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. 2024 Feb 21;17(2):e14401. doi: 10.1111/1751-7915.14401

Electroacupuncture at ST36 modulates the intestinal microecology and may help repair the intestinal barrier in the rat model of severe acute pancreatitis

Huimin Xu 1, Qian Wen 1, Hangqi Hu 1, Sihao Yang 2, Lingyun Lu 1, Xiangyun Hu 3, Hao Li 1, Xianhao Huang 1, Ning Li 1,✉
PMCID: PMC10880739  PMID: 38381095

Abstract

Severe acute pancreatitis (SAP) onset and development are closely associated with intestinal barrier injury. Evidence from clinical practice and research has shown that electroacupuncture (EA) at the Zusanli (ST36) acupoint can improve intestinal barrier function and abdominal symptoms in patients with SAP; however, the specific mechanisms of action remain unclear. This study aimed to observe the changes in the intestinal microbiota and metabolites in SAP rats and to explore the effect of EA at ST36 on intestinal barrier injury in SAP rats. 16S rRNA gene sequencing combined with microbial diversity analysis, short‐chain fatty acids (SCFAs)‐targeted metabolomics, immunohistochemistry, immunofluorescence, western blotting, and other techniques were used to explore the mechanism of EA at bilateral ST36 acupoints on SAP‐related intestinal barrier injury. Our results showed that EA at ST36 could repair intestinal barrier injury by modulating intestinal microecology, thereby reducing intestinal inflammation, restoring intestinal function, and ultimately alleviating the prognosis of SAP. Our study provides new insights into the mechanisms and treatment of intestinal barrier injury in patients with SAP from the perspectives of microbiota and SCFAs regulation.

Short abstract

Electroacupuncture at ST36 modulates the intestinal microecology and may help repair the intestinal barrier in the rat model of severe acute pancreatitis.

INTRODUCTION

Acute pancreatitis (AP) is a common abdominal emergency with a global annual incidence of approximately 34 cases per 100,000 individuals, which has shown a consistent upward trend in recent years (Xiao et al., 2016). This condition involves calcium signalling, premature trypsinogen activation, and autophagy, all of which damage the pancreas. In addition, intestinal barrier injury and microbiota changes can cause endotoxin and bacterial translocation into the blood circulation and/or distant organs, leading to a systemic inflammatory response syndrome (SIRS) and multiple organ dysfunction syndromes (MODS), both of which have been implicated in the development of severe acute pancreatitis (SAP). The mortality rate of SAP has been estimated in the range of 36%–50% (Banks et al., 2013; Ge et al., 2020; van den Berg et al., 2021). Overall, this evidence suggests that the intestinal barrier may improve intestinal function in patients with SAP, leading to better outcomes.

A growing body of research has described the influence of intestinal microbiota disorders on SAP. In 2018, the Clinical Guidelines of the American Gastroenterological Association proposed that the treatment approach to AP should shift from the previously recommended “fasting and resting” of the gastrointestinal and pancreatic systems to early “awakening and maintenance of intestinal function” (Crockett et al., 2018). Some evidence suggests that AP is associated with remarkable changes in the intestinal microbiota (Thomas & Jobin, 2020; van den Berg et al., 2021; Zhu et al., 2019). Moreover, disorders of the intestinal microbiota may be closely associated with AP severity. The disrupted intestinal microbiota associated with AP damages the intestinal barrier function in two ways. On the one hand, the increase in bacterial pathogen levels increases the inflammatory response rate mediated by tumour necrosis factor‐inflammatory response‐α (TNF‐α) and decreases the expression levels of tight junctions between cells, directly damaging the intestinal epithelial cells (Zheng et al., 2019). On the other hand, a decrease in beneficial bacteria volume reduces the competitive colonization activities of intestinal epithelial cells and dramatically increases the risk of bacterial pathogens invading the intestinal mucosa (Tan et al., 2015). Furthermore, other studies have found that butyric acid derived from the intestinal microbiota can stabilize the expression of hypoxia‐inducible factors and their target genes, thereby enhancing mechanical barrier function (Kelly et al., 2015). In addition, butyric acid protects the intestinal barrier by inhibiting the expression of Claudin‐2 via a subunit‐dependent mechanism of the interleukin‐10 (IL‐10) receptor (Zheng et al., 2017). Overall, significant changes in the intestinal microorganisms composition during AP could damage the mucosal layer and bacterial membrane barrier of the intestinal mucosa, aggravating the severity of AP and damaging intestinal barrier function. Consequently, restoring intestinal microecology may be an effective way to alleviate the progression of SAP, reduce its fatality rate, and improve the prognosis of SAP.

In traditional Chinese medicine, electroacupuncture (EA) is widely used to treat diseases such as constipation (Liu, Wu, et al., 2021), chronic prostatitis (Sun et al., 2021), and urinary leakage (Liu et al., 2017). Evidence from long‐term clinical practice and research has shown that EA at the Zusanli acupoint (ST36) can effectively relieve the abdominal symptoms of patients with SAP, reduce the fatality rate, and shorten the duration of hospitalization. Several studies have found (Guo et al., 2014; Wang et al., 2022) that EA at ST36 and other acupoints can reduce intestinal mucosal permeability, reduce intestinal epithelial cell necrosis, and protect the intestinal barrier in patients with AP. It has been suggested that acupuncture therapy may help regulate the intestinal microbiota composition, increasing the abundance of certain species through multiple pathways and targets, supporting intestinal mucosal immune function, and protecting intestinal barrier structure and function (Bai et al., 2021; Wei et al., 2019; Yaklai et al., 2021).

In our study, we aim to establish a rat model of L‐ornithine‐induced SAP. The rats were then subjected to EA therapy. Cecal contents of each group were collected for 16S rDNA sequencing and short‐chain fatty acids (SCFAs) analysis. Serum and pancreatic and intestinal tissue samples were collected to evaluate the inflammatory response, pathological changes, and intestinal barrier function. Finally, the effect of EA on the mortality rate was evaluated. Our study aimed to investigate whether EA at ST36 repairs intestinal barrier injury and reduces inflammation by modulating intestinal microecology to improve SAP prognosis.

EXPERIMENTAL PROCEDURES

Animals and ethics

Eight‐weeks‐old male Sprague–Dawley rats (200–250 g) were obtained from Byrness Weil Biotech, Ltd. (Chengdu, China). All rats were housed in a certified SPF animal facility with normative cages at a temperature of 23 ± 2°C on a 12/12‐h light–dark cycle. They had access to standard rodent chow and water ad libitum for 1‐week acclimatization. Before conducting the study, we conducted a pilot study. In a pilot study, we used an SAP model induced by two intraperitoneal injections of L‐ornithine. Twenty‐four hours after the L‐ornithine injection, we collected samples from surviving rats and observed the pathological manifestations of the pancreas, which included interstitial oedema, diffuse pancreatic acinar cell necrosis, extensive inflammatory cell infiltration, haemorrhage, and increased pancreatic histopathology scores in all rats. Moreover, we observed an increase in systemic inflammation indicators, including lipase, amylase, TNF‐α, IL‐6, and LL‐37 levels, in the SAP rats. These findings indicate a successful establishment of the SAP model.

The rats were randomly assigned to four groups: control group (CON), control rats with EA group (CON_EA), SAP model rats without EA group (SAP), and SAP model rats with EA group (SAP_EA) (n = 8 per group). The SAP and SAP_EA groups were intraperitoneally injected with two doses of 30% L‐ornithine (3.0 g/kg, pH:7.3–7.4, Sigma, St Louis, MO, USA) at 1‐h intervals; 24 h later, the SAP model was successfully produced. Equivalent doses of saline were administered to the CON and CON_EA groups. Postoperatively, all rats were placed in a cage with a heating pad for at least 6 h to ensure survival and then sent back to the animal experiment centre.

All animal procedures were performed in strict accordance with the guidelines of the CPCSEA and the World Medical Association Declaration of Helsinki. The study was approved by the Institutional Animal Care and Treatment Committee of Sichuan University in China (permit number: 20211228A).

EA intervention

After the rat models were established, the two EA‐exposed groups (CON_EA and SAP_EA) were treated with EA four times for 12 h each until all animals were sacrificed after 72 h. The animals were gently placed in a specific fixator tailored to expose both the immobile hindquarters and ST36 acupoints. EA was administrated with continuous‐mode stimulation for 30 min, an electrical current of 1.0 mA, and a frequency of 3 Hz for 2 s (sparse wave) or 6 Hz for 3 s (dense wave), controlled by a stimulator (Model 3800, A‐M Systems). The EA was placed on the hindlimb ST36 by obliquely inserting pairs of 0.23 × 13 mm unipolar stainless‐steel needles 7 mm deep in each spot, which was situated approximately 4 mm below the rat knee joint and about 2 mm lateral to the rat anterior tubercle of the tibia. The positive and negative electrodes were connected to two acupuncture needles separated by 1 mm, both of which were inserted at ST36.

Sample collection

After 72 h of the L‐ornithine‐induced SAP model being established, the rats in each group were intraperitoneally injected with 3% pentobarbital (20 mg/kg) for anaesthesia. Blood samples were collected from the heart, centrifuged at 3000 rpm at 4°C for 15 min, and the supernatant was stored in a freezer at −80°C for subsequent serum analysis. The rat pancreas was removed along the duodenum and placed in a 4% paraformaldehyde fixative. Then, 75% alcohol and iodophor were used for repeated disinfection of the cecum and its surroundings. High‐temperature sterilization was used to disinfect the eye, and forceps were used to lift the free end of the cecum. This disinfection method was repeated. A small incision (approximately 65 mm) was made using eye scissors. Sterile gloves were then changed to avoid contact with the incision end of the cecum and the entrance end of the sterile freezing tube. The cecal contents were quickly extruded into the sterile freezing tubes (processed using high‐temperature sterilization). After temporary storage in liquid nitrogen, the samples were transferred to the −80°C freezer for preservation for subsequent microbiological and metabolic‐related testing (Piccolo et al., 2018). The colon and ileum were removed from the intestines, and the mesentery was removed. The intestines were washed with sterile saline until no excretion was observed. The cleaned intestinal tissue was cut into sections measuring 2, 2, and 4 cm. The 4 cm intestinal segments were stored in a −80°C freezer. The 2 cm intestinal segments were placed in a neutral 10% formalin solution.

GC–MS analysis

Cecal contents (25 mg) were collected for SCFAs extraction, which was initially mixed with 900 μL methanol and 100 μL 2‐ethylbutyric acid (1000 μg/mL) as an internal standard. These mixtures were homogenized with mill beads under 50 Hz at ‐10℃ for 3 min, then implemented ultrasound under 40 kHz for 30 min in ice bath. The supernatant was obtained by centrifuging the samples at 13,000 g for 15 min at 4°C, and 1 μL sample was installed into the GC/MSD (8890B‐5977B, Agilent, Santa Clara, CA, USA) under these conditions: helium, 99.999%; constant flow rate, 1 mL/min; inlet temperature, 260°C; initial temperature, 80°C; increased to 120°C with 40°C/min, and then increased to 200°C with 10°C/min, and finally held at 230°C for 6 min. Data acquisition was performed using the full scan mode with a range of m/z 30–300 and finally identified and quantified using Masshunter software (v10.0.707.0, Agilent).

DNA extraction, 16S rRNA gene amplification, and sequencing

Microbial genomic DNA was extracted from cecal contents using the E.Z.N.A.® soil DNA kit (Omega Bio‐tek, Norcross, GA, USA). DNA quality was assessed using a NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA). Amplifications of the V3‐V4 hypervariable region of the bacterial 16S rRNA gene were conducted using primer pairs 338F and 806R. PCR products were detected by 2.0% agarose gel electrophoresis. PCR products were purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The amplification products were quantified using a Quantus fluorometer (Promega). These purified amplicons were further prepared and sequenced on an Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA) by Shanghai Majorbio Bio‐Pharm Technology Co., Ltd. (Shanghai, China). Raw paired‐end raw data from the original DNA fragments were aggregated using FLASH (v.1.2.7). Quality filtering, length trimming, and sequence truncation were processed by OTU clustering; taxonomy assignments were performed using UPARSE (v.7.1) at a 97% similarity cutoff, and chimeras were identified and removed. The taxonomy of each OTU representative sequence was analysed using the RDP Classifier (v.2.2) against the 16S rRNA database (Silva, v.138) at 70% confidence. Microbiome data were analysed using a web platform provided by Shanghai Majorbio Biopharm Technology Co., Ltd.(Shanghai, China). The Kruskal‐Wallis rank sum test was used to assess any significant differences in α diversity between the four groups. A principal coordinate analysis (PCoA), based on the Bray_Curtis analysis, was conducted to compare community distributions. Based on the results of the OUT division and classification status identification, the composition and abundance distribution of each sample at different classification levels of phyla and genera were obtained and clustered. The correlations concerning the degree of association among different genera and SCFAs were analysed using Spearman correlation tests (using R version 3.3.1) with the pheatmap package, using setting parameters (|r| ≥ 0.6 and a p‐value of <0.5).

Enzyme‐linked immunosorbent assay (ELISA)

Blood samples from each group were centrifugated at 1500 g for 15 min to obtain the supernatant. Serum pancreatic amylase and lipase activities were measured by West China‐Frontier Pharma Tech (Chengdu, China). Enzyme‐linked immunosorbent assay kits (NEOBIOSCIENCE, Shenzhen, China; Jiangsu Meimian Industrial Co., Ltd., Jiangsu, China) were used for detecting serum TNF‐α, IL‐6, and LL‐37 levels. A chromogenic Limulus amebocyte lysate assay (XIAMEN BIOENDO Technology Co., Ltd., Xiamen, China) was used to measure serum endotoxin levels.

Histopathology analysis

Fresh pancreatic tissues and intestinal tissues flushed with saline were immersed in 10% neutral buffered formalin for at least 48 h, then paraffin‐embedded, and cut into 5‐μm sections. The sections were stained with haematoxylin and eosin (H&E) and blinded for an assessment using a Zeiss Axio Imager A2 light microscope (Zeiss, Germany) set up with Zeiss ZEN software. Neutrophil infiltration into the intestinal tissue of the rats was assessed by immunohistochemical staining with anti‐myeloperoxidase (MPO) antibodies (Proteintech, 22,225‐1‐SAP). For immunofluorescence staining, primary antibodies anti‐Occludin (1:800, 27,260‐1‐SAP, ProteinTach), anti‐ZO‐1 (1:2000, 21773‐1‐SAP, ProteinTach), and anti‐LL‐37 (1:100, A15653, Abclonal) were used.

Western blot

Total protein content extracted from intestinal tissues was evaluated using radioimmunoprecipitation assay lysis buffer with a protease inhibitor cocktail and quantified using a BCA protein assay kit (Beyotime Biotechnology, China). Proteins were resolved by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and blotted onto PVDF membranes and fluoride membranes. The primary antibodies against β‐actin, Occludin, ZO‐1, and LL‐37 were 1:1000, MPO (1:750), β‐actin (1:50,000), and HRP goat anti‐rabbit IgG antibody (1:10,000). Protein bands were visualized using an enhanced chemiluminescence reagent and quantified using the Western Blot Imager (Bio‐Rad, Hercules, CA, USA). The densitometric outcomes of immunoblotting signals were expressed as the ratio to β‐actin and finally normalized to those obtained from the sham operation rats.

Statistical analysis

Values are expressed as mean ± standard deviation, and statistical analysis was performed using the GraphPad Prism 9.2.2 software. The normality of the distribution was verified with Levene's test. Normally distributed variables were analysed with a one‐way ANOVA for multiple comparisons, followed by a two‐tailed Student's t test. Non‐normally distributed variables were analysed with the Kruskal‐Wallis test for multiple comparisons, followed by the Mann–Whitney U test. p‐values of <0.05 were considered indicative of statistically significant results.

RESULTS

EA reduced the severity of SAP in rats

H&E staining was used to determine whether the SAP model was established. After the intraperitoneal injection of L‐ornithine in the SAP model group, severe interstitial oedema, diffuse pancreatic acinar cell necrosis, extensive inflammatory cell infiltration, haemorrhage, and increased pancreatic histopathology scores were observed, compared to those in the control group (Figure 1A,B). Moreover, we observed an increase in the levels of systemic inflammatory indicators in SAP rats (Figure 1C–G). This finding indicates the successful establishment of the SAP model. In the CON_EA group, pancreatitis markers were unaffected. Histological damage in the pancreas of the SAP_EA rats was significantly alleviated compared to that in the SAP rats, as evidenced by the decreased inflammatory infiltration, oedema, and acinar necrosis scores (Figure 1A,B). Pancreatic amylase and lipase levels, and serum TNF‐α and IL‐6 levels (Figure 1C–F), were reduced in the SAP_EA rats, and the antimicrobial peptide LL‐37 (Figure 1G) concentration in the serum samples was increased in the SAP_EA rats, confirming the anti‐inflammatory and protective effects of EA supplementation in SAP. In summary, EA reduced the severity of SAP.

IGURE 1.

IGURE 1

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Pancreatic histopathology findings and inflammatory factors levels in L‐ornithine‐induced SAP. (A) Haematoxylin and eosin staining of animal pancreatic tissue after the injection of normal saline and L‐ornithine (magnitude: 100 μm; scale: 100×). (B) Pathological scores of the pancreas in four groups(n = 8). (C, D) Serum levels of amylase and lipase in four groups (n = 8). (E–G) Serum levels of TNF‐α, IL‐6, and LL‐37 in four groups (n = 4). The data are presented as means with standard deviations. ****p < 0.0005, **p < 0.05.

EA modulated intestinal microbiota abundance in SAP rats

The progression of SAP is closely related to intestinal dysbiosis (Signoretti et al., 2017). Accumulating evidence suggests that acupuncture can improve the composition of the intestinal microbiota (An et al., 2022; Wang et al., 2023; Xu et al., 2023). Therefore, we collected the cecal samples from rats to evaluate the impact of EA on the microbiota in L‐ornithine‐induced SAP models by using 16S rDNA gene sequencing. The alpha diversity results showed that there was no significant difference in the intestinal bacterial richness measured by the Chao index among the four groups of rats at the OTU level (Figure 2A). For the Shannon index, alpha diversity was higher in the SAP group than in the CON groups, indicating that the microbiome in the cecum segment was disturbed in the SAP model (Figure 2B). The increase in diversity in the SAP group was partially relieved by the administration of EA. Moreover, macrobiotic diversity demonstrated that the microbiota composition of the CON_EA and SAP groups differed, with an increase in alpha diversity in the CON_EA groups, suggesting that EA may modulate the intestinal microbiota under different conditions. In addition, principal coordinates analysis (PCoA) demonstrated that the composition of the intestinal microbiota in these groups was distinct (Figure 2C).

FIGURE 2.

FIGURE 2

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Effect of EA at ST36 on intestinal microbiota in rats with SAP. (A) Chao 1 index of four groups (n = 5). (B) Shannon index of four groups (n = 5). (C) Comparisons of cecal content community composition were analysed based on the Bray_Curtis distance algorithm of PCoA principal coordinates (n = 5). (D) Dominant genera in cecal contents of rats in four groups changed at the genus level (n = 5). (E) Microbes and relative abundance of intestinal flora in the cecal contents of rats in four groups at the phylum level (n = 5). (F) Ratio of Firmicutes to Bacteroidota counts (n = 4).

We further explored the variations in the microbiome abundance at different taxonomic levels. Taxonomically, the differences in the microbial distributions of the four groups were visualized by classification from phylum to genus (Figure 2D,E). Firmicutes and Bacteroidota were the most abundant taxa at the phylum level (Figure 2E). The results of the relative abundance analysis showed a profound decrease in Firmicutes and a simultaneous increase in Actinobacteriota levels in the cecal content of the SAP rats, as demonstrated by previous studies (Han et al., 2022; Pushalkar et al., 2018), while the level of Actinobacteriota in the SAP_EA rats was slightly alleviated (Figure 2E). In addition, the CON_EA rats, compared to the saline injection group, were enriched with Bacteroidetes, which are primarily involved in the fermentation of the carbohydrates and the synthesis of SCFAs such as acetate and antimicrobial proteins by the host Paneth cells. Finally, at the phylum level, the Firmicutes/Bacteroidota ratio differed between the SAP and SAP_EA groups (Figure 2F).

At the genus level, Lactobacillus was the predominant bacteria in the cecal content of the CON group as compared with the other four groups (Figure 2D). However, the proportion of bacteria in the SAP group was reversed, and that in the SAP_EA group was recovered. Compared to previous experiments, a shift from Lactobacillus to norank_f_Muribaculaceae was observed in the cecum in the CON and SAP groups (Figure 2D). Moreover, 16S rRNA gene amplicon information of the supplemented SAP_EA group showed that the genera of the two dominant probiotics (Lactobacillus and Bifidobacterium) accounted for approximately 50% of the total bacteria, which was different from those present in the SAP rats (norank_f_Muribaculaceae and Bifidobacterium) (Figure 2D). The results demonstrated that the microbiota composition in the SAP and CON groups differed significantly, with a change in alpha diversity that was partially recovered by EA.

SCFAs of SAP rats after EA treatment tended to increase

Targeted metabolomic determination and analysis of the concentrations of SCFAs demonstrated that butanoic acid, acetic acid, propanoic acid, isobutyric acid, valeric acid, and hexanoic acid from cecal contents were profoundly depleted in the L‐ornithine‐induced SAP rats compared to those in the normal saline control rats (Figure 3A–G). The isohexanoic acid content noticeably increased in the SAP group, although the difference was not statistically significant (Figure 3H). Notably, butyric acid production was significantly reduced in the SAP group. Although the measured SCFAs were not statistically significant, there was a trend toward increasing SCFAs in the SAP_EA group rats treated with EA, including an increase in butyric acid levels (Figure 3B). No significant differences were observed between the CON and CON_EA groups. Butyric acid, an important intestinal microbiota‐derived metabolite, is a carbon source for the colonic enterocytes, which may protect against intestinal barrier injury. Butyric acid can modulate the assembly of tight junctions, leading to the enhancement of the intestinal barrier. It can tighten the intestinal epithelial cell barrier and reduce intestinal barrier permeability by inducing the anti‐inflammatory cytokine IL‐10RA‐dependent inhibition of the claudin‐2 protein (Zheng et al., 2017). In addition, it is closely involved in modulating intestinal pathogenesis, offering a phylactic role against intrusive pathogens (Wang et al., 2020). Therefore, increasing butyric acid levels after EA treatment may improve outcomes in rats with SAP.

FIGURE 3.

FIGURE 3

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Effect of EA at ST36 on SCFAs metabolism in the intestinal microbiota of rats with SAP. (A–H) Comparison of the concentrations of eight common SCFAs in cecal contents of rats in four groups (n = 5). (I) Heatmap of correlations among intestinal microbiota and SCFAs levels. ***p < 0.001, **p < 0.01, *p < 0.05.

We compared the Spearman correlation coefficients between SCFAs and the intestinal microbiota (Figure 3I). At the genus level, Romboutsia and Lachnospiraceae_UCG‐006 levels were positively correlated with butanoic acid, hexanoic acid, valeric acid, isobutyric acid, and isovaleric acid levels (all p < 0.05). Acetic acid level was positively correlated with Lactobacillus level (p < 0.05). The butanoic acid level was negatively correlated with Bifidobacterium, Faecalibaculum, and Allobaculum levels (all p < 0.01).

EA reduced intestinal inflammation and repair intestinal injury in SAP rats

Considering the critical role of intestinal homeostasis in the progression of SAP, we evaluated intestinal injury in an experimental SAP model. Rats in the SAP group exhibited significant macroscopic pancreatic and intestinal lesions at necropsy 72 h after the first injection. H&E staining revealed marked intestinal damage characterized by epithelial cells exfoliation, inflammatory infiltration, villi oedema, and crypt atrophy, distortion, and damage in the SAP rats, which were ameliorated in the SAP_EA rats (Figure 4A). The intestines of the CON and CON_EA rats showed no lesions, and no difference was observed in the histological scores (Figure 4B,C). We observed consistent changes in intestinal permeability in the four groups compared with those in serum endotoxin levels in both saline control rats (CON vs. CON_EA, p > 0.05) and L‐ornithine model rats (SAP vs. SAP_EA, p < 0.05) (Figure 4D).

FIGURE 4.

FIGURE 4

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Effects of EA at ST36 on intestinal tissue injury and inflammation in rats with SAP. (A) Typical images of pathological sections of the terminal ileum and colon of four groups (magnitude: 100 μm; scale: 100×). (B) Four groups of Chui's score (n = 8). (C) Four groups of colon pathology scores (n = 8). (D) Serum levels of endotoxin in four groups (n = 4). (E) Typical immunohistochemistry MPO findings in the intestinal tissues samples of four groups (magnitude: 100 μm; scale: 100×). The experimental values are presented as means and standard deviations. ****p < 0.0005, ***p < 0.005.

To assess the ability of EA to reduce the degree of acute intestinal inflammation in rats with SAP, immunohistochemical analysis of myeloperoxidase was performed to detect the sequestration activity of neutrophils in intestinal tissues with severe morphological damage in the colon. Rats with SAP induced by L‐ornithine injection showed a significant increase in the level of MPO in colonic tissue compared with normal saline controls, but EA administration prevented this increase, suggesting the recovery of SAP‐associated intestinal inflammation and injury in SAP_EA rats (Figure 4E).

EA improves intestinal barrier function in SAP rats

To study the effect of EA on intestinal barrier function, we combined immunostaining and immunoblotting to estimate the mechanical and chemical properties of the barriers in the SAP rats. Fluorescent staining diagram (Figure 5A,B,D,E) showed occludin (green) and ZO‐1 (green) at the paracellular junctions of the intestinal epithelial cells, and the antimicrobial peptide LL‐37 (green) was observed at the mucosal surface of the colonic epithelial layer. Intestinal specimens from the SAP group showed weak occludin, ZO‐1, and LL‐37 protein expression in intestinal epithelial cells. The differential distribution of tight junctions and endogenous chemical barrier proteins indirectly reflects the extent of intestinal barrier destruction. However, their levels showed a trend toward improvement with EA treatment compared to the effects observed in the SAP group.

FIGURE 5.

FIGURE 5

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Effect of EA at ST36 on the intestinal barrier in rats with SAP. (A, B) Immunofluorescence staining of ZO‐1, occludin and LL‐37 protein in ileum tissues samples of four groups of animals (magnitude: 100 μm; scale: 100×). (C) Typical western blot images of ZO‐1, occludin and LL‐37 proteins in the ileum of four groups. (D, E) Immunofluorescence staining results of ZO‐1, occludin and LL‐37 protein in the colon tissues samples of four groups of animals (magnitude: 100 μm; scale: 100×); (F) Typical western blot images of ZO‐1, occludin and LL‐37 proteins in ileum of four groups. The values are presented as means and standard deviations.

Western blotting results (Figure 5C,F) showed that the expressions of occludin, ZO‐1, and LL‐37 in the colon of rats was attenuated in the SAP group, which was notably increased with EA treatment. Finally, the expression of MPO in colon returned to within the normal range after EA, as demonstrated by our previous immunohistochemistry results (Figure 4E). The immunoblotting results were consistent with the merged fluorescence images.

DISCUSSION

SAP is a potentially life‐threatening disease associated with severe comorbidities, high mortality rates, and prolonged hospitalization (Xiao et al., 2016). It is characterized by oxidative stress, inflammatory processes, and intestinal dysfunction, including damage to the intestinal barrier and subsequent bacterial translocation, which contributes to SAP severity (Cen et al., 2018; Li et al., 2020; Martens et al., 2018). Accumulating evidence indicates that the intestinal microbiota participates in the progression of SAP; however, given limited therapeutic strategies, further studies are needed to elucidate what may constitute effective management of SAP‐associated intestinal lesions.

Acupuncture has shown positive effects on intestinal microbiome composition and general human health. Thus, we established an SAP rat model to explore the impact of EA on SAP and its associated intestinal barrier injury. Our findings showed that EA may have protective effects against SAP and intestinal lesions, given following outcomes: decreased serum biomarkers and cytokines levels, pancreatic and intestinal microanatomical changes, antimicrobial peptide expression, and tight junction disruption in inflamed intestines. These effects are potentially mediated by the modulation of the intestinal microbiota.

Our study is aimed to investigate the effect of EA on the intestinal microbiota of SAP rats after the administration of a basic amino acid (L‐ornithine). We observed no significant change in the alpha diversity of the cecal microbiota in the SAP rats induced by 30% L‐ornithine, as indicated by the Shannon index of the SAP rats, which was similar to that of the control rats. Moreover, we observed that EA stimulation tended to increase the intestinal microbiota diversity in rats. Furthermore, we discovered that EA likely modulated the bacterial community structure during SAP. In our study, Lactobacillus levels, considered to be a beneficial bacterium, increased in rats after EA compared with the SAP group. Lactobacillus may constrain the growth of pathogenic microorganisms and protect the host intestinal epithelium (e.g., immune modulation). Moreover, Ruminococcus levels, which is considered a putatively aggressive bacterium of critical importance in Crohn's disease (Henke et al., 2019), decreased after treatment with EA in the SAP rats. Studies on the inflammatory properties of Ruminococcus found that they could produce many metabolites in the form of glucomannan polysaccharides, which can trigger the immune system, such as TNF‐α (Chua et al., 2018). Most importantly, TNF‐α is an inflammatory biomarker of Crohn's disease (Claßen et al., 2022). Blautia is also thought to be not only the potentially protective microbe but also less abundant in UC hosts (Liu, Mao, et al., 2021). The results of this study are consistent with those of other studies confirming the protective effect of the host intestinal microbiome against lower‐abundance microbes, which can be enriched by SAP as well as from invading pathogens. These results suggest that the treatment of the hindlimb ST36 region with EA can drive the restoration of the intestinal microbiota in a favourable direction in SAP. In addition, although the administration of EA changed the microbiome composition in both desirable and undesirable directions, compared to the changes observed in the CON group, EA in other areas or with varying settings of stimulation still had a positive effect on intestinal microbes. Further research may help clarify whether selective EA can modulate the diversity, composition, structure, and function of the intestinal microbiota. In addition, intestinal microbe‐ derived metabolites, such as SCFAs, modulate the host immune system and contribute to intestinal mucosal homeostasis via metabolic signalling pathways (Chen & Vitetta, 2020; Schauber et al., 2003). We administered intraperitoneal injections of basic amino acids to normal rats, revealing that a 30% L‐ornithine solution attenuated the production of SCFAs in the SAP group, which was the first report in the field of L‐ornithine‐induced SAP research. In our study, targeted metabolomic analysis of eight common SCFAs showed that, compared to those in the control group, the concentration of SCFAs in the cecum of SAP model rats decreased to different degrees, with a significant depletion of butyric acid. After EA treatment at the ST36, the concentration of SCFAs, especially that of butyric acid, in the cecum of the rats showed different degrees of increase compared to those in the SAP group. The butyric acid level was especially depleted in rats with necrotizing pancreatitis progression compared to that in the control rats (van den Berg et al., 2021). A growing body of evidence has demonstrated that supplementation with butyric acid may have a protective effect, with a noticeable increase in the expression of genes involved in paracellular junctions and a reduction in bacterial dissemination, serum endotoxin content, and mortality rates in AP. This approach promotes intestinal barrier repair, thus preventing increased intestinal penetration (Pan et al., 2019; van den Berg et al., 2021). An increase in beneficial bacteria levels in the host gut microbiota is accompanied by an increase in the SCFA content. SCFAs, coupled with butyric acid secreted following EA treatment, help reduce inflammation and improve intestinal barrier injury in patients with SAP. Supplementing with butyric acid in mice with AP has been shown to enhance intestinal barrier integrity, reduce pancreatic tissue necrosis and serum lipopolysaccharide‐binding protein levels, and alleviate oxidative stress in the pancreatic tissue (Xia et al., 2023). SCFAs may help regulate mucus production, provide fuel for epithelial cells, maintain intestinal barrier integrity, and promote the expression of tight junction protein (Blaak et al., 2020; Peng et al., 2009; Zhao et al., 2018). Although no significant differences were found in the various SCFAs, EA tended to increase their concentrations. Therefore, we speculated that EA may affect the intestinal barrier, regulate changes in the intestinal microbiota and fatty acid metabolism, maintain normal intestinal permeability, and thus potentially counteract the pathological and clinical progression of SAP. Low‐intensity EA in the hindlimb ST36 regions can reduce intestinal villus swelling, inflammatory cell infiltration, and epithelial defects, and it can activate the neutrophils biomarker (MPO) overexpression in these tissues. Moreover, the intestinal barrier impairment associated with SAP is characterized by the downregulating of tight junction proteins expression, excessive intestinal permeability, and endotoxin dissemination (De Beaux & Fearon, 1996; Sonika et al., 2017). Impaired intestinal barrier function may facilitate the translocation of microbial components, such as lipopolysaccharide (LPS), from the intestinal lumen into the circulation. However, the epithelium‐derived antimicrobial peptide LL‐37 is one of the most critical components of host defence on the human colonic mucosa surface and is modulated by SCFAs separately through distinct pathways. Our findings suggested that EA increased the expression of tight junctions markers (occludin and ZO‐1) and antimicrobial peptides (LL‐37) and decreased serum endotoxin levels in L‐ornithine‐induced SAP rats. Moreover, previous studies have shown that increased proinflammatory cytokines levels, including TNF‐α and IL‐6, may contribute to increased tight junction permeability and intestinal injury (Tatiya‐Aphiradee et al., 2018). Herein, we observed lower levels of IL‐6 and TNF‐α after EA treatment in the SAP_EA rats. These results suggest that EA may have a protective effect against intestinal injury in rats with SAP by upregulating the expression of antibacterial peptides, repairing the intestinal barrier, and restoring barrier permeability.

CONCLUSION

EA at ST36 may modulate the intestinal microbiota and its metabolic derivatives; increase the expression of intestinal barrier‐related proteins occludin, ZO‐1, and LL‐37; repair the injury of the SAP intestinal barrier; reduce intestinal tissue inflammation; and reduce bacterial endotoxin transition into the blood, thus alleviating pancreatic and systemic inflammatory responses and improving the prognosis of SAP.

AUTHOR CONTRIBUTIONS

Huimin Xu: Data curation (equal); methodology (equal); writing – original draft (equal); writing – review and editing (equal). Qian Wen: Conceptualization (supporting); methodology (equal); Data curation (equal); writing – review and editing (supporting). Hangqi Hu: Data curation (equal); methodology (equal); writing – original draft (equal). Sihao Yang: Writing – original draft (supporting); writing – review and editing (supporting). Lingyun Lu: Conceptualization (supporting). Xiangyun Hu: Methodology (supporting). Hao Li: Investigation (supporting). Xianhao Huang: Investigation (supporting). Ning Li: Writing – review and editing (supporting).

ACKNOWLEDEMENTS

This study was supported by the National Natural Science Foundation of China (No. 82104591).

FUNDING INFORMATION

No funding information provided.

CONFLICT OF INTEREST STATEMENT

The author declare that they have no competing interests.

ETHICS STATEMENT

This study was approved by the Ethics Committee of Animal Experiments of the West China Hospital of Sichuan University for Animal Care and Use.(Approval number: 20211228A).

Xu, H. , Wen, Q. , Hu, H. , Yang, S. , Lu, L. , Hu, X. et al. (2024) Electroacupuncture at ST36 modulates the intestinal microecology and may help repair the intestinal barrier in the rat model of severe acute pancreatitis. Microbial Biotechnology, 17, e14401. Available from: 10.1111/1751-7915.14401

Huimin Xu, Qian Wen and Hangqi Hu are contributed equally to this work.

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