Electroacupuncture effect the neuromuscular functionality in rats with intensive care unit-acquired weakness

doi:10.21203/rs.3.rs-3763951/v1

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Electroacupuncture effect the neuromuscular functionality in rats with intensive care unit-acquired weakness

https://doi.org/10.21203/rs.3.rs-3763951/v1

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ICU-Acquired Weakness (ICU-AW) is a multifaceted condition capable of impeding the recuperation of critically ill patients afflicted with sepsis. Acupuncture therapy has demonstrated notable effectiveness across a spectrum of ailments, particularly in the initial stages of rehabilitating critically ill patients. This study aims to assess the potential of electro-acupuncture stimulation in enhancing muscular functionality within a rat model of ICU-AW, with implications for ICU-AW treatment. We created a rat model of acquired weakness induced by septic shock using intraperitoneal injection of lipopolysaccharide (LPS) at a dose of 5 mg/kg, in conjunction with immobilization. This investigation monitored alterations in rat motor function, body weight, the levels of inflammatory markers including IL-6, TNF-α, SIRT3, and CAF in rat serum, and their fluctuations preceding and following the onset of ICU-AW. Additionally, we assessed muscle tissue ATP levels to appraise mitochondrial functionality in ICU-AW rats and analyzed the expression of proteins related to signaling pathways and autophagy within muscle tissue. Our study implies that electro-acupuncture stimulation can activate the AMPK-PGC1α-SIRT3 signaling pathway, ensuring consistent autophagy and calpain levels, consequently ameliorating both inflammation and muscular function in ICU-AW rats.

Sepsis-related intensive care unit-acquired weakness

AMPK-PGC1α-SIRT3

Electroacupuncture

Mitochondria

Autophagy

Intensive care unit-acquired weakness (ICU-AW) is a prevalent neurological and muscular complication within the realm of critical care medicine. According to statistical data, up to 90% of severe sepsis patients manifest clinical indications of muscle atrophy, notably ICU-AW [1]. ICU-AW is a prolonged condition, resulting in significant impediments to functional mobility, with potential persistence for several years post-discharge [2]. This condition substantially impacts patients' quality of life. Despite the recognition of various contributing factors, including inflammatory responses, protein metabolism imbalances, mitochondrial dysfunction, and oxidative stress [3–6], our understanding of the regulatory elements implicated in skeletal muscle atrophy remains limited.

The neuromuscular junction (NMJ), serving as a "bridge" between motor neuron terminals and skeletal muscles, plays a crucial role in the development of ICU-AW. Previous research has indicated that motor neurons can release neuron-aggregating proteins into the synaptic cleft at the NMJ [7]. During NMJ remodelling, the aggregated proteins at the synaptic cleft are cleaved by neuraminidase, resulting in the generation of C-terminal agrin fragments (CAFs). CAFs can dissolve and circulate in the bloodstream. When muscle atrophy occurs, particularly with a reduction in the number of muscle fibres, NMJ remodelling takes place, leading to a significant increase in circulating CAF levels. Using circulating CAF levels as a diagnostic tool for ICU-AW holds promise and practical application [7, 8].

Current research has established the pivotal role of the AMPK-PGC1α-SIRT3 signaling pathway in the growth and development of skeletal muscles [9]. AMPK, a serine/threonine kinase, orchestrates cellular metabolic activities to fulfil the body's energy demands. PGC1α functions as the principal regulator of mitochondrial biogenesis [10], and SIRT3, a downstream target gene of PGC1α, predominantly resides within mitochondria and is intimately intertwined with cellular energy metabolism and oxidative stress. The AMPK-PGC1α-SIRT3 signaling pathway assumes critical functions in maintaining energy homeostasis, governing skeletal muscle pathophysiology, and modulating exercise function. Nevertheless, it remains uncertain whether this signaling pathway plays a significant role in the development of ICU-AW.

Autophagy was historically deemed an adverse contributor to muscle atrophy. However, in critically ill patients during their ICU stay, autophagy fails to be sufficiently activated [11]. Suppressed autophagy levels lead to the accumulation of anomalous mitochondria and intracellular components. Dysfunctional organelles persist without engaging in degradation metabolism, ultimately resulting in muscle atrophy and weakness [12, 13]. Considering the potential pivotal role of mitochondrial autophagy in ICU-AW, the investigation of methods to trigger its activation presents a promising avenue for countering ICU-AW.

At present, specific drugs and treatment methods for ICU-AW are lacking, and modern medicine emphasizes a comprehensive approach, with early mobilization being a pivotal aspect of ICU-AW management. However, recent research has affirmed that premature or excessive functional exercise may impose increased cardiopulmonary stress on patients, potentially leading to adverse events [14]. Electroacupuncture therapy for ICU-AW has garnered substantial recognition through extensive clinical validation, as it has demonstrated the ability to enhance muscle strength and partially improve neuromuscular function [15, 16]. Nevertheless, the precise mechanism underlying electroacupuncture therapy for ICU-AW remains unclear. Therefore, this study seeks to explore the mechanism of electroacupuncture therapy for ICU-AW using a rat model induced by sepsis-related immobilization.

Animal preparation

We selected healthy male SD rats, SPF-grade, aged 6 to 8 weeks, with a body weight of 200 ± 20 g. These rats were provided by Nanjing Kaisjia Biological Technology Co., Ltd. (Licence Number: SYXK (Su) 2021-0025). The rats were housed in a meticulously maintained animal laboratory with a humidity level of 45 ± 5%, a temperature maintained at 24 ± 2°C, and a 12-hour light-dark cycle with proper ventilation. Their diet consisted of pelleted feed supplied by the Experimental Animal Center of Nanjing University of Chinese Medicine. Following a 7-day acclimatization period in the same controlled environment, the rats were included in the experimental procedures. In this study, each individual rat served as an experimental unit, and random numbers were generated using the "=RAND()" function in Microsoft Excel.

ICU-AW model preparation

Rats were intraperitoneally injected with lipopolysaccharide (LPS) at a dosage of 5 mg/kg (Escherichia coli 0111: B4, L2630, Sigma, USA) to establish a sepsis model, concurrently placing the rats in a fixed position to induce immobilization (n = 24). Sepsis in rats was characterized by reduced activity, drowsiness, piloerection, periorbital white discharge, and tremors (according to the rat sepsis scoring scale for reference) [15, 16].However, if a rat died, neither serum nor tissue collection was performed. The control group of rats received an intraperitoneal injection of the same volume of normal saline (n = 8). All experimental rats were subjected to a consistent daily feeding regimen, where they received a standardized 5% glucose saline solution at a rate of 5 ml/kg·h. Additionally, their mental status and state of immobilization were closely monitored.

Electroacupuncture intervention

The acupoint locations and needling techniques were referenced from "Names and Locations of Acupoints Commonly Used in Laboratory Animals". The Zusanli acupoint (ST36) is situated on the outer side of the hind knee joint in rats, approximately 5 mm below the head of the fibula, and was needled to a depth of 7 mm. The Sanyinjiao acupoint (SP6), located 10 mm directly above the tip of the inner ankle in the hind limb, was needled to a depth of 4–5 mm. In the nonmeridian, nonacupoint group, acupoints adjacent to ST36 and SP6 were needled, each 1–2 mm away from the respective points (with the requirement that the selected acupoints are not on the normal meridian lines). Needling procedures were conducted using filiform needles (0.12 mm×10 mm) attached to an electroacupuncture device (G6805-II, Qingdao Xinsheng Co., Ltd., Qingdao, China). The stimulation intensity was adjusted to induce slight limb tremors in the rats (1–2 mA). Stimulation was administered in a sparse-dense wave pattern (2 Hz/100 Hz) with an output voltage of 2 V. Electroacupuncture treatment was administered for 20 minutes daily over a total period of 7 days. Rats in the sham group and SI group were immobilized for 20 minutes at the same time each day without receiving electroacupuncture.

Collection of experimental specimens

Following the conclusion of electroacupuncture therapy, a 24-hour fasting period with access to water was observed for all rats. On the subsequent day, rats were anaesthetized with isoflurane to facilitate sample collection. Blood samples were drawn from the cardiac apex. After a 2-hour resting period, the blood samples underwent centrifugation at 4500 × g for 15 minutes, and the resulting supernatant was collected and preserved at -80°C until the analysis of biochemical parameters. Muscle specimens from the gastrocnemius and diaphragm were dissected, weighed, and then divided into two portions. One portion was immersed in a 10% formalin solution (Sigma‒Aldrich, USA) for histological analysis, while the other portion was rapidly frozen in liquid nitrogen and stored at -80°C for subsequent assessments of ATP concentration and Western blot analysis of tissue proteins.

Muscle Strength and Weight Evaluation

Muscle strength was assessed on three occasions: before modelling, prior to electroacupuncture treatment, and before tissue collection. A specialized grip strength meter for experimental rodents (YSL-13A, Jinan, China) was employed to measure the muscle strength of each rat group. This test engaged all forelimb muscles in generating force. Rats were gently removed from their cages and placed horizontally on the grid to assess their firm grip with both front paws. Subsequently, they were pulled away from the grid, and the maximum force applied by each rat was recorded by the sensor. Only measurements that reflected the rat's resistance to the experiment were considered, with those involving the use of a single front paw or hind limb or any rat turning during the pulling process being excluded. Any instance of instability in gripping, turning, or struggling resulted in data exclusion, and remeasurement was conducted. Each rat underwent five measurements, with the highest and lowest values discarded, and the average of the remaining three values was recorded as the rat's grip strength.

The body weight of the rats was measured using an experimental weight scale before the initiation of the experiment, prior to electroacupuncture treatment, and before tissue collection.

Immunofluorescence

Fresh muscle tissue samples were promptly frozen in liquid nitrogen, embedded in optimal cutting temperature (OCT) compound, and sliced into 8 µm sections using a Leica CM1950 cryomicrotome (Leica, Wetzlar, Germany). These sections were then fixed in 4% paraformaldehyde for 10 minutes, followed by washing with PBS and blocking in a buffer solution containing 5% bovine serum albumin (BSA) at 37°C for 30 minutes. Next, a combination of primary antibodies (anti-fast myosin skeletal heavy chain, anti-slow skeletal myosin heavy chain, Abcam) was applied to the sections, which were incubated at 4°C for 16 hours. After rinsing with heated PBS, a mixture of Alexa Fluor 488/594 secondary antibodies was added, and the sections were incubated in darkness at room temperature for 1 hour. Finally, DAPI (Solarbio) was used to stain the sections, and they were examined under an Olympus FV10i confocal microscope (Tokyo, Japan). The degree of colocalization was measured in a minimum of 8 distinct fields from at least 3 rats, and the results were represented by the average integrated optical density, which was analysed using ImagePro 6.0 (Bethesda, MD, USA).

Adenosine Triphosphate Level Measurement in Muscles

Fresh rat diaphragm and gastrocnemius muscle samples were obtained, rinsed in precooled PBS to eliminate residual blood and nearby blood vessels, patted dry with absorbent paper, and precisely weighed. Subsequently, rat tibialis anterior muscle tissue was minced. An equal volume of PBS, with a weight-to-volume ratio of 1:9 (e.g., 1 g of muscle tissue per 9 ml of PBS), was added to the minced skeletal muscle. The mixture was then meticulously homogenized in a glass homogenizer on ice, with further disruption using an ultrasonic disruptor. Following this, the homogenate was centrifuged at 5000 × g for 10 minutes, and the resulting supernatant was collected for analysis. An enhanced ATP assay kit was employed to determine ATP concentrations in the tissue using a multifunctional microplate reader, adhering to the manufacturer's instructions.

Enzyme-linked immunosorbent assay (ELISA)

All reagents and samples were retrieved from the refrigerator ahead of time, allowing them to reach room temperature for 30 minutes. An ELISA kit (Catalog Number 5) was used to measure the levels of TNF-α, IL-6, CAF, and SIRT3 in rat serum according to the manufacturer's instructions. The specific assay kits used were as follows: TNF-α (E-EL-R2856c), IL-6 (E-EL-R0015c), CAF (ELA08127r), and SIRT3 (ELA06555r).

Western blot analysis (WB)

The proteins were extracted using a prepared RIPA lysis buffer containing a cocktail of protease and phosphatase inhibitors. After being centrifuged at 12,000 rpm for 10 min at 4℃, the protein concentration of the lysates was measured using a BCA protein assay (Thermo Scientific, # 23225) and was adjusted to the same final concentration. Equal amounts of protein (20 µg) from each sample were separated using a 10% Tris-Gly PAGE and transferred to polyvinylidene fluoride (PVDF, Millipore) membranes. After undergoing blocking with 5% non-fat milk in tris-buffered saline containing 0.1% Tween-20 (TBST) for 2 h at room temperature, the membranes were incubated at 4℃ overnight with the following specific primary antibodies:AMPK (AF6423;1:1000 dilution), PGC1 (AF5395;1:1000 dilution), SIRT3 (AF5135;1:1000 dilution), SOD (AF5144;1:1000 dilution), Calpain-1 (DF6306;1:1000 dilution), and autophagy-related proteins ATG7 (DF6130;1:1000 dilution), P62 (AF5384;1:1000 dilution), and Beclin-1 (AF5128;1:1000 dilution). Then, the membranes were washed four times in TBST and incubated with the HRP-conjugated goat anti-rabbit secondary antibody (A0208, Beyotime; 1:5000 dilution) at room temperature for 2 h.The immunoreactive proteins were visualized using an enhanced chemiluminescent (ECL) substrate (180–501, Tanon) and an imaging system (GelView 6000Plus, Guangzhou Biolight Biotechnology Co., Ltd., Guangzhou, China).The band intensities were quantified using ImageJ software and normalized to the loading control.

Statistical analysis

GraphPad Prism V5 and ImageJ software were employed for data visualization and statistical analysis. We initially assessed the quantitative data for normal distribution. Normally distributed quantitative data are expressed as the mean ± standard deviation (x ± S), and intergroup comparisons were performed using one-way analysis of variance (ANOVA), followed by Fisher's least significant difference (LSD) post hoc test. In instances of heteroscedasticity, a rank-sum test was utilized, with statistical significance defined as P < 0.05.

Serum levels of CAF and SIRT3 hold diagnostic significance for ICU-AW

When muscle atrophy occurs, especially with a decrease in the number of muscle fibres, neuromuscular junctions undergo remodelling, and a significant generation of circulating CAFs is observed. Using serum CAF levels as a diagnostic tool for muscle atrophy holds promise and practical value. We observed changes in CAF levels in the serum of ICU-AW rats in our study. Compared to that in the sham group, the serum CAF concentration in the SI group was elevated (P < 0.05; Fig. 1a). Simultaneously, alterations in the serum levels of the SIRT3 signaling pathway were observed, with results indicating a decrease in SIRT3 concentration in the SI group (P < 0.05; Fig. 1b).

Electroacupuncture therapy has the potential to mitigate weight and muscle strength loss in ICU-AW rats

At the outset of the experiment, there was no significant difference in the weights of animals among the various groups (P > 0.05; Fig. 2a). The rats in the Sham group maintained robust health, characterized by shiny fur, heightened activity levels, and regular bowel movements. In contrast, the induction of the ICU-AW model in the SI group resulted in rats displaying a more emaciated appearance, reduced food consumption, weight loss, decreased activity, dry and shedding fur, and loose stools. Following electroacupuncture treatment, rats in the SI EA group exhibited increased food intake, slight weight gain, enhanced activity levels, improved fur condition, and some degree of stool consistency improvement. Conversely, rats in the Sham EA group did not display significant enhancements in their overall condition (Fig. 2b).

The grip strength test results for the rats are presented in Table 1. It is evident that the baseline grip strength of rats in all groups with immobilized forelimbs was similar (P > 0.05), as depicted in Fig. 3a. However, a significant decline in grip strength was observed after the induction of the ICU-AW model (P < 0.05). Following electroacupuncture treatment, there was a notable recovery in rat grip strength compared to the SI group (P < 0.05), whereas the Sham EA group exhibited no significant change, as shown in Fig. 3b.

Table 1 Effect of EA on the grip strength of ICU-AW rats

Group	Baseline Grip Strength(N)	Model Grip Strength(N)	Final Grip Strength(N)
Sham	786.18±33.02	760.46±29.47 *	758.02±30.74
SI	761.86±67.16	562.36±32.92	560.53±29.41 *
SI EA	746.81±25.93	556.02±37.99	699.06±35.44 #
Sham EA	777.78±80.73	535.45±29.81	594.51±37.24

Data are expressed as the means ± SD; * P < 0.05 compared with the sham group; # P < 0.05 compared with the SI EA group.

Electroacupuncture demonstrates the potential to enhance muscle quality in rats with ICU-AW

Muscle strength and quality may not necessarily align, and muscle atrophy might occur without changes in muscle-specific strength, and vice versa. Therefore, we assessed muscle physiological function by evaluating muscle cross-sectional area to consider changes in muscle quality.

We conducted a more detailed analysis of alterations in muscle fibre types. The findings revealed that, when compared to the Sham group, the rats in the SI group exhibited a shift in muscle fibre composition, with their gastrocnemius and diaphragm muscles transitioning from slow-twitch to fast-twitch fibres. This observation implies that ICU-AW is associated with reduced exercise endurance in rats. Conversely, in the SI EA group, there were no noteworthy changes in muscle fibre types when compared to the Sham group (Fig. 4 a-d).

Electroacupuncture therapy has the potential to enhance mitochondrial function in rats with ICU-AW

Due to the close correlation between the severity of ICU-AW and the amount of ATP synthesis, we observed changes in ATP. ATP generation assays revealed that, compared to the Sham group, ATP generation significantly decreased in the muscle tissues of rats in the SI group (P<0.05). Conversely, when compared to the SI group and the Sham EA group, rats in the SI EA group exhibited a significant increase in ATP generation (P<0.05; Fig. 5 a and b).

Electroacupuncture therapy has the potential to mitigate inflammation in rats with ICU-AW

We employed ELISA to quantify the concentrations of inflammatory factors (TNF-α and IL-6) within the skeletal muscles of rats. When contrasted with the Sham group, rats in the SI group exhibited a notable elevation in serum TNF-α and IL-6 levels (P < 0.05). Conversely, the SI EA group demonstrated significantly lower levels of TNF-α and IL-6 than the SI group (P< 0.05; Fig. 6 a and b).

The activation of the AMPK-PGC1α-SIRT3 signaling pathway by electroacupuncture affects protein expression in the gastrocnemius and diaphragm muscles of rats with ICU-AW

To delve deeper into the mechanism by which electroacupuncture enhances mitochondrial function and mitigates inflammation in ICU-AW, Western blot analysis revealed notable differences. Compared to the sham group, rats in the SI group exhibited a downregulation in the expression levels of AMPK, PGC1α, SIRT3, and SOD2 proteins within both the gastrocnemius and diaphragm muscles (Fig. 7 a-j). Additionally, the expression of autophagy-related proteins, specifically ATG7, P62, and Beclin-1, decreased (Fig. 8 a-h), while the calcium-dependent protease Calpain-1 exhibited an increase in expression (Fig. 9 a-d). These findings underscore the suppression of the AMPK-PGC1α-SIRT3 signaling pathway in ICU-AW rats, resulting in inadequate downstream autophagy levels and an upregulation in Calpain-1 expression, ultimately contributing to muscle atrophy. In comparison to the SI group, the SI EA group exhibited an elevation in the protein levels of AMPK, PGC1α, SIRT3, and SOD2 within the gastrocnemius muscle of rats. Moreover, there was an increased expression observed in autophagy-related proteins, namely, ATG7, P62, and Beclin-1, alongside a reduction in the expression of the calcium protease calpain-1. These findings strongly imply that electroacupuncture sustains the equilibrium of autophagy and calcium protease levels, subsequently enhancing the structural and functional aspects of the gastrocnemius and diaphragm muscles in ICU-AW rats through the activation of the AMPK-PGC1α-SIRT3 signaling pathway.

ICU-AW represents a significant complication in critically ill patients within the intensive care unit (ICU). Its occurrence is intricately linked to variables such as the underlying disease, timing of admission, and severity of patients' conditions. Furthermore, diverse risk factors contribute to its manifestation. ICU-AW profoundly impacts the post-ICU quality of life of survivors and escalates the financial burden associated with rehabilitative interventions [18]. To date, the exploration of the pathophysiological mechanisms underlying ICU-AW and the quest for specific therapeutic targets have emerged as pressing interdisciplinary scientific inquiries. As a result, this study endeavors to establish a rat model of sepsis-induced acquired weakness and investigate the involvement of the AMPK-PGC1α-SIRT3 signaling pathway at the in vivo level during the development of ICU-AW.

NMJ plays a pivotal role as the intermediary connection between motor neuron terminals and skeletal muscles. Its integrity is indispensable for preserving the equilibrium of motor neurons and muscle fibres. When influenced by factors such as inflammation, inhibition, and malnutrition, the NMJ can undergo structural destabilization, resulting in muscle atrophy and other associated manifestations. During the process of NMJ remodelling, soluble fragments generated by localized cleavage of aggregating proteins by neural pancreatic enzymes, referred to as CAFs, enter the bloodstream, leading to elevated CAF levels above the norm [8]. This phenomenon has been validated through experimentation utilizing a rat model of ICU-AW. Additionally, alterations in serum SIRT3 levels have the potential to offer diagnostic insights into ICU-AW. SIRT3 predominantly resides within mitochondria, engaging in activities encompassing mitochondrial damage repair, oxidative stress response, apoptosis, autophagy, and a variety of other physiological functions [19]. Prior research has indicated that reduced SIRT3 expression is associated with diverse conditions, including cardiovascular diseases [20], neurodegenerative disorders [21], and diabetic cardiomyopathy [22]. In the present study, a similar trend of decreased serum SIRT3 levels was observed in the SI group of rats when assessing serum SIRT3 levels in the ICU-AW model rats. These findings collectively suggest that alterations in serum SIRT3 and CAF markers could serve as potential indicators of the presence of ICU-AW.

The pathogenesis of ICU-AW is intricate, and it is challenging to attribute its development and patterns to a single influencing factor. In this study, we established a model of sepsis-related acquired weakness by intraperitoneal injection of lipopolysaccharide in combination with immobilization. Prior to electroacupuncture treatment, we assessed the inflammatory markers TNF-α and IL-6, revealing a significant elevation in TNF-α and IL-6 levels in the SI group compared to the sham group (P < 0.05). Histological examination using HE staining revealed pronounced muscle fibre atrophy in the SI group, accompanied by varying degrees of interfiber spacing widening and a substantial reduction in muscle fibre diameter values (P < 0.05). These findings confirm the successful creation of ICU-AW, underscoring the utility of sepsis combined with immobilization as a straightforward and effective modelling approach for elucidating the mechanisms underlying ICU-AW.

In our study, we observed that in the SI group of rats, during the onset of ICU-AW, the AMPK-PGC1α-SIRT3 signaling pathway was suppressed. This downregulation of SIRT3 levels hindered the expression of SOD2, resulting in an escalation of reactive oxygen species (ROS) within rat mitochondria, subsequently leading to heightened oxidative stress. When ROS production reached significant levels, autophagy was impeded, consequently causing mitochondrial dysfunction. Furthermore, microscopic examination revealed a shift in skeletal muscle fibre composition from slow-twitch to fast-twitch fibres within the SI group of rats, implying a reduction in skeletal muscle endurance in ICU-AW rats. To summarize, during the progression of ICU-AW, skeletal muscle mitochondrial dysfunction, alterations in muscle fibre morphology, diminished autophagy levels, and elevated calcium protease levels may all be linked to the inhibition of the AMPK-PGC1α-SIRT3 signaling pathway.

The research findings demonstrate the efficacy of electroacupuncture therapy in enhancing muscle function in ICU-AW rats. It is hypothesized that this improvement is linked to the activation of the AMPK-PGC1α-SIRT3 signaling pathway by electroacupuncture. Electroacupuncture treatment initiates the activation of nuclear AMPK, subsequently leading to an increase in nuclear PGC1α expression. PGC1α, in turn, phosphorylates SIRT3, thereby activating it. The upregulation of SIRT3 promotes the clearance of damaged mitochondria, enhances mitochondrial function, and induces skeletal muscle mitochondrial autophagy, ultimately alleviating ICU-AW. Electroacupuncture stimulation, as an effective diagnostic and therapeutic approach for delaying and managing muscle atrophy, presents notable advantages.

We chose SP6 and ST36 as the acupuncture points for this study for several compelling reasons. First, Sanyinjiao acupoint (SP6), as documented in the "Zhenjiu Jiayi Jing" in the acupuncture literature, is situated along the Spleen meridian of the Foot Taiyin. Positioned on the inner aspect of the lower leg, three inches above the inner ankle malleolus, and behind the medial border of the tibia, this acupoint plays a crucial role in supporting spleen health, nourishing blood, and regulating liver and kidney functions. Zusanli acupoint (ST36), based on the "Ling Shu · Ben Shu," belongs to the stomach meridian of Foot Yangming. It is located three inches below the outer knee joint and one horizontal finger breadth from the anterior crest of the tibia. ST36 is renowned for its capacity to strengthen muscles and bones, enhance circulation, and serve as a pivotal acupoint in the treatment of lower limb weakness. These two acupoints align with the guidance in the "Huangdi Neijing" that emphasizes focusing on the Yangming meridian for addressing flaccidity syndrome. Second, previous research has demonstrated that electroacupuncture administered at these acupoints can effectively mitigate inflammatory responses [23, 24], maintain autophagy stability, and consequently lead to an improvement in skeletal muscle function. Last, it has been reported that stimulating these acupoints can activate the AMPK signaling pathway, thereby ameliorating mitochondrial dysfunction and reducing oxidative stress [25, 26].

Our research findings demonstrate that electroacupuncture therapy has the capacity to activate the AMPK-PGC1α-SIRT3 signaling pathway. This activation, in turn, contributes to the stabilization of autophagy and calcium protease levels, ultimately preserving the typical structure and function of skeletal muscles. Electroacupuncture, as a treatment modality for ICU-AW, presents a promising therapeutic avenue with potential clinical relevance.

ICU

Intensive Care Unit

ICU-AW

Intensive Care Unit-Acquired Weakness

LPS

Lipopolysaccharide

NMJ

Neuromuscular junction

CAF

C-terminal agrin fragments

ST36

Zusanli acupoint

SP6

Sanyinjiao acupoint

OCT

Optimal Cutting Temperature

BSA

Bovine Serum Albumin

ELISA

Enzyme-linked immunosorbent assay

LSD

Least Significant Difference

Sham

control group

Sepsis with Immobilization group

SI EA

electroacupuncture group

Sham EA

Electroacupuncture nonacupoint group

ROS

Reactive Oxygen Species.

Acknowledgements

This research was supported by the Nanjing Health Science and Technology Development Special Fund(YKK21209), Nanjing Lishui District Hospital of Traditional Chinese Medicine (LZY202120), Jiangsu Provincial Graduate Scientific Research and Practice Innovation Program Project (SJCX23_0816).

Authors’ contributions

Xing-Xing Hu: Conceptualization. Ren-Jun Yu and Hong-Lei Lu: Methodology, manuscript writing, data collection and analysis. Yu-Hao Zhang, Lei Chen and Chen-Wei Lv: Text checking and language polishing. Hai-Bin Ni and Xing-Xing Hu: Review and editing. All authors contributed to the article and approved the submitted version.

Availability of data and materials

The data used to support the findings of this study are available from the corresponding author upon request.

Funding

This research was supported by the Nanjing Health Science and Technology Development Special Fund(YKK21209),NanJing LiShui District Hospital Of Traditional Chinese Medicine (LZY202120),Jiangsu Provincial Graduate Scientific Research and Practice Innovation Program Project(SJCX23_0816).

Ethics approval and consent to participate

This study was conducted in strict accordance with the recommendations outlined in the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health. The animal care and experimental procedures used in the current study were approved by the Animal Ethics Committee of Nanjing University of Traditional Chinese Medicine (Permit number: AEWC-20200708-1). Every effort was made to minimize the suffering of animals.

Competing interests

The authors declare no competing interests.

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The authors declare no competing interests.

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Electroacupuncture effect the neuromuscular functionality in rats with intensive care unit-acquired weakness

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Abstract

Figures

Introduction

Methods

Animal preparation

ICU-AW model preparation

Electroacupuncture intervention

Collection of experimental specimens

Muscle Strength and Weight Evaluation

Immunofluorescence

Adenosine Triphosphate Level Measurement in Muscles

Enzyme-linked immunosorbent assay (ELISA)

Western blot analysis (WB)

Statistical analysis

Results

Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

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