Carotid baroreceptor stimulation attenuates obesity-related hypertension through sympathetic-driven IL- 22 restoration of intestinal homeostasis

Abstract

Background

Gut microbiota and its metabolites, as well as the intestinal barrier, play important roles in the development of obesity-related hypertension. Sympathetic nerves are critical for intestinal homeostasis. Carotid baroreceptor stimulation (CBS) has been shown to exert protective effects against hypertension via sympathetic tone reduction. This study aimed to reveal the effects of CBS treatment on intestinal homeostasis and its underlying mechanisms in obesity-related hypertension.

Methods

An animal model of obesity-related hypertension was established with Sprague–Dawley rats by a high-fat diet and 10% fructose solution for 13 weeks. CBS devices were implanted at the 5 th week. The effects of CBS on body weight, blood pressure, gut microbiota, intestinal autonomic nerve, intestinal barrier, and type 3 innate lymphoid cells (ILC3 s) were investigated.

Results

CBS treatment significantly reduced blood pressure and body weight in rats with obesity-related hypertension. In addition, CBS obviously improved gut microbial dysbiosis and intestinal barrier damage. Interestingly, after an 8-week CBS intervention, the obesity-related hypertensive rats exhibited a dramatic decrease in sympathetic nerve distribution and norepinephrine concentration, as well as an increase in IL- 22 production by ILC3 s in the intestine.

Conclusions

CBS increased IL- 22 production in ILC3 s to alleviate gut microbial dysbiosis and intestinal barrier destruction, thus improving obesity-related hypertension in rats.

Introduction

Over 70% of obese individuals develop hypertension by midlife, making obesity-related hypertension (OH) the leading phenotype of resistant hypertension in metabolic syndrome [1]. Current antihypertensive treatments fail to control 40–50% of OH cases due to persistent gut-derived neuroimmune activation [2]. Three interlinked mechanisms connect obesity to hypertension: sympathetic nervous system (SNS) hyperactivation causing renal sodium retention, gut-derived pro-inflammatory metabolites impairing vascular function, and intestinal barrier breakdown enabling endotoxin translocation [3, 4]. This triad forms a self-sustaining loop, where neural dysfunction disrupts microbial ecology, driving neuroinflammatory cascades that evade conventional therapies.

Emerging evidence highlights pro-inflammatory cytokines such as TNF-α, IL- 6, and IL- 1β as key mediators linking gut microbiota dysbiosis and OH pathogenesis. These cytokines exacerbate systemic insulin resistance, endothelial dysfunction, and structural damage to intestinal tight junction proteins, establishing a vicious cycle by altering gut microbial composition [5, 6]. Recent advances position the gut as a central regulator of OH through three pathways: microbial metabolite-mediated endocrine signaling, epithelial barrier dysfunction allowing bacterial translocation, and immune activation involving adaptive and innate responses [7]. ILC3-derived IL- 22 plays a critical role in counter-regulating these processes by suppressing pro-inflammatory Th17 cell differentiation and enhancing regulatory T cell function, thus maintaining intestinal homeostasis [8].

Intestinal sympathetic nerves exhibit bidirectional immunomodulation. Chronic sympathetic activation promotes inflammation via β-adrenergic receptor-dependent immunosuppression, while acute noradrenaline release triggers β2-AR/STAT3 signaling in ILC3 s, upregulating barrier-protective IL- 22 [9, 10]. Precise modulation of sympathetic tone, rather than complete blockade, may be necessary for optimal intestinal homeostasis.

Current OH therapies are ineffective in addressing gut-derived pathophysiology and may worsen microbial dysbiosis [11]. This gap motivates exploring neuromodulation strategies that target both neural drivers and peripheral effectors of OH. Carotid baroreceptor stimulation (CBS) is a promising technique with key advantages: (1) It maintains physiological baroreflex adaptability through phase-specific activation. (2) It avoids irreversible nerve damage unlike renal denervation. (3) Our prior research demonstrated its benefits for cardiometabolic parameters in obesity [12,13,14]. In the present study, we hypothesized that CBS could reduce chronic sympathetic overexcitation and restore intestinal homeostasis in OH rats, and the mechanism may involve CBS promoting IL- 22 production by ILC3 s.

Materials and methods

Animals and study protocol

Six-week-old male Sprague–Dawley rats were purchased from Hunan SJA Laboratory Animal Co., Ltd (Changsha, Hunan, China). The rats were housed under temperature-controlled conditions and a 12-h light/dark cycle with free access to drinking water and food. All procedures were reviewed and approved by the Institutional Animal Care and Use Committees at Renmin Hospital of Wuhan University (IACUC Issue No. WDRM20220303 A).

After 1 week of adaptation, 28 rats were randomly assigned to two groups. OH models were established by administering a high-fat high-fructose diet (HFHFD) for a duration of 13 weeks. HFHFD consisted of a high-fat diet (D12492, Beijing HFK Bioscience, China) and 10% fructose solution. The control group was fed a normal diet (ND) consisting of standard chow diet (D12450B, Beijing HFK Bioscience, China) and tap water. At the 5 th week, the rats in the OH group underwent either sham operation (OH-sham, n = 7) or CBS implantation (OH-CBS, n = 7). Similarly, the control group was divided into C-CBS group (n = 7) and C-sham group (n = 7), with or without CBS implantation.

The fecal contents of the rats were collected at the 0 th, 4 th, and 12 th weeks. At the end of the experiments, after 24 h urine collection, the rats were euthanized under fasting conditions. Then, blood and small intestine samples were collected for further detection.

Blood pressure and weight measurement

Blood pressure was measured noninvasively weekly from 8 to 10 am using tail-cuff plethysmography (CODA; Kent Scientific Corporation, United States). The average of five independent blood pressure measurements was taken at each session.

Body weight was measured weekly at room temperature, and visceral adipose tissue was resected and weighed at the end of the experiments.

CBS device implantation

The CBS device (custom-designed and custom-made by Three Lion Technologies Co., Ltd., Wuhan, China) implantation method was based on a protocol previously reported by our group [12]. The CBS device includes a stimulator unit and a metallic electrode tip. Under anesthesia, rats underwent a neck incision to uncover the right carotid sinus. The electrode tip was placed above the right common carotid artery near the internal carotid sinus, and the stimulator unit was implanted subcutaneously.

Bacterial 16S rRNA gene sequencing

Fecal DNA was extracted from the samples using a quick-DNA fecal microbe kit (Zymo Research, Irvine, CA). Primers compatible with Pfu DNA Polymerase (TransGen Biotech, China) were used to amplify bacterial 16S V4–V5 variable regions. The NovaSeq platform was used for double-ended sequencing of the 16S rRNA gene. QIIME2 (2020.11) and R (v3.2.0) software packages were utilized for analyzing the sequence data [15]. Sequencing data were deposited in the Sequence Read Archive (https://www.ncbi.nlm.nih.gov/sra/PRJNA1100318).

Metabolic analysis

Serum levels of short-chain fatty acids (SCFAs), trimethylamine N-oxide (TMAO), and their precursors were detected using UPLC–MS/MS (Santa Clara, CA, USA), and the final data were normalized based on serum volume [16].

RNA extraction and real-time quantitative PCR

Total RNA was isolated from small intestinal tissue with TRIzol (TAKARA) and converted to cDNA using the HiScript Reverse Transcriptase Kit (VAZYME). Quantitative real-time PCR was conducted with SYBR Green Master Mix (VAZYME), and results were analyzed using the qTOWER 2.2 system (Analytik Jena). The primer sequences are shown in Supporting Information Table S2.

Histological assessment

After sacrificing the rats, small intestinal (The ileum located 10–15 cm away from the cecum) tissue was fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 8–10 µm slices. The sections were deparaffinized and stained with hematoxylin and eosin (H&E), Masson’s trichrome, and Alcian blue periodic acid-Schiff (AB-PAS). Stained sections were examined and imaged using a microscope (PHILIPS, Netherlands).

Immunofluorescence staining

Paraffin sections of small intestinal (The ileum located 10–15 cm away from the cecum) tissue underwent antigen retrieval in 10 mM citrate buffer for 20 min. After being blocked with 3% BSA for 30 min, the slices were incubated with primary antibodies at 37 °C for 1 h. Following three washes in PBS, secondary antibodies were applied and incubated at 37 °C for 2 h. The slices were washed three times with PBS and labeled with DAPI before fluorescence signals were detected using a confocal laser microscope (Nikon, Tokyo, Japan). The details of the antibodies are shown in Supporting Information Table S3.

Western blots

Protein levels in small intestinal tissue samples were determined using BCA protein quantitative detection kit (G2026 - 200 T, Servicebio). After SDS–PAGE, proteins were transferred onto a nitrocellulose membrane, blocked with 5% milk, and incubated with primary antibodies at 4 °C overnight. The membranes were incubated with secondary anti-rabbit antibodies for 1 h at room temperature. After washing 3 times in TBST, signals were developed with enhanced chemiluminescence.

Enzyme-linked immunosorbent assay (ELISA)

The levels of norepinephrine (NE) and IL- 22 in the small intestine (The ileum located 10–15 cm away from the cecum) and plasma intestinal fatty acid-binding protein (I-FABP) were measured using the Rat NE ELISA Kit (E-EL- 0047, eBioscince), Rat IL- 22 ELISA Kit (E-EL-R2440, Elabscience) and Rat IFABP/FABP2 ELISA Kit (E-EL-Ro572c, eBioscince), respectively, following the provider’s guidance. The data were analyzed by the FLUOstar Omega (BMG LABTECH).

Tissue preparation and flow cytometry

Small intestinal (The ileum located 10–15 cm away from the cecum) tissue was placed in precooled PBS at 4 °C. Mesentery, adipose tissue, and Peyer’s lymph nodes were removed, and the tissue was washed twice with PBS. The tissue was digested in a solution with gentle shaking at 37 °C for 20 min, then discarded the supernatant. It was chopped, treated with 0.25% collagenase I, and shaken for 2 h at 37 °C. After filtering through 100 μm and 70 μm filters, the filtrate was centrifuged at 1000 rpm for 5 min at 4 °C, and the supernatant was discarded. The pellet was resuspended in 30% Percoll and centrifuged at 2000 rpm for 10 min at 4 °C. The middle layer was collected, washed with PBS, and centrifuged again at 2000 rpm for 10 min at 4 °C. The supernatant was discarded, and the pellet was resuspended in RPMI 1640 complete medium with 1% antibiotics and 20% fetal bovine serum.

Single-cell suspensions were subjected to cell surface staining with flow antibodies (CD45-PEcy7, CD3-APC, and CD90-PE). The cells were processed using eBioscience^™ Foxp3/Transcription Factor Staining Buffer (Thermo Fisher) and stained for the cellular transcription factor RORγt–FITC. Flow cytometry analysis was performed on a BD Fortessa flow cytometer and FACSDiva software (BD Biosciences), and the data were analyzed using FlowJo 9.0. ILC3 markers include CD45⁺, CD3⁻, CD90⁺, RORγt⁺. Antibodies used in this study are listed in Table S3.

Biochemical parameters

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), uric acids (UA), urea, creatinine (Cr), cystatin C (Cys-C), triglycerides (TG), total cholesterol (TCh), low-density lipoprotein cholesterol (LDL-Ch), high-density lipoprotein cholesterol (HDL-Ch), free fatty acid (FFA) and fasting blood glucose (Glu) levels, as well as urine levels of Cr, microalbumin (mAlb) and sodium (Na), were examined using Siemens automatic biochemical analytical instrument (ADVIA1800, Germany).

Statistical analysis

The statistical significance was analyzed using SPSS 19.0 software. Non-normal distributed data were analyzed using the Kruskal–Wallis test. Normal distributed data were assessed with two-way ANOVA test, followed by the LSD post-hoc. A p value of less than 0.05 was considered to indicate statistical significance. All data were presented as the mean ± SEM. At least 3 independent repeats were conducted for each experimental design.

Results

CBS controls body weight gain and reduces blood pressure in obesity-related hypertensive rats

As shown in Fig. 1b–d, HFHFD significantly induced weight gain, visceral fat accumulation, and obesity in rats. CBS effectively mitigated visceral fat deposition and controlled weight gain in obesity-related hypertensive rats. These findings were consistent with other obesity-related indicators (Table S1). There were no significant differences observed in food intake, water intake, and caloric intake between the OH-CBS and OH-sham groups, nor between the C-CBS and C-sham groups (Table S1). These results indicated that CBS ameliorated obesity without affecting caloric intake. Moreover, the rats in OH-CBS group exhibited reduced systolic blood pressure, diastolic blood pressure, and mean arterial pressure as compared to the OH-sham group (Fig. 1e–g), indicating a favorable antihypertensive effect of CBS on obesity-related hypertensive rats.

Fig. 1

CBS controls the body weight gain and lowers blood pressure in obesity-related hypertensive rats. a Schematic representation of the study design. SD rats were fed with an HFHFD, or ND and sacrificed at 13 weeks. Fecal contents were collected at 0, 4 and 12 weeks. The CBS was implanted at 5 weeks. b Representative images of rat body size. c Comparison of visceral fat mass among the 4 groups. d Body weight change in rats. e Systolic blood pressure. f Diastolic blood pressure. g Mean arterial pressure. C-sham group, n = 7; C-CBS group, n = 6; OH-sham group, n = 7; OH-CBS group, n = 7. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 versus OH-Sham; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 versus C-sham. FC: Fecal Collection; CBS: Carotid Baroreceptor Stimulation; HFHFD: High-Fat, High-Fructose Diet; ND: Normal Diet

The levels of TCH, TG, and FFA were elevated in the OH-sham group compared to the C-sham and C-CBS groups, whereas HDL-CH levels were lower. CBS administration resulted in a reduction in serum TG concentration in obesity-related hypertensive rats. Urinary sodium excretion was decreased in the OH-sham group compared to the C-sham and C-CBS groups. However, CBS increased sodium excretion in obesity-related hypertensive rats (Table 1).

Table 1 Changes in biochemical parameters, renal function and 24-h urinary sodium excretion after CBS treatment

CBS affects the gut microbial diversity and composition in obesity-related hypertensive rats

There were no significant differences in Shannon diversity or Chao 1 richness among all groups after 4 weeks of HFHFD feeding (Fig. 2a, b). Before the experiments, there was no statistically significant difference in the beta diversity of gut microbiota in rats with or without OH. However, at the 4 th week, a notable difference in the beta diversity of gut microbiota emerged between obesity-related hypertensive rats and control rats (analysis of similarities, R = 0.79, P = 0.001; Fig. 2c). The phylum-level analysis revealed that the ratio of Firmicutes/Bacteroidetes and the relative abundance of Proteobacteria in the fecal samples from obesity-related hypertensive rats were significantly higher when compared with those from control rats at week 4, while no significant differences were observed between obesity-related hypertensive rats and control rats at week 0 (Fig. 2d). LEfSe analysis revealed differences in the taxa between obesity-related hypertensive rats and control rats at the 4 th week (Fig. 2g). The findings imply that alterations in diet have a discernible impact on the gut microbiota of rats.

Fig. 2

Effects of HFHFD on gut microbial diversity and composition. a, b α-Diversity Chao1 index and Shannon index (n = 8–14). c Principal coordinate analysis (PCoA) score plot based on unweighted UniFrac metrics. d Relative abundances of the top 20 dominant phyla. e Relative abundances of the top 20 dominant genera. f Heatmap of the relative abundance of the top 50 dominant genera for each sample. g Cladogram generated from LEfSe analysis

The OH-CBS group exhibited higher alpha-diversity scores compared to the OH-sham group (Fig. 3a, b). The Jaccard distance index of the OH-sham group was significantly distinct from that of the C-sham group. In addition, a notable disparity in the Jaccard distance was observed between the OH-CBS and OH-sham groups (Fig. 3c). At the phylum level, the OH-sham and OH-CBS groups showed higher relative abundance of Proteobacteria, Actinobacteria, and the Firmicutes to Bacteroidetes ratio compared to the C-sham group. Conversely, a lower relative abundance of Tenericutes was observed (Fig. 3d). At the genus level, there was minimal disparity between the C-sham and C-CBS groups, with the highest abundance of Lactobacillus observed in the C-sham group and the lowest in the OH-CBS group (Fig. 3e). LEfSe analysis revealed significant differences in taxa between the OH-CBS and OH-sham groups (Fig. 3g). Several taxa of Firmicutes, including members of the genera Enterococcus, SMB53, Clostridium, Oscillospira, and Roseburia, were positively correlated with visceral fat mass, body weight, and blood pressure. The abundances of Prevotella, [Prevotella], and unidentified_S24_7 exhibited a significant negative correlation with visceral fat mass and body weight (Fig. 3h, i).

Fig. 3

CBS affects the gut microbial diversity and composition of obesity-related hypertensive rats. a, b α-Diversity Chao1 index and Shannon index (n = 4–7). c Principal coordinate analysis (PCoA) score plot based on unweighted UniFrac metrics. d Relative abundances of the top 20 dominant phyla. e Relative abundances of the top 20 dominant genera. f Heatmap of the relative abundance of the top 50 dominant genera for each sample. g Cladogram generated from LEfSe analysis. h Bar graph of the linear discriminant analysis (LDA) score distribution showing genera with LDA scores greater than the set value of 3. i Heatmap of Spearman correlation analysis of the 30 microbes associated with obesity-related parameters and blood pressure. The Spearman rank correlation coefficient (r) ranged from − 1 (blue) to 1 (red). *P < 0.05, **P < 0.01, ***P < 0.001 A, B Statistical comparison: Kruskal–Wallis test

CBS exerts beneficial effects on gut microbiota and related metabolites in obesity-related hypertensive rats

Several gut microbiota metabolites in the serum were quantified. The levels of acetic acid and total SCFAs in the serum were lower in the OH-sham group compared to the C-sham and C-CBS groups. CBS increased acetic acid and total SCFAs levels in the serum of obesity-related hypertensive rats (Fig. 4a). TMAO and creatinine levels were higher in the OH-sham group compared to the C-sham group. Importantly, CBS significantly reduced serum TMAO levels in obesity-related hypertensive rats (Fig. 4b).

Fig. 4

CBS exerts beneficial effects on gut microbiota and related metabolites in obesity-related hypertensive rats. a Concentration of SCFAs in the serum of each group (n = 4–6). b Concentration of TMAO and its precursors in the serum of each group (n = 4–6). c Heatmap of the Spearman correlation analysis of the top 50 microbes associated with SCFAs and TMAO. The Spearman rank correlation coefficient (r) ranged from − 0.6 (blue) to 0.6 (red). d Relative abundance of genera between groups. *P < 0.05, **P < 0.01, versus OH-Sham; ^#P < 0.05, ^##P < 0.01, versus C-sham. A, B, D statistical comparison: Kruskal–Wallis test

The abundances of SMB53, Clostridium, Oscillospira, Allobaculum, and Roseburia were higher in the OH-sham group compared to the C-sham and C-CBS groups. In addition, CBS reduced the abundance of these bacteria in obesity-related hypertensive rats. Furthermore, there was a positive correlation between the abundance of Oscillospira, Allobaculum, and Roseburia with serum caproic acid levels (Fig. 4c, d). In the OH-sham group, there was a decrease in the abundance of Faecalibacterium and unidentifited_S24 - 7 compared to the C-sham group. However, CBS increased the abundance of Faecalibacterium, [Eubacterium], [Ruminococcus], Akkermansia, and unidentifited_S24 - 7 in obesity-related hypertensive rats. There was an inverse correlation between Faecalibacterium and [Ruminococcus] abundances with serum TMAO concentrations. The serum’s caproic acid content was positively correlated with [Eubacterium] abundance and negatively correlated with unidentifited_S24 - 7 abundance (Fig. 4c, d).

CBS attenuates gut pathological alterations and protects intestinal barrier function in obesity-related hypertensive rats

The OH-sham group showed significantly higher collagen-positive area and intestinal wall thickness compared with the C-sham and C-CBS groups while exhibiting a lower goblet cell count and villi length. The OH-CBS group had more goblet cells, longer villi, and thinner intestinal walls than the OH-sham group. CBS treatment resulted in improved intestinal pathological changes in obesity-related hypertensive rats. (Fig. 5a–h). Western blot results revealed a significant decrease in the expression of tight junction markers, claudin- 1 and occludin, in the OH-sham group compared to the C-sham group. However, there was a remarkable increase in claudin- 1 and occludin protein expression following CBS treatment in obesity-related hypertensive rats (Fig. 5i–k). The levels of I-FABP in the plasma were higher in the OH-sham group compared to the C-sham group, while they were lower in the OH-CBS group compared to the OH-sham group (Fig. 5l). CBS restored intestinal barrier integrity and reduced intestinal permeability in obesity-related hypertensive rats.

Fig. 5

CBS attenuates gut pathological alterations and protects intestinal barrier function in obesity-related hypertensive rats. a, e Representative Masson trichrome staining images of small intestinal tissue sections and quantification of the percent fibrotic area (scale bar = 50 µm, images were captured at X400 magnification, n = 6–7). b, f Representative H&E-stained images of small intestinal tissue and quantification of muscular thickness (scale bar = 50 µm, images were captured at X400 magnification, n = 6–7). c, g Representative H&E-stained images of small intestinal tissue and quantification of villus length (scale bar = 100 µm, images were captured at X200 magnification, n = 6–7). d, h Representative AB-PAS staining images of small intestinal tissue sections and quantification of the number of goblet cells per villus (scale bar = 100 µm, images were captured at X200 magnification, n = 6–7). i, j, k Immunoblot analysis of tight junction proteins Claudin- 1 and Occludin in small intestinal tissue tissues (n = 4). l Plasma concentrations of intestinal fatty acid-binding protein (I-FABP). Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

CBS alleviates intestinal autonomic imbalance and reduces intestinal inflammation in obesity-related hypertensive rats

As shown in Fig. 6a–d, the expression levels of tyrosine hydroxylase (TH) in the intestine were higher in the OH-sham group compared to the C-sham group. Conversely, there was no significant difference observed in choline acetyltransferase (ChAT) expression. Following CBS treatment in obesity-related hypertensive rats, TH expression in the intestine decreased significantly, while ChAT expression remained unchanged. Notably, elevated norepinephrine levels were detected in the intestine of the OH-sham group when compared to the C-sham group. In contrast, norepinephrine levels were lower in the OH-CBS group than those observed in the OH-sham group, consistent with variations shown in TH expression. Next, we detected the mRNA expression of inflammatory cytokines in the intestine. When compared with the C-sham group, the mRNA level of IL- 22 in the intestine was downregulated in the OH-sham group, while there were no significant differences in the mRNA levels of IL- 17 A, IL- 6, IL- 5, TNF-α, and IL- 10. After CBS treatment, both IL- 22 and IL- 10 mRNA expressions were upregulated in the intestines of obesity-related hypertensive rats (Fig. 6e–j). Conversely, a significant correlation was observed between IL- 22 and serum metabolites, as well as gut microbiota associated with OH (Fig. 6k, l). Obesity-related hypertensive rats exhibit an augmented distribution of sympathetic nerves and diminished levels of IL- 22 mRNA in their intestines compared to controls. CBS was found to attenuate norepinephrine levels and the dispersion of sympathetic nerves in the gut of obesity-related hypertensive rats while concurrently elevating IL- 22 mRNA expression in their intestine.

Fig. 6

CBS alleviates intestinal autonomic imbalance and reduces intestinal inflammation in obesity-related hypertensive rats. a Representative images of tyrosine hydroxylase immunofluorescence (red) and acetyltransferase immunofluorescence (green), scale bar = 50 µm, images were captured at X400 magnification, n = 6. b, c Quantitative analysis of the immunofluorescence results (n = 6, two visual fields were randomly selected from each section for statistical analysis). d Small intestinal norepinephrine (NE) concentration (n = 6–7). e–j Relative mRNA expression of IL- 22, IL- 17 A, IL- 5, TNF-α, IL- 6, and IL- 10 in the small intestine (n = 6). k, l Heatmap of the correlation network between inflammatory factor mRNA content and genus, TMAO, and SCFAs levels. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

CBS increases IL- 22 production by ILC3 s, improves intestinal mucus barrier, and reduces lipid absorption

The production of IL- 22 in the intestine is primarily attributed to ILC3 s. Therefore, the effects of CBS on ILC3 s function and its effector were examined. Flow cytometry results confirmed that the percentage of ILC3 s in the intestine has no significant difference between C-sham and OH-sham groups. However, the percentage of IL- 22-producing ILC3 s in the intestine was reduced in the OH-sham group compared to the C-sham group. CBS treatment increased the percentage of IL- 22-producing ILC3 s in the intestine, while the total percentage of ILC3 s in CD4⁺ lymphocytes remained unchanged in obesity-related hypertensive rats (Fig. 7a–c). The protein levels of IL- 22 were lower in the intestinal tissue of the OH-sham group compared to both the C-sham and C-CBS groups, while they were higher in the OH-CBS group than in the OH-sham group (Fig. 7d). IL- 22 affects the intestinal mucus barrier and lipid absorption. We found mucin 2 (MUC2) protein and mRNA levels were significantly lower in the OH-sham group compared to the C-sham group (Fig. 7e, f). Moreover, the mRNA expression levels of regenerating islet-derived protein III gamma (Reg III γ), were lower in the OH-sham group compared to both the C-sham and C-CBS groups. Conversely, CD36 and fatty acid binding protein 1 (Fabp1) expression were significantly higher in the OH-sham group than that in both the C-sham and C-CBS groups. After CBS treatment, MUC2 and Reg III γ levels increased, while CD36 and Fabp1 levels decreased in the intestines of obesity-related hypertensive rats. (Fig. 7e–i). CBS increased IL- 22 expression in the intestines of obesity-related hypertensive rats, enhanced intestinal mucus barrier function, and reduced lipid absorption. Therefore, increasing IL- 22 secretion from ILC3 s may contribute to the effects of CBS on the intestinal barrier and microbiota.

Fig. 7

CBS increases IL- 22 production by ILC3 s, improves intestinal mucus barrier, and reduces lipid absorption. a Logical plot of the flow cytometry gate (CD45⁺, CD3⁻, CD90⁺, RORγt⁺). b, c Ratio of ILC3 and IL- 22⁺ILC3 in the intestine was detected by flow cytometry (n = 5). d IL- 22 protein levels in small intestinal tissue (n = 5). e Representative images of MUC2 immunofluorescence (green), scale bar = 50 µm, images were captured at X400 magnification, n = 6. f–i Relative mRNA expression of MUC2, RegIIIγ, CD36, Fabp1 in small intestinal tissue (n = 6). Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001

Discussion

In the present study, it was found that OH was accompanied by dyslipidemia, impaired urinary sodium excretion, excessive sympathetic activation in the intestine, gut microbial dysbiosis, and impaired intestinal barrier function. CBS promoted urinary sodium excretion, reduced norepinephrine levels, attenuated intestinal sympathetic nerve distribution, rebalanced intestinal autonomic nervous activity, blunted intestinal inflammatory response, increased IL- 22 production by ILC3 s, restored intestinal barrier integrity, and balanced gut microbial dysbiosis. Hence, it could be speculated that CBS may ameliorate OH in rats through the mechanisms mentioned above.

Our findings demonstrate the beneficial effects of CBS on intestinal homeostasis and hypertension. Moreover, its modulation of SNS activity suggests possible benefits beyond hypertension, such as improved metabolic and inflammatory profiles. However, potential side effects should also be considered. For example, increasing serum uric acid levels in OH rats and decreasing intestinal tight junction protein levels in normal rats. Future research should focus on identifying any long-term risks associated with CBS and optimizing its application for safe clinical use.

Gut microbiota and metabolites in OH

Multiple lines of evidence suggest that obesity and obesity-related diseases are associated with profound gut dysbiosis [17]. Palmu et al. [18] reported that different strains of Lactobacillus species had varying effects on blood pressure, with some lowering it, while others increased it. In addition, these species were linked to higher 24-h urinary sodium excretion. Sun et al. [19] revealed a negative correlation between systolic blood pressure and the α diversity of gut microbiota. Our findings align with these reports, as CBS markedly enhanced the α-diversity scores of gut microbiota, reduced the abundance of Lactobacillus, increased 24-h urinary sodium excretion, and lowered blood pressure. In addition, CBS reversed obesity-induced alterations in the abundance of key bacterial genera, which may contribute to its antihypertensive effects.

In addition, SCFAs and TMAO are critical gut microbial metabolites that influence host metabolism and blood pressure [20, 21]. SCFAs supplementation has been shown to lower blood pressure, improve insulin sensitivity, and reduce fat accumulation, whereas elevated TMAO levels are linked to hypertension and cardiovascular risks [21,22,23]. Jama et al. [23, 24] provided clinical evidence that acetate and butyrate reduced blood pressure in untreated patients with hypertension. In this study, CBS treatment increased SCFAs, particularly acetate, and decreased TMAO levels, supporting the hypothesis that CBS mitigates hypertension by modulating gut microbial metabolism. Future studies could examine the precise signaling pathways linking CBS-induced microbial changes to metabolic outcomes.

Intestinal barrier function and intestinal immunity in OH

Recent studies have highlighted the role of intestinal immunity in obesity and hypertension, particularly in relation to the intestinal barrier [24, 25]. It has been observed that high-fat diet-induced obesity leads to a reduction in the number of ILC3 s in the colon, which in turn compromises intestinal barrier function and increases permeability [26]. ILC3 s are capable of producing IL- 22, a cytokine that regulates intestinal epithelial cell growth and permeability, mucus production, and antimicrobial peptide (AMPs) synthesis [27]. In addition, IL- 22 also influences metabolism by suppressing lipid transporter expression [28]. IL- 22-deficient mice have weakened barrier function and altered microbiota composition, which can be rescued by IL- 22 treatment [29]. Mar et al. [30] revealed that IL- 22 remodeled the gut microbiome by regulating microbiome composition, function, and AhR ligand production in gut following exogenous IL- 22 treatment in mice.

The composition of the immune system in the intestines is altered in obesity, which serves as a focal point for the crosstalk between intestinal barrier function and gut microbiota [31]. Our results confirm that CBS restores intestinal barrier integrity by promoting IL- 22 production, upregulating tight junction proteins, and reducing inflammatory responses. The augmentation of IL- 22 suggests a neuroimmune regulatory mechanism through which CBS enhances gut epithelial integrity. Future research should explore whether exogenous IL- 22 administration can replicate CBS effects, potentially opening new therapeutic avenues.

Effect of SNS on intestinal immunity and gut microbiota

The SNS plays a dual role in intestinal immunity and gut microbiota regulation [32]. Excessive sympathetic activation in obesity contributes to gut dysbiosis and inflammation [33]. Regional sympathetic blockade with thoracic epidural anesthesia has been shown to attenuate endotoxin-induced intestinal epithelial injury, reduce intestinal permeability, and protect the epithelial barrier by attenuating the inflammatory response [34].

SNS has a direct impact on intestinal immunity and is correlated with the gut microbiota. Hypothalamic neuroinflammation increases sympathetic nerve activity, leading to intestinal dysbiosis. Inhibiting neuroinflammation reduces sympathetic nerve activity, ameliorates intestinal dysbiosis, and attenuates pathological changes in the intestinal wall of hypertensive rats [35]. Our findings demonstrate that CBS decreases norepinephrine levels and sympathetic nerve distribution, thereby reducing inflammatory responses and restoring intestinal homeostasis.

Neuroimmune regulation is associated with the intestinal homeostasis. It has been demonstrated that ILC3 s highly express genes related to α2a- and α1b-adrenergic receptors [36]. Frederic J. de Sauvage’s team showed that sympathetic nerves promoted intestinal epithelial cell repair following irradiation-induced injury by stimulating IL- 22 production from ILC3 s through β-adrenergic receptor signaling [9]. Three independent research groups have demonstrated that intestinal ILC3 s express high levels of genes involved in circadian regulation [37,38,39]. Similarly, it is widely recognized that autonomic nervous system activity has a circadian rhythm. These data strengthen the role of CBS in IL- 22 secretion by ILC3 s, highlighting the need for future research to uncover the exact molecular mechanisms underlying this process.

Limitation

This study contained several limitations. First, plasma IL- 22 levels were not measured, making it unclear whether the effect of CBS on IL- 22 is local or systemic. Second, we did not record nerve electrical signals for sympathetic activity; instead, we recorded the NE level and TH staining. Third, while our findings establish a strong link between CBS and gut microbiota alterations, further investigations are needed to explore the crosstalk between CBS -intestinal modulations and blood pressure.

Conclusion

CBS significantly reduces blood pressure in obese rats and exerts profound effects on intestinal homeostasis. Increased secretion of IL- 22 from ILC3 s appears to be a crucial mechanism underlying these effects. By modulating gut microbiota composition, enhancing SCFA production, and reducing sympathetic activation, CBS emerges as a promising non-pharmacological intervention for obesity-related hypertension.