Effects of electroacupuncture at Baihui (GV20) and Yintang (GV29) on endoplasmic reticulum stress in depressive rats caused by chronic unpredictable mild stress

GUO Zhuo (郭卓), WU Yunchu (吳云楚), XU Shuting (徐舒亭)

1 College of Integrated Chinese and Western Medicine of Hebei Medical University, Shijiazhuang 050031, China

2 Shijiazhuang Hospital of Traditional Chinese Medicine, Hebei Province, Shijiazhuang 050011, China

Abstract

Keywords: Acupuncture Therapy; Electroacupuncture; Point, Baihui (GV20); Point, Yintang (GV29); Depression; Endoplasmic Reticulum Stress; Apoptosis; Rats

Depression is a common and serious neuropsychiatric disorder.It is also one of the main causes of the global disease burden that affects more than 300 million people of different ages[1-2].Common symptoms and signs of depression include loss of interest in daily activities, difficulty concentrating and making decisions,fatigue, sleep problems, overeating or anorexia,pessimism, despair, persistent sorrow, and anxiety[3].Untreated depression can lead to suicidal thoughts or suicide attempts.The pathology of depression is not clear.However, major depression disease is considered a multifactorial disease caused by the interaction of social, psychological, and biological aspects.At present,there is no exact pathological theory that can scientifically and independently explain its pathogenesis.

Escitalopram (ESC) is a highly selective serotonin reuptake inhibitor.In the aspect of inhibition of serotonin reuptake, ESC is at least 100 times more active than enantiomers, and its inhibition of serotonin transporters is twice that of citalopram.Because of its remarkable effect and fewer side effects, it is favored by doctors and patients with depression at home and abroad.In addition to a good antidepressant effect, ESC oxalate also has a certain curative effect on generalized anxiety, panic disorder, posttraumatic stress, and cognitive impairment.Therefore, it has become the first-line drug for antidepressant treatment[4-6].

Acupuncture and moxibustion treatment is an important part of traditional Chinese medicine[7].Previous research has shown that electroacupuncture(EA) can effectively stimulate points through electric currents.Such an approach is safe and effective without the risk of addiction, which makes it advantageous for the treatment of depression[8].EA has strong antidepression-like effects[9].In an 8-week controlled clinical trial research, EA, as a treatment for depression, had a better effect on the improvement of anxiety/somatization and feelings of despair than selective serotonin reuptake inhibitors (SSRI)[10].Another clinical research reported that EA and fluoxetine had similar effects on depression[11]; compared with fluoxetine, EA was associated with a faster effect, higher response rate,and greater improvements.It was also reported that EA at Baihui (GV20) could regulate aberrant amygdala networks in patients with depression, providing further imaging evidence to support the regulatory mechanisms of EA on depression[12].Therefore, more researchers proposed that EA treatment could protect the central nervous system and be taken as an alternative for depression[13].

Apoptosis is mediated by different pathways in different environments or stimulation conditions.Over recent years, endoplasmic reticulum stress (ERS) has been identified as a new way to mediate apoptosis.Research suggests that chronic stress can mediate neuronal apoptosis, reduce the number of neurons in the brain through ERS, and then reduce synaptic connections, finally inducing depression-like behaviors[14].Meanwhile, numerous studies have confirmed that the occurrence and development of many diseases are closely related to ERS and apoptosis[14-15].JNK pathway and caspase-12 pathway are two important pathways in ERS-induced apoptosis,and caspase-12 is a unique caspase protein in the endoplasmic reticulum (ER).The hippocampus belongs to the limbic system, closely associated with cognition,learning, feeling, and memory.It is an important brain region that mediates stress responses.It has been found that when rats manifest depression-like behaviors, the number of neuronal apoptosis in the hippocampus CA3 area significantly increases[16].Therefore, it has become a new way to treat depression by promoting hippocampus neuron regeneration and inhibiting neuronal apoptosis.

This study used chronic unpredictable mild stress(CUMS) to establish a depression model and explore whether EA at Baihui (GV20) and Yintang (GV29) can effectively improve depression by inhibiting cell apoptosis by regulating the ERS pathway.

1 Materials and Methods

1.1 Experimental animals

A total of 32 male Sprague-Dawley rats, with a body mass of (180±30) g, were purchased from Hebei Experimental Animal Center [Experimental Certificate No.SCXK(Ji) 2018-004-2011043].Feeding conditions:temperature 23-25 ℃, light/dark cycle 12 h, humidity 40%-60%, free intake of food and water.The implementation of this experiment was in accordance with the requirements of animal ethics and was approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University (Approval No.IACUC-Hebmu-2022033).All operations followed the guidelines for the care and use of laboratory animals advocated by the National Institutes of Health and the“3R” principle of animal experiments with reduction,refinement, and replacement as the core.

1.2 Grouping and intervention

1.2.1 Grouping and modeling

Before treatment, rats were adaptively fed for 1 week.After the open field test (OFT), 32 rats were randomly divided into 4 groups: a control group, a CUMS group,an EA group, and an ESC group.In the control group,rats were group housed in cages (4 rats in each cage).They had free access to water and food for 28 d.No model induction or treatment was performed.The other three groups received daily CUMS stimulation[17].Briefly, rats in the CUMS, ESC, and EA groups were exposed to various mild stressors: swimming at 4 ℃ for 5 min; fasting for 24 h; banning water for 24 h;perversion of light and dark cycle for 24 h; wet pad for 24 h; bound stress for 6 h; clipping tail for 1 min.The stimulus sequence was randomly arranged, and three kinds of stimulations were selected once a day.Any stimulus was used at least one time and no more than 4 times.The intervention lasted for 28 d.The timeline of the experimental procedure is shown in Figure 1.

Figure 1 Schematic diagram of the timeline for CUMS, ESC, and EA intervention and behavioral testing schedule

1.2.2 Drug treatment

Rats in the ESC group were administered escitalopram oxalate (Sichuan Kunlun Pharmaceutical.Co., Ltd., China) 1 h before CUMS stimulation by gavage until the end of the experiment.The drug was freshly prepared escitalopram suspension with distilled water and was administered 10 mg/(kg·bw) daily.Meanwhile,considering the stimulating effect of gavage, the CUMS group and EA group were also treated with gavage of normal saline [10 mg/(kg·bw)] each day.

1.2.3 EA treatment

During EA administration, rats were fixed on the rat fixator.Baihui (GV20) and Yintang (GV29) were selected for EA pretreatment (Figure 2).Baihui (GV20) is located above the apex auriculate, on the midline of the head,and Yintang (GV29) at the midpoint between the two eyes.Sterilized disposable stainless-steel needles(0.20 mm in diameter and 25 mm in length,manufactured by Suzhou Tianxie Acupuncture Instruments Co., Ltd., China) were inserted obliquely as deep as 5 mm for both points.Following the insertion,electrodes were connected to the handle of needles(XS-998B04 EA apparatus manufactured by Nanjing Xiaosong Medical Instrument Research Institute, China)with 1 mA, 2 Hz, and a discontinuous wave.EA pretreatment was applied 1 h before CUMS stimulation,15 min per session, and 1 session daily for 28 d.

1.3 Observation indicators and detection methods

1.3.1 Body mass

All rats’ body mass was measured on day 0 and day 28 of the experiment.

1.3.2 OFT

Rats were individually placed in the center of an open field apparatus (50 cm × 50 cm × 50 cm, Gene&I, China),which was divided into 4×4 equal squares.A single rat was gently placed in the center of the floor and allowed to familiarize itself with the apparatus for 3 min.The sessions were recorded using an overhead digital video camera.The total distance, duration, central area distance, and duration of central area were analyzed by TopScan v2.0 software (Clever Sys, Incorporated, USA).

1.3.3 Sucrose preference test (SPT)

Before the test, all rats were trained to adapt to drinking water containing 1% sucrose.The SPT was carried out on day 0 and day 28[18]to calculate the sucrose preference rate of the rats.Sucrose preference rate = Sucrose consumption ÷ Total liquid consumption × 100%.

1.3.4 Western blotting (WB)-stain-free blot

After the OFT, the hypothalamus tissues of rats were weighed and operated on ice.RIPA lysate was added at a ratio of 200 μL of lysate per 20 mg tissue.1% PMSF solvent (1 mol/L) was added within a few minutes of the lysate addition.After fully grinding and splitting, the regimen was centrifuged at 10 000 r/min for 5 min.The supernatant was taken and the protein was quantified using the BCA method.SDS-polyacrylamide gels electrophoresis (TGX and TGX Stain-Free FastCast acrylamide kits, Bio-Rad, USA) was performed, and PVDF membrane was transferred.Afterward, the total protein image was taken with an imager.The PVDF membrane was then put into a fast-sealing solution,incubated at room temperature for 15-30 min, and rinsed with TBST 3 times, 15 min each time.Primary antibody: GADD153 polyclonal antibody (1:50, Item No.15204-1-AP, Proteintech, USA), anti-caspase-12 antibody (1:5 000, Item No.ab62484, Abcam, UK), and glucose-regulated protein 78 (GRP78) antibody (1:5 000,Item No.GTX102580, GeneTex, USA) were added and incubated overnight at 4 ℃ in a refrigerator.The next day, the regimens were rewarmed for 30 min and rinsed with TBST 3 times, 15 min each time.HRP-labeled secondary antibody (1:10 000) was added and incubated at room temperature for 1 h, then rinsed with TBST 3 times, 15 min each time.Images were developed and taken in the chemiluminescence imager using a hypersensitive ECL chemiluminescence solution.Subsequently, the Image lab was used for the quantitative analysis of total protein normalization.The stain-free blot was selected to obtain the normalization factor and the normalized gray value through the comparison with the total protein.

1.3.5 TdT-mediated dUTP-biotin nick end labeling(TUNEL) assay

TUNEL assay was used for the cell apoptosis assay.TUNEL assay kit (Servicebio?, China) was used to treat the prefrontal cortex according to the manufacturer’s instructions.After the slices were slightly dried, buffer was added to the tissues in the circle, and the buffer was incubated at room temperature for 10 min.Took appropriate amount of TDT enzyme, dUTP, and buffer in the TUNEL kit according to the number of slices and tissue size and mixed at a ratio of 1:5:50.Prepared this reaction solution according to demand before use.Added this mixture to objective tissue placed in a flat wet box, incubated at 37 ℃ for 2 h.Be sure to keep the wet box moist by adding water DAPI counterstain in the nucleus: washed 3 times with PBS (pH 7.4) in a Rocker device, 5 min each.Then incubated with DAPI solution at room temperature for 10 min, kept in a dark place.Washed 3 times with PBS (pH 7.4) in a Rocker device,5 min each.Threw away liquid slightly, then covered slip with anti-fade mounting medium.Microscopic examination and image collection were conducted using the fluorescence microscope.DAPI emits blue light at an ultraviolet excitation wavelength of 330-380 nm and emits green at an emission wavelength of 420 nm.

1.4 Statistical analysis

The SPSS version 25.0 software was used for analysis.

2 Results

2.1 EA treatment improved CUMS-induced depressionlike behaviors

There was no significant difference in the total distance, central area distance, and time in the central area of OFT among the four groups at the beginning of the study (P＞0.05), and there was no significant difference in SPT among the four groups (P＞0.05).At the end of the study (day 28), compared with the control group, rats in the CUMS group were characterized by body mass descent or negative growth(P＜0.01).Meanwhile, CUMS exposure significantly reduced the total distance, central area distance, and time in the central area of OFT as compared with the control group (P＜0.01).Furthermore, the sucrose preference rate in the CUMS group was significantly decreased compared with the control group (P＜0.01).Compared with the CUMS group, the application of EA pretreatment improved the body mass, motor activities,and exploration intention in the OFT, and the pleasure experience in SPT of rats (P＜0.05 or P＜0.01).See Figure 3 and Figure 4.

Figure 3 Trials of rats in OFT before and after the CUMS

Figure 4 Effects of EA and ESC on the CUMS-induced depression-like behaviors (n=8)

2.2 Expression levels of protein GRP78, caspase-12,and C/EBP homologous protein (CHOP) in the hippocampus

Compared with the control group, the expression levels of GRP78, caspase-12, and CHOP in the hippocampus were significantly increased after CUMS exposure (P＜0.05).On day 28, compared with the CUMS group, EA and ESC pretreatment reduced the GRP78 and CHOP expression in the hippocampus(P＜0.05), but failed to reduce the caspase-12 expression,though a lower trend was shown in the chart (P＞0.05).See Figure 5.Data were expressed as mean ± standard error.Statistical analysis of data was carried out by one-way analysis of variance, followed by the least-significant difference post hoc test, and Kruskal-Wallis H test was used for pairwise comparisons.P＜0.05 indicated statistically significant.

Figure 5 Effects of EA and ESC on the ERS markers in the hippocampus of CUMS-induced rats (n=3)

2.3 Expression levels of GRP78, caspase-12, and CHOP in the prefrontal cortex

Compared with the control group, the expression levels of caspase-12 and CHOP in the prefrontal cortex were significantly increased after CUMS exposure(P＜0.05).On day 28, compared with the CUMS group,EA and ESC pretreatment reduced caspase-12 and CHOP expression in the prefrontal cortex (P＜0.05).The expression level of GRP78 was relatively high in the CUMS group, but there was no statistical significance(P＞0.05).See Figure 6.

Figure 6 Effects of EA and ESC on the ERS markers in the cortex of CUMS-induced rats (n=3)

2.4 Apoptosis-positive area in the prefrontal cortex of each group

Compared with the control group, the cardiomyocyte apoptosis-positive area (%) increased in the prefrontal cortex after CUMS exposure (P＜0.05).Whereas, on day 28, compared with the CUMS group, EA and ESC pretreatment reduced the rate of TUNEL-positive cell area in the prefrontal cortex (P＜0.05).See Figure 7 and Figure 8.

Figure 7 Apoptosis-positive area in the prefrontal cortex of each group

Figure 8 Comparison of the apoptosis-positive area in the prefrontal cortex of each group (n=3)

3 Discussion

Although global research has been focusing on depression for more than 50 years, the exact etiology and pathological mechanism research still lacks breakthroughs, and effective treatments are demanded.Therefore, depression continues to be a major problem,perplexing the medical community.“Stressing out” has gained a new connotation in the research of brain neuroscience and mental disorders.When stimulation is characterized by overload, conflict, and uncontrollability, it becomes a stressor.The body needs to adapt to various stressors in the environment, so individual stress is affected by adverse experiences,genetic factors, and behavioral reactions.When the brain senses stressors, physiological and behavioral responses are activated, resulting in an allostatic load[19].Allostatic load involves excessive exposure to the nerve,endocrine, and immune stress mediators and then participates in pathological processes such as depression[20-22].The allostatic load caused by stresses has an important role in the occurrence and pathological process of depression.

CUMS, as an unpredictable chronic stressor, can induce a series of depression-like behaviors in rats,including slow gain or even decline of body mass,decreased sucrose preference rate, and total distance,central area distance, and time in the central area in OFT, thus suggesting that the depressive rats showed a decreased sensitivity to sugar water reward and absence of pleasure, and affected or disturbed spontaneous activities and exploration behaviors.

The ER is the main site responsible for protein synthesis, folding, and calcium storage in cells and is very sensitive to stress.The state of misfolded and unfolded proteins gathering in the cavity and calcium ion balance disorder is known as ERS[23].The existing research results have indicated that perturbations of ER homeostasis lead to ERS, and ERS helps restore normal ER function by restoring the protein-folding capacity of the ER.This biological defense mechanism is imperative for preventing depression, and excessive or persistent ERS eventually causes cell apoptosis.When damage occurs in the hippocampus, amygdale, striatum, and other areas of the neurons, it participates in the development of depression[24].The present study showed that ERS and cell apoptosis occurred in the hippocampus and prefrontal cortex, and the molecular mechanisms in the hippocampus and prefrontal cortex are an important topic in depression research.

Previous studies have shown that ERS is the key mechanism in neurodegenerative diseases and depression.ERS results in the accumulation of unfolded proteins, which promotes the dissociation of BiP(GRP78) from three transmembrane sensing PERK, IRE1,and ATF6 at the same time[14-15,25-26].After CUMS exposure, the present study found a significant increase in the expression of ERS-related markers GRP78 and CHOP in the hippocampus and caspase-12 and CHOP in the prefrontal cortex.This is consistent with the findings of XU X, et al[27].ERS-mediated apoptotic pathway has been generally recognized as an important mechanism for cell apoptosis and CHOP, one of the specific proapoptotic molecules under ERS[28].PERK-CHOP pathway is the main pathway that mediates ERS and drives apoptosis.PERK is an ER transmembrane sensor that exists in the ER lumen.When sustained and severe ERS occurs, the activated PERK induces CHOP expression, which in turn initiates the CHOP-mediated apoptotic pathway, eventually leading to apoptosis[29].caspase-12 is a key molecule mediating ERS apoptosis.During ERS, IRE1 α activates the recruited TRAF2,cleaves, and caspase-12, then activates the caspase family to cause caspase-mediated apoptosis[30].In the present research, TUNEL detection showed significant cell apoptosis in the prefrontal cortex after CUMS.

Su Wen of Huang Di Nei Jing (Essential Questions of Yellow Emperor’s Inner Canon) says that the key to acupuncture is to concentrate on understanding the deficiency and excess of the five Zang organs and the changes in the pulse condition of three body parts and nine pulse taking sites, emphasizing the necessity of“Xing Shen” (inducing resuscitation).Baihui (GV20)occupies the “Yang position” to mobilize the Yang Qi of the whole body.Located between the two eyebrows,Yintang (GV29) is the gathering place of the essence, Qi,and spirit of the human body and can relieve convulsion,induce resuscitation, restore consciousness, relieve stuffy nose, and improve acuity of vision.The present study suggests the hypothesis that EA at Baihui (GV20)and Yintang (GV29) has a specific alleviatory effect on depressive behaviors, as it reduces the accumulation of unfolded proteins in the hippocampus to alleviate the side effects of ERS, and inhibits the apoptosis caused by ERS, so as to alleviate the brain injuries and improve depression.A study on SSRI drugs showed that ESC significantly reduced the protein and mRNA levels of GRP78, CHOP, and caspase-12 in CUMS-induced rats[14].According to the results of the present research, EA pretreatment and ESC have the same antidepressant effect.EA has been shown to be able to reverse apoptosis and have a protective effect on hippocampal CA3 regions[31], including down-regulating presynaptic glutamate synthesis and release, blocking postsynaptic excitatory amino acid receptors, and terminating pathological chain reaction caused by excessive excitatory receptors to inhibit glutamate release[32-33].In addition, there are many studies on the impact of EA on the downstream pathways of ERS[34-35].

In the present study, EA failed to reduce the expression of caspase-12 in the hippocampus and GRP78 in the prefrontal cortex induced by CUMS, which may be due to the minimal effect on caspase-12 mediated apoptosis pathway and unfolded protein response.The expression of caspase-12 in the hippocampus and GRP78 in the prefrontal cortex treated with ESC is inconsistent with previous studies,possibly due to the following reasons.Firstly, the distribution and concentration of caspase-12 and GRP78 vary in different brain regions.Secondly, there may be individual differences in the expression of caspase-12 and GRP78 in the depressed brain.Although the expression of caspase-12 and GRP78 is not significant in WB, the relationship between apoptosis-related proteins and depression should not be ignored.Therefore, this study preliminarily revealed that EA at Baihui (GV20) and Yintang (GV29) could regulate ER homeostasis, inhibit cell apoptosis, and improve depressive symptoms.

In addition, the stain-free total protein normalization method was used for WB detection in our study.Compared with the traditional housekeeping protein(HKP) normalization, this method has two advantages.One is better reflecting the change trend of protein expression, and the other is to simplify the operation process of WB.The study on the stain-free total protein normalization method shows more accurate and stable total protein than the method with a single HKP as the internal reference and improves the reliability of the data[36-37].The linear detection range of total protein is wider than that of HKP, and it is more stable in the whole experiment and system.Therefore, it can effectively eliminate the influence of experimental conditions and systems on the results and more truly reflect the change in target protein expression.Therefore, the total protein normalization method is used to ensure that the experimental results are more accurate[38-39].

In summary, the present study demonstrated that ERS and cell apoptosis in rats’ hippocampus and frontal cortex mediated the onset of depressive symptoms after CUMS induction.Importantly, the present findings suggest that EA can significantly mitigate behavioral deficits elicited by CUMS by adjusting ER homeostasis and inhibiting cell apoptosis.

Conflict of Interest

The authors declare that there are no conflicts of interest associated with this manuscript.

Acknowledgments

This work was supported by Scientific Research Program of Hebei Provincial Administration of Traditional Chinese Medicine (河北省中醫(yī)藥管理局科研計(jì)劃項(xiàng)目, No.2023130); Youth Science and Technology Innovation Talent Support and Cultivation Program Project of Hebei Medical University (河北醫(yī)科大學(xué)青年科技創(chuàng)新人才托舉培育計(jì)劃項(xiàng)目, No.PJZR202010).

Statement of Human and Animal Rights

This study has been approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University (Approval No.IACUC-Hebmu-2022033).The treatment of animals in this experiment conformed to the ethical criteria.

Received: 29 October 2022/Accepted: 18 May 2023