Acupuncture Exerts Antidepressant Effects in Rats of Chronic Unpredictable Mild Stress: The Involvement of Inflammation in Amygdala and Brain-Spleen Axis

doi:10.21203/rs.3.rs-1630267/v1

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Research Article

Acupuncture Exerts Antidepressant Effects in Rats of Chronic Unpredictable Mild Stress: The Involvement of Inflammation in Amygdala and Brain-Spleen Axis

https://doi.org/10.21203/rs.3.rs-1630267/v1

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Background

Acupuncture has shown antidepressant effects in rats with chronic unpredictable mild stress (CUMS). However, the mechanisms of antidepressant effects of acupuncture still need to be explored. In the study, acupuncture was applied to a rat depression model of CUMS, high-mobility group box 1(HMGB1) / toll-like receptor 4 (TLR4) and brain-spleen axis were assessed.

Methods

Male Sprague Dawley (SD) rats were exposed to CUMS with two stressors per day for 28 days. In the meantime, manual acupuncture (at GV16 and GV23 acupoints, once every other day) and fluoxetine gavage (2.1 mg/kg, 0.21 mg/mL) were administered daily post-CUMS stressors. Behavioral tests and biological detection methods were conducted in sequence to evaluate depression-like phenotype in rats.

Results

The results showed CUMS induced depression-like behaviors, hyper-activation of HMGB1/TLR4 signaling pathway, elevated inflammation in amygdala and peripheral blood, and hyperactivation of hypothalamic-pituitary-adrenal (HPA) axis. These changes could be reversed by acupuncture to varying extents.

Conclusion

Acupuncture ameliorated depression-like symptoms induced by CUMS, possibly via regulating inflammation mediated by HMGB1/TLR4 signaling pathway in amygdala and peripheral blood circulation, thus improving behavioral outcomes through brain-spleen axis and HPA axis regulation.

Acupuncture

Depression

Inflammation

Amygdala

Brain-spleen axis

HMGB1/ TLR4 signaling pathway

Systemic immunity

Depression is an emotional disorder mainly with significant and persistent depressive mood and pleasure deficiency(1). The disease has posed a threat to human health with high prevalence, high recurrence rate and high disability rate(2).

Currently, selective serotonin reuptake inhibitors (SSRIs), particularly fluoxetine, provide better tolerance in patients compared to other antidepressant drugs (ADDs) and are commonly used to treat depression(3, 4). However, SSRIs have been questioned due to many adverse effects, like headache, dizziness, tremor and sexual dysfunction(5, 6), inefficacy in about 50% of patients and heavy financial burden during longtime treatment(7–10). Given this, increasing number of patients are unwilling to take ADDs for treatment. Therefore, natural alternative therapies, such as acupuncture, have been increasingly accepted. To have acupuncture function adequately, there is a need to further explore the neurobiological underpinnings of acupuncture on depression.

The amygdala plays a critical role in regulating emotional behaviors such as depression(11), learning ability and cognitive function(12, 13). The spleen is a secondary lymphoid organ that plays an important role in generating immune responses. Interestingly, there exists a neuronal pathway, i.e., brain-spleen axis, connecting corticotropin-releasing-hormone (CRH)-expressing neurons in the central amygdala (CeA) and paraventricular nucleus (PVN) of the hypothalamus with the splenic nerve in the spleen(14). It is hypothesized that the brain-spleen axis is an emerging integrative system involved in depression via modulating systemic immunity(14, 15). Additionally, the hypothalamic-pituitary-adrenal (HPA) axis is the primary neuroendocrine hormonal pathway by which the central nervous system (CNS) regulates the immune system(16). Since the underlying initiation and development mechanisms of depression has not been elucidated so far, these studies may shed some light on it.

Increasing number of evidences indicate that immune responses, with inflammation and neuroinflammation included, directly affects neurodegeneration in CNS, which is closely related to the development of brain and psychiatric disorders(17, 18). High-mobility group box 1 (HMGB1) is critical in inducing and promoting the occurrence of inflammation and neuroinflammation(19, 20). Studies indicate that HMGB1 can be up-regulated when stressed, accompanied with the increasing expression of its receptor in toll-like receptor 4 (TLR4)(21, 22). TLR4 is an immune regulator of neuroimmune and neuroendocrine interactions in stress and depression(23). After HMGB1 binds TLR4, inflammatory responses in the brain and periphery are induced(24–26).

Acupuncture is an effective alternative therapy for depression, with fewer adverse effects and lower cost. Manual acupuncture combined SSRIs treatment was reported to ameliorate symptoms and quality of life for patients with moderate to severe depression(27). Some researchers found that acupuncture could maintain internal homeostasis through acupuncture at normal rats(28). Additionally, acupuncture has a general lower risk of adverse effect than that in ADDs(29). Though acupuncture plays a certain role in antidepressant, what is to be further studied is how acupuncture works mechanistically(30), especially how acupuncture affects inflammation, brain-spleen axis and HPA axis. Previous studies show that acupuncture corrects pro-inflammatory factors in specific brain regions of rats induced by CUMS as fluoxetine did(31). Further, electroacupuncture (EA) reverses inflammation and neuroinflammation in rodents’ hippocampi(32), and depresses hyperactivity of HPA axis(33) in CUMS rats. However, there are a few studies focusing on the effects of acupuncture on the brain-spleen axis.

Our previous studies illustrate that acupuncture may reverse depression-like behavior by regulating intestinal microbes, neurotransmitters(34), NO-cGMP signaling pathway in hippocampus and plasma(35), and reducing oxidative stress products via regulating the Nrf2/HO-1 signaling pathway(36). Others have explored the effects of acupuncture on rodents’ brain regions associated with depression other than the hippocampus, such as the cingulate cortex, motor cortex, insular cortex, thalamus, and hypothalamus(30, 37). However, inflammation in amygdala, brain-spleen axis, and the relationship among them in the development of depression have not been clearly elucidated. Further, it is still unknown how acupuncture works on depression via the above possible mechanism.

Herein, we investigated the effects of simultaneous stimulation of acupuncture at Fengfu (GV16) and Shangxing (GV23) acupoints in rats exposed to CUMS stressors on HMGB1-induced inflammation in the amygdala and peripheral blood. This research provides insight into the integrative biology between the HPA axis and the brain-spleen axis.

Animals

Forty specific-pathogen-free (SPF) male Sprague Dawley (SD) rats (130-150g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (qualified No. SCXK 2017-0005, Shanghai, China), and raised in the Animal Laboratory of Medical College of Xiamen University with a SPF laboratory environment. They were housed in plastic cages (3 or 4 rats per cage) with dry corncob padding with temperature of 23±2°C and relative humidity of 65±5%, exposed to light from 08:00 to 20:00 daily, and given free access to adequate water and food (except when indicated). All experiments and protocols complied with international animal experimental ethics and requirements, and were approved by the Animal Ethics Committee of Xiamen University (license No. XMULAC20210062).

Groups

All animals were allowed to adapt to the new environment for 7 days before the conduction of formal experiments. Then rats were weighed and 36 of 40 rats were selected to ensure a consistent baseline. Afterwards, the 36 rats were randomly divided into four groups (9 rats in each group): control group (CON), chronic unpredictable mild stress (CUMS) group (CUMS), acupuncture + CUMS group (AP), and fluoxetine + CUMS group (FX). Over the next 28 days, except for rats in the control group, fed as adaptive feeding, other rats were performed with CUMS (Supplementary Table 1), and the latter two groups were treated with acupuncture (20 min per time, once every other day) and intragastric fluoxetine (once daily) respectively between 14:00 and 15:00 p.m. when CUMS procedure finished(38). See the Figure 1A for all specific procedures.

Chronic unpredictable mild stress model procedure

The CUMS protocol, originally developed by Paul Willner, has been a widely used, reliable, and effective rodent model of depression(39, 40). The protocols were selectively referred to the study of Franklin and her colleagues(25) with minor changes. Rats in CUMS, AP and FX groups were all exposed to CUMS protocols that consisted of a random combination of two stressors per day lasting for 28 days when the same stressors not used again in 3 days (Supplementary Table 1). To ensure the unpredictability of molding, the same stressor did not appear continuously. Survival was closely monitored throughout the procedure.

Intervention procedure

Manual acupuncture

Manual acupuncture was administered at the Fengfu (GV16) and Shangxing (GV23) acupoints with location referring to Experimental Acupuncture Science(41) and shown in Figure 1B. Fengfu lies in the concave joint of the pillow bone top at the back of the pillow bone, i.e. the middle point of the back edge of both ears. Shangxing is located at the midpoint of the Baihui (GV20, the intersecting point of the tip of the two ears and anterior midline) and the Yintang (GV29, at the midpoint between the two medial ends of the eyebrow). Disposable aseptic steel filiform needles (0.25 mm diameter, 13 mm length; Hanyi, Changchun Aikang Medical Instrument Co., Ltd) were inserted into the two acupoints of the skin, with an oblique angle (30°) towards apex nasi of each rat, and the depth of 5 mm, and were twisted at a frequency of 100 times/min and an amplitude of -90 to 90 degree for 5 s. To keep the filiform needles in right location, we prepared an auxiliary needling cloth headset for rat (patent No. ZL2019224218143) avoiding rats’ tempering in advance (Figure 1B) and then kept it on rats’ heads for 20 min. The needles were twisted another 5 s at same frequency and amplitude as mentioned above. Acupuncture procedure was conducted in each rat once every other day for 28 days.

Fluoxetine gavage

Rats in FX group were intragastrically administered with fluoxetine (2.1 mg/kg with the concentration of 0.21 mg/mL distilled water; PHR1394-1G, Sigma-Aldrich, USA) at a volume of 10 mL/kg once a day for 28 days(34).

Behavioral testing

Rats’ body weight assessing their appetite and nutritional status were measured on days 0, 7, 14, 21, and 28. All rats were subjected to behavioral tests on days 29 (OFT), 30 (EPM), 31 (FST), and 32-34 (SPT) after molding and intervention procedure.

Open field test (OFT)

The OFT is designed for the evaluation of spontaneous locomotor activity in rodents(42). A black and brightly lit box (100 cm × 100 cm × 40 cm, length × width × height) with the bottom equally divided into sixteen grids (four grids in the center called the central zone, surrounded by twelve peripheral zones, allowing to evaluate the exploration of internal and external area) was prepared. Firstly, each rat was gently placed into the center for 5 min when the spontaneous moving of rats was videotaped by a tracking system (Smart 3.0). Then the number of grids crossed for the assessment of rats’ locomotor activity(43) (forelegs and one hind leg of an animal in one grid for one crossing to be accounted) as well as defecation times evaluating the level of depression and anxiety(44) were written down by two observers blind to grouping. Lastly, each rat was put back its home cage and the box was thoroughly cleaned with 75% ethanol and kept dry between trials.

Elevated plus maze test (EPM)

The EPM is originally developed to evaluate anxiety-like behavior of rodents(45). The test was performed in a black apparatus consisted of two opposite open arms (50 cm × 10 cm), a central area (10 cm × 10 cm) and two opposite closed arms (50 cm × 10 cm × 40 cm), elevated 60 cm from the ground. Based on rodents’ exploratory nature(46), animals were placed in the central area where the arms crossed, facing the open arms where each rat could see over the edge and allowed to explore for 5 min. Exploratory behavior of each rat was monitored by a high-resolution infrared camera, connected with a tracking system (Smart 3.0), above the apparatus. Then time spent in the open and closed arms(47) were measured and each rat was returned back to their home cages. The apparatus was cleaned with 75% ethanol and allowed time to dry prior to the next run.

Forced swimming test (FST)

Immobility in FST reflects behavioral despair in rodent(48). We made minor changes and recorded immobility duration total (%) for detection. Briefly, all rats were placed into a plastic cylinder (diameter = 21 cm, height = 60 cm) filled with freshwater (depth = 45 cm; 23 ± 1℃). Rats’ immobility time was followed and recorded by a numeric tripod-fixed camera connected with a tracking system (Smart 3.0) through a 5 min testing session. The immobility was determined by the absence of movement except slight actions to keep the head above the water and maintain balance. Immobility duration in a total 5 min was calculated.

Sucrose preference test (SPT)

SPT has been widely applied to assess anhedonia and depression(49). The sucrose powder (R015039) was purchased from Rhawn (Yien Chemical Technology Co., Ltd) in Shanghai, China. Firstly, there was a habituation period of 1% sucrose solution for 48 h, when rats were provided with two bottles of 1% sucrose solution (250 g bottle weight, the same below) for the first 24 h, and a bottle of pure water and a bottle of 1% sucrose solution for the second 24 h with exchanging location every 12 h to avoid side-bias. Deprivation of water and food was conducted for 12 h. Afterwards, animals went through formal testing for 12 h(34), during which they were given free access to food and two bottles of different fluid as in the second 24 h and the consumption of pure water and 1% sucrose solution measured by the weight loss of corresponding bottles. Sucrose preference rate of each rat was calculated as follows: sucrose preference rate (%) = sucrose intake/total fluid consumption×100%.

Sample collecting procedure

Rats were euthanized by intraperitoneally injecting 10% chloral hydrate (3 mL/kg)(50) combined with immediate bloodletting to gather amygdaloid nucleus, spleens and blood from the abdominal aorta. Blood samples were collected in blood collection tubes with separating gel and coagulant (YA1271, Solarbio, Beijing, China), allowed to stand at 4℃ for over 4 h and centrifuged at 3000 rpm for 15 min to collect serum. Amygdaloid nucleus was promptly separated from the brain. Spleens were removed, and their weight and length were measured. All samples collected except blood were quickly frozen in liquid nitrogen and then put at -80℃ for subsequent biochemical analysis.

Immunofluorescence (IF)

Brain matrix was cut into 2 mm coronal sections. After fixed in 4% paraformaldehyde, amygdala tissue was embedded in paraffin and sliced into 5-μm-thick sections. Amygdala sections were deparaffinized and rehydrated, then treated with EDTA antigen retrieval buffer (pH 8.0) to retrieve antigen. After that, the following steps were performed: antibody blocking, incubated with primary antibody (HMGB1 antibody, 1:100, GB11103; IBA1 antibody, 1:100, GB13105-1; GFAP antibody, 1:100, GB11096) followed by secondary antibody (Cy3 conjugated Goat Anti-Rabbit IgG (H+L), 1:300, GB21303), nucleus counterstained by DAPI (G1012), incubated with spontaneous fluorescence quenching reagent, and PBS (pH 7.4) washing three times, 5-min each between steps. Lastly, slips were covered with anti-fluorescence solution and observed under a confocal microscope (×200 magnification, Nikon DS-U3, Tokyo, Japan) to search for the presence of immune-positive neurons and collect images. The %area of HMGB1, IBA1 and GFAP (portion of positive area in a total visual area) was calculated using the “area fraction” in Fiji ImageJ software (National Institutes of Health, Bethesda, MD)(51). There were 3 samples in each group for immunofluorescence staining. A total of 3 nonoverlapping visual fields in amygdala were randomly selected in each section respectively. Quantification was carried out by experimenters blinded to the study. Mean value of %area obtained under three different visual fields of one section stood for the %area of the section. The antibodies and reagents used for IF were all purchased from Servicebio company in Wuhan, China.

Western blotting (WB)

Tissue samples (100 mg per rat) were ground and centrifuged for collecting supernatants form tissue homogenates. After that, protein concentration was determined by a BCA protein assay kit (QB214754, Thermo Scientific, MA, USA). Proteins were separated on prepared 10% SDS-PAGE gel, and then separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (K5NA8023B, Amersham, Sweden) by electrophoresis. After transfer, the PVDF membrane was soaked in a blocking reagent containing 5% nonfat dry milk and incubated at a room temperature for 1h, with corresponding primary antibodies (anti-HMGB1, 1: 1000, 10829, Proteintech, Chicago, USA; anti-TLR4, 1: 1000, 19811, Proteintech; anti-NKG2D, 1: 1000, Ab203353, Abcam, Cambridge, USA; anti-β-actin, 1: 1000, 20536, Proteintech) and shaken at 4 ℃ till next day. After washed, the membrane was incubated with HRP-labeled rabbit secondary antibody (1:5000, SA00001, Proteintech) solution at room temperature for 1 h. Washed again, the membrane interacted with enhanced chemiluminescent to develop color, and was exposed and developed in a dark room. Protein band density was quantified by ImageJ software (National Institutes of Health, Bethesda, MD). The gray value of the target strip was divided by the gray value of the internal parameter (β-actin). Lastly, the ratios in other groups were compared with that of control group respectively for normalization and shown in the results. Every sample was detected 3 times, and cropped strips were shown in figures. The original strips were provided as supplementary materials.

Reverse transcription-polymerase chain reaction (RT-PCR)

Before detection, mRNA coding sequences of HMGB1, TLR4 and NKG2D were obtained from Consensus Coding Sequence database of the National Center for Biotechnology Information (NCBI). Based on these sequences, specific primers of proteins were designed as Supplementary Table 2. Firstly, tissue samples (50 mg per rat) were ground, lysed with 1 mL of Trizol reagent for extraction of total RNA under the instructions of RNA extraction kit (RN03, Aidley Biotechnology Co. Ltd, China), including quantifying the concentration of RNA by measuring optical density value (OD value) with ultramicro spectrophotometer (NanoVue Plus, General Electric Company, Boston, USA) and the concentration range was 500-2000 ng/μL. Secondly, 1 μL of RNA was reversely transcribed to cDNA using Oligo (dT). Then 25 ng of the cDNA was used for the preparation and amplification of PCR system according to the instrument of reverse transcription kit (FSQ-101, TOYOBO, Japan). Finally, 1.5 μL of PCR product was detected by electrophoresis (120 V) on 1% agarose gel for 10 min, and the gel was imaged for assessing the integrity of RNA. Temperature cycle was conducted as following conditions: 94 ℃, 5 min; denaturation temperature 94℃, 30 s; annealing temperature 56 ℃, 30 s; extension temperature 72 ℃, 30 s; 72 ℃, 10 min; 4 ℃, 5 min; one cycle includes denaturation, annealing and extension; there were 35 cycles conducted. ImageJ software (National Institutes of Health, Bethesda, MD) was used for gray-scale analysis, where target mRNA result was compared with that of referring β-actin.

Enzyme-linked immunosorbent assay (ELISA)

Serum and supernatants from amygdala homogenates were collected for ELISA. Standard dilution solution of various concentration was prepared previously. All protocols of kit utilization were conducted under guidance of the manufactures of corresponding ELISA kits. Except for TNFα (JL17113, Jianglai biology, Shanghai, China) and CORT (H094, Jiancheng, Nanjing, China) ELISA kit, others (CRP, E-EL-R0506c; IL6, E-EL-R0015c; IL1β, E-EL-R0012c; CRH, E-EL-R0270c; ACTH, E-EL-R0048c) were all products of Elabscience company in Wuhan, China. After that, the OD value of each well was measured at a wavelength of 450 nm in 15 min, and the quantities of serum and supernatants from amygdala homogenates were calculated according to the standard curve.

Statistical analysis

Normality and homogeneity of variance in groups were confirmed before data analysis. One-way analysis of variance (ANOVA) was performed when data confirmed to normality and homogeneity of variance. Brown-Forsythe test and Welch ANOVA tests were applied when the variance was uneven. Nonparametric test (Kruskal-Wallis test) was applied when data did not conform to normal. Dunnett’s method was used for multiple comparisons. Data in figures are expressed as mean ± standard error of the mean (x̄ ± s). A P value <0.05 was of statistical significance. All data were analyzed using GraphPad Prism 8.2.0 (GraphPad software Inc., San Diego, California, USA).

Acupuncture ameliorated depression-like symptoms in CUMS rats.

Behavioral test results were shown in Fig. 1 and Fig. 2. Compared with the control group, our results showed significant reduction in rats’ weight growth (F _{(3.000, 21.32)} = 32.38, P < 0.0001, Fig. 1C and D), sucrose intake (F _{(3.000, 27.43)} = 13.58, P < 0.05, Fig. 2A), number of grids crossed (F _{(3.000, 19.21)} = 12.74, P < 0.001, Fig. 2C) and time in open arms (P < 0.01, Fig. 2G) but more immobility duration total (F _{(3.000, 13.48)} = 10.96, P < 0.01, Fig. 2B), fecal excretion times (F _{(3.000, 23.83)} = 12.47, P < 0.01, Fig. 2F), and time in closed arms (P < 0.001, Fig. 2H) after CUMS. No significance between intervention groups and control one (P > 0.05). The alteration of activity track of rats in OFT and EPM have been also shown in Fig. 2D and E. Whereas, compared with CUMS group, acupuncture group showed more weight gain from the 21st day (F _{(3.000, 21.32)} = 32.38, P < 0.05, Fig. 1C and D), sucrose consumption (F _{(3.000, 27.43)} = 13.58, P < 0.01, Fig. 2A), grids crossed(F _{(3.000, 19.21)} = 12.74, P < 0.01, Fig. 2C), and time in open arms (P < 0.001, Fig. 2G), but less immobility duration total (F _{(3.000, 13.48)} = 10.96, P < 0.05, Fig. 2B), fecal excretion times (F _{(3.000, 23.83)} = 12.47, P < 0.001, Fig. 2F) and time in closed arms (P < 0.01, Fig. 2H). There was no statistical significance between groups of acupuncture and fluoxetine (P > 0.05). The above indicated a beneficial effect of acupuncture on depression-like behavior of CUMS rats as that of fluoxetine.

Acupuncture depressed activation of HMGB1/ TLR4 signaling pathway in amygdala of CUMS rats.

HMGB1/ TLR4 signaling pathway in amygdala were mainly detected with WB analyses and RT-PCR as illustrated in Fig. 3 and Supplementary Table 3. HMGB1 was also visualized by IF. Overall, compared with the control, protein and mRNA concentrations of HMGB1(F _{(3.000, 7.019)} = 8.568, P < 0.05, Fig. 3B; F _{(3.000, 6.591)} = 10.36, P < 0.01, Fig. 3E) and TLR4 (F _{(3.000, 4.575)} = 5.003, P = 0.0678, Fig. 3C; P < 0.05, Fig. 3F) in amygdala were increased in CUMS group. There was no statistical significance between intervention groups and control group (P > 0.05). These increased levels in the pathway were reduced respectively by acupuncture to varying extents: protein and mRNA levels of HMGB1(F _{(3.000, 7.019)} = 8.568, P = 0.0844, Fig. 3B; F _{(3.000, 6.591)} = 10.36, P = 0.0814, Fig. 3E) and TLR4 (F _{(3.000, 4.575)} = 5.003, P = 0.2980, Fig. 3C; P = 0.6388, Fig. 3F). In addition, the %area of HMGB1 was increased visibly (F _{(3.000, 4.901)} = 15.54, P = 0.0541, Fig. 3G and H) after stress but reduced by acupuncture (F _{(3.000, 4.901)} = 15.54, P < 0.05, Fig. 3G and H). All the above indexes had no statistical significance between acupuncture groups and fluoxetine groups (P > 0.05). Though the effect of acupuncture on TLR4 was light, there existed a trend of alleviating inflammation via HMGB1/ TLR4 signaling pathway under acupuncture intervention in general.

Acupuncture reversed structural alteration and elevated inflammation in spleens of CUMS rats.

We measured the length and weight of spleens in rats in Fig. 4A-C. Rats under CUMS condition had shorter (F _{(3.000, 14.16)} = 6.393, P < 0.01, Fig. 4C) and lighter (F _{(3.000, 13.35)} = 4.284, P = 0.0662, Fig. 4B) spleens than they in CON. Acupuncture could improve the length (F _{(3.000, 14.16)} = 6.393, P = 0.2586, Fig. 4C) and weight (F _{(3.000, 13.35)} = 4.284, P = 0.1115, Fig. 4B) of spleens marginally. No significance was observed between groups of intervention and control (P > 0.05), or between groups of acupuncture and fluoxetine (P > 0.05).

Further investigations of HMGB1/TLR4 pathway and pro-inflammatory factors in spleens were measured with WB (Fig. 4D-H and Supplementary Table 3). Compared with the control, the protein concentration of HMGB1(F _{(3, 8)} = 9.370, P < 0.01, Fig. 4E), TLR4 (F _(3,8) = 11.56, P < 0.001, Fig. 4G), IL1β (F _{(3, 8)} = 10.77, P < 0.01, Fig. 4H) and TNFα (F _(3,8) = 10.11, P < 0.01, Fig. 4F) were significantly elevated in CUMS group. No statistical significances between intervention groups and control group were observed (P > 0.05). These increased levels were reduced respectively by acupuncture: HMGB1 (F _{(3, 8)} = 9.370, P < 0.05, Fig. 4E), TLR4 (F _(3,8) = 11.56, P < 0.05, Fig. 4G), IL1β (F _{(3, 8)} = 10.77, P = 0.0549, Fig. 4H) and TNFα (F _(3,8) = 10.11, P < 0.05, Fig. 4F). There was no significance between groups of acupuncture and fluoxetine (P > 0.05). These indicated acupuncture could affect spleens both functionally and structurally, similarly fluoxetine.

Acupuncture decreased pro-inflammatory factors in amygdala and serum of CUMS rats.

To explore whether acupuncture reduced inflammation, pro-inflammatory factors, such as IL6, IL1β, TNFα and CRP in amygdala and serum were detected with ELISA. As shown in Fig. 5A-H, CUMS markedly increased the levels of CRP (F _{(3.000, 14.56)} = 6.496, P < 0.05, Fig. 5A; F _{(3.000, 19.68)} = 10.27, P < 0.01, Fig. 5E), IL6 (6; F _{(3.000, 19.01)} = 14.16, P < 0.001, Fig. 5B; F _{(3.000, 13.53)} = 16.69, P < 0.01, Fig. 5F), TNFα (F _{(3.000, 19.14)} = 13.81, P < 0.001, Fig. 5C; F _{(3.000, 17.00)} = 23.93, P < 0.001, Fig. 5G) and IL1β (F _{(3, 20)} = 7.590, P < 0.001, Fig. 5D; P < 0.001, Fig. 5H) in amygdala and serum. Acupuncture could significantly decrease the level of IL6 (F _{(3.000, 19.01)} = 14.16, P < 0.01, Fig. 5B), TNFα (F _{(3.000, 19.14)} = 13.81, P < 0.05, Fig. 5C) and IL1β (F _{(3, 20)} = 7.590, P < 0.05, Fig. 5D) in amygdala, CRP (F _{(3.000, 19.68)} = 10.27, P < 0.01, Fig. 5E), IL6 (F _{(3.000, 13.53)} = 16.69, P < 0.05, Fig. 5F), TNFα (F _{(3.000, 17.00)} = 23.93, P < 0.01, Fig. 5G) and IL1β(P < 0.05, Fig. 5H) in serum. Acupuncture did not affect CRP levels (F _{(3.000, 14.56)} = 6.496, P = 0.1435, Fig. 5A) in amygdala. There was no significance between the groups of control and intervention (P > 0.05), or between the groups of acupuncture and fluoxetine (P > 0.05). These alterations indicated that acupuncture reduced inflammatory environments in peripheral blood and amygdala, similar to fluoxetine positive controls.

Acupuncture depressed the hyperactivation of HPA axis in CUMS rats.

We also detected the concentrations of CRH, ACTH and CORT using ELISA to test the activation of HPA axis. In Fig. 6A-F, the expression of CRH (F _{(3.000, 18.83)} = 7.954, P = 0.0517, Fig. 6A; F _{(3, 20)} = 9.871, P < 0.001, Fig. 6D), ACTH (F _{(3, 20)} = 2.591, P < 0.05, Fig. 6B; F _{(3, 20)} = 8.190, P < 0.001, Fig. 6E) and CORT (F _{(3.000, 15.81)} = 7.659, P < 0.01, Fig. 6C; F _{(3, 20)} = 9.995, P < 0.001, Fig. 6F) in amygdala and serum were elevated in CUMS rats. There was no significance between control groups and two intervention groups (P > 0.05). Interestingly, acupuncture could reduce the level of CRH (F _{(3.000, 18.83)} = 7.954, P < 0.01, Fig. 6A; F _{(3, 20)} = 9.871, P < 0.01, Fig. 6D) in amygdala and serum, ACTH (F _{(3, 20)} = 8.190, P < 0.05, Fig. 6E) and CORT (F _{(3, 20)} = 9.995, P < 0.01, Fig. 6F) in serum and CORT (F _{(3.000, 15.81)} = 7.659, P = 0.0623, Fig. 6C) in amygdala. No significance between acupuncture group and fluoxetine group could be seen from the above indicators (P > 0.05). These indicated that acupuncture depressed the hyperactivation of HPA axis of CUMS rats as fluoxetine did.

Acupuncture changed the activation of microglia and astrocytes in amygdala of CUMS rats.

To visualize the expression of IBA1 and GFAP in amygdala, immunofluorescence was performed. As Fig. 7 and Supplementary Table 3 show, compared with the control group, the %area of IBA1 and GFAP in the model group were (F _{(3, 8)} = 2.808, P = 0.5632, Fig. 7C) and (F _{(3, 8)} = 2.380, P = 0.2659, Fig. 7D); compared with the model group, the %area of IBA1 and GFAP in AP group were P = 0.2084 and P = 0.1435, respectively. There existed the same effect between groups of acupuncture and fluoxetine (P > 0.05).

Acupuncture ameliorated immunity depression in spleens of CUMS rats.

To probe into the effects of acupuncture on immune response in spleen, the concentration of NKG2D was detected using WB and RT-PCR as demonstrated in Fig. 8 and Supplementary Table 3. As we can observe, when compared with the control, the expression of NKG2D protein (F _{(3.000, 4.674)} = 17.04, P < 0.05, Fig. 8C) and its mRNA (P < 0.05, Fig. 8D) were both obviously decreased by CUMS. Acupuncture slightly reversed the increased protein level of NKG2D (F _{(3.000, 4.674)} = 17.04, P = 0.0865, Fig. 8C), but did not affect its mRNA (P = 0.5227, Fig. 8D). No significances between groups of acupuncture and fluoxetine (P > 0.05) were observed.

We conducted a model of depression induced by 28-days of CUMS stressors in rats to investigate the mechanisms involved. Our results indicated acupuncture at GV16 and GV23 acupoints could reverse inflammation and depression-like behaviors.

Acupuncture has been used for the treatment of neuropsychiatric disorders, such as depression. GV16 and GV23 are two of acupoints(52), lying in Governor Vessel (GV) which is capable of gathering and conveying energy to brain for energizing the whole body(53). Additionally, they are all on the top, available of adjusting brain function(54). Some researchers reviewed a high use frequency of the two points in clinical trials(55). In the theory of traditional Chinese medicine, there exists synergy and complementarity in simultaneous stimulation at different acupoints, which can also enhance safety and curative effect(56). Our previous studies found that acupuncture at an acupoint combination of GV23 and PC7 or a combination of GV16 and GV23, exerts antidepressant effect in CUMS rats(34–36). Accordingly, GV16 and GV23 were selected and applied in this study. Fluoxetine exerted anti-inflammatory and antioxidant effect whether in patients with depression(57) or in rats of chronic social isolation model of depression(58). Given these, we took fluoxetine as a positive control to better investigate antidepressant mechanisms of acupuncture. The results showed that acupuncture at GV16 and GV23 effectively reversed depression-like phenotypes in CUMS rats as fluoxetine did. Considering that there was no significant difference between groups of control and control + fluoxetine or between groups of control and control + acupuncture based on previous work of us and other researchers(59), neither a control + acupuncture nor a control + fluoxetine was performed in these studies following the 3R principle of animal welfare.

Previous reports indicate depression-like behaviors and pro-inflammatory cytokines are observed in rodents under chronic stress, which is consistent with elevated serum inflammatory factors in patients with depressive disorders(60–62). The CUMS model is a popular and reliable rodent model of depression(63). Core symptoms of depression induced by the CUMS model, were detected by OFT, EPM, FST and SPT. Our study showed increasing levels in immobility duration, defecation times and time in closed arms but decreased weight growth, number of grids crossed, sucrose preference rate and time in open arms in CUMS rats. These indicated that CUMS induced poor autonomy ability, strong anhedonia, despair and depression in rats. These changes were all improved significantly by acupuncture as fluoxetine did. Therefore, acupuncture might begin to work from the 21st day of administration based on body weight result. This may prove that there is a gradually ameliorating effect of acupuncture on depression-like behaviors, and that a longer timepoint of intervention of acupuncture may be required to treat depression clinically.

Neurobiological abnormalities play essential roles in modulating depression, including alterations in inflammation, the HPA axis, neurogenesis, monoaminergic systems and interaction among them(64, 65). Neurons expressing CRH in CeA and PVN of the hypothalamus end in the splenic nerve of the spleen. Interestingly, when CRH-expressing neurons are activated in the amygdala the splenic nerve could also be activated, stimulating the formation of antibody producing plasma cell differentiation(14). These studies suggest a possible pathway between brain-spleen axis and humoral immunity(14, 66). Amygdala, a key brain region associated with emotion, learning and memory, is closely related to the development of depression(12). Therefore, amygdala was selected as the target brain region in the study to explore the inflammation induction in amygdala and peripheral blood.

HMGB1, a nucleoprotein for DNA replication, transcription and repair, is released into cytoplasm and involves in the initiation of inflammation and neuroinflammation under stress condition(67). HMGB1 can silence stress-induced inflammatory diseases after being inhibited(24, 26). HMGB1 also plays a role as a damage associated molecular pattern (DAMP), capable of binding to its pattern recognition receptors (PRRs) and thus leading to inflammation and neuroinflammation when released(20). TLR4 is one of its PRRs(20). Besides, HMGB1 could promote the action of other DAMPs as an internal cellular stress, thus further increasing inflammation and neuroinflammation(25). Previous studies reported that the expression of HMGB1 and TLR4 were both upregulated in the brains of patients with depression(21) and in stress-induced rats(68). In this study, the expression of HMGB1 and TLR4 were elevated under CUMS in rats’ amygdalae and spleens with downregulating effects of acupuncture.

HMGB1 binding to TLR4 promotes a series of inflammatory response, such as the upregulation of interleukin 1 beta (IL1β) and tumor necrosis factor alpha (TNFα)(24, 26). Similar to our study, inflammation is thought to positively correlate with depression. Particularly the upregulation of pro-inflammatory factors, such as IL1β, IL6, TNFα and C reactive protein (CRP), participate in the occurrence of depression as reported in both clinical(69) and experimental research(70, 71). We observed that the expression of IL1β, IL6, TNFα and CRP increased in the amygdala and serum of rats post CUMS as the previous. Additionally, increased expression of IL1β and TNFα was also observed in rats’ spleens. Yet these changes were all corrected by acupuncture and fluoxetine. Furthermore, pro-inflammatory cytokines tend to activate glial cells to expand inflammation. In the study, ionized calcium binding adaptor molecule 1 (IBA1) and glial fibrillary acidic protein (GFAP) were used as the former(72, 73) to mark the activated phenotype of microglia and astrocytes, respectively. We observed increased trends of %area (the portion of positive area in a total vision) of microglia and astrocytes in model rats, which were downregulated by acupuncture slightly. Although significance was not reached, we suspected, the small sample size might account for it.

PVN, one end of brain-spleen axis, is a critical neuroendocrine organ in the activation of HPA axis(74). Meanwhile, the CRH-expressing neurons in CeA may affect HPA axis and exert their stress capacity by affected CRH. Reports demonstrate increasing mRNA of CRH in amygdala of rats under stress(75), similar to our study. Furthermore, the level of adrenocorticotropic hormone (ACTH) and corticosterone (CORT) in amygdala and serum were detected and are both important components in HPA axis. The HPA axis is a primary hormonal pathway by which the CNS regulates the immune system, through the hormones of the neuroendocrine stress response(16). The activity of HPA axis was reflected by an increased concentration of CORT in serum(76), and sustained release of pro-inflammatory factors may give rise to its expression levels based on previous report(77)and our results. Although there are few reports about ACTH or CORT in amygdala, our report results indicated similar trends of the HPA axis in the amygdala and the peripheral blood, the details of which require further studies. In line with this, some researchers insert ACTH analogues into amygdala and find that (ACTH) directly acts on amygdala neurons and down-regulates the expression of CRH gene(78, 79).

However, it is worth noting that HPA hyperactivation may in turn disrupt the normal functions of the glucocorticoid receptor (GR), and thus increasing the levels of anti-inflammatory cytokines and decreasing the levels of pro-inflammatory cytokines to protect the body from more inflammatory damage(80, 81). This may be why we observed natural killer group 2 member D (NKG2D) reduced expression levels, which is a critical molecule activating peripheral immune response(82, 83). We found that NKG2D acted as a non-specific cytotoxic marker of leukocytes and was reduced in CUMS, suggesting leukocyte subset reduction and potential deficiency. Furthermore, our results indicated that rats under CUMS had shorter and lighter spleens, which might indicate impaired immune function(84). Importantly, acupuncture could reverse these changes. Chronic stress can induce not only HPA axis hyperactivation but also immune suppression(85–87). In addition, the time of collecting samples was 6 days post-CUMS, which is when we observed the hyperactivation of HPA axis(88). This may be due to continuous TLR4 activity post-stress removal, a process that requires further studies(23).

This study indicated that simultaneous stimulation of acupuncture at GV16 and GV23 can exert antidepressant effects mainly by inhibiting inflammation via HMGB1/TLR4 signaling pathway in the brain-spleen axis (Fig. 9). Certainly, there are some shortages in our research, for example the small sample size and exploration of acupuncture on brain-spleen axis is still rudimentary. Nonetheless of these shortcomings, we report CUMS-induced stress in rats results in elevated depressive behaviors and inflammation, that could be successfully treated with acupuncture.

CUMS

chronic unpredictable mild stress

HMGB1

high mobility group box 1

TLR4

toll-like receptor 4

HPA

hypothalamic-pituitary-adrenal

CRH

corticotropin-releasing hormone

ACTH

adrenocorticotropic hormone

CORT

corticosterone

CRP

C reactive protein

IL6

interleukin 6

IL1β

interleukin 1β

TNFα

tumor necrosis factorα

GFAP

glial fibrillary acidic protein

IBA1

ionized calcium binding adaptor molecule 1

NKG2D

natural killer group 2 member D

Governor Vessel

Pericardium Meridian

CeA

central amygdala

PVN

paraventricular nucleus

DAMP

damage associated molecular pattern

PRR

pattern recognition receptor

OFT

open field test

EPM

elevated plus maze test

FST

forced swim test

SPT

sucrose preference test

immunofluorescence

western blotting

RT-PCR

reverse transcription-polymerase chain reaction

ELISA

enzyme-linked immunosorbent assay.

Ethical Approval

and Consent to participate

Consent for publication

Not applicable.

Competing interests

The authors declare that there is no conflict of interest.

Funding

The study was supported by grants from the Shenzhen Science and Technology Program (No. JCYJ20210324121800002).

Authors’ Contributions

Every author participated in the designing, conduction and data collection of the study. WC and YC contributed to the experimental conduction, results interpretation. WC finished manuscript writing. JS and TT performed data analysis. ZH, JL and SY assisted in the experimental conduction. XM, PL, WC and SH made revisions to the manuscript. XM approved the final manuscript.

Acknowledgments

This study was supported by the Shenzhen Science and Technology Program (No. JCYJ20210324121800002) and the Innovation and Entrepreneurship Club of the Department of Traditional Chinese Medicine of Xiamen University.

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No competing interests reported.

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Acupuncture Exerts Antidepressant Effects in Rats of Chronic Unpredictable Mild Stress: The Involvement of Inflammation in Amygdala and Brain-Spleen Axis

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