Effects of manual acupuncture on microglial polarization and the Sirt1/NF-κB pathway in a rat model of post-stroke depression

Abstract

Background:

In the treatment of post-stroke depression (PSD), acupuncture has emerged as a therapeutic option; however, the exact mechanisms underlying its efficacy remain unclear. Prior research suggests acupuncture may improve PSD symptoms by regulating M1/M2 microglial polarization, reducing the release of inflammatory factors and inflammatory-associated damage.

Objective:

The aim of the present study was to explore whether manual acupuncture (MA) can mediate microglial polarization via the silence information regulator/nuclear factor κB (Sirt1/NF-κB) pathway, thereby reducing inflammatory responses in the brains of rats exhibiting PSD and improving depressive behavior.

Methods:

Thirty male rats were divided into a middle cerebral artery occlusion (MCAO) group, PSD group, PSD + MA group, PSD + MA + Sirt1 inhibitor group, and PSD + MA + NS (normal saline) group. Acupuncture was applied 6 days a week for 4 weeks at GV26, GV20 and bilateral PC6 and SP6. In the PSD + MA + Sirt1 inhibitor group, nicotinamide (NAM), a specific inhibitor of Sirt1, was slowly injected into the frontal lobe with a stereotactic injector 1 h before acupuncture. Behavioral tests were conducted after modeling and intervention, including the sucrose preference test (SPT) and open field test (OFT), and body weight was monitored. Following the tests, specimens of the dorsolateral frontal lobe were taken to evaluate molecular markers. Immunofluorescence staining was employed to determine the expression levels of M1 microglial markers CD16/Iba1 and M2 microglial markers CD206/Iba1 in the dorsolateral prefrontal cortex. mRNA expression of CD16/intrinsic nitric oxide synthase (iNOS) and CD206/arginase (Arg)-1 was detected by q-RT-PCR. Western blot analysis was used to quantify protein expression of Sirt1, total p65 and phosphorylated p65 (p-p65) proteins. Interleukin (IL)-1β, IL-6, IL-10 and tumor necrosis factor (TNF)-α levels were quantified using ELISA.

Results:

Depression-like behavior of PSD model rats was reduced by MA. Acupuncture promoted transformation of microglia from M1 to M2 in the prefrontal lobe, thereby decreasing expression of pro-inflammatory factors (IL-1β, IL-6 and TNF-α) and increasing expression of anti-inflammatory IL-10 after PSD. In addition, expression of Sirt1 in the prefrontal lobe increased following MA, while expression of p65/p-p65 was reduced, indicating activation of the Sirt1/NF-κB pathway. These findings suggest that M1 to M2 microglial polarization was closely related to Sirt1/NF-κB pathway activation following MA.

Conclusion:

MA promoted the transformation of microglia from M1 to M2 in the prefrontal lobe of PSD rats, reduces the inflammatory response and improves depression-like behavior in rats. These effects were associated with activation of the Sirt/NF-κB pathway.

Keywords

acupuncture microglial polarization neuroinflammation post-stroke depression Sirt1/NF-κB pathway

Introduction

Stroke is the second leading cause of mortality worldwide,¹ and causes physical impairment and psychological distress among survivors. Post-stroke depression (PSD), a common complication, presents with an indifferent or tired mood and poses diagnostic and therapeutic challenges.² It is associated with higher mortality rates than other stroke-related complications.³ Despite increasing amounts of research into PSD, the underlying pathogenesis remains unclear. Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis is one of the proposed mechanisms,⁴ while a strong inflammatory response in the brain following stroke is also considered to be a major contributing factor.⁵ Changes in neurotransmitters, such as decreased 5-hydroxytryptamine (5-HT) levels,⁶ as well as increased glutamate levels,⁷ have been linked to the onset of PSD.

Acupuncture is a widely used therapeutic modality in China and has shown some promise in treating PSD. A meta-analysis of 17 randomized controlled trials demonstrated that acupuncture may have similar or even superior efficacy than antidepressants, with an arguably better safety profile.⁸ Scalp, ear and periorbital acupuncture have all been reported to be effective at treating depression.⁹ While most studies have attributed the effectiveness of acupuncture to its anti-neuroinflammatory effects, the precise underlying mechanisms remain incompletely understood. Acupuncture can modulate various inflammatory factors such as interleukin (IL)-1β, IL-18, IL-6 and tumor necrosis factor (TNF)-α in the brain and serum, suggesting mitigated inflammation.^10–12 Furthermore, acupuncture exerts bidirectional regulation of microglia and astrocytes, which are critical mediators of inflammation associated with depression.^13,14

The inflammatory environment caused by microglial activation is a significant pathogenic factor in PSD. Patients with depression exhibit high microglial activation in the brain,¹⁵ and the pronounced neuroinflammatory reaction in the brain after a stroke can trigger depression.⁵ Following damage to the central nervous system (CNS), microglia can become activated and differentiate into various phenotypes that play either a pro-inflammatory or anti-inflammatory role.¹⁶ Studies have demonstrated that M1 microglia can elicit pro-inflammatory responses by releasing IL-1, IL-6 and TNF-α, whereas M2 microglia secrete anti-inflammatory cytokines such as IL-4, IL-10 and transforming growth factor (TGF)-β, as well as neurotrophic factors like brain-derived neurotrophic factor (BDNF), which can suppress inflammation and promote tissue repair.¹⁷

Silence information regulator (Sirt)1 is a histone deacetylase that depends on nicotinamide adenine dinucleotide.¹⁸ It plays a role in various signal transduction pathways and participates in neuroprotection, cellular aging and apoptosis, glycolipid metabolism, inflammatory oxidative stress responses and other processes.^19,20 Sirt1 activates the microglial M1/M2 polarization process,²¹ reducing inflammatory reactions in the brain after injury.²² The expression of Sirt1 in the brain of individuals with severe depression is significantly decreased.²³ Due to bidirectional communication between the CNS and peripheral nervous system via cytokines, sensory fibers and sympathetic and parasympathetic nerve pathways, as well as regulation of peripheral immune responses by the hypothalamic-pituitary-adrenal (HPA) axis, immune system activation and inflammation can form a feedback system, thus triggering the development of PSD.²⁴ Sirt1 protein is closely related to inflammation.²⁵ Due to its widespread expression in the CNS, activation and upregulation of its expression can regulate the inflammatory immune pathway by deacetylating inflammatory target genes such as nuclear factor (NF)-kB and Forkhead box protein (Fox)O1, thereby attenuating the inflammatory pathway in CNS injury.²⁶ NF-κB is a key mediator of inflammatory response and one of the essential pathways for microglial activation leading to neuroinflammation.²⁷ Sirt1 can act on subunit RelA/p65 of NF-κB, reduce its binding to nuclear inflammatory genes through deacetylation, and reduce the production of inflammatory factors such as TNF-α and IL-1β, resulting in a neuroprotective and antidepressant effect.^28,29 Recent research has found that the Sirt1/NF-κB pathway is related to microglial M1/M2 polarization in ischemic brain tissue.³⁰ Previous studies have shown that electroacupuncture (EA) can attenuate inflammation after ischemic stroke by inhibiting NF-κB-mediated activation of microglia,³¹ and improve cognitive ability in patients with Alzheimer’s disease by regulating microglial M2 polarization.³² However, it remains to be determined whether acupuncture can regulate microglia M1/M2 polarization through the Sirt1/NF-κB pathway, thus potentially reducing the production of inflammatory factors and improving symptoms of PSD. Herein, we aimed to examine the role of Sirt1 in the effects of manual acupuncture (MA) in a rat model of PSD induced by middle cerebral artery occlusion (MCAO) and chronic unpredictable mild stress (CUMS), using the Sirt1-specific inhibitor nicotinamide (NAM).

Methods

Experimental animals

Forty-one male Sprague-Dawley (SD) rats (aged 5–7 weeks and weighing 220 ± 10 g) were obtained from the Experimental Animal Center of the Institute of Radiology, Chinese Academy of Medical Sciences. The rats were housed in a controlled environment at a temperature of 24 ± 1°C and 50% humidity under a 12 h light-dark cycle. Food and water were provided ad libitum. The Experimental Animal Ethics Committee of the Institute of Radiology, Chinese Academy of Medical Sciences, approved the experimental procedures. The study complied with the National Institutes of Health (NIH) Guide for Care and Use of Laboratory Animals (NIH, Bethesda, MD, USA) and is reported in line with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines.

Stroke modeling

The MCAO model was employed to induce ischemic stroke in all 41 rats. Anesthesia was induced with 4% isoflurane in oxygen and maintained with 1.5%–1.8% isoflurane. Following skin preparation, the surgical area was disinfected with povidone-iodine and then de-iodized with alcohol, after which the right cervical muscle was bluntly separated and the right external carotid artery was ligated. A notch was made 5 mm from the bifurcation of the common carotid artery (CCA) and a monofilament nylon thread (RWD, Shenzhen, China) was inserted into the skull through the internal carotid artery (ICA). The nylon filament was carefully advanced into the ICA to occlude the MCA and induce focal cerebral ischemia, after which the ICA was ligated before suturing the neck incision. During the MCAO modeling process, two rats died due to surgical complications, and an additional four rats died within 3 days of the surgery, likely due to severe cerebral ischemia. No rats were excluded for failure to meet the predefined inclusion criteria for successful MCAO induction. Six rats that underwent successful MCAO modeling were randomly selected by drawing lots and formed the MCAO group (no depression), while the remaining rats continued on to PSD modeling.

PSD modeling

One week after the induction of the MCAO model, the depression model was established using CUMS in the other 29 surviving rats. Various forms of stress were randomly arranged daily for 3 weeks, including wet bedding, ice water swimming, tail clipping, water prohibition, tail suspension, black and white inversion, tilting of the cage, fasting and behavioral restriction. Before the CUMS procedure, the sucrose preference test (SPT) and open field test (OFT) were performed to assess baseline behavioral changes. After 3 weeks of CUMS, the SPT and OFT were repeated to evaluate depression-like behaviors. The criteria for successful depression modeling included reduced activity and irritability, with significantly lower SPT and OFT scores compared to the MCAO group. During the CUMS procedure, three rats died due to severe stress-related complications and two rats were excluded due to failure to meet the predefined criteria for successful PSD modeling.

Experimental groups

The 24 surviving, successfully PSD-modeled rats were randomly assigned to one of four additional experimental groups using a computer-generated randomization sequence: (1) PSD group (PSD model rats with no additional treatment); (2) PSD + MA group (PSD model rats treated with MA); (3) PSD + MA + NAM group (PSD model rats treated with MA and Sirt1-specific inhibitor (NAM) in normal saline (NS) vehicle); and (4) PSD + MA + NS group (PSD model rats treated with MA and NS vehicle only). The final analysis included five groups (n = 6 rats per group) with a total of 30 rats completing the experimental protocol.

Acupuncture manipulation

The intervention was started 4 days after the PSD model had been successfully established. Rats in the PSD + MA, PSD + MA + NAM and PSD + MA + NS groups received MA at PC6 (on the medial side of the forelimb, between the ulna and radius, about 3 mm from the wrist joint), GV26 (1 mm below the nose tip, at the middle of the nasolabial groove), SP6 (10 mm straight up the inner ankle from the tip of the hind limb) and GV20 (median parietal bone), located as detailed in the “Atlas of Animal Acupuncture and Moxibustion Points” developed by the Experimental Acupuncture Branch of the Chinese Acupuncture and Moxibustion Society. Needles were inserted: perpendicularly at PC6 to a depth of 1 mm and twisted/lifted for 1 min; perpendicularly/upward at GV26 to a depth of 1 mm and pecked for 1 min; perpendicularly at SP6 to a depth of 5 mm and lifted/inserted for 30 s; and anteriorly at GV20 to a depth of 2 mm and twisted for 1 min. Following manipulation, the needles were retained for 30 min and the procedure was repeated 6 days a week for 4 consecutive weeks. Rats in the MCAO and PSD groups were fed typically and subjected to the same handling/fixation procedures as those in the MA-treated groups but without additional treatment.

Intracerebroventricular infusion

Using a stereotactic injection instrument, rats in the PSD + MA + NAM group underwent administration of the Sirt1-specific inhibitor NAM (1 mg/kg; SN8120; Solarbio, Beijing, China) into the frontal lobe. Under isoflurane anesthesia (as detailed above), the injection was performed 1 h before every other MA session approximately 4.4 mm anterior to and 1.2 mm lateral to the anterior fontanelle and to a depth of 4.5 mm at a rate of 0.5 L/min. The microinjection needle was slowly removed 10 min after the injection and the scalp was sutured. This procedure was performed once every other day, three times a week for 4 weeks. The PSD + MA + NS group was used as a control for this injection and received the same amount of NS (vehicle) only. The injection method and acupuncture parameters were identical to those used in the PSD + MA + NAM group.

Behavioral assessment of depression

To assess depressive behavior in rats across the five groups following the experiment, body weight measurement, SPTs and OFTs were conducted. The SPT was conducted by providing rats with 1% sucrose water and purified water on opposite sides of the cage. The rats were allowed to drink freely and the sucrose and purified water positions were alternated every 12 h. After 48 h of adaptive drinking water, fasting and water prohibition for 24 h, sucrose and purified water were given again. The percentage of sucrose water consumption was calculated as follows: (sucrose water consumption ÷ (sucrose water consumption + purified water consumption)) × 100%. The OFT used an 80 cm × 80 cm × 60 cm open box. The floor of the box was divided into 25 sections with equally sized areas. The rats were allowed to move freely and their horizontal and vertical movements were observed for 5 min. The rats’ horizontal locomotor activity was scored based on the number of times the three feet of the unaffected limbs crossed into adjacent sections, with 1 point assigned for each crossing. This three-foot modification was applied in order to accommodate the characteristics of the stroke model (unilateral limb dysfunction). The vertical activity score was assigned 1 point when the rat’s two front paws left the bottom and fell back.

Immunofluorescence and image analysis

Following the acupuncture intervention and completion of the behavioral assessments, rats from all five groups underwent cervical dislocation under inhalational isoflurane anesthesia (4% induction, 1%–2% maintenance) after ensuring the loss of corneal reflexes. After confirming death by cessation of respiration and cardiac activity, the brain was excised within 2–3 min. The dorsolateral prefrontal cortex (DLPFC) tissue was harvested. Immunofluorescence staining was employed to determine the expression levels of M1 (CD16/Iba1) and M2 (CD206/Iba1) microglia in the dorsolateral prefrontal cortex. Dorsolateral prefrontal tissue was isolated, fixed in a 4% paraformaldehyde solution for 24 h, dehydrated in 30% sucrose + 4% paraformaldehyde for 48 h and then embedded in optical cutting temperature (OCT) compound for continuous coronal sectioning at a thickness of 10 μm. The sections were subjected to antigen retrieval using a sodium citrate solution (10 mM, pH 6.0) and were subsequently sealed then incubated with rabbit primary antibodies against Iba1 (1:600; DF7217, Affinity, Jiangsu, China) plus either CD16 (1:600; 16559-1-AP, Proteintech, Chicago, IL, USA) or CD206 (1:600; DF4149, Affinity) overnight at 4℃. The next day, goat anti-rabbit fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (1:1000; #4412; Cell Signaling Technology, Danvers, MA, USA) were added, and the samples were incubated in the dark at 37℃ for 40 min. Next, 4′,6-diamidino-2-phenylindole (DAPI) was used for nuclear staining and the samples were sealed with an anti-fluorescence quenching agent before being examined under a Nikon Eclipse Ti inverted fluorescence microscope (Nikon, Tokyo, Japan) at 200× magnification. Five high-power fields were randomly selected for imaging. The average optical density (AOD), defined as the integrated optical density (IOD) per unit area (IOD/area), was measured for the target cells using image analysis software and the mean AOD value was calculated for each sample.

Quantitative real-time polymerase chain reaction

mRNA expression of M1 microglial markers CD16 and inducible nitric oxide synthase (iNOS) and M2 microglial markers CD206 and arginase 1 (Arg-1) was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The dorsolateral prefrontal tissue was separated, placed into a 1.5 mL enzyme-free tube, to which was added 1 mL of TRIzol™ reagent (Thermo Fisher Scientific, Waltham, MA, USA), and fully ground with a tissue crusher. After addition of 200 µL chloroform, the tubes were shaken vigorously for 15 s and left at room temperature for 10 min. After centrifugation at 4℃ and 12,000 g for 15 min, 400 µL of the upper aqueous phase was transferred into a new centrifuge tube, to which was added an equal volume of isopropanol (precooled to 4℃), stored at −20℃ for 15 min and then centrifuged at 4℃ and 12,000 g for 10 min. The supernatant was discarded and 1 mL of 75% ethanol (precooled to 4℃) was added to wash the RNA precipitation. A NanoDrop 2000 (Thermo Fisher Scientific) was used to determine the OD value and concentration of the extracted RNA, which was reversed transcribed into cDNA according to the manufacturer’s instructions. Real-time qPCR was performed using the following primers: CD16 forward 5′-TAGGGATAACCAGGCTCTAC-3′ reverse 5′-CGTGGGTGTCTTTTGCTGTA-3′; iNOS forward 5′-ATGGCTCCTTCAAAGAGGCA-3′ reverse 5′-CTATTTCCTTTGTTACGGCTTCCA-3′; CD206 forward 5′-GGTTCCGGTTTGTGGAGCAG-3′ reverse 5′-TCCGTTTGCATTGCCCAGTA-3′; Arg-1 5′-TCCTTAGAGATTATCGGAGCG-3′ reverse 5′-GTCTTTGGCAGATATGCAGG-3′; and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 5′-TTCAGCTCTGGGATGACCTT-3′ reverse 5′-TGCCACTCAGAAGACTGTGG-3′. The PCR amplification conditions were as follows: pre-denaturation at 95℃ for 15 s, 95℃ for 5 s, 60℃ for 20 s, 72℃ for 10 s (for 40 cycles). At the end of the reaction, the 2-ΔΔ CT method was used to analyze the results.

Western blotting

Western blot analysis was used to assess the expression levels of Sirt1, total p65 and phosphorylated p65 (p-p65) proteins in the dorsolateral prefrontal lobe from three animals per group. Dorsolateral prefrontal tissues were isolated and 100 μL/mg tissue lysate (R0020, Solarbio) was added to the samples. The supernatant was collected after homogenization using a tissue crusher and centrifugation at 12,000 g for 20 min. The protein concentration of the sample was determined by constructing a standard protein curve using the bicinchoninic acid (BCA) method. Sodium dodecyl sulfate polycacrylamide gel electrophoresis (SDS-PAGE) gel was prepared and protein samples from each group were loaded into each well for electrophoresis. Proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked in 5% milk at room temperature for 3 h. Subsequently, the membranes were incubated with mouse Sirt1 (1:1000; #8469, Cell Signaling Technology), rabbit p65 (1:1000; #8242, Cell Signaling Technology) and rabbit p-p65 (1:1000; #3033, Cell Signaling Technology) primary antibodies at 4℃ for 12 h, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (1:3000; bs-0295G-HRP, Bioss, Beijing, China) or goat anti-mouse ((1:3000; bs-0296G-HRP, Bioss) immunoglobulin (Ig)G secondary antibodies at 37℃ for 2 h. The PVDF membranes were then washed and exposed to a luminescent solution (P0018M, Beyotime, Shanghai, China). After development, they were dried and imaged using a Bio-Rad ChemiDoxXRS imaging system (Hercules, CA, USA). Grayscale value analysis was carried out using Quantity One software (Bio-Rad) and the relative expression levels of each target protein were calculated with GAPDH (UM4002, Youkang, Tianjin, China) as the internal reference. Expression levels of the MCAO, PSD + MA, PSD + MA + NAM and PSD + MA + NS groups were normalized to the PSD group as a reference.

Enzyme-linked immunosorbent assay

Inflammatory cytokines were quantified using enzyme-linked immunosorbent assay (ELISA) kits for IL-1β (MM-0047R1, Meimian, Jiangsu, China), IL-6 (MM-0190R1, Meimian), IL-10 (MM-0195R1, Meimian) and TNF-α (MM-0180R1, Meimian) according to the manufacturer’s instructions. Prior to ELISA, samples were thawed on ice, homogenized in ice-cold lysis buffer and centrifuged at 12,000 g for 20 min at 4°C. Briefly, chromogenic substrate was added and the absorbance was measured at 450 nm using an enzyme marker. The standard curve was generated, and the sample content was calculated based on regression analysis of the standard curve.

Statistical analysis

Graphpad Prism version 9.4 (La Jolla, CA, USA) was used to analyze the data. Data were summarized as mean ± standard error of the mean (SEM). Groups were compared using one-way analysis of variance (ANOVA) followed by post hoc test of least significant difference for pairwise comparisons. p < 0.05 was considered statistically significant.

Results

Acupuncture alleviated depression in rats

As depicted in Figure 1, the PSD group weighed significantly less than MCAO controls (p < 0.001; Figure 1(a)), exhibited a reduced sucrose-preference index (p < 0.001; Figure 1(b)), and displayed pronounced deficits in open-field locomotion, with decreased horizontal activity (p = 0.0018; Figure 1(c)) and markedly lower vertical activity (p < 0.001; Figure 1(d)). The body weight of rats in the PSD + MA group was significantly greater than that of the PSD group (p < 0.001). The acupuncture intervention effectively reduced sugar preference among rats (Figure 1(b)). In the open-field experiment (Figure 1(c) and (d)), the number of horizontal grid crossings and standing times of rats in the PSD + MA group were significantly greater than those in the PSD group (p < 0.001). Consistent with our hypothesis, the PSD + MA + NAM group exhibited significantly poorer performance in in the SPT and OFT (Figure 1(b)–(d)). However, the body weight of the PSD + MA + NAM group was higher than that of the PSD group, with statistically significant results (p = 0.016), suggesting a complex impact of post-stroke depression on rat body weight.

Acupuncture promoted the transformation of microglia from M1 to M2

Figure 2 shows the results of immunofluorescence double staining for microglia, in which the pan-microglial marker Iba-1, M1 microglial marker CD16 and M2 microglial marker CD206 were used to observe the polarization of microglia. M1 polarization (CD16 AOD) was higher in PSD versus MCAO groups (p < 0.001; Figure 2(a)) and was attenuated in the PSD + MA group relative to the PSD group (p < 0.001). By contrast, M2 polarization (CD206 AOD) was lower in PSD versus MCAO groups (p = 0.0459; Figure 2(b)) but increased in PSD + MA versus PSD and MCAO groups (both p < 0.001), indicating a shift toward an anti-inflammatory phenotype. Other than the effect on the polarization of microglia, acupuncture seemed to have an inhibitory effect on neuroinflammation overall.

Figure 3 shows the results of mRNA detection of M1 microglia markers CD16 and iNOS, as well as M2 microglia markers CD206 and Arg-1, by RT-PCR. M1 markers CD16 and iNOS mRNA were upregulated in the PSD group relative to the MCAO group (p < 0.001 and p < 0.01, respectively) and were both significantly downregulated in PSD + MA versus PSD groups (p < 0.001; Figure 3(a) and (b)). By contrast, the levels of CD206 and Arg1 were significantly increased in the PSD + MA group (p < 0.001; Figure 3(c) and (d)). Meanwhile, CD16 and iNOS mRNA expression in the PSD + MA group was significantly lower than the MCAO group (p < 0.001), and CD206 and Arg1 mRNA expression in the PSD + MA group was significantly improved (p < 0.001). The results demonstrate that stroke induced inflammatory responses in related brain regions and that the onset of PSD was associated with persistent inflammatory injury; importantly, acupuncture was able to reverse this inflammatory response.

Acupuncture inhibited the expression of NF-κB by increasing the activity of Sirt1

Figure 4 shows the results of Western blot analysis to investigate the expression of Sirt1 and subunits p-65 and p-p65 of NF-κB. Our findings indicated that the levels of p65 and p-p65 in the PSD + MA group were significantly decreased compared to the PSD group (p = 0.008 and p < 0.001, respectively; Figure 4(a) and (b)). Moreover, the level of Sirt1 in the PSD + MA group was significantly increased compared to the PSD group (p < 0.001; Figure 4(c)). To determine whether Sirt1 affects the activity of NF-κB, we injected Sirt1 inhibitor into the rats’ brains and our results showed that the regulatory effect of acupuncture was reversed, and the levels of p65 and p-p65 were significantly increased in PSD + MA versus PSD + MA + NAM groups (p = 0.009 and p = 0.010, respectively; Figure 4(a) and (b)). Levels of Sirt1, p65 and p-p65 did not significantly differ between the PSD + MA + NS and PSD + MA groups (p > 0.05). These observations support our hypothesis that acupuncture can up-regulate Sirt1 expression in the brain-related regions, activate the Sirt1/NF-κB pathway, and exert a marked antidepressant and anti-inflammatory effect. Furthermore, we observed that the levels of p65 and p-p65 in the PSD group were significantly higher, and the level of Sirt1 was significantly lower, than the MCAO group (p = 0.0175, p = 0.027 and p = 0.0256, respectively), indicating that post-stroke depression was closely related to the decreased expression of Sirt1 in the brain. In addition, the expression level of p-p65 was higher and that of Sirt1 was lower in MCAO versus PSD + MA groups (p = 0.033 and p = 0.0046, respectively). This suggests that neuroinflammatory reactions are closely related to the occurrence of depression, and the reduction of related molecules after depression will also lead to continuous neuroinflammation.

Acupuncture regulated inflammatory reactions through the Sirt1/NF-κB pathway

Figure 5 shows the expression levels of four inflammatory factors (IL-1β, IL-6, TNF-α and IL-10) as measured by ELISA. Our results indicate that PSD markedly exacerbated neuroinflammation compared with MCAO alone: levels of the pro-inflammatory cytokines IL-1β (p < 0.001; Figure 5(a)), IL-6 (p < 0.001; Figure 5(b)) and TNF-α (p < 0.001; Figure 5(c)) were all significantly elevated in PSD versus MCAO groups. MA robustly suppressed these increases (all p < 0.001 for PSD + MA vs PSD groups) and restored each cytokine toward MCAO values (all p < 0.001 vs PSD group). Co-treatment with the Sirt1 inhibitor nicotinamide fully reversed the anti-inflammatory effects of acupuncture, producing IL-1β, IL-6 and TNF-α levels that were indistinguishable from PSD (all p < 0.001 in (PSD + MA + NAM vs PSD + MA groups). By contrast, injection of vehicle (NS) alone did not alter the suppressive action of MA on any pro-inflammatory cytokine (p > 0.05 for PSD + MA + NS vs PSD + MA groups). Furthermore, the anti-inflammatory cytokine IL-10 was significantly reduced in PSD versus MCAO groups (p < 0.001; Figure 5(d)). Acupuncture restored IL-10 to above baseline (p < 0.001 for PSD + MA vs both PSD and MCAO groups), whereas NAM injection attenuated this effect (p < 0.001 for PSD + MA + NAM vs PSD + MA groups) but still maintained IL-10 above untreated PSD levels (p < 0.001 vs PSD group). This anti-inflammatory effect of acupuncture was reversed following intracerebral injection of a Sirt1 inhibitor (p < 0.001), suggesting that the anti-inflammatory effects of acupuncture at least partially involve the Sirt1/NF-κB pathway.

Blocking Sirt1 reduced the antidepressant and anti-inflammatory effects of acupuncture via the Sirt1/NF-κB pathway

Upon injection of a Sirt1 inhibitor into the brain, the observed antidepressant effect of acupuncture in rats was significantly attenuated. Specifically, the PSD + MA + NAM group demonstrated significantly worse outcomes in terms of body weight (p = 0.015), sugar preference (p < 0.001) and activity frequency (p < 0.001) as compared to the PSD + MA + NS group (Figure 1). Further analysis indicated that the conversion of microglia M1 to M2 was prominently reduced in the PSD + MA + NAM group as compared to the PSD + MA group (all p < 0.001) and PSD + MA + NS group (p < 0.001 for CD16 and p = 0.0015 for CD206; Figure 2). In addition, the expression of mRNA related to microglial polarization was altered (Figure 3). Notably, the anti-inflammatory and antidepressant effects of acupuncture, as measured by the reduction of pro-inflammatory factors and increase in anti-inflammatory factors, were also significantly downregulated after Sirt1 inhibition (all p < 0.0001; Figure 5). These findings suggest that acupuncture exerts its therapeutic effects primarily via the Sirt1/NF-κB pathway to regulate microglia polarization and inflammatory factors.

Discussion

The location of intracranial injury after stroke is closely associated with the occurrence of PSD. Lesions in the left frontal lobe or basal ganglia have a significantly higher probability of leading to depression than lesions in other locations.^33–35 Furthermore, depression severity is positively correlated with the proximity of the lesion margin to the left hemisphere frontal lobe within the first 6 months after stroke.³⁶ Therefore, we selected the left lateral prefrontal lobe as the target area in this study.

The pathogenesis of PSD is related to functional and cognitive impairments caused by the location and severity of brain injury.³⁷ The high expression of pro-inflammatory cytokines at the injury site after stroke can lead to the occurrence of PSD.³⁸ Previous studies have demonstrated the correlation between increased inflammatory factors (such as IL-1β, TNF-α and IL-18) at the injury site and the occurrence of PSD.³⁹

Microglia mediate neuroinflammation in neurodegenerative diseases.¹⁷ After stroke, the disruption of brain homeostasis leads to the activation of microglia,⁴⁰ which polarize into different functional phenotypes. M1 microglia produce pro-inflammatory factors, including TNF-α, IL-1β and IL-6.⁴¹ By contrast, M2 microglia produce angiogenic and anti-inflammatory factors, such as IL-10, transforming growth factor (TGF)-ß, insulin-like growth factor and vascular endothelial growth factor (VEGF).⁴² The percentage of activated microglia in the dorsal anterior cingulate cortex of depression patients is increased.⁴³

Moreover, the depression model extensively activates microglia in the brain and up-regulates pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) in multiple brain regions.⁴⁴ The activation of microglia after stroke injury may induce PSD. Promoting M1/M2 polarization of microglia is essential in regulating depression-related neuroinflammation. A previous study demonstrated that acupuncture can reverse the production of pro-inflammatory factors by M1 microglia and promote the polarization of M1 to M2.⁴⁵ Our research has further demonstrated that acupuncture reduces microglial activation in PSD rats and significantly promotes the conversion of activated M1 microglia to M2.

Sirt1, a class III histone deacetylase (HDAC III) homologous to yeast silencing information regulator 2 (Sir2),⁴⁶ exhibits potent antioxidant and anti-inflammatory effects.^47–49 It has been demonstrated that Sirt1 can reduce the acetylation of histone H3K9 in the promoters of IL-6 and TNF-α and block their expression.⁵⁰ Activation of Sirt1 inhibits neuroinflammation induced by microglial polarization,³⁰ and can down-regulate p65 via deacetylation, thereby inhibiting the expression of inflammatory cytokines mediated by NF-κB.⁵¹ The Sirt1/NF-κB pathway can regulate microglial polarization, reducing cognitive impairment and hippocampal neuroinflammation,⁵² and down-regulating neuroinflammation in depression.⁵³ Prior research has shown that electroacupuncture can inhibit NF-κB-mediated microglial activation and reduce inflammation after ischemic stroke.³¹ The aim of this study was to investigate whether the anti-inflammatory effects of acupuncture are realized through the regulation of microglial polarization via the Sirt1/NF-κB pathway. Our results support this hypothesis, as acupuncture promoted the transformation of M1 microglia into M2, downregulated the expression of pro-inflammatory factors in the prefrontal lobe, and upregulated the expression of anti-inflammatory factors. When Sirt1 expression was inhibited, the anti-inflammatory and antidepressant effects of acupuncture were significantly reversed, and NF-κB inhibition was downregulated, which suggests that acupuncture activated the Sirt1/NF-κB pathway to regulate the polarization of microglia. The selection of traditional acupuncture point locations for this study was taken from part of the “Xingnao Kaiqiao” acupuncture method, which is supported by clinical and mechanistic evidence in the treatment of brain diseases,^54–56 and international standards have been introduced. This combination has also been utilized in previous clinical studies.⁵⁷

Our research is not without limitations. First, we found that, although the anti-inflammatory effect of acupuncture was significantly downregulated after Sirt1 inhibition, it was still substantially higher than that of the PSD group. This indicated that acupuncture intervention may activate other signaling pathways and play a synergistic anti-inflammatory role. The anti-inflammatory effect in the PSD + MA group was significantly better than that of the MCAO group, which supports this assertion. The functionality of other signaling pathways still have research value. Second, the location we observed was the prefrontal lobe of rats, and there is still a relative lack of research on other brain regions such as the hippocampus. Specific injuries in certain areas are still correlated with the occurrence of PSD. The development of a targeted brain injury model may be helpful in expanding our understanding of this aspect.

Conclusion

Acupuncture treatment ameliorated depressive behavior in a rat model of PSD and facilitated the transformation of microglia from M1 to M2 in the prefrontal cortex. This intervention also suppressed microglial activation and decreased the expression of inflammatory mediators following PSD. Furthermore, acupuncture elevated Sirt1 activity in the prefrontal cortex and downregulated the expression of p65 and p-p65. These findings suggest that acupuncture activates the Sirt1/NF-κB pathway to exert its anti-inflammatory and antidepressant effects.

Footnotes

Contributors

ML contributed to the conception and design of this work and took part in the experimental studies. XY, DR and JZ performed the experiments. YW performed the data analysis and statistical analysis. SD and LS performed the literature research. YD and ML guided this study. All authors read and approved the final version of the manuscript accepted for publication.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship and/or publication of this article: This work was supported by the Tianjin Municipal Diversified Investment Fund for Applied Basic Research, Tianjin Municipal Bureau of Science and Technology (grant no. 21JCQNJC01160) and the Tianjin Education Commission Research Program Project (grant no. 2021KJ150).

Ethics statement

The animal study was reviewed and approved by the Experimental Animal Ethics Committee of the Institute of Radiology, Chinese Academy of Medical Sciences (IRM-DWLL-2022216), and complied with the Guidelines for the Care and Use of Experimental Rats.

ORCID iD

Xinming Yang

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