Effects of Acupuncture at ST36 and SP6 on Activity Levels and Pain Thresholds Through Interleukin-1, Interleukin-6, Cortisol, and Matrix Metalloproteinase-13 Modulation in an Osteoarthritis Rat Model

Abstract

Objectives

Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage degradation, inflammation, and altered stress-related responses. This preclinical study examined the effects of acupuncture at ST36 (stomach 36), SP6 (spleen 6), and their combination on pain thresholds, locomotor function, and inflammation and stress-related biomarkers in a monosodium iodoacetate (MIA)-induced OA rat model.

Methods

Thirty male Wistar rats were randomized into five groups a healthy control, an OA control, and three acupuncture-treated groups (ST36, SP6, and ST36 + SP6). Acupuncture-treated groups underwent eight 15-min sessions of acupuncture per day. Pain thresholds and locomotor activity were assessed before and after treatment. Joint tissue was analyzed for interleukin (IL)-1, IL-6, cortisol, and matrix metalloproteinase-13 (MMP-13) levels. Group differences were analyzed using appropriate parametric or nonparametric tests, and the associations between biomarkers and behavioral outcomes were assessed using Pearson correlation analysis.

Results

The acupuncture-treated rats showed increased pain thresholds (~5.3 g to 9.93–11.60 g, p < 0.05) and higher locomotor activity compared with those in the OA controls. IL-1, IL-6, cortisol, and MMP-13 levels were reduced from pretreatment levels in all acupuncture groups. Biomarker levels were negatively correlated with pain thresholds (r = −0.526 to −0.686) and locomotor activity (r = −0.537 to −0.893; all p < 0.01).

Conclusion

In rats with MIA-induced OA, acupuncture was associated with improved pain thresholds and locomotor outcomes and changes in inflammation and stress-related biomarker levels, providing exploratory preclinical evidence linking biomarker modulation with functional responses.

INTRODUCTION

Osteoarthritis (OA) is a multifactorial joint disorder involving biomechanical, structural, and inflammatory processes. It is characterized by progressive cartilage degradation and pathological changes in joint tissue [1]. Clinically, OA manifests as joint pain, stiffness, swelling, and reduced mobility, leading to substantial impairment in physical function and quality of life [2]. Globally, OA affects approximately 9.6% of adults, with an estimated 27 million cases in the United States alone [3]. OA prevalence increases markedly with age, affecting up to 65% of those aged > 60 years in Indonesia, with knee OA being the most common presentation [4].

OA pathogenesis is driven by mechanical stress and cellular responses that shift chondrocyte activity toward a catabolic phenotype, resulting in increased production of matrix-degrading enzymes such as matrix metalloproteinase-13 (MMP-13) [5]. This process is accompanied by inflammatory signaling mediated by cytokines, including interleukin (IL)-1, IL-6, and tumor necrosis factor alpha (TNFα), which collectively contribute to extracellular matrix disruption, cartilage degradation, and pain [6]. Oxidative stress and the activation of proinflammatory pathways such as nuclear factor kappa B (NF-κB) further worsen joint damage and structural alterations, including osteophyte formation [7, 8]. In parallel, neuroendocrine factors may influence OA-related inflammation, as cortisol, which is an endogenous anti-inflammatory hormone, has been shown to modulate cytokine production and cartilage catabolism [9-12].

Although pharmacological therapies are commonly used to manage OA symptoms, their long-term use is associated with gastrointestinal, hepatic, and cardiovascular adverse effects. Consequently, nonpharmacological interventions such as acupuncture have been explored in both clinical and preclinical settings, with experimental studies suggesting generally favorable safety profiles [13]. Preclinical evidence indicates that acupuncture may exert analgesic and anti-inflammatory effects through the modulation of neurotransmitter release and neuroimmune signaling pathways [14]. In animal models of OA, stimulation of the ST36 (stomach 36, also known as Zusanli) and SP6 (spleen 6, also known as Sanyinjiao) acupuncture points has been associated with reduced inflammatory responses and altered neuroendocrine activity. These effects are considered to involve pathways related to NF-κB, mitogen-activated protein kinase (MAPK), transforming growth factor beta (TGF-β), phosphoinositide 3-kinase protein kinase B (PI3K Akt), endogenous opioids, and stress-related hormone regulation [15-19]. Preclinical studies have suggested that the combined stimulation of ST36 and SP6 may exert synergistic effects on cytokine expression, stress-related hormones, and neuroimmune-associated pathways [20]. Rat models are widely used in OA research because of their suitability for experimental manipulation and reproducibly inducing joint degeneration using monosodium iodoacetate (MIA) [21]. Accordingly, the present study investigates the effects of acupuncture on ST36, SP6, and their combination in an experimental rat model of OA focusing on the inflammatory and hormonal biomarkers IL-1, IL-6, MMP-13, and cortisol. By integrating biological mediators with behavioral and functional outcomes in a preclinical setting, this study aimed to obtain exploratory mechanistic insights into acupuncture-related effects relevant to OA pathophysiology.

MATERIALS AND METHODS

1. Study design

In accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines, we conducted a preclinical experimental animal study of the effects of acupuncture on pain thresholds; locomotor activity; and IL-1, IL-6, MMP-13, and cortisol expression levels in a rat model of OA. Healthy adult male Wistar rats (N = 25) aged 6-8 weeks and weighing 200-250 g were housed in pairs under standard laboratory conditions with a 12:12-h light–dark cycle, an ambient temperature of approximately 25℃, and an ad libitum access to food and water. Environmental enrichment was provided through standard bedding and group housing. All animals underwent a 1-week acclimatization period prior to the experimental procedures. Rats were randomly assigned to one of five groups using a simple randomization procedure (n = 5 per group): negative control (K−, saline injection), positive control (K+, MIA-induced OA without treatment), OA with acupuncture at ST36 and SP6 (K + A), OA with acupuncture at ST36 only (K + B), and OA with acupuncture at SP6 only (K + C). Sample size was determined based on previous studies using comparable OA models and outcome measures, following ethical principles to minimize animal use.

OA-like pathology was induced by intra-articular injection of MIA into the right knee joint of all rats except those in the negative control group. The animals were monitored daily for general health, mobility, and signs of distress, with predefined humane endpoints including severe movement impairment, inability to access food or water, and marked deterioration of health. Acupuncture treatment was administered once daily for seven consecutive days following OA induction. No analgesic agents were provided to avoid interference with pain-related assessments. Pain thresholds and locomotor function were evaluated after completion of the treatment protocol. At the study endpoint, all animals were euthanized by trained personnel via cervical dislocation in accordance with the institutional and national ethical guidelines. Tissue from the knee joints was collected for histological and molecular analyses. IL-1, IL-6, and cortisol levels were measured using enzyme-linked immunosorbent assay (ELISA). MMP-13 expression was assessed by immunohistochemistry. No blinding was implemented during allocation, outcome assessment, or data analysis, which is acknowledged as a methodological limitation.

2. Osteoarthritis induction

OA was induced by intra-articular injection of 2 mg of MIA (Sigma, St. Louis, MO, USA) dissolved in 25 μL of sterile saline into the right patellofemoral joint. Rats were anesthetized with isoflurane, and the right knee was shaved, disinfected, and positioned supine with the hind limb flexed at 90° at the knee. The patellar ligament was palpated, and MIA was injected medially using a 0.5-inch needle. Control rats received 25 μL of sterile saline following the same procedure. OA development was confirmed 7 days after injection by gross anatomical examination of the knee joint [22].

3. Electroacupuncture procedure

Electroacupuncture (EA) stimulation was applied to induce analgesia in the rats with MIA-induced knee OA. No additional analgesics were administered during the experimental period to avoid interference with the pain-related outcome assessments. The treatment was administered unilaterally on the right hind limb using sterile, single-use stainless steel acupuncture needles (0.18 × 7 mm, 0.25 cun [body inch]; Huan Qiu, China) [23]. The acupuncture points used were ST36, located 3.5 mm inferior to the fibular head, and SP6, located 5 mm above the peak of the medial malleolus. The needle was inserted perpendicularly to a depth of approximately 2 mm at each point [20].

EA was administered using a KWD-808 electrical stimulator (China) at alternating frequencies of 2/100 Hz and an intensity of 0.5-1.5 mA. This was gradually adjusted until local muscle twitching was observed. Each session lasted 15 min, with a total of eight daily sessions conducted between 09:00 and 17:00 h (Greenwich Mean Time +7) [23]. During stimulation, the rats were gently restrained, with the needled leg immobilized inside a polyvinyl chloride tube to limit movement. The acupuncture points were located based on established skeletal landmarks that closely resemble those in human anatomy [24].

4. Measurement of the pressure–pain threshold

The pain sensitivity of the right knee joint was evaluated during the fifth week using the pressure–pain threshold (PPT) test following a 1-week adaptation period. PPT was measured using the Randall–Selitto apparatus (Ugo Basile, Varese, Italy) [25]. Rats were placed in a sock-like restrainer with the right hind limb exposed. The rounded tip of the transducer probe was applied laterally to the knee at a pressure rate of 48 g/s. The threshold was defined as the force required to elicit a hind limb withdrawal reflex or vocalization. Three measurements were taken at 3-min intervals, and the average PPT was calculated after excluding the highest and lowest values.

5. Treadmill-based mobility assessment

Locomotor limitations were assessed using a treadmill test conducted in the fifth week following eight daily sessions of electroacupuncture. The test was conducted in all groups, including the negative control, the positive control, and the three treatment groups. Each rat was placed on the treadmill for 10 min, and the number of steps was recorded using a step counter. These measurements were conducted in the morning to minimize stress, with rats tested alternately. None were forced to perform physical activity.

6. Measurement of IL-1 and IL-6 levels in articular cartilage tissue using ELISA

The levels of IL-1 and IL-6 in the articular cartilage tissue samples were measured using commercial rat ELISA kits. These were the Rat IL-1 ELISA Kit (cat. no. E0119Ra) and the Rat IL-6 ELISA Kit (cat. no. E0135Ra), both from BT-LAB (Shanghai, China). The articular cartilage samples were collected from the right knee joint of rats after eight sessions of acupuncture treatment. All assay procedures were conducted following the manufacturer’s protocol.

Briefly, 100 μL of each sample was added to micro-ELISA plate wells and incubated at 37℃ for 90 min. After aspiration, 100 μL of biotinylated detection antibody was added for 1 h. The sample was then washed, and 100-μL of horseradish peroxidase conjugate was added for 30 min. The wells were washed again, and 90 μL of substrate reagent was added for 15 min at 37℃. This reaction was stopped with 50 μL of stop solution. The optical density (OD) was measured at 450 nm using a microplate reader (Biogear), and the IL-1 and IL-6 concentrations were calculated from the OD values.

7. Histopathological and immunohistochemical analysis

MMP-13 expression in the cartilage was assessed by immunohistochemistry. The tissue sections were deparaffinized, rehydrated, and incubated overnight at 4℃ with rabbit polyclonal anti-MMP-13 antibody (bs-10581R, Bioss Antibodies, MA, USA). After washing with phosphate-buffered saline, the sections were incubated for 20 min at 37℃, mounted with Entellan, and air-dried. Histological evaluation was performed using an Olympus BX53 microscope (Tokyo, Japan), beginning at 10× magnification to divide each specimen into four quadrants, followed by 40× magnification to count chondrocytes showing brown immunoreactive staining. The final MMP-13 score was calculated as the mean number of positive cells across the quadrants.

8. Data analysis

Data were analyzed using SPSS version 22 (IBM Corp., Armonk, NY, USA). Normality was assessed using the Shapiro–Wilk test, with p > 0.05 indicating a normal distribution. Homogeneity of variance was evaluated using Levene’s test, with p > 0.05 considered homogeneous. For normally distributed and homogeneous data, one-way analysis of variance was used, followed by Duncan’s post hoc test. Non-normally distributed data were subjected to transformation. If normality was not achieved, the Kruskal–Wallis test was used, followed by pairwise Mann–Whitney U comparisons. To examine the associations between inflammatory and hormonal biomarkers (IL-1, IL-6, MMP-13, and cortisol) and behavioral outcomes (pain threshold after treatment and locomotor activity), two-tailed Pearson correlation analysis was performed. p-values < 0.05 were considered statistically significant.

RESULTS

1. Validation of the MIA-induced osteoarthritis model

After 7 days of OA induction, gross pathological examination of the rats’ knee joints revealed thinning and discoloration of the cartilage layer. A reduced number of chondrocytes and increased infiltration of inflammatory cells, including neutrophils and monocytes, were observed. In addition, synovial cell proliferation was observed in the affected joints, and subchondral bone sclerosis and surface erosion were evident, indicating structural alterations following OA induction (Fig. 1).

Figure 1.

Representative hematoxylin and eosin–stained section of a rat knee joint 7 days after monosodium iodoacetate-based induction of osteoarthritis. Histopathological features include cartilage thinning, reduced chondrocyte density, inflammatory cell infiltration, and subchondral bone changes. Scale bar = 4 mm.

2. Effects of acupuncture on pain threshold

Pain threshold measurements were conducted 7 days after OA induction. A paired t-test showed that the negative control group (K−), in which the rats did not undergo OA induction or acupuncture treatment, had the highest mean pain threshold values, measured at 13.27 ± 1.23 g before treatment and 12.60 ± 0.72 g after treatment, with no significant difference (p = 0.106). In contrast, the positive control group (K+), in which the rats underwent OA induction but no acupuncture, exhibited the lowest mean pain threshold values, at 5.17 ± 1.40 g (pretreatment) and 5.33 ± 0.67 g (post-treatment), also with no significant difference (p = 0.824). These findings indicate that, without treatment, MIA-induced OA was associated with sustained reductions in pain thresholds during the observation period (Table 1).

Table 1.

Paired t-test comparison of pressure–pain thresholds (PPT) in healthy controls, an untreated osteoarthritic rat model, and before and after acupuncture treatment in osteoarthritic model rats

Group	Mean analgesy meter measurement		Significance
	Pre	Post
K- (Normal saline injection)	13.27 ± 1.23 g	12.60 ± 0.72 g	0.106
K+ (MIA injection)	5.17 ± 1.40 g	5.33 ± 0.67 g	0.824
K + A (MIA injection + ST36 + SP6 acupuncture)	5.37 ± 0.70 g	10.97 ± 0.61 g	0.000
K + B (MIA injection + ST36 acupuncture)	5.37 ± 0.80 g	11.60 ± 1.55 g	0.007
K + C (MIA injection + SP6 acupuncture)	5.63 ± 0.61 g	9.93 ± 1.91 g	0.018

MIA, monosodium iodoacetate; SP6, spleen 6 acupuncture point; ST36, stomach 36 acupuncture point.

The three experimental groups showed significant increases in their pain thresholds following acupuncture therapy. The PPT of the K + A group increased from 5.37 ± 0.70 g to 10.97 ± 0.61 g (p = 0.000), indicating the most optimal therapeutic effect. The PPT of the K + B group increased from 5.37 ± 0.80 g to 11.60 ± 1.55 g (p = 0.007), and that of the K + C group increased from 5.63 ± 0.61 g to 9.93 ± 1.91 g (p = 0.018). Overall, acupuncture treatment following OA induction was associated with increased pain thresholds (Fig. 2).

Figure 2.

Pressure–pain thresholds before and after acupuncture treatment in healthy controls, an untreated osteoarthritic rat model, and before and after acupuncture treatment in osteoarthritic model rats. Data are presented as mean ± standard deviation. Statistical significance was determined using a one-way analysis of variance followed by Duncan’s post hoc test. p < 0.05 versus the positive control group (K+). K−, negative control; K+, positive control; K + A, ST36 + SP6; K + B, ST36 only; K + C, SP6 only.

3. Effects of acupuncture on locomotor activity

The rats that received acupuncture exhibited higher movement counts than those in the untreated OA group. Among the treatment groups, the K + A group showed the highest locomotor activity, with 1,326.6 ± 131.01 movements per 10 min. The K + B and K + C groups also demonstrated increased locomotor activity, with 1,132.2 ± 129.92 and 1,225.8 ± 104.90 movements per 10 min, respectively, compared with the K+ group (Fig. 3).

Figure 3.

Movement activity during a 10-min observation period in an untreated osteoarthritis rat model, healthy control rats, and three experimental groups of osteoarthritis model rats following acupuncture treatment as an indicator of movement limitation. Data are presented as mean ± standard deviation. Statistical analysis was performed using a one-way analysis of variance followed by Duncan’s multiple range test. Lowercase letters indicate significant differences between groups (p < 0.05). K−, negative control; K+, positive control; K + A, ST36 + SP6; K + B, ST36 only; K + C, SP6 only.

4. Effects of acupuncture on matrix metalloproteinase-13 expression on joint tissue

Immunohistochemical analysis showed that acupuncture was associated with changes in the expression of MMP-13 in joint tissue 7 days after MIA-based induction of OA (Fig. 4). The K+ group exhibited the highest MMP-13 expression at 25.46% ± 0.61%, reflecting increased MMP-13 immunoreactivity following OA induction. The K− group showed the lowest expression at 18.39% ± 0.55%. Acupuncture was associated with reduced MMP-13 expression in all treatment groups. The K + A group showed an expression level of 22.20% ± 0.49%, comparable to that of the K + C group at 22.55% ± 0.42%, both of which were significantly lower than that in the K+ group. The K + B group exhibited slightly higher expression at 23.86% ± 0.60%, although this was still lower than that in the K+ group.

Figure 4.

Matrix metalloproteinase-13 expression in the articular cartilage of an untreated osteoarthritis rat model, healthy control rats, and three experimental groups of osteoarthritis model rats following acupuncture treatment. (A) Representative immunohistochemical staining. The brown stain indicates positive MMP-13 expression (scale bar = 60 μm). (B) Quantitative analysis of the MMP-13-positive area (mean ± standard deviation). Different letters indicate significant differences between groups (p < 0.05). K−, negative control; K+, positive control; K + A, acupuncture to the ST36+SP6 points; K + B, acupuncture to the ST36 point only; acupuncture to the K + C, SP6 point only; MMP-13, matrix metalloproteinase-13.

5. Effect of acupuncture on cortisol levels

Cortisol levels differed significantly between groups following OA induction (Fig. 5). The K+ group exhibited the highest mean cortisol levels (12.09 ± 1.26 ng/mL). In contrast, the acupuncture-treated groups (K + A, K + B, and K + C) showed lower cortisol levels, ranging from 1.38 to 1.85 ng/mL, with no significant differences between the treatment groups. The K− group showed intermediate cortisol levels (6.34 ± 0.81 ng/mL), which were significantly different from those of both the K+ and the acupuncture-treated groups.

Figure 5.

Cortisol levels in an untreated osteoarthritis rat model, healthy control rats, and three experimental groups of osteoarthritis model rats following acupuncture treatment. Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way ANOVA followed by Duncan’s multiple range test. Different letters indicate significant differences between groups (p <0.05). ANOVA, analysis of variance; K−, negative control; K+, positive control; K + A, acupuncture to the ST36 + SP6 points; K + B, acupuncture to the ST36 point only; acupuncture to the K + C, SP6 point only.

6. Effects of acupuncture on joint tissue proinflammatory cytokine levels

The IL-1 and IL-6 levels differed between groups (Fig. 6). The K+ group exhibited the highest IL-1 (7.79 ± 0.55 pg/mL) and IL-6 (7.12 ± 0.66 ng/mL) levels, whereas the K− group showed intermediate levels (IL-1: 3.98 ± 0.33 pg/mL; IL-6: 3.69 ± 0.51 ng/mL). All three acupuncture-treated groups (K + A, K + B, and K + C) showed lower IL-1 (1.65-1.83 pg/mL) and IL-6 (1.63-1.76 ng/mL) levels than the K+ group. No significant differences were observed between the treatment groups.

Figure 6.

Serum levels of interleukin (IL)-1 (A) and IL-6 (B) in an untreated osteoarthritis rat model, healthy control rats, and three experimental groups of osteoarthritis model rats following acupuncture treatment. Data are presented as mean ± standard deviation. Statistical analysis was performed using one-way analysis of variance followed by Duncan’s multiple range test. Different letters indicate significant differences between groups (p < 0.05). K−, negative control; K+, positive control; K + A, acupuncture to the ST36 + SP6 points; K + B, acupuncture to the ST36 point only; acupuncture to the K + C, SP6 point only.

7. Associations between inflammatory markers and behavioral outcomes

Pearson correlation analysis was performed to examine associations between inflammatory and hormonal biomarkers and post-treatment PPT in rats with MIA-induced OA (n = 25). Significant negative correlations were observed between pain thresholds and all measured biomarkers. IL-1 levels showed a moderate negative correlation with PPT (r = −0.526, p = 0.007), whereas IL-6 demonstrated a stronger negative correlation (r = −0.686, p < 0.001). Similarly, MMP-13 expression (r = −0.670, p < 0.001) and cortisol levels (r = −0.645, p < 0.001) were negatively correlated with PPT. These findings indicate that higher levels of inflammation and stress-related biomarker levels are associated with lower pain thresholds following acupuncture treatment of OA (Table 2).

Table 2.

Pearson correlations between inflammatory and hormonal biomarkers and behavioral outcomes in a monosodium iodoacetate-induced osteoarthritis rat model

Biomarker	Pain threshold (r, p)	Locomotor activity (r, p)
IL-1	−0.526, p = 0.007	−0.678, p < 0.001
IL-6	−0.686, p < 0.001	−0.893, p < 0.001
MMP-13	−0.670, p < 0.001	−0.537, p = 0.006
Cortisol	−0.645, p < 0.001	−0.890, p < 0.001

IL, interleukin; MMP-13, matrix metalloproteinase-13; r, Pearson’s correlation coefficient.

Locomotor activity was also significantly negatively correlated with inflammation and stress hormone biomarkers. IL-1 levels were negatively correlated with locomotor activity (r = −0.678, p < 0.001), whereas IL-6 levels showed a very strong negative correlation (r = −0.893, p < 0.001). MMP-13 expression exhibited a moderate negative correlation with locomotor activity (r = −0.537, p = 0.006). Cortisol levels demonstrated a similarly strong negative correlation (r = −0.890, p < 0.001). PPT after treatment was positively correlated with locomotor activity (r = 0.760, p < 0.001), indicating that animals with higher pain thresholds tended to exhibit greater locomotor performance. All correlation coefficients and their corresponding p-values are summarized in Table 2.

DISCUSSION

This study provides preclinical evidence that acupuncture is associated with the modulation of inflammatory mediators in an experimental rat model of OA. OA is a multifactorial disease, in which mechanical loading, joint biomechanics, and joint alignment play critical causal roles. Inflammatory processes are generally considered contributory or downstream components rather than primary drivers of disease progression [26]. Acupuncture treatment was associated with reduced IL-1, IL-6, cortisol, and MMP-13 levels along with improvements in behavioral and functional outcomes related to pain responses and mobility. Correlation analyses demonstrated significant associations between inflammatory and hormonal biomarkers and behavioral outcomes, indicating that changes in these mediators were closely associated with pain sensitivity and locomotor performance. Collectively, our results suggest associations between acupuncture treatment, biomarker modulation, and improved behavioral outcomes in this preclinical OA model. Further studies incorporating pathway-specific analyses are necessary to clarify the molecular mechanisms underlying these observations.

This study also demonstrated that MIA injection reliably induces OA in rats, as evidenced by cartilage thinning, chondrocyte degeneration, and subchondral bone erosion. These histopathological alterations recapitulate key features of OA progression observed in experimental models. They are also consistent with previous findings on MIA-induced joint damage [27, 28]. In MIA-induced OA rats, increased synovial cell proliferation, potentially associated with fibroblast subpopulations such as FAPα+THY1−, indicates active joint remodeling under experimental conditions in the absence of overt inflammation [29]. In this experimental rat model, acupuncture was associated with significant improvements in pain-related responses, as reflected by increased PPT in treated rats. The highest improvement was observed in rats receiving combined ST36 and SP6 stimulation, suggesting a potential synergistic contribution of these acupoints to pain modulation in OA rats. These observations are consistent with those of previous preclinical studies, in which acupuncture at ST36 and SP6 was reported to influence neuroinflammatory and anti-inflammatory responses, although such mechanisms were not evaluated in the present study [30, 31]. Previous preclinical studies have also reported ST36 stimulation to be associated with reduced IL-1β expression and increased IL-10 levels and SP6 stimulation to be associated with the levels of other anti-inflammatory cytokines, including IL-4 and TGF-β [32, 33].

In addition to reductions in pain-related behavior, acupuncture was associated with pronounced changes in locomotor performance in the MIA-induced OA rats. The highest recovery of locomotor function was observed in rats that received combined ST36 and SP6 stimulation, which, in some instances exceeded that of the negative control group under the present experimental conditions. Notably, locomotor activity is a composite behavioral outcome influenced by pain perception, motivation, and stress responsiveness rather than the structural integrity of the joint alone. Therefore, activity levels exceeding those of healthy controls should not be interpreted as functional normalization or supraphysiological recovery. Considering the timing of locomotor assessment relative to the administration of acupuncture, these findings may reflect transient post-treatment effects. Acupuncture-induced peripheral nerve stimulation may have acutely enhanced neuromuscular excitability and motor unit recruitment. This could have resulted in short-term increases in motor output resembling postactivation potentiation-like effects, without sustained changes in muscle or joint function [34]. These observations are consistent with those of previous preclinical studies in which electroacupuncture has been reported to lead to improvements in motor-related outcomes in animal models, potentially through enhanced circulation, reduced muscle stiffness, and neuromuscular modulation [35, 36]. At the molecular level, acupuncture was associated with reduced expression of MMP-13, a key enzyme in cartilage degradation. The SP6 and ST36 + SP6 groups exhibited the most pronounced reductions in MMP-13 expression, suggesting that SP6 stimulation may have contributed to modulation of cartilage catabolic activity in this experimental model. Considering the established role of MMP-13 in cartilage matrix breakdown, these findings support a previously reported association between acupuncture treatment and the attenuation of catabolic processes within the OA joint [37, 38].

In the present study, acupuncture-treated rats exhibited markedly lower circulating cortisol levels than both untreated OA and healthy control rats. In some cases, these reductions extended below the baseline levels observed in untreated control rats, prompting careful consideration of the underlying neuroendocrine context of this finding. Consistent with this result, previous studies have shown that acupuncture or EA can inhibit hypothalamic–pituitary–adrenal (HPA) axis activity and reduce plasma corticosterone levels in rat models of chronic stress and anxiety [39]. However, such reductions should not be interpreted solely as evidence of therapeutic benefit, as cortisol reflects dynamic HPA axis activity and is highly sensitive to experimental context, handling, and sampling timing. Nevertheless, this finding suggests an association with altered HPA axis activity in rats under the present experimental conditions. Previous studies have reported that EA at ST36, which was anatomically mapped from the human ST36 to the rat hind paw for experimental purposes, was associated with the attenuation of stress-induced elevations in HPA axis-related hormones, including adrenocorticotropic hormone and corticosterone. These effects were not observed following sham point stimulation, indicating acupoint-specific responses [40-42]. However, the physiological significance of cortisol reductions in animal models remains to be fully elucidated, and these findings should be interpreted strictly within their preclinical context. In parallel, our acupuncture-treated OA rats exhibited reduced levels of the proinflammatory cytokines IL-1 and IL-6, further supporting an association between acupuncture and the modulation of inflammatory markers in this experimental model. Rather than indicating definitive suppression of inflammatory signaling pathways, these changes suggest a potential shift toward an altered inflammatory profile in OA rats, consistent with previous preclinical studies describing the anti-inflammatory and antioxidative properties of acupuncture in animal models [43, 44].

Despite its strengths, including a well-characterized OA model and multimodal outcome assessment, this study had several limitations. The findings from an animal model may not directly translate to clinical settings because of interspecific physiological differences. In addition, the molecular pathways underlying the observed biomarker changes were not directly examined, as no gene expression or pathway-specific analyses were performed. Furthermore, MMP-13 expression was assessed solely by immunohistochemistry. Although this approach enabled the localization and semiquantitative evaluation of protein expression in joint tissue, we did not perform complementary molecular analyses such as western blotting or quantitative polymerase chain reaction, which may have provided additional validation of our findings. Moreover, we did not assess key biomechanical factors that are known to play dominant causal roles in OA, such as joint loading, joint alignment, and compartment-specific stress distribution. Finally, the relatively small sample size (n = 5 per group) may limit the statistical power of our analyses and the robustness of the observed associations. Therefore, our findings should be interpreted cautiously and considered exploratory and hypothesis-generating rather than indicative of causal relationships.

Additional methodological limitations should also be acknowledged. We did not assess endogenous opioid activity, which is widely implicated in acupuncture-induced analgesia and conducted no pharmacological blockade experiments using opioid antagonists such as naloxone. Consequently, the causal relationships between changes in inflammatory and neuroendocrine markers and the observed analgesic effects could not be definitively established. The observed immunoendocrine alterations may represent secondary responses to opioid-mediated analgesia rather than primary mechanistic drivers. Furthermore, we did not assess outcomes under blinded conditions, which may have introduced observer bias, particularly in our behavioral measurements. Finally, the physiological significance of the marked cortisol reduction observed in this study warrants further investigation.

CONCLUSION

This preclinical study demonstrated that stimulation of the ST36 and SP6 acupuncture points, both alone and in combination, is associated with improved pain-related responses and locomotor activity in a rat model of OA. This is accompanied by changes in inflammatory and neuroendocrine marker levels. These findings provide exploratory evidence linking biomarker alterations to functional outcomes in an experimental context.