2, 24 = 19.84, p < 0.0001; for group: F 1, 24 = 14.82, p = 0.0008; for phase × time: F 2, 24 = 0.7181, p = 0.4979; Fig. 2 C). These results indicate that HPH effectively reduced the neuronal excitability of spinal dorsal horns following intra-plantar injection of capsaicin. HPH attenuates capsaicin-induced both c-Fos expression and p-PKCε immunoreactivity in spinal dorsal horn Expression of c-Fos (a marker of neuronal activation) and p-PKCε (a signaling mediator implicated in nociceptive sensitization) in the spinal dorsal horn was estimated to investigate the analgesic mechanism of HPH in capsaicin-induced pain model. Experiment was performed in the following groups: ( 1 ) Naive (no intraplantar capsaicin injection); ( 2 ) CAP-Veh (intraperitoneal saline prior to capsaicin injection); ( 3 ) CAP-HPH (intraperitoneal HPH at 3.6 mL/kg before capsaicin injection). Either saline or HPH was administered 30 minutes before capsaicin injection. Capsaicin injection increased the number of c-Fos positive nuclei in the superficial dorsal horn (CAP-Veh group) compared to naive group (p < 0.01; F 2, 13 = 7.03, p = 0.0085), whereas this increase was prevented by HPH treatment (CAP-HPH vs. naive group, p < 0.05, Fig. 3 A, B). Similarly, significantly enhanced expression of p-PKCε was observed in the capsaicin-induced pain model compared with naive group (p < 0.01, F 2, 8 = 8.725, p = 0.0098), which was attenuated by HPH pretreatment (p < 0.05, Fig. 3 C, D). These results indicate that HPH mitigated both neuronal activation and PKCε phosphorylation in the spinal dorsal horn following capsaicin injection. HPH-dexamethasone combination achieves analgesia comparable to high-dose dexamethasone in the capsaicin-induced pain model To investigate whether combined administration could reduce the requirement for high-dose dexamethasone (5 mg/kg), multiple treatment groups were established, and primary and secondary hyperalgesia were measured separately. To evaluate the effect of HPH combined with dexamethasone (DEX) or DEX alone, rats were divided into four groups: ( 1 ) CAP-Veh (capsaicin + saline), ( 2 ) CAP-DEX 1 mg/kg (capsaicin + low dose dexamethasone (1 mg/kg)), ( 3 ) CAP-DEX 5 mg/kg (capsaicin + high dose dexamethasone (5 mg/kg)), and ( 4 ) CAP-DEX 1mg/kg + HPH (capsaicin + dexamethasone 1 mg/kg + HPH 3.6 mL/kg). DEX and/or HPH were administered 30 minutes before capsaicin injection. Behavioral tests were conducted at 0.5 h before capsaicin (-0.5 h) and 0.5, 1, 1.5, 2 and 24 h after capsaicin injection. While capsaicin injection lowered MWTs compared to the value before capsaicin (p < 0.0001), pretreatment with dexamethasone at a high dose of 5 mg/kg significantly alleviated primary hyperalgesia in capsaicin-injected rats (CAP-DEX 5mg/kg) compared to control group (Fig. 4 A; CAP-Veh; p < 0.05 at 2h; for time: F 5, 168 = 30.90, p < 0.0001; for group: F 3, 168 = 8.099, p < 0.0001; for time × group: F 15, 168 = 0.9032, p = 0.5617). However, administration of dexamethasone at a lower dose of 1 mg/kg failed to inhibit primary and secondary hyperalgesia (Fig. 4 A, B). On the other hand, co-treatment of a low dose dexamethasone (1 mg/kg) with HPH effectively suppressed the development of primary and secondary hyperalgesia, compared to control group (CAP-Veh; p < 0.05). It suggests that treatment of HPH combined with a low dose dexamethasone produced similar analgesic effects as high-dose dexamethasone. HPH co-administration mitigates dexamethasone-induced body weight loss To see potential systemic side effects of HPH and/or dexamethasone, rats were divided into five groups: ( 1 ) Saline, ( 2 ) HPH 3.6 mL/kg, ( 3 ) DEX 1 mg/kg, ( 4 ) DEX 5 mg/kg, ( 5 ) HPH 3.6 mL/kg + DEX 1mg/kg. Animals received once-daily intraperitoneal injections of the assigned treatment for five consecutive days (Day 0–4), followed by two drug-free days (Days 5–6). Body weight was recorded at baseline (prior to the first injection), daily before each injection, and again on Day 7 (72 hours after the last dose) to assess recovery (Fig. 5 A). HPH (3.6 mL/kg) group showed a steady increase in body weight over the observation period, consistent with vehicle-treated group (Saline). It is noted that dexamethasone treatment at either 5mg/kg or 1 mg/kg caused profound body weight losses from Day 2, reaching ~ 88% of baseline by Day 4 (p < 0.0001; for day: F 5, 150 = 29.18, p < 0.0001; for group: F 4, 150 = 98.69, p < 0.0001; for day × group: F 20, 150 = 13.59, p < 0.0001). Even after two drug-free days, body weight remained below 90% of baseline on Day 7 compared with the Saline group. Although rats receiving combined treatment of HPH and dexamethasone treatment also exhibited weight loss during the dosing phase, the body weight partially recovered after drug discontinuation, reaching ~ 95% of baseline by Day 7, in contrast to the persistent loss observed with dexamethasone alone. These results indicate that dexamethasone induces pronounced body weight loss as a systemic side effect, while cotreatment with HPH partially mitigates this catabolic effect. Discussion The present study demonstrates the analgesic effect of human placental hydrolysate (HPH) in a capsaicin-induced pain model. Pretreatment with HPH effectively alleviated both primary and secondary hyperalgesia and suppressed neuronal activity by inhibiting PKCε activation. Moreover, combined administration of HPH with dexamethasone produced a comparable antinociceptive effect while permitting a reduced dose of dexamethasone and attenuating dexamethasone-induced body weight loss. These results suggest that HPH can suppress capsaicin-induced primary and secondary hyperalgesia while minimizing the adverse effects of dexamethasone monotherapy. The analgesic efficacy of HPH was evaluated in capsaicin-induced pain model. The capsaicin-induced pain model represents an acute form of inflammatory and neurogenic pain ( 31 ), as capsaicin activates TRPV1 receptors on primary sensory neurons, leading to neurogenic inflammation, peripheral sensitization ( 32 ), and subsequent central sensitization ( 33 ). In this model, pretreatment with HPH effectively alleviated both primary and secondary hyperalgesia. Since primary hyperalgesia reflects peripheral sensitization at the site of injury, whereas secondary hyperalgesia reflects spinal cord and beyond, the ability of HPH to suppress both types of hyperalgesia suggests that it may exert dual actions on peripheral nociceptor activity and central pain processing. HPH contains various bioactive components, including peptides and amino acids, which exert anti-inflammatory, antioxidant, and tissue-protective effects ( 13 , 17 ). As the bioactive components of HPH generated the analgesic efficacy observed in this study, identification the specific components responsible for the analgesic effect will be an important subject of future investigation. Given that HPH alleviated secondary hyperalgesia at the behavioral level, we next examined spinal cord excitability using VSDI to determine whether these effects were associated with changes in dorsal horn neuronal activity. The reduction of neuronal responses following HPH treatment suggests that HPH dampens stimulus-evoked hyperexcitability within the dorsal horn, a hallmark of central sensitization ( 34 , 35 ). Although VSDI does not resolve single-cell activity, its strength lies in capturing spatiotemporal patterns of neuronal population dynamics, thereby providing network-level evidence for the central actions of HPH. Together, these findings support the notion that HPH exerts its analgesic effects not only through peripheral modulation but also by influencing spinal pain circuitry, consistent with previous reports highlighting the importance of reducing dorsal horn hyperactivity in inflammatory pain ( 36 , 37 ). IHC analyses further supported the central actions of HPH by demonstrating reductions in c-Fos and PKC expression in the spinal dorsal horn. c-Fos is widely recognized as a marker of neuronal activation in pain research ( 38 ), and studies have shown its robust upregulation in the dorsal horn following inflammatory or capsaicin-induced pain ( 39 ), reflecting enhanced nociceptive input and central sensitization ( 40 ). In line with these observations, the suppression of c-Fos expression by HPH in our study suggests that the treatment attenuates excessive neuronal activation under acute inflammatory pain conditions. Similarly, PKC, particularly the PKCε isoform, has been implicated in excitatory synaptic plasticity ( 41 ) and the maintenance of central sensitization through the phosphorylation and trafficking of AMPA ( 42 ) and NMDA receptors ( 43 ). Previous studies have reported that enhanced PKC activity in inflammatory and neuropathic pain models, contributing to spinal hyperexcitability ( 44 , 45 ). Our finding that HPH reduced PKC expression therefore indicates that inhibition of PKC-dependent signaling may represent a key mechanism by which HPH counteracts spinal hyperexcitability. Together, these results suggest that HPH suppressed secondary hyperalgesia by inhibiting PKC-dependent signaling and central sensitization in spinal dorsal horn neurons. Because the clinical use of dexamethasone is limited by dose-dependent adverse effects ( 46 , 47 ), we investigated whether combining HPH with dexamethasone could provide effective analgesia while minimizing corticosteroid exposure. In the capsaicin-induced pain model, our results showed that combining HPH with dexamethasone achieved analgesic effects equivalent to high-dose dexamethasone, but with a reduced corticosteroid requirement. At the primary site, the combination achieved efficacy similar to 5 mg/kg dexamethasone, whereas at the secondary site the analgesic effect appeared more sustained. The comparable effect at the primary site likely reflects the strong anti-inflammatory action of dexamethasone in suppressing peripheral sensitization ( 48 ), which is mainly driven by TRPV1 activation and the release of inflammatory mediators ( 49 ). In contrast, the more prolonged effect at the secondary site may indicate that HPH provides additional modulatory actions relevant to central pain processing, thereby complementing the effects of dexamethasone. Importantly, this combination strategy also offers a pharmacological advantage by reducing the glucocorticoid burden. Corticosteroid-related adverse effects, including metabolic disturbances, osteoporosis, immunosuppression, and alterations in body weight, are well recognized to be dose- and duration-dependent ( 24 , 50 ). This concept is consistent with the principles of multimodal analgesia, in which combining agents with distinct but complementary mechanisms enhances therapeutic benefit while minimizing toxicity ( 51 ). Such a strategy could be particularly valuable in clinical contexts requiring repeated or prolonged corticosteroid administration, where the cumulative risk of side effects poses a major limitation. To further evaluate systemic tolerability, we examined body weight changes as a representative adverse effect. In normal rats without capsaicin treatment, repeated dexamethasone administration resulted in a pronounced reduction in body weight, highlighting its catabolic actions ( 52 , 53 ). Co-administration of HPH partially mitigated this effect, allowing recovery after treatment discontinuation. These results indicate that HPH not only sustains the analgesic efficacy or dexamethasone but also alleviates systemic adverse effects, thereby contributing to an improved safety profile. Nonetheless, several limitations should be acknowledged. First, we tested the treatment only in a single model of acute capsaicin-induced hyperalgesia, which may limit the generalizability of our findings to chronic inflammatory or neuropathic pain conditions. Second, the evaluation of side effects was incomplete. Apart from body weight, we did not assess other corticosteroid-related toxicities such as hyperglycemia, osteoporosis, or immune suppression. Comprehensive toxicity studies will be required to establish the long-term safety of HPH. Third, although we observed reduced spinal c-Fos and p-PKCε expression, inflammatory mediators and the bioactive constituents of HPH were not directly analyzed. Thus, the precise components and mechanisms underlying the analgesic effect remain unclear. Future studies should identify the active constituents and validate their interaction with glucocorticoid signaling to support standardized application and clinical translation of HPH. Conclusion Our findings demonstrate that HPH could suppress both primary and secondary hyperalgesia induced by capsaicin, reflecting the inhibition of peripheral and central sensitization, respectively. Its combination with dexamethasone provides effective analgesia while reducing corticosteroid-associated toxicity. These results highlight HPH as a promising adjunct for corticosteroid-based pain management. Importantly, the ability of HPH to sustain analgesic efficacy while minimizing systemic side effects implies potential value in optimizing current therapeutic strategies. Abbreviations aCSF artificial Cerebrospinal fluid AMPA α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ANOVA Analysis of Variance CAP Capsaicin CFA Freund’s adjuvant c-Fos cellular Proto-oncogene DAPI 4′,6-diamidino-2-phenylindole DEX Dexamethasone DNCB Dinitrochlorobenzene HPH Human placental hydrolysate MWT Mechanical Withdrawal Threshold NMDA N-methyl-D-aspartate PBS Phosphate-buffered saline PKC Protein Kinase C p-PKCε phosphorylated Protein Kinase C epsilon SEM standard error of the mean TRPV1 Transient Receptor Potential Vanilloid 1 VSDI Voltage sensitive dye imaging Veh Vehicle Declarations Ethic approval and consent to participate All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University College of Medicine (IACUC No. [2024-0001]) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition) and applicable Korean regulations. Every effort was made to minimize animal suffering and the number of animals used. Consent for publication Not applicable Competing interests Authors KJ, MI and JWK were employed by the company Green Cross Wellbeing Corporation. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This research was supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health & Welfare, the Ministry of Food and Drug Safety (RS-2023-00253560) and Green Cross Wellbeing, Korea. Author Contribution BL and TWK drafted the original manuscript and contributed to the study methodology and data analysis. EJ contributed to the study methodology and project administration. GN and HYK reviewed and edited the original manuscript and contributed to the study methodology and investigation. KJ, MI and JWK supervised the study and reviewed the manuscript. THC conceived the original idea for this study. SC and DAS conceptualized and supervised the study, oversaw project administration, and reviewed and edited the manuscript. All authors read and approved the final manuscript. Acknowledgements Not applicable Data Availability Data is available from the corresponding author upon reasonable request References Mills, S. E. E., Nicolson, K. P. & Smith, B. H. Chronic pain: a review of its epidemiology and associated factors in population-based studies. Br. J. Anaesth. 123 (2), e273–e83 (2019). Lurie, J. M. & Javaid, A. Visualizing Global Chronic Pain. Anesth. Analg . 138 (4), 918–919 (2024). Steinmetz JD, Culbreth GT, Haile LM, Rafferty Q, Lo J, Fukutaki KG, et al. 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Impact of Intraoperative Dexamethasone on Perioperative Blood Glucose Levels: Systematic Review and Meta-Analysis of Randomized Trials. Anesth. Analg . 139 (3), 490–508 (2024). Kim, H. Y. et al. Electroacupuncture suppresses capsaicin-induced secondary hyperalgesia through an endogenous spinal opioid mechanism. Pain 145 (3), 332–340 (2009). Shin, E. H. et al. Effects of Human Placenta Extract (Laennec) on Ligament Healing in a Rodent Model. Biol. Pharm. Bull. 42 (12), 1988–1995 (2019). Beaudry, F., Girard, C. & Vachon, P. Early dexamethasone treatment after implantation of a sciatic-nerve cuff decreases the concentration of substance P in the lumbar spinal cord of rats with neuropathic pain. Can. J. Vet. Res. 71 (2), 90–97 (2007). Nan, G. et al. Vinpocetine alleviates chemotherapy-induced peripheral neuropathy by reducing oxidative stress and enhancing mitochondrial biogenesis in mice. Biomed. Pharmacother . 190 , 118434 (2025). Ruscheweyh, R. & Sandkühler, J. 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PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking. Neuron 47 (3), 407–421 (2005). Zhou, M. H. et al. Protein Kinase C-Mediated Phosphorylation and α2δ-1 Interdependently Regulate NMDA Receptor Trafficking and Activity. J. Neurosci. 41 (30), 6415–6429 (2021). Sluka, K. A. & Audette, K. M. Activation of protein kinase C in the spinal cord produces mechanical hyperalgesia by activating glutamate receptors, but does not mediate chronic muscle-induced hyperalgesia. Mol. Pain . 2 , 13 (2006). Park, J. S. et al. Persistent inflammation induces GluR2 internalization via NMDA receptor-triggered PKC activation in dorsal horn neurons. J. Neurosci. 29 (10), 3206–3219 (2009). Mattano, L. A. et al. Effect of alternate-week versus continuous dexamethasone scheduling on the risk of osteonecrosis in paediatric patients with acute lymphoblastic leukaemia: results from the CCG-1961 randomised cohort trial. Lancet Oncol. 13 (9), 906–915 (2012). Low, Y., White, W. D. & Habib, A. S. Postoperative hyperglycemia after 4- vs 8-10-mg dexamethasone for postoperative nausea and vomiting prophylaxis in patients with type II diabetes mellitus: a retrospective database analysis. J. Clin. Anesth. 27 (7), 589–594 (2015). Dionne, R. A., Gordon, S. M., Rowan, J., Kent, A. & Brahim, J. S. Dexamethasone suppresses peripheral prostanoid levels without analgesia in a clinical model of acute inflammation. J. Oral Maxillofac. Surg. 61 (9), 997–1003 (2003). Basbaum, A. I., Bautista, D. M., Scherrer, G. & Julius, D. Cellular and molecular mechanisms of pain. Cell 139 (2), 267–284 (2009). Laurent, M. R. et al. Prevention and Treatment of Glucocorticoid-Induced Osteoporosis in Adults: Consensus Recommendations From the Belgian Bone Club. Front. Endocrinol. (Lausanne) . 13 , 908727 (2022). Bauer, H. C. et al. Assessment of preemptive analgesia with ibuprofen coadministered or not with dexamethasone in third molar surgery: a randomized double-blind controlled clinical trial. Oral Maxillofac. Surg. 17 (3), 165–171 (2013). Won Jahng, J. et al. Dexamethasone reduces food intake, weight gain and the hypothalamic 5-HT concentration and increases plasma leptin in rats. Eur. J. Pharmacol. 581 (1), 64–70 (2008). Gensler, L. S. Glucocorticoids: complications to anticipate and prevent. Neurohospitalist 3 (2), 92–97 (2013). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9079119","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":606508218,"identity":"9421f80e-a2ef-4d36-a31e-d5add87b3043","order_by":0,"name":"Baoji Lu","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Baoji","middleName":"","lastName":"Lu","suffix":""},{"id":606508219,"identity":"f3a0bd4b-04c6-46fd-84e3-95ad4c1c28a6","order_by":1,"name":"Tae Woo Kim","email":"","orcid":"","institution":"College of Medicine, Ewha Womans University Seoul Hospital","correspondingAuthor":false,"prefix":"","firstName":"Tae","middleName":"Woo","lastName":"Kim","suffix":""},{"id":606508224,"identity":"d520ec82-a68f-4afd-a7f6-3a2113ce73ce","order_by":2,"name":"Eun-ee Jung","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Eun-ee","middleName":"","lastName":"Jung","suffix":""},{"id":606508227,"identity":"0028ca0d-585b-417c-ac99-dacf3446b45a","order_by":3,"name":"Guanghai Nan","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Guanghai","middleName":"","lastName":"Nan","suffix":""},{"id":606508229,"identity":"bf0bbb08-0599-43f2-939d-c70580185ee6","order_by":4,"name":"Jae-Won Kim","email":"","orcid":"","institution":"Research and Development Center, Green Cross Wellbeing Corporation","correspondingAuthor":false,"prefix":"","firstName":"Jae-Won","middleName":"","lastName":"Kim","suffix":""},{"id":606508230,"identity":"8402f730-aa3f-484b-8b11-ce9b427ca8b2","order_by":5,"name":"Kyeongsoo Jeong","email":"","orcid":"","institution":"Research and Development Center, Green Cross Wellbeing Corporation","correspondingAuthor":false,"prefix":"","firstName":"Kyeongsoo","middleName":"","lastName":"Jeong","suffix":""},{"id":606508235,"identity":"37a61398-140f-423c-bcac-8f54af7b36f4","order_by":6,"name":"Minju Im","email":"","orcid":"","institution":"Research and Development Center, Green Cross Wellbeing Corporation","correspondingAuthor":false,"prefix":"","firstName":"Minju","middleName":"","lastName":"Im","suffix":""},{"id":606508237,"identity":"4ea91bfb-d166-4fd7-a70c-044d373a2523","order_by":7,"name":"Hee Young Kim","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Hee","middleName":"Young","lastName":"Kim","suffix":""},{"id":606508247,"identity":"0050a4bf-8342-4f03-896d-af199c347ee9","order_by":8,"name":"Tae Hwan Cho","email":"","orcid":"","institution":"Cho Orthopaedic Surgery Clinic","correspondingAuthor":false,"prefix":"","firstName":"Tae","middleName":"Hwan","lastName":"Cho","suffix":""},{"id":606508249,"identity":"203bf2bf-2241-49ed-8df7-b629b7947152","order_by":9,"name":"Seungsoo Chung","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYHACNhAhx8DAQ6IWY9K1JDYQrUW+//Czx7w7atM33Mg9/IGhxo6wFoMbaebGvGeO5264kZcmwXAsmQgtEjxs0rxtx4BacsyAjjxAjMPOgLWkG9zIMf7A8I8ILQwHckBaahKAWgwkGNuI0AL0i5nk3LYDhjPPvDGTSOwjwi+gEJN421Ynz3cc6LAP34gIMSg4zKAAclIC0RoYGOoY5BtIUD4KRsEoGAUjCwAAaM84s/lWxQEAAAAASUVORK5CYII=","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Seungsoo","middleName":"","lastName":"Chung","suffix":""},{"id":606508251,"identity":"408fbba0-239b-4b20-b989-61669b212db1","order_by":10,"name":"Dong Ah Shin","email":"","orcid":"","institution":"Yonsei University College of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"Ah","lastName":"Shin","suffix":""}],"badges":[],"createdAt":"2026-03-10 04:53:18","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9079119/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9079119/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104876884,"identity":"f31f55f0-fd99-4bdc-9c42-05b351ddccff","added_by":"auto","created_at":"2026-03-18 08:43:57","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":317037,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalgesic effect of human placental hydrolysate (HPH) in a capsaicin-induced pain model. (A) \u003c/strong\u003eSchematic representation of pain induction. Capsaicin (CAP; 20 μg in 20 μL olive oil) was injected intradermally into the left hind paw. The needle was inserted at the heel (site X) and advanced to the injection site (site I). Mechanical sensitivity was assessed at two distinct sites: the primary hyperalgesia site (P), and the secondary hyperalgesia site (S).\u003cstrong\u003e (B) \u003c/strong\u003eExperimental timeline for drug administration and von Frey filament behavioral test (BT).\u003cstrong\u003e (C-D) \u003c/strong\u003eEffects of different concentrations of HPH on capsaicin-induced primary\u003cstrong\u003e (C) \u003c/strong\u003eand secondary\u003cstrong\u003e (D) \u003c/strong\u003ehyperalgesia (n = 6 in CAP-Veh group; n = 5 in CAP-HPH 1.8 mL/kg group; n = 6 in CAP-HPH 3.6 mL/kg group). \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05 vs. CAP-Veh group, \u003csup\u003e##\u003c/sup\u003ep \u0026lt; 0.01 vs. -0.5h, two-way repeated ANOVA followed by Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9079119/v1/12412ad1116afb0015a75119.jpg"},{"id":104876825,"identity":"f4133bed-6a30-44bf-b404-bb7693029a40","added_by":"auto","created_at":"2026-03-18 08:43:46","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":455032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in neuronal activity in the spinal dorsal horn of the capsaicin-induced pain model following HPH treatment. (A) \u003c/strong\u003eRepresentative image showing electrical stimulation intensity-dependent neuronal activity in the spinal dorsal horn following HPH or vehicle treatment.\u003cstrong\u003e (B-C) \u003c/strong\u003eComparison of peak amplitudes before and during the treatment of vehicle\u003cstrong\u003e (B) \u003c/strong\u003eor HPH\u003cstrong\u003e (C) \u003c/strong\u003e(n = 5 per group). \u003csup\u003e*\u003c/sup\u003eP \u0026lt; 0.05 vs. vehicle group, two-way repeated ANOVA followed by Bonferroni’s post hoc test.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9079119/v1/1c5bbcd2099cc0a281fca026.jpg"},{"id":104876854,"identity":"8638f4a9-797a-4634-9214-fc67e98d4c0a","added_by":"auto","created_at":"2026-03-18 08:43:55","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":469177,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges in pain-related molecular markers in the rat spinal cord following HPH treatment. (A) \u003c/strong\u003eRepresentative immunofluorescence images of c-Fos expression in the spinal dorsal horn.\u003cstrong\u003e (B) \u003c/strong\u003eComparison of c-Fos positive cells in the spinal dorsal horn among the groups (n = 4 in Naïve group, n = 6 in CAP-Veh and CAP-HPH groups).\u003cstrong\u003e (C) \u003c/strong\u003eRepresentative immunofluorescence images of p-PKC expression in the spinal dorsal horn.\u003cstrong\u003e (D) \u003c/strong\u003eComparison of p-PKC area positive cells in the spinal dorsal horn among the groups (n = 5 in Naïve group, n = 3 in CAP-Veh and CAP-HPH groups). \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01,\u0026nbsp;\u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05, one-way ANOVA followed by Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9079119/v1/6e44fb92edac5db334db18f9.jpg"},{"id":104876852,"identity":"0318c232-95c2-445e-999a-478e59e4983b","added_by":"auto","created_at":"2026-03-18 08:43:54","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":236268,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnalgesic effect of dexamethasone with or without HPH in the capsaicin-induced pain model. (A-B) \u003c/strong\u003eEffects of different concentrations of dexamethasone and combined treatment (HPH + dexamethasone 1 mg/kg) applied to the hind paw on capsaicin-induced primary \u003cstrong\u003e(A) \u003c/strong\u003eand secondary\u003cstrong\u003e (B) \u003c/strong\u003ehyperalgesia (n = 8 per group). \u003csup\u003e**\u003c/sup\u003eP \u0026lt; 0.01, \u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05 vs. CAP-Veh group, two-way repeated ANOVA followed by Bonferroni’s post hoc test.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9079119/v1/c7fb9bee93b493b3b4149ac0.jpg"},{"id":104876901,"identity":"dcea4e8c-46db-4520-96c9-df80e5afc396","added_by":"auto","created_at":"2026-03-18 08:44:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":165562,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eChanges of body weight following repeated dexamethasone or combined HPH and dexamethasone treatment. (A) \u003c/strong\u003eExperimental timeline for drug treatment and the measurement of body weight.\u003cstrong\u003e (B) \u003c/strong\u003eBody weight changes over time following treatment (n = 6 per group). \u003csup\u003e****\u003c/sup\u003eP \u0026lt; 0.0001, \u003csup\u003e***\u003c/sup\u003ep \u0026lt; 0.001, \u003csup\u003e**\u003c/sup\u003ep \u0026lt; 0.01, \u003csup\u003e*\u003c/sup\u003ep \u0026lt; 0.05 vs. Saline group, as determined using two-way repeated ANOVA followed by Tukey’s post hoc test.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9079119/v1/50cf43453155b6ccb8a5c798.jpg"},{"id":104877000,"identity":"ca92fb1f-dfb3-443d-a0d9-74e86ac0a1be","added_by":"auto","created_at":"2026-03-18 08:44:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2520062,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9079119/v1/a6199be0-fd34-4b3f-a9d6-d60cfcdd72c8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Analgesic effects of human placental hydrolysate on capsaicin-induced hyperalgesia in rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003ePain, especially inflammatory pain, represents a major clinical challenge worldwide, affecting up to 20% of the global population and imposing substantial personal and socioeconomic burdens (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Such pain conditions often lead to disability, reduced quality of life, and high healthcare costs (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Corticosteroids are widely prescribed for their potent anti-inflammatory and analgesic properties and remain a mainstay in the management of pain associated with inflammation (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, their long-term use is restricted by serious side effects, including metabolic disturbances (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), osteoporosis (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), and immune suppression (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e), which limit their clinical utility. These limitations underscore the need for adjunctive strategies that preserve therapeutic efficacy while reducing systemic toxicity.\u003c/p\u003e \u003cp\u003eHuman placental hydrolysate (HPH) contains a complex mixture of biologically active substances, including amino acids, peptides, nucleotides (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e) that contribute to pharmacological effects. The antioxidant and anti-inflammatory effects of HPH are relatively well known in inflammatory conditions, especially in liver diseases, based on findings from both experimental and clinical studies (\u003cspan additionalcitationids=\"CR12 CR13 CR14\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). In the patients with osteoarthritis, HPH has also been clinically applied to alleviate pain symptoms (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). While some clinical studies have shown promise in the treatment of pain conditions like osteoarthritis, shoulder impingement syndrome and chronic back pain (\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e), the mechanism underlying analgesic effect of HPH remains unclear.\u003c/p\u003e \u003cp\u003e Capsaicin, the pungent compound in chili peppers, induces pain by activating transient receptor potential vanilloid 1 (TRPV1) channel on nociceptive fibers, producing a burning sensation and local inflammation. Intradermal injection of capsaicin results in primary hyperalgesia at the site of application, characterized by increased sensitivity to thermal and mechanical stimuli due to peripheral sensitization of nociceptors. Secondary hyperalgesia also develops in surrounding uninjured areas and reflects central sensitization, leading primarily to enhanced mechanical sensitivity. This model is particularly useful for determining whether the analgesic effects of compound are mediated through peripheral or central site of action (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this context, previous \u003cem\u003ein vitro\u003c/em\u003e and animal studies provide mechanistic evidence supporting the potential of HPH in alleviating pain (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Specifically, HPH significantly attenuated inflammatory pain behaviors and reduced pro-inflammatory cytokine expression (TNF-α, IL-1β, IL-6) in a complete Freund\u0026rsquo;s adjuvant (CFA)-induced inflammatory pain model in mice (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e), implicating its ability to modulate pathways involved in nociceptive sensitization. Additionally, HPH suppressed inflammatory responses in a dinitrochlorobenzene (DNCB)-induced atopic dermatitis mouse model and reduced cytokine levels, further supporting its anti-inflammatory and neuro-modulatory potentials (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Collectively, these preclinical findings provide a mechanistic rationale for investigating whether HPH can attenuate pain underlying capsaicin-induced hyperalgesia, which is driven by peripheral inflammatory sensitization.\u003c/p\u003e \u003cp\u003eThe present study investigated whether HPH could alleviate capsaicin-induced primary hyperalgesia and secondary hyperalgesia and compared its efficacy with dexamethasone, a standard corticosteroid used for pain relief (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e) but limited by systemic side effects (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Furthermore, we examined whether combining HPH with a low dose of dexamethasone could provide a synergistic analgesic effect while reducing corticosteroid-associated adverse outcomes such as body weight loss. Behavioral assessments and molecular analyses were performed to explore potential mechanisms underlying the analgesic effects of HPH.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003eChemicals\u003c/p\u003e \u003cp\u003eCapsaicin (M2028; Sigma-Aldrich, St. Louis, MO, USA), olive oil (O1514; Sigma-Aldrich, St. Louis, MO, USA), dexamethasone disodium phosphate (08806505003025; Jeil Pharmaceutical Co., Ltd., Daegu, Korea), and human placental hydrolysate (HPH; Laennec\u0026reg;, GC Wellbeing, Seoul, Korea) were used.\u003c/p\u003e \u003cp\u003eExperimental animals\u003c/p\u003e \u003cp\u003eAdult male Sprague\u0026ndash;Dawley rats (8 weeks; 250\u0026ndash;300 g; Orient Bio, Sungnam, Gyonggi, Korea) were used. The rats were housed in groups of three in plastic cages with soft bedding under a 12-h light/dark cycle with free access to food and water. All experimental procedures involving animals were conducted in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University Health System (permit no.: 2024-0001).\u003c/p\u003e \u003cp\u003eCapsaicin-induced pain model\u003c/p\u003e \u003cp\u003eThe procedure for capsaicin injection in rats was performed as described previously (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Briefly, rats were lightly anesthetized with isoflurane (Kyongbo Pharmaceutical CO., LTD, Asan, Republic of Korea), and a 30-gauge needle (with a Hamilton syringe) was inserted through the skin at the heel (designated as site X in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) and advanced to the central plantar area of the left hind paw (site I), which was used consistently across all experiments. At this location, capsaicin (20 \u0026micro;g in 20 \u0026micro;l of olive oil) was administered via intradermal injection slowly. After the procedure, rats were returned to their home cage and closely observed until they recovered fully from anesthesia (typically within 5 minutes).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDrug administration\u003c/p\u003e \u003cp\u003eTwo different doses of HPH were chosen and administered intraperitoneally: 3.6 mL/kg as the reference therapeutic dose and 1.8 mL/kg as a half-dose to evaluate dose dependency, based on previous studies (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). Dexamethasone was administered intraperitoneally at 1 mg/kg (low dose) or 5 mg/kg (high dose). Saline was used as the control.\u003c/p\u003e \u003cp\u003eBehavioral assessment of capsaicin-induced pain\u003c/p\u003e \u003cp\u003eAs previously described (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), mechanical hypersensitivity was assessed using the up-down method with a series of von Frey filaments (Stoelting, Chicago, IL, USA) applied to the injection site to evaluate primary hyperalgesia and to an adjacent area to evaluate secondary hyperalgesia. Baseline assessment was performed at 0.5 h before HPH application (-0.5h). Following capsaicin injection (0h), behavioral test was conducted at 0.5, 1, 1.5, 2, and 24 h post-injection. The 50% mechanical withdrawal threshold (MWT) was calculated, and all behavioral test was conducted in a blinded manner.\u003c/p\u003e \u003cp\u003eVoltage sensitive dye imaging (VSDI) in spinal dorsal horns\u003c/p\u003e \u003cp\u003eVoltage sensitive dye imaging (VSDI) was carried out as performed in our laboratory (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In brief, rats were deeply anesthetized with urethan (1.25 g/kg, intraperitoneally) after 30 minutes of capsaicin injection and transcardially perfused with ice-cold solution containing 213 mM sucrose, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 10 mM MgSO₄, 0.5 mM CaCl₂, 26 mM NaHCO₃, and 11 mM glucose. The L4-L5 spinal cord segments were surgically extracted and immediately immersed in ice-cold artificial cerebrospinal fluid (aCSF) solution for 5 minutes. The spinal cord was mounted on an agarose block and sectioned into 400-\u0026micro;m thick transverse slices using a vibratome (Leica Biosystems Inc., Buffalo Grove, IL, USA). These sections were immediately placed in interface chambers perfused with oxygenated aCSF containing: 126 mM NaCl, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM MgCl₂, 2 mM CaCl₂, 26 mM NaHCO₃, and 10 mM glucose, continuously bubbled with 95% O₂/5% CO₂ to maintain pH 7.2. After a 1-hour recovery period at room temperature under continuous aCSF perfusion, sections were incubated for 1 h with voltage-sensitive dye (di-2-ANEPEQ, 50 \u0026micro;g/mL in aCSF; Molecular Probes, Eugene, OR, USA). For optical recordings, a concentric bipolar microelectrode (30213, FHC, Bowdoin, Maine, USA) was positioned within the region of dorsal horn (laminae I\u0026ndash;II) using an optical microscope (Olympus Optical Co. Ltd., Tokyo, Japan) equipped with a 10\u0026times; objective and 0.35\u0026times; projection lens. Electrical stimulation consisted of square pulses (2 ms duration, 5-second interstimulus interval) delivered through a stimulus isolation unit (World Precision Instruments, Sarasota, FL, USA), with intensity adjusted to evoke reliable responses. Neuronal activity was captured using a high-resolution CCD camera-based optical imaging system (Brainvision Inc., Tokyo, Japan) configured with a dichroic mirror, 510\u0026ndash;555 nm excitation filter, and 590 nm emission filter. A 150 W tungsten-halogen lamp provided fluorescence excitation. The imaging field encompassed 184 \u0026times; 124 pixels. Fluorescence measurements were acquired over 943.5 ms periods using the MiCAM02 optical imaging device (Brainvision Inc.) at a temporal resolution of 3.7 ms per frame. Signals were averaged over 20 trials to improve signal-to-noise ratio. Fluorescence changes were normalized by calculating the fractional change (ΔF/F) relative to baseline fluorescence for each pixel. Signal amplitude and spatial extent of activation were analyzed using spatial filtering (9 \u0026times; 9 pixels) and cubic filtering (3 \u0026times; 3 pixels).\u003c/p\u003e \u003cp\u003eAfter the baseline responses were recorded, the slice was either maintained in aCSF (vehicle) or treated with HPH. For HPH treatment in vitro, lyophilized HPH was dissolved in aCSF and transferred into the chamber. The slice was incubated with HPH for 30 minutes. Following incubation, the same electrical stimulation protocol (0.3, 0.6, 0.9 mA) was applied, and the post-treatment VSD responses were recorded. All data acquisition and analysis were performed using BV Analyzer software (Brainvision Inc.). Optical signals were quantified as percentage fluorescence change (%ΔF/F) within circular areas (radius\u0026thinsp;=\u0026thinsp;5 pixels) positioned specifically in the dorsal horn region. Both temporal dynamics and spatial patterns of activation were systematically analyzed.\u003c/p\u003e \u003cp\u003eImmunohistochemistry for c-Fos or PKC expression in spinal dorsal horns\u003c/p\u003e \u003cp\u003eAs performed in our laboratory (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e), rats were deeply anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally) and perfused with 150 mL of 0.01M phosphate-buffered saline (PBS, pH 7.4), followed by 500 mL of 4% paraformaldehyde in 0.1M phosphate buffer (pH 7.4). The L5 spinal cord segment was dissected out and post-fixed in 4% paraformaldehyde for 2 hours at 4\u0026deg;C, then cryoprotected in 30% sucrose solution for 12 hours at 4\u0026deg;C. Tissue sections (40 \u0026micro;m thickness) were prepared using a freezing microtome (Cryostat 1720; Leitz, Mannheim, Germany). Free-floating sections were washed three times in 0.01 M PBS before blocking for 1 hour at room temperature with 10% normal goat serum (ab7481; Abcam, Cambridge, MA) or donkey serum (ab7475; Abcam) in PBS containing 0.3% Triton X-100.\u003c/p\u003e \u003cp\u003eFor c-Fos labeling, primary antibody incubation was performed at room temperature using rabbit anti-Fos antibody (1:1000, ab190289; Abcam). Following three-time PBS washes, sections were incubated for 2 hours at room temperature with Alexa Fluor 488-conjugated goat anti-rabbit IgG secondary antibody (1:200; AB_2576217; Invitrogen, Thermo Fisher Scientific, Waltham, MA) in 0.3% Triton X-100 PBS solution. For phosphorylated protein kinase C (P-PKC) staining, a similar protocol was followed on separate sections: a goat monoclonal anti-phospho-PKC (p-PKCε (Ser 729): sc-12355, 1:500, Santa Cruz Biotechnology, Dallas, TX) was used as primary, and an Alexa Fluor 647\u0026ndash;conjugated donkey anti-goat IgG (AB_2535864; Invitrogen) as secondary. In all cases, nuclei were counterstained with DAPI (4\u0026prime;,6-diamidino-2-phenylindole, blue fluorescence, Sigma-Aldrich, St. Louis, MO 63103 USA) before mounting the sections. Finally, sections were mounted onto glass slides and cover slipped with an anti-fade mounting medium.\u003c/p\u003e \u003cp\u003eImmunofluorescence imaging was performed using a Zeiss LSM 710 confocal laser scanning microscope (Axio Examiner.Z1, Jena, Germany). For each rat, multiple sections of the L5 dorsal horn were imaged, and the number of c-Fos\u0026ndash;positive nuclei was counted in a standardized region of the superficial dorsal horn (laminae I\u0026ndash;III) on the side of capsaicin injection. P-PKCε immunoreactivity was quantified by calculating the percentage of neurons (identified by DAPI-stained nuclei) that showed clear PKC phosphorylation (red cytoplasmic staining) in the dorsal horn. Quantification was performed using ImageJ (National Institutes of Health, Bethesda, MD, USA).\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were conducted using GraphPad Prism 9.0 (GraphPad Software Inc., San Diego, CA, USA). Behavioral and body weight data were analyzed by two-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s multiple comparisons test. Optical signal intensities were compared by two-way ANOVA, followed by Bonferroni\u0026rsquo;s post hoc test. Immunohistochemistry data were analyzed using one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test. Data are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). P values less than 0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cp\u003eHPH alleviates capsaicin-induced hyperalgesia in a dose-dependent manner\u003c/p\u003e \u003cp\u003eTo evaluate the analgesic effect of HPH in the capsaicin-induced pain model, capsaicin was injected into X site and primary hyperalgesia at the injection site (site P) and secondary hyperalgesia at the base of the third-fourth toes (site S) were measured (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Rats were randomly assigned to three groups: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) CAP-Veh (capsaicin\u0026thinsp;+\u0026thinsp;saline), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) CAP-HPH 1.8 mL/kg (capsaicin\u0026thinsp;+\u0026thinsp;HPH 1.8 mL/kg), (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) CAP-HPH 3.6 mL/kg (capsaicin\u0026thinsp;+\u0026thinsp;HPH 3.6 mL/kg). HPH was administered 0.5 h before capsaicin injection. Behavioral tests were performed at 0.5 h before capsaicin injection and 0.5, 1, 1.5, 2 and 24 h after capsaicin injection (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Thirty minutes after intraplantar injection of capsaicin, the mean paw withdrawal threshold (MWT) rapidly dropped from a pre-injection value (log value\u0026thinsp;=\u0026thinsp;5.1) of approximately 12 g to around 1 g (CAP-Veh) (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; for time: F\u003csub\u003e5, 84\u003c/sub\u003e = 17.85, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for group: F\u003csub\u003e2, 84\u003c/sub\u003e = 14.01, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for time \u0026times; group: F \u003csub\u003e10, 84\u003c/sub\u003e = 0.7767, p\u0026thinsp;=\u0026thinsp;0.6508, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), indicating development of primary hyperalgesia by intradermal capsaicin. This reduction remained statistically significant for about 2 hours (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), followed by a gradual recovery over 24 hours. Intraperitoneal administration of HPH attenuated capsaicin-induced changes of MWTs at both primary and secondary sites in a dose-dependent manner, compared to the CAP-Veh group. CAP-HPH (3.6 mL/kg) group significantly reduced primary hyperalgesia at 1 and 2 h after capsaicin injection (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC) and suppressed secondary hyperalgesia up to 24 hr (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD) compared with the vehicle (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It showed that HPH, particularly at 3.6 mL/kg, effectively attenuated both primary and secondary hyperalgesia induced by capsaicin, with significant analgesic effects evident from 1 h and persisting up to 24 h.\u003c/p\u003e \u003cp\u003eHPH reduces neuronal activity in the spinal cord dorsal horn of the capsaicin-induced pain model\u003c/p\u003e \u003cp\u003eTo investigate the effect of HPH on the neuronal excitability of spinal dorsal horns, VSDI was performed on spinal cord slices from capsaicin-injected rats. Animals were divided into Veh and HPH groups. Neuronal activity was recorded before and during vehicle or HPH treatment. Based on the previous study (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e), the electrical stimulation intensities of 0.3, 0.6, 0.9 mA were applied. Neuronal signals and the activated areas increased in a stimulation intensity-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). While no significant differences were observed between the pre- and vehicle treatment phases at any stimulation intensity in the Veh group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). On the other hand, HPH treatment reduced stimulation-induced peak amplitudes at 0.9 mA compared with the values before HPH treatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; for phase: F\u003csub\u003e2, 24\u003c/sub\u003e = 19.84, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for group: F\u003csub\u003e1, 24\u003c/sub\u003e = 14.82, p\u0026thinsp;=\u0026thinsp;0.0008; for phase \u0026times; time: F\u003csub\u003e2, 24\u003c/sub\u003e = 0.7181, p\u0026thinsp;=\u0026thinsp;0.4979; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These results indicate that HPH effectively reduced the neuronal excitability of spinal dorsal horns following intra-plantar injection of capsaicin.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHPH attenuates capsaicin-induced both c-Fos expression and p-PKCε immunoreactivity in spinal dorsal horn\u003c/p\u003e \u003cp\u003eExpression of c-Fos (a marker of neuronal activation) and p-PKCε (a signaling mediator implicated in nociceptive sensitization) in the spinal dorsal horn was estimated to investigate the analgesic mechanism of HPH in capsaicin-induced pain model. Experiment was performed in the following groups: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Naive (no intraplantar capsaicin injection); (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) CAP-Veh (intraperitoneal saline prior to capsaicin injection); (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) CAP-HPH (intraperitoneal HPH at 3.6 mL/kg before capsaicin injection). Either saline or HPH was administered 30 minutes before capsaicin injection. Capsaicin injection increased the number of c-Fos positive nuclei in the superficial dorsal horn (CAP-Veh group) compared to naive group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; F\u003csub\u003e2, 13\u003c/sub\u003e = 7.03, p\u0026thinsp;=\u0026thinsp;0.0085), whereas this increase was prevented by HPH treatment (CAP-HPH vs. naive group, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, B). Similarly, significantly enhanced expression of p-PKCε was observed in the capsaicin-induced pain model compared with naive group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, F\u003csub\u003e2, 8\u003c/sub\u003e = 8.725, p\u0026thinsp;=\u0026thinsp;0.0098), which was attenuated by HPH pretreatment (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, D). These results indicate that HPH mitigated both neuronal activation and PKCε phosphorylation in the spinal dorsal horn following capsaicin injection.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHPH-dexamethasone combination achieves analgesia comparable to high-dose dexamethasone in the capsaicin-induced pain model\u003c/p\u003e \u003cp\u003eTo investigate whether combined administration could reduce the requirement for high-dose dexamethasone (5 mg/kg), multiple treatment groups were established, and primary and secondary hyperalgesia were measured separately. To evaluate the effect of HPH combined with dexamethasone (DEX) or DEX alone, rats were divided into four groups: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) CAP-Veh (capsaicin\u0026thinsp;+\u0026thinsp;saline), (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) CAP-DEX 1 mg/kg (capsaicin\u0026thinsp;+\u0026thinsp;low dose dexamethasone (1 mg/kg)), (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) CAP-DEX 5 mg/kg (capsaicin\u0026thinsp;+\u0026thinsp;high dose dexamethasone (5 mg/kg)), and (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) CAP-DEX 1mg/kg\u0026thinsp;+\u0026thinsp;HPH (capsaicin\u0026thinsp;+\u0026thinsp;dexamethasone 1 mg/kg\u0026thinsp;+\u0026thinsp;HPH 3.6 mL/kg). DEX and/or HPH were administered 30 minutes before capsaicin injection. Behavioral tests were conducted at 0.5 h before capsaicin (-0.5 h) and 0.5, 1, 1.5, 2 and 24 h after capsaicin injection. While capsaicin injection lowered MWTs compared to the value before capsaicin (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), pretreatment with dexamethasone at a high dose of 5 mg/kg significantly alleviated primary hyperalgesia in capsaicin-injected rats (CAP-DEX 5mg/kg) compared to control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA; CAP-Veh; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 at 2h; for time: F\u003csub\u003e5, 168\u003c/sub\u003e = 30.90, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for group: F\u003csub\u003e3, 168\u003c/sub\u003e = 8.099, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for time \u0026times; group: F\u003csub\u003e15, 168\u003c/sub\u003e = 0.9032, p\u0026thinsp;=\u0026thinsp;0.5617). However, administration of dexamethasone at a lower dose of 1 mg/kg failed to inhibit primary and secondary hyperalgesia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, B). On the other hand, co-treatment of a low dose dexamethasone (1 mg/kg) with HPH effectively suppressed the development of primary and secondary hyperalgesia, compared to control group (CAP-Veh; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). It suggests that treatment of HPH combined with a low dose dexamethasone produced similar analgesic effects as high-dose dexamethasone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHPH co-administration mitigates dexamethasone-induced body weight loss\u003c/p\u003e \u003cp\u003eTo see potential systemic side effects of HPH and/or dexamethasone, rats were divided into five groups: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) Saline, (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) HPH 3.6 mL/kg, (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) DEX 1 mg/kg, (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) DEX 5 mg/kg, (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e) HPH 3.6 mL/kg\u0026thinsp;+\u0026thinsp;DEX 1mg/kg. Animals received once-daily intraperitoneal injections of the assigned treatment for five consecutive days (Day 0\u0026ndash;4), followed by two drug-free days (Days 5\u0026ndash;6). Body weight was recorded at baseline (prior to the first injection), daily before each injection, and again on Day 7 (72 hours after the last dose) to assess recovery (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). HPH (3.6 mL/kg) group showed a steady increase in body weight over the observation period, consistent with vehicle-treated group (Saline). It is noted that dexamethasone treatment at either 5mg/kg or 1 mg/kg caused profound body weight losses from Day 2, reaching\u0026thinsp;~\u0026thinsp;88% of baseline by Day 4 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for day: F\u003csub\u003e5, 150\u003c/sub\u003e = 29.18, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for group: F\u003csub\u003e4, 150\u003c/sub\u003e = 98.69, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; for day \u0026times; group: F\u003csub\u003e20, 150\u003c/sub\u003e = 13.59, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). Even after two drug-free days, body weight remained below 90% of baseline on Day 7 compared with the Saline group. Although rats receiving combined treatment of HPH and dexamethasone treatment also exhibited weight loss during the dosing phase, the body weight partially recovered after drug discontinuation, reaching\u0026thinsp;~\u0026thinsp;95% of baseline by Day 7, in contrast to the persistent loss observed with dexamethasone alone. These results indicate that dexamethasone induces pronounced body weight loss as a systemic side effect, while cotreatment with HPH partially mitigates this catabolic effect.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe present study demonstrates the analgesic effect of human placental hydrolysate (HPH) in a capsaicin-induced pain model. Pretreatment with HPH effectively alleviated both primary and secondary hyperalgesia and suppressed neuronal activity by inhibiting PKCε activation. Moreover, combined administration of HPH with dexamethasone produced a comparable antinociceptive effect while permitting a reduced dose of dexamethasone and attenuating dexamethasone-induced body weight loss. These results suggest that HPH can suppress capsaicin-induced primary and secondary hyperalgesia while minimizing the adverse effects of dexamethasone monotherapy.\u003c/p\u003e \u003cp\u003eThe analgesic efficacy of HPH was evaluated in capsaicin-induced pain model. The capsaicin-induced pain model represents an acute form of inflammatory and neurogenic pain (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e), as capsaicin activates TRPV1 receptors on primary sensory neurons, leading to neurogenic inflammation, peripheral sensitization (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e), and subsequent central sensitization (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). In this model, pretreatment with HPH effectively alleviated both primary and secondary hyperalgesia. Since primary hyperalgesia reflects peripheral sensitization at the site of injury, whereas secondary hyperalgesia reflects spinal cord and beyond, the ability of HPH to suppress both types of hyperalgesia suggests that it may exert dual actions on peripheral nociceptor activity and central pain processing. HPH contains various bioactive components, including peptides and amino acids, which exert anti-inflammatory, antioxidant, and tissue-protective effects (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). As the bioactive components of HPH generated the analgesic efficacy observed in this study, identification the specific components responsible for the analgesic effect will be an important subject of future investigation.\u003c/p\u003e \u003cp\u003eGiven that HPH alleviated secondary hyperalgesia at the behavioral level, we next examined spinal cord excitability using VSDI to determine whether these effects were associated with changes in dorsal horn neuronal activity. The reduction of neuronal responses following HPH treatment suggests that HPH dampens stimulus-evoked hyperexcitability within the dorsal horn, a hallmark of central sensitization (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Although VSDI does not resolve single-cell activity, its strength lies in capturing spatiotemporal patterns of neuronal population dynamics, thereby providing network-level evidence for the central actions of HPH. Together, these findings support the notion that HPH exerts its analgesic effects not only through peripheral modulation but also by influencing spinal pain circuitry, consistent with previous reports highlighting the importance of reducing dorsal horn hyperactivity in inflammatory pain (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIHC analyses further supported the central actions of HPH by demonstrating reductions in c-Fos and PKC expression in the spinal dorsal horn. c-Fos is widely recognized as a marker of neuronal activation in pain research (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e), and studies have shown its robust upregulation in the dorsal horn following inflammatory or capsaicin-induced pain (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e), reflecting enhanced nociceptive input and central sensitization (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). In line with these observations, the suppression of c-Fos expression by HPH in our study suggests that the treatment attenuates excessive neuronal activation under acute inflammatory pain conditions. Similarly, PKC, particularly the PKCε isoform, has been implicated in excitatory synaptic plasticity (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e) and the maintenance of central sensitization through the phosphorylation and trafficking of AMPA (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e) and NMDA receptors (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Previous studies have reported that enhanced PKC activity in inflammatory and neuropathic pain models, contributing to spinal hyperexcitability (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Our finding that HPH reduced PKC expression therefore indicates that inhibition of PKC-dependent signaling may represent a key mechanism by which HPH counteracts spinal hyperexcitability. Together, these results suggest that HPH suppressed secondary hyperalgesia by inhibiting PKC-dependent signaling and central sensitization in spinal dorsal horn neurons.\u003c/p\u003e \u003cp\u003eBecause the clinical use of dexamethasone is limited by dose-dependent adverse effects (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e), we investigated whether combining HPH with dexamethasone could provide effective analgesia while minimizing corticosteroid exposure. In the capsaicin-induced pain model, our results showed that combining HPH with dexamethasone achieved analgesic effects equivalent to high-dose dexamethasone, but with a reduced corticosteroid requirement. At the primary site, the combination achieved efficacy similar to 5 mg/kg dexamethasone, whereas at the secondary site the analgesic effect appeared more sustained. The comparable effect at the primary site likely reflects the strong anti-inflammatory action of dexamethasone in suppressing peripheral sensitization (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e), which is mainly driven by TRPV1 activation and the release of inflammatory mediators (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). In contrast, the more prolonged effect at the secondary site may indicate that HPH provides additional modulatory actions relevant to central pain processing, thereby complementing the effects of dexamethasone. Importantly, this combination strategy also offers a pharmacological advantage by reducing the glucocorticoid burden. Corticosteroid-related adverse effects, including metabolic disturbances, osteoporosis, immunosuppression, and alterations in body weight, are well recognized to be dose- and duration-dependent (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). This concept is consistent with the principles of multimodal analgesia, in which combining agents with distinct but complementary mechanisms enhances therapeutic benefit while minimizing toxicity (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e). Such a strategy could be particularly valuable in clinical contexts requiring repeated or prolonged corticosteroid administration, where the cumulative risk of side effects poses a major limitation.\u003c/p\u003e \u003cp\u003eTo further evaluate systemic tolerability, we examined body weight changes as a representative adverse effect. In normal rats without capsaicin treatment, repeated dexamethasone administration resulted in a pronounced reduction in body weight, highlighting its catabolic actions (\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Co-administration of HPH partially mitigated this effect, allowing recovery after treatment discontinuation. These results indicate that HPH not only sustains the analgesic efficacy or dexamethasone but also alleviates systemic adverse effects, thereby contributing to an improved safety profile.\u003c/p\u003e \u003cp\u003eNonetheless, several limitations should be acknowledged. First, we tested the treatment only in a single model of acute capsaicin-induced hyperalgesia, which may limit the generalizability of our findings to chronic inflammatory or neuropathic pain conditions. Second, the evaluation of side effects was incomplete. Apart from body weight, we did not assess other corticosteroid-related toxicities such as hyperglycemia, osteoporosis, or immune suppression. Comprehensive toxicity studies will be required to establish the long-term safety of HPH. Third, although we observed reduced spinal c-Fos and p-PKCε expression, inflammatory mediators and the bioactive constituents of HPH were not directly analyzed. Thus, the precise components and mechanisms underlying the analgesic effect remain unclear. Future studies should identify the active constituents and validate their interaction with glucocorticoid signaling to support standardized application and clinical translation of HPH.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur findings demonstrate that HPH could suppress both primary and secondary hyperalgesia induced by capsaicin, reflecting the inhibition of peripheral and central sensitization, respectively. Its combination with dexamethasone provides effective analgesia while reducing corticosteroid-associated toxicity. These results highlight HPH as a promising adjunct for corticosteroid-based pain management. Importantly, the ability of HPH to sustain analgesic efficacy while minimizing systemic side effects implies potential value in optimizing current therapeutic strategies.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eaCSF\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eartificial Cerebrospinal fluid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eAMPA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eα-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eANOVA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnalysis of Variance\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCAP\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCapsaicin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eCFA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFreund\u0026rsquo;s adjuvant\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ec-Fos\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecellular Proto-oncogene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDAPI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4\u0026prime;,6-diamidino-2-phenylindole\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDEX\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDexamethasone\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDNCB\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDinitrochlorobenzene\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHPH\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eHuman placental hydrolysate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMWT\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMechanical Withdrawal Threshold\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNMDA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eN-methyl-D-aspartate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePKC\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eProtein Kinase C\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ep-PKCε\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphorylated Protein Kinase C epsilon\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003estandard error of the mean\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTRPV1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTransient Receptor Potential Vanilloid 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eVSDI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVoltage sensitive dye imaging\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eVeh\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVehicle\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cb\u003eEthic approval and consent to participate\u003c/b\u003e \u003c/p\u003e \u003cp\u003e All animal procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Yonsei University College of Medicine (IACUC No. [2024-0001]) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition) and applicable Korean regulations. Every effort was made to minimize animal suffering and the number of animals used.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCompeting interests\u003c/strong\u003e \u003cp\u003eAuthors KJ, MI and JWK were employed by the company Green Cross Wellbeing Corporation. The remaining authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis research was supported by the Korea Medical Device Development Fund grant funded by the Korea government (the Ministry of Science and ICT, the Ministry of Trade, Industry and Energy, the Ministry of Health \u0026amp; Welfare, the Ministry of Food and Drug Safety (RS-2023-00253560) and Green Cross Wellbeing, Korea.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eBL and TWK drafted the original manuscript and contributed to the study methodology and data analysis. EJ contributed to the study methodology and project administration. GN and HYK reviewed and edited the original manuscript and contributed to the study methodology and investigation. KJ, MI and JWK supervised the study and reviewed the manuscript. THC conceived the original idea for this study. SC and DAS conceptualized and supervised the study, oversaw project administration, and reviewed and edited the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eNot applicable\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is available from the corresponding author upon reasonable request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMills, S. E. E., Nicolson, K. P. \u0026amp; Smith, B. H. Chronic pain: a review of its epidemiology and associated factors in population-based studies. \u003cem\u003eBr. J. Anaesth.\u003c/em\u003e \u003cb\u003e123\u003c/b\u003e (2), e273\u0026ndash;e83 (2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLurie, J. M. \u0026amp; Javaid, A. Visualizing Global Chronic Pain. \u003cem\u003eAnesth. Analg\u003c/em\u003e. \u003cb\u003e138\u003c/b\u003e (4), 918\u0026ndash;919 (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSteinmetz JD, Culbreth GT, Haile LM, Rafferty Q, Lo J, Fukutaki KG, et al. Global,regional, and national burden of osteoarthritis, 1990\u0026ndash;2020 and projections to 2050: a systematic analysis for the Global Burden of Disease Study 2021. The Lancet Rheumatology. 2023;5(9):e508-e22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAnselmo, A. et al. A Systematic Review on the Neuropsychological Assessment of Patients with LBP: The Impact of Chronic Pain on Quality of Life. \u003cem\u003eJ. Clin. Med.\u003c/em\u003e ;\u003cb\u003e13\u003c/b\u003e(20). (2024).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRhen, T. \u0026amp; Cidlowski, J. A. Antiinflammatory action of glucocorticoids\u0026ndash;new mechanisms for old drugs. \u003cem\u003eN Engl. J. Med.\u003c/em\u003e \u003cb\u003e353\u003c/b\u003e (16), 1711\u0026ndash;1723 (2005).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoutinho, A. E. \u0026amp; Chapman, K. E. The anti-inflammatory and immunosuppressive effects of glucocorticoids, recent developments and mechanistic insights. \u003cem\u003eMol. Cell. Endocrinol.\u003c/em\u003e \u003cb\u003e335\u003c/b\u003e (1), 2\u0026ndash;13 (2011).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBarker, H. L., Morrison, D., Llano, A., Sainsbury, C. A. R. \u0026amp; Jones, G. C. Practical Guide to Glucocorticoid Induced Hyperglycaemia and Diabetes. \u003cem\u003eDiabetes Ther.\u003c/em\u003e \u003cb\u003e14\u003c/b\u003e (5), 937\u0026ndash;945 (2023).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSeibel, M. J., Cooper, M. S. \u0026amp; Zhou, H. 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Glucocorticoids: complications to anticipate and prevent. \u003cem\u003eNeurohospitalist\u003c/em\u003e \u003cb\u003e3\u003c/b\u003e (2), 92\u0026ndash;97 (2013).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Human placental hydrolysate, capsaicin, neurogenic inflammatory pain, analgesia, PKCε, dexamethasone toxicity","lastPublishedDoi":"10.21203/rs.3.rs-9079119/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9079119/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eAim\u003c/h2\u003e \u003cp\u003eTo explore the analgesic potential of HPH, we examined whether HPH could reduce primary and secondary hyperalgesia in a rat model of capsaicin-induced pain and whether co-administration with dexamethasone could provide enhanced analgesia with fewer side effects.\u003c/p\u003e\u003ch2\u003eMethod\u003c/h2\u003e \u003cp\u003eHPH was administered in capsaicin-induced pain model. Primary and secondary hyperalgesia were assessed using von Frey filaments. Voltage-sensitive dye imaging (VSDI) was conducted in spinal cord slices to examine neuronal excitability. Immunohistochemistry was performed to evaluate c-Fos and phosphorylated PKCε (p-PKCε) expression. Body weight was monitored as an indicator of corticosteroid-associated toxicity in rats treated with dexamethasone alone or in combination with HPH.\u003c/p\u003e\u003ch2\u003eResult\u003c/h2\u003e \u003cp\u003eHPH dose-dependently attenuated capsaicin-induced both primary and secondary hyperalgesia, suppressed spinal dorsal horn hyperactivity, and reduced expression of c-Fos and p-PKCε. Combination of HPH with low-dose dexamethasone produced analgesia comparable to high-dose dexamethasone while reducing corticosteroid-related systemic toxicity, as reflected by body weight loss.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eHPH effectively alleviated capsaicin-induced neurogenic inflammatory pain and enhances the therapeutic profile of dexamethasone. Co-treatment with low-dose dexamethasone may provide synergistic analgesic benefits while minimizing systemic toxicity, supporting HPH as a promising adjunctive therapy for corticosteroid-based pain management.\u003c/p\u003e","manuscriptTitle":"Analgesic effects of human placental hydrolysate on capsaicin-induced hyperalgesia in rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-18 08:42:15","doi":"10.21203/rs.3.rs-9079119/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-04-08T18:00:44+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-02T02:09:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"28552446022443009014799683988589263752","date":"2026-03-24T19:10:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-03-20T02:24:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"158050967679706932511139512113886165173","date":"2026-03-14T11:04:32+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-13T16:22:26+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-03-13T15:14:25+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-10T13:54:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-10T13:53:32+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2026-03-10T04:44:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d029069d-7e47-42ac-831b-1fdd136c5632","owner":[],"postedDate":"March 18th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":64537684,"name":"Health sciences/Diseases"},{"id":64537685,"name":"Biological sciences/Drug discovery"},{"id":64537686,"name":"Health sciences/Medical research"},{"id":64537687,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-04-28T10:08:05+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-18 08:42:15","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9079119","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9079119","identity":"rs-9079119","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}