Dendrobium officinale polysaccharides attenuates spasmolytic polypeptide-expressing metaplasia via dual modulation of gastric epithelial cell fate and macrophage polarization

Abstract

BACKGROUND

Spasmolytic polypeptide-expressing metaplasia (SPEM) may serve as an important strategy for preventing intestinal-type gastric cancer, inhibition of STAT6 to inhibit M2 macrophage polarization has been shown to suppress SPEM-associated gene expression and to impede SPEM.

AIM

To further investigate whether Dendrobium officinale polysaccharides (DOP) inhibits the early stages of metaplasia in SPEM by downregulating interleukin (IL)-33 and IL-13 expression, consequently attenuating the IL-13/STAT6 signaling pathway in M2 subtype macrophages.

METHODS

Preventive administration of DOP was conducted for a duration of two weeks prior to model induction. The SPEM mouse model was established through intraperitoneal injections of high concentration of tamoxifen (250 mg/kg) administered over three consecutive days, followed by continued treatment for an additional week. Gastric tissue samples were processed for hematoxylin and eosin staining as well as alcian blue-periodic acid-Schiff staining. Serum levels of IL-13 and IL-33 were quantified using enzyme-linked immunosorbent assay. Immunofluorescence (IF) staining and real-time polymerase chain reaction analyses were employed to evaluate the expression of parietal cell ATPase alpha polypeptide (H+/K+ exchanging alpha polypeptide, ATP4a), the chief cell marker Mist1, the mucous neck cell marker GS II, and the SPEM marker trefoil factor 2. Furthermore, IF staining was utilized to examine the co-expression of aquaporin 5 with gastric intrinsic factor, as well as aquaporin 5 with the proliferation marker Ki67. Both IF and real-time polymerase chain reaction analyses were also performed to assess macrophage polarization markers, including iNOS, CD206, and CD163. Lastly, the impact of DOP on the IL-13/STAT6 signaling pathway was investigated within the SPEM model and in bone marrow-derived macrophages.

RESULTS

DOP was recognized as a protective agent against SPEM. It enhanced the expression of ATP4a and GS II while decreasing the differentiation of chief cells. Mechanistically, DOP inhibited M2 macrophage polarization via the IL-13/STAT6 signaling pathway in both in vivo and in vitro settings, thereby mitigating the onset and progression of SPEM.

CONCLUSION

DOP mitigates SPEM by promoting the activity of parietal and mucous neck cells, suppressing the differentiation of chief cells, and inhibiting M2 macrophage polarization through the IL-13/STAT6 signaling pathway.

Key Words: Dendrobium officinale polysaccharides; Gastric epithelial cell; Spasmolytic polypeptide-expressing metaplasia; M2 macrophage polarization; IL-13/STAT6 signaling pathway; Chief cells

Core Tip: Dendrobium officinale polysaccharides upregulated the expression of ATP4a and GS II, while concurrently reducing the differentiation of chief cells. Furthermore, it inhibited M2 macrophage polarization via the IL-13/STAT6 signaling pathway and was identified as a protective agent against spasmolytic polypeptide-expressing metaplasia.

INTRODUCTION

Intestinal metaplasia (IM) and spasmolytic polypeptide-expressing metaplasia (SPEM) are two types of parallel metaplasia[1]. In particular, SPEM is characterized by the presence of pyloric adenoid glands in the gastric corpus, which are located at the base of the metaplastic glands after injury and are defined by co-expression of multiple markers, such as GS II-lectin, trefoil factor 2 (TFF2), Muc6, CD44v9 and aquaporin 5 (AQP5)[2,3]. Single-cell transcriptional analyses of SPEM arising from acute drug injury and chronic inflammation in the stomach[4]. It has been suggested that the SPEM cells may not only contribute to the epithelial regeneration in response to injury, but can also enter a pro-carcinogenic programme leading to dysplasia, that are considered crucial for the development of gastric cancer[1,5]. Therefore, as an initial precancerous lesion, early intervention to block the progression of SPEM to IM may serve as an important strategy for preventing intestinal-type gastric cancer[6].

The development and advancement of SPEM are closely linked to the infiltration of CD163-positive M2 macrophages within the gastric mucosa. An increased presence of M2 macrophages facilitates the recruitment of eosinophils, thereby sustaining the inflammatory milieu and accelerating SPEM development[7]. M2 macrophages exhibit phenotypic similarities to tumor-associated macrophages and are involved in immune regulation, tissue repair, wound healing, angiogenesis, and the promotion of tumor progression[8]. Research utilizing advanced SPEM models has demonstrated that the T-helper 2 cytokine interleukin (IL)-33, secreted by gastric epithelial cells, induces the release of IL-13 from group 2 innate lymphoid cells via the ST2 receptor[9], subsequently IL-13 promotes macrophage polarization, and accelerates SPEM progression[10]. This process is driven by the IL-4/IL-13-STAT6 signaling axis, which initiates a cascade of molecular events governing M2 macrophage polarization[11]. Consequently, inhibition of STAT6 has been shown to suppress SPEM-associated gene expression and to impede SPEM maturation[12].

Several established methods exist to induce SPEM in mice. Pharmacological agents such as DMP-777, L635, and high-dose tamoxifen can cause acute SPEM accompanied by epithelial cell damage[7,8]. AQP5 has emerged as a novel lineage-specific marker for SPEM cells, initially localized at the base of metaplastic glands. And also, it is upregulated early during chief cell reprogramming and is promoted by IL-13[3]. Recent studies also suggest that AQP5 functions as a gastric cancer stem cell marker in both mouse and human tumors[9]. Consequently, it is highly important to investigate the SPEM model concerning AQP5 function, the transdifferentiation of chief cells, and their interrelationships.

Traditional Chinese medicine demonstrates distinct advantages and considerable potential in inhibiting the carcinogenesis of the gastric mucosa, with particular emphasis on the pathological mechanisms underlying SPEM[13]. Weifuchun capsule (WFC) is a traditional Chinese polyherbal formulation recognized for its properties in tonifying the spleen, replenishing qi, promoting blood circulation, and detoxification. Comprising Red Ginseng, Isodon amethystoides, and Aurantii Fructus, it is clinically employed in the management of chronic atrophic gastritis and gastric cancer[14,15]. Additionally, formulations such as Zuojin capsule[16], Weiwei decoction[17], and Yiwei Xiaoyu granules[18], have been reported to ameliorate SPEM. In November 2023, Dendrobium officinale was officially added to the national list of substances recognized as both medicine and food. Dendrobium officinale polysaccharides (DOP) possess anti-cancer, anti-inflammatory, and immunomodulatory properties[19]. Our research team has previously demonstrated that DOP can inhibit the occurrence and progression of IM[20]. Notably, we discovered that DOP modulates macrophage polarization by converting M2 subtype macrophages into M1 subtypes via the STAT6/PPAR-γ and JAGGED1/NOTCH1 signaling pathways, thereby reducing apoptosis and preventing cellular migration, which suggests its potential to inhibit gastric cancer development[21]. Therefore, this study aims to further examine whether DOP exerts an inhibitory effect on the early stages of metaplasia in SPEM by downregulating IL-33 and IL-13 levels, thereby dual modulation of gastric epithelial cell fate and macrophage polarization via the IL-13/STAT6 signaling pathway.

MATERIALS AND METHODS

Materials and reagents

Fifty specific pathogen-free (SPF)-grade male C57BL/6J mice, weighing 18-20 g, 6 weeks old, purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. Weifuchun tablets (WFC), Hangzhou Huqingyutang Pharmaceutical Co., Ltd. Tamoxifen, MCE, HY-13757A; F4/80 Monoclonal Antibody, Invitrogen, 14-4801-85; Mouse IL-13 ELISA Kit, Jianglai Bio, JL20247; Mouse IL-33 ELISA Kit, Jianglai Bio, JL20264; Mouse IL-13 Antibody, R&D Systems, MAB413; CD206 Rabbit PolyAb, Proteintech, 18704-1-AP; F4/80 Monoclonel Antibody, Invitrogen,14-4801-82; iNOS Polyclonal antibody, Proteintech, 18985-1-AP; anti-STAT6 antibody, Abcam, ab32520; anti-STAT6 (phospho Y641) antibody, Abcam, ab263947; CD163 Antibody Panel, Abcam, ab182422; Arginase-1 Rabbit mAb, CST, 93668T; anti-Beta Actin rabbit antibody, Abclonal, AC038; Goat Anti-Rabbit IgG, ZSGB, ZB-5301; High-Purity RNA Extraction Kit, TransGen biotech, 501-01-V2; TB Green^® Premix Ex Taq™ II, TaKaRa, RR820A; PrimeScript™ RT reagent Kit with gDNA Eraser, TaKaRa, RR047A; Murine M-CSF, Peprotech, 315-02; Recombinant Murine IL-13, Peprotech, 210-13; F4/80 Monoclonal Antibody (BM8), eFluor™ 450, eBioscience™ Invitrogen, 48-4801-82; CD206 (MMR) Monoclonal Antibody (MR6F3), APC, eBioscience™ Invitrogen, 17-2061-82.

Extraction of DOP

The methods were reported before[22]. A measured amount of Dendrobium officinale was extracted twice by refluxing with water at 50 times its volume, each extraction lasting 2 hours. The resulting decoctions were filtered, combined, and concentrated. The solvent was removed under reduced pressure to yield a solution with a concentration of 0.2 g/mL, which was then mixed with pure ethanol in a 1:5 ratio. Following a 24-hour standing period, the mixture was subjected to centrifugation at 4000 rpm for 15 minutes, after which the supernatant was discarded. This process was repeated twice. Subsequently, the precipitate was sequentially washed with ethanol and acetone, fully dissolved in 80% ethanol, and then subjected to deproteinization using the trichloroacetic acid/acetone protein precipitation technique. Following this, the solution was filtered through a 3 kDa ultrafiltration membrane and freeze-dried to obtain the final product, with a DOP content of 83%.

Animal experiments

Fifty SPF-grade male C57BL/6J mice, aged 6 weeks and weighing 18-20 g, were kept in an SPF-grade animal facility under controlled conditions of 22 ± 2 °C temperature, 50% ± 5% humidity, and a 12-hour light/dark cycle, with unrestricted access to food and water. After a one-week acclimatization period, the animals were grouped for the subsequent experiments. A SPEM mouse model was then established by intraperitoneal injection of a high dose of tamoxifen. Prior to the establishment of the SPEM model, except for the control and SPEM model groups, the preventive administration groups received DOP (200 mg/kg and 100 mg/kg) and positive medicine Weifuchun (1.44 g/kg) for two weeks (n = 10 per group). After two weeks, tamoxifen was administered intraperitoneally at 250 mg/kg once daily for three consecutive days to induce the SPEM model[23,24]. Treatment continued for one week during model induction. After three weeks, mice were anesthetized with 0.3% sodium pentobarbital at 0.1 g/mL, and gastric tissue was collected for analysis and measurement.

Hematoxylin and eosin staining and alcian blue-periodic acid-Schiff staining

Hematoxylin and eosin staining, along with alcian blue-periodic acid-Schiff (AB-PAS) staining, were conducted to identify pathological alterations and to characterize mucin expression in the gastric tissue of the SPEM mouse model. Gastric tissues were first fixed in 4% paraformaldehyde, then dehydrated using a graded series of ethanol, cleared in xylene, and embedded in paraffin. Thin sections of 5 μm were cut with a microtome. For hematoxylin and eosin staining, the slides were deparaffinized and rehydrated, then treated with hematoxylin to stain the nuclei, followed by eosin to highlight the cytoplasm and extracellular matrix, using standard procedures. For AB-PAS staining, the deparaffinized sections were first treated with alcian blue (pH 2.5) to stain acidic mucins blue. Subsequently, the sections were oxidized with periodic acid and then exposed to Schiff’s reagent to detect neutral mucins and certain glycoproteins, which develop a magenta color.

Enzyme-linked immunosorbent assay

Centrifuge serum samples at 1000 revolutions per minute using a centrifuge with a radius of 15 cm for a duration of 15 minutes at a temperature of 4 °C. Subsequently, collect the supernatant for analysis. Measure the levels of IL-13 and IL-33 in the serum according to the instructions provided with enzyme-linked immunosorbent assay kit.

Immunofluorescence staining

To detect the expression of SPEM-related markers in the gastric tissue of the SPEM mouse model, frozen sections were used for immunofluorescence (IF) staining to detect the expression of SPEM markers TFF2, exogenous lectin GS II (lectin GS II, GS II), parietal cell marker hydrogen/potassium ion exchange ATPase alpha polypeptide (H+/K+ exchanging alpha polypeptide, ATP4a), and chief cell marker Mist1 in the SPEM mouse model. Additionally, the co-expression of the SPEM marker AQP5 with the chief cell marker gastric intrinsic factor (GIF) and the proliferation marker tumour proliferation antigen (Ki67 antigen, Ki67) was examined in the SPEM mouse model. Observations and photography were performed under a fluorescence microscope.

Real-time polymerase chain reaction

Total RNA was isolated from gastric tissue utilizing the Eastep^® Super Total RNA Extraction Kit. The RNA concentration was determined using a NanoDrop spectrophotometer. Complementary DNA was synthesized via reverse transcription and subsequently analyzed by real-time polymerase chain reaction (RT-PCR) employing a fluorescent quantitative PCR kit. PCR primers were designed with the Primer-BLAST tool from the NCBI database to ensure specificity. All primers were synthesized by Sangon Biotech (Shanghai, China) and are detailed in the Supplementary material. Glyceraldehyde 3-phosphate dehydrogenase served as the internal reference gene, and each reaction was conducted in triplicate. Relative mRNA expression levels were calculated using the 2^-ΔΔCT method.

Western blot

Proteins from gastric tissue were extracted using RIPA buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were determined and standardized across all samples utilizing a BCA Assay Kit. Subsequent to separation by SDS-PAGE, the proteins were transferred onto PVDF membranes. These membranes were incubated overnight at 4 °C with primary antibodies against CD163, CD206, ARG-1, iNOS, IL-13, STAT6, and phosphorylated STAT6, each diluted at 1:1000, followed by incubation with the appropriate secondary antibodies. Protein bands were detected through chemiluminescence. To account for loading variability, β-actin was employed as an internal control. The intensity of the target protein bands was quantified using Image Lab 6.0 software.

Bone marrow-derived macrophages cell culture

Bone marrow-derived macrophages (BMDMs) were isolated and cultured following previously described methods[25]. Briefly, bone marrow cells were collected from the femurs and tibias and flushed out using phosphate-buffered saline. To prepare BMDMs, the cells were cultured in glucose-free RPMI-1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. For differentiation into M0 BMDMs, the cells were treated with 20 ng/mL M-CSF for 7 days. Next, to drive M2 polarization, M0 BMDMs were exposed to 30 ng/mL IL-13 for 72 hours. After the treatment, the cells were collected and assessed for BMDM immunomodulatory effects using flow cytometry. For macrophage polarization analysis, BMDMs were harvested, washed, and resuspended in phosphate-buffered saline supplemented with 1% bovine serum albumin. The cells were then stained with fluorochrome-conjugated antibodies against F4/80, CD86, and CD206. Following a 30-minute incubation with the antibodies at 4 °C, cells were washed and analyzed by flow cytometry. FlowJo software (v10.10.1) was used to analyze the resulting data.

The effect of DOP on IL-13/STAT6 signal pathway on BMDMs cell

After the cells were treated with IL-13 for 72 hours and identified as M2-type macrophages, DOP was administered at concentrations of 100 mg/L, 200 mg/L, and 400 mg/L. All cells were incubated at 37 °C with 5% CO₂ for 1 hour before being collected for subsequent experiments. Test by flow cytometry and PCR.

Statistical analysis

The data are shown as the mean ± SD and analyzed with SPSS 23.0 (IBM Corp.). For two groups of measurement data that met the assumptions of normality and equal variances, an independent sample t-test was used. When comparing more than two groups with homogeneity of variance, one-way analysis of variance was applied, followed by the least significant difference test or Dunnett’s test. For two or multiple groups that did not meet the normal distribution and homogeneity of variance assumptions, non-parametric Kruskal-Wallis tests were used. A P value of < 0.05 was considered statistically significant.

RESULTS

Effects of DOP on mouse body weight, pathological improvement, mucus types, and levels of IL-13 and IL-33 in the SPEM model

As illustrated in Figure 1A, compared to the control group, mice in the SPEM model group experienced a significant decrease in body weight from day 15 to day 21 (P < 0.01). Treatment with DOP notably increased the body weight of mice within the induced SPEM model during the same period (P < 0.05). Figure 1B depicts the pathological morphology of gastric tissues in the control group, where the gastric mucosal epithelial cells were orderly arranged, with intact glandular structures such as gastric pits and simple columnar epithelium, predominantly composed of numerous parietal and chief cells. Parietal cells (PCs) displayed variability in size and shape, basophilic cytoplasm, and pink staining, and were widely distributed. In contrast, the SPEM model group exhibited relatively orderly gastric mucosal epithelial cells; however, there was evident parietal cell loss. Mild edema of the gastric mucosa was observed, accompanied by disorganized and enlarged stromal glands. Localized epithelial hyperplasia was present in both the mucosa and submucosa, along with dilation of glandular lumens. Treatment with WFC and DOP resulted in more orderly and densely arranged gastric mucosal cells and a reduction in parietal cell loss.

Figure 1 The Dendrobium officinale polysaccharides effects on mouse body weight, pathological improvement, mucus types and levels of interleukin-13 and interleukin-33 on spasmolytic polypeptide-expressing metaplasia model. A: Mouse body weight; B: Pathological improvement; C: Mucus types; D and E: Levels of interleukin-13 and interleukin-33. The data are represented as mean ± SD, n = 6. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; IL: Interleukin.

As shown in Figure 1C, AB-PAS staining demonstrated that the gastric mucosa of mice in the SPEM model group contained numerous blue-stained lesions at the base of the gastric glands, indicating substantial secretion of acidic glycoproteins and proteoglycans by epithelial cells. In contrast, no significant blue-stained lesions were observed in the WFC and DOP groups, where cells were neatly arranged and glandular structures remained intact. These findings suggest that the epithelial cells predominantly secreted neutral glycoprotein mucus, with relatively low levels of acidic glycoprotein and proteoglycan mucus.

Figure 1D and E reveal that, relative to the control group, the SPEM model group exhibited a significant elevation in serum IL-13 and IL-33 levels (P < 0.001 and P < 0.01, respectively). Preventive administration of both WFC and DOP significantly reduced serum IL-13 and IL-33 concentrations.

Examination of markers associated with PCs, chief cells, and SPEM

As illustrated in Figure 2A-D, IF analysis showed a significant reduction in the expression of the parietal cell marker ATP4α and the chief cell marker Mist1 in the gastric mucosa of the SPEM model group (P < 0.001 and P < 0.05, respectively). Following treatment with DOP, ATP4α expression increased significantly (P < 0.05), whereas the proportion of Mist1-positive cells in the gastric tissue decreased slightly, but not significantly. Furthermore, we investigated the expression of SPEM markers TFF2, GS II, and Gpx2. IF analysis, presented in Figure 2E-H, revealed a significant increase in the expression of markers TFF2 and GS II in mouse gastric mucosal cells within the SPEM model group (P < 0.001). This elevated expression was notably reduced after treatment with both high-dose DOP (H-DOP) and low-dose DOP (L-DOP). Additionally, RT-PCR analysis of gene expression mirrored the IF results, as shown in Figure 2I. Specifically, ATP4α expression was significantly upregulated after DOP treatment (P < 0.05), indicating a restoration of parietal cell function. As shown in Figure 2J, RT-PCR results showed that the mRNA levels of TFF2 and Gpx2 were significantly upregulated in the SPEM group (P < 0.001) and significantly downregulated in the H-DOP group (P < 0.001 and P < 0.05, respectively).

Figure 2 Analysis of parietal, chief cell and spasmolytic polypeptide-expressing metaplasia markers. A: Immunofluorescence (IF) analysis of the parietal cell marker ATP4α; B: IF analysis of the chief cell marker Mist1; C: Statistical analysis of IF results (ATP4α) by using ImageJ software; D: Statistical analysis of IF results (Mist1) by using ImageJ software; E and F: IF analysis of spasmolytic polypeptide-expressing metaplasia markers trefoil factor 2 and GS II; G and H: Statistical analysis of IF results (TFF2 and GS II) by using ImageJ software; I and J: Real-time polymerase chain reaction analysis of ATP4α, Mist1, TFF2 and Gpx2. The data are represented as mean ± SD, IF analysis, n = 3. Real-time polymerase chain reaction analysis, n = 6. ^aP < 0.05, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides.

Analysis of AQP5, Ki67, and GIF expression

The co-expression of AQP5 and Ki67 in mouse gastric mucosal cells is illustrated in Figure 3A and B. Compared to the control group, the area exhibiting dual positive expression of AQP5⁺ and Ki67⁺ in gastric tissue was significantly elevated (P < 0.001) in the SPEM model group, indicating an increased proliferative capacity within the SPEM model. Subsequent to treatment, both H-DOP and L-DOP significantly reduced the dual positive expression of AQP5⁺Ki67⁺ (P < 0.001 and P < 0.05, respectively). Additional analysis of AQP5 gene expression confirmed the IF findings presented in Figure 3C, revealing a significant increase in expression in the model group (P < 0.05) and a marked decrease after treatment (P < 0.01).

Figure 3 The co-expression of aquaporin 5 and Ki67, gastric intrinsic factor and Ki67 in mouse gastric mucosal cells. A: Immunofluorescence (IF) analysis the co-expression of aquaporin 5 (AQP5) and Ki67; B: Statistical analysis of IF results (Ki67+ AQP5+) by using ImageJ software; C: Real-time polymerase chain reaction analysis of AQP5; D: IF analysis of the co-expression of gastric intrinsic factor and Ki67; E: Statistical analysis of IF results (gastric intrinsic factor + AQP5+) by using ImageJ software. ^aP < 0.05, ^cP < 0.001, vs control; ^dP < 0.05, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; AQP5: Aquaporin 5; GIF: Gastric intrinsic factor.

Meanwhile, as shown in Figure 3D and E, the GIF⁺ expression area was significantly reduced in SPEM model mice (P < 0.001), whereas AQP5⁺ expression area was significantly increased (P < 0.001). After DOP intervention, both H-DOP and L-DOP groups showed a significant increase in the GIF⁺ area (P < 0.001, P < 0.01), and the H-DOP group exhibited a marked reduction in the AQP5⁺ area in gastric tissue (P < 0.05). A negative correlation was observed between AQP5⁺ and GIF⁺ expression. These results indicate that co-staining reveals a decrease in GIF and an increase in AQP5 in the model, which is a hallmark of the SPEM model. Simultaneous co-staining with AQP5 and Ki67 suggests that SPEM cells possess unlimited proliferative capacity, providing a biological basis for cancer development.

Analysis of macrophages

As depicted in Figure 4A-D, fluorescence analysis revealed a significant increase in the co-localization of the M2 macrophage marker (CD163) with the macrophage marker F4/80 in the SPEM model (P < 0.001), whereas the co-localization of the M1 macrophage polarization marker (iNOS) with F4/80 was minimally detected (P < 0.001). Upon administration of DOP, there was a notable decrease in M2 phenotype macrophages accompanied by an increase in M1 phenotype macrophages, particularly evident in the H-DOP (P < 0.01). Additionally, gene expression analysis of CD206 and CD163, as shown in Figure 4E and F, corroborated the IF results, indicating a significant upregulation in the model group (P < 0.001, P < 0.05) followed by a marked reduction after DOP treatment.

Figure 4 Analysis of M2-macrophages in gastric tissue by immunofluorescence and real-time polymerase chain reaction. A: Immunofluorescence (IF) analysis of the M2 macrophage polarization marker F4/80+ CD163+; B: Statistical evaluation of the IF results (F4/80+ iNOS+) conducted using Image J 6.0 software; C: IF analysis of the M1 macrophage polarization marker F4/80+ iNOS+; D: Statistical evaluation of the IF results (F4/80+ CD163+) conducted using Image J 6.0 software; E and F: Real-time polymerase chain reaction analysis of CD206 and CD163 expression levels. The data are represented as mean ± SD, Sample sizes: IF analysis, n = 3; real-time polymerase chain reaction analysis, n = 6. ^aP < 0.05, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides.

Western blot analysis the M2 macrophage polarization and IL-13/STAT6 signal pathway

Western blot analysis was conducted to evaluate the impact of DOP on the protein expression of CD163, ARG-1, CD206, and iNOS in the gastric tissue of SPEM model mice. As shown in Figures 5A and B, the levels of CD163, ARG-1, and CD206 were significantly higher than in the control group (P < 0.05), while iNOS expression was significantly decreased (P < 0.01). Administration H-DOP resulted in a marked downregulation of ARG-1 protein expression, alongside an upregulation of iNOS expression (P < 0.05). Furthermore, both H-DOP and L-DOP demonstrated significant inhibitory effects on CD163 and CD206 expression (P < 0.05). These results indicate that DOP suppresses M2 macrophage polarization, thereby potentially delaying the progression of the SPEM model. Further investigate possible pathways of M2 polarization. In comparison to the control group, as shown in Figure 5C and D, the protein expression level of IL-13 in the gastric tissue of SPEM mice was significantly elevated in the model group (P < 0.01). Following preventive treatment, the protein expression levels of IL-13 in the gastric tissues of both the positive control drug WFC group, H-DOP and L-DOP groups were significantly reduced compared to the SPEM model group (P < 0.05). Additionally, the p-STAT6/STAT6 ratio was significantly increased in the model group relative to the normal group (P < 0.05). After preventive administration, this ratio was significantly decreased in H-DOP groups compared to the SPEM model group (P < 0.05), indicating that DOP effectively down-regulates STAT6 phosphorylation. These results indicate that DOP inhibits the IL-13/STAT6 signaling pathway, potentially through mechanisms involving macrophage polarization.

Figure 5 Western blot analysis of M2 macrophage polarization and the IL-13/STAT6 signaling pathway. A: Western blot analysis of M2 macrophage polarization markers; B: Relative protein quantification (CD206, ARG1, CD163, iNOS) performed using Image Lab 6.0; C: Western blot analysis of interleukin-13 and STAT6 protein expression; D: Relative protein quantification (interleukin-13, p-STAT6/STAT6) performed using Image Lab 6.0. The data are represented as mean ± SD, n = 3. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, vs spasmolytic polypeptide-expressing metaplasia model. Ctrl: Control; SPEM: Spasmolytic polypeptide-expressing metaplasia; WFC: Weifuchun capsule; H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; IL: Interleukin.

The effect of DOP on IL-13/STAT6 signal pathway on BMDMs cell

Flow cytometry results demonstrated that the number of F4/80⁺CD206⁺ positive cells in the M2 group, as shown in Figure 6A and B, which was significantly higher than in the M0 group (P < 0.001). Following DOP intervention, both the H-DOP and M-DOP groups exhibited a significant reduction in F4/80⁺CD206⁺ positive cells compared to the M2 group (P < 0.05 and P < 0.01, respectively). Compared to the M0 group, as shown in Figure 6C, the M2 group showed a significant increase in mRNA and protein expression of CD163 and CD206, accompanied by a decrease in iNOS expression. After DOP administration, both the M-DOP and H-DOP groups displayed a marked decrease in CD163 and CD206 expression and an increase in iNOS levels relative to the M2 group. Additionally, in the M2 model, the expression levels of ARG1 and TGM2 proteins were elevated (P < 0.01 and P < 0.05, respectively). Following DOP treatment, both protein levels decreased significantly (Figure 6D and E). While the mRNA levels of IL-33, IL-13, IL-13Rα1, IL-13Rα2, and ST2 were significantly elevated in the M2 group, DOP treatment reduced their expression. The M2 group also showed significantly increased IL-13 protein expression (P < 0.01) and an elevated p-STAT6/STAT6 ratio (P < 0.01). DOP-treated groups decreased IL-13 protein levels and p-STAT6/STAT6 ratios (Figure 6F and G).

Figure 6 The effect of Dendrobium officinale polysaccharides on IL-13/STAT6 signal pathway on bone marrow-derived macrophages cell. A: Flow cytometry analysis of M2 macrophage polarization; B: Flow cytometry result analysis by flowjo software; C: Genes expression of macrophage marker and interleukin (IL)-13, IL-13Ra1, IL-13Ra2, ST2 by real-time polymerase chain reaction; D: Protein expression of macrophage marker; E: Relative protein quantification (macrophage marker) performed using Image Lab 6.0; F: Protein expression of IL-13, STAT6 and p-STAT6; G: Relative protein quantification (IL-13, STAT6 and p-STAT6) performed using Image Lab 6.0. The data are represented as mean ± SD, n = 3. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, vs control; ^dP < 0.05, ^eP < 0.01, ^fP < 0.001, vs spasmolytic polypeptide-expressing metaplasia model. H-DOP: High-dose Dendrobium officinale polysaccharides; L-DOP: Low-dose Dendrobium officinale polysaccharides; M-DOP: Middle-dose Dendrobium officinale polysaccharides; IL: Interleukin.

DISCUSSION

Dendrobium contains various bioactive components, including polysaccharides, alkaloids, bibenzyls, flavonoids, and phenanthrenes, which have inhibitory effects on multiple types of tumors. Among these, research on Dendrobium polysaccharides is more extensive; these polysaccharides have been shown to possess anti-tumor, liver-protective, and anti-glycation properties[26]. Among the Dendrobium species, Dendrobium officinale is the most valuable and is known as the “gold of medicinal herbs”. DOP is the main active component of Dendrobium officinale[27]. Our research team has previously confirmed that DOP can inhibit the occurrence and progression of IM[20]. Therefore, this study aims to further investigate whether DOP can also suppress the earlier stage of metaplasia, specifically SPEM.

Historically, the development of SPEM has been associated with the loss of PCs, wherein differentiated chief cells undergo direct reprogramming within the gastric environment[6,28]. The transcription factor MIST1 has been identified as a novel marker of human gastric chief cells, with its expression notably diminished in metaplasia, dysplasia, and carcinoma[29]. Additionally, ATP4α is essential for the acid-secretory function of PCs, the normal development and cellular homeostasis of the gastric mucosa, and the maintenance of the typical architecture of secretory membranes[30]. Our results, derived from fluorescent immunostaining and gene expression analyses targeting MIST1 and ATP4α, indicate that treatment with DOP induces an upregulation of ATP4α expression, which confers a protective effect against parietal cell loss in SPEM, thereby supporting the preservation of parietal cell function. Simultaneously, the SPEM model demonstrated a moderate decrease in MIST1 expression; however, post-treatment levels of MIST1 remained largely stable, suggesting the need for further research into the mechanisms governing chief cell differentiation.

AQP5, recognized as a novel marker of SPEM, contributes to SPEM expression[3]. Moreover, existing literature identifies GIF as a stomach tissue-specific gene, with protein expression uniquely localized to mature mouse chief cells[31]. Consequently, we assessed the co-expression of AQP5 and GIF, revealing a synergistic co-expression pattern that further supports the notion that chief cells progressively transition into SPEM cells upon differentiation, consistent with prior studies[31,32]. Administration of DOP resulted in a reduction in the expression levels of both markers, indicating an inhibitory effect on the initiation and progression of SPEM. Additionally, to elucidate the oncogenic characteristics of SPEM cells, we examined the co-expression of AQP5 and Ki67. Our observations indicated that regions exhibiting pronounced AQP5 expression also demonstrated significantly elevated Ki67 expression, aligning with previously reported findings[3].

During the process of SPEM, the activities of cytokines such as IL-33 and IL-13, along with cell types including M2 macrophages and eosinophils, are closely interconnected, thereby facilitating chronic gastritis-associated and more widespread metaplasia[7]. The population of M2 macrophages (characterized by F4/80+ and CD163+ markers) was found to be elevated at the gland base in wild-type mice treated with DMP-777 or tamoxifen[9]. Fluorescent immunohistochemistry and gene expression analyses revealed a significant increase in M2 macrophage markers, CD206 and CD163, within the SPEM model. Following administration of DOP, a marked reduction in M2 macrophages was observed, accompanied by an increase in M1 macrophages, particularly pronounced in the H-DOP group. Furthermore, gene expression analysis indicated a corresponding rise in markers associated with M1 macrophages. To elucidate the mechanisms underlying M2 macrophage polarization, BMDMs were isolated to investigate how DOP modulates M2 macrophage polarization.

The IL-33/IL-13 signaling pathway serves as the principal mechanism in SPEM[33]. IL-13 interacts with its receptors, IL-13Rα1 and IL-13Rα2, predominantly mediating its effects via the JAK/STAT signaling cascade[34]. The activation and upregulation of STAT6 constitute the primary processes facilitating the recruitment and expansion of T-helper 2 cells, which subsequently drive the polarization of M2 macrophages[35]. In our study, the expression levels of IL-13, IL-13RA1, and IL-13Rα2 were elevated in BMDM-induced M2 macrophages, accompanied by an increased p-STAT6/STAT6 ratio. Conversely, treatment with DOP resulted in reduced IL-13 protein levels and decreased p-STAT6/STAT6 ratios. These findings indicate that DOP may inhibit M2 macrophage polarization through modulation of the IL-13/STAT6 signaling pathway.

Our experimental findings demonstrate that DOP effectively inhibits the initiation and progression of SPEM. The primary mechanism involves DOP upregulating the expression of ATP4α in PCs, reducing the levels of the SPEM marker TFF2 and the mucous neck cell marker GS II, and concurrently inhibiting the differentiation of chief cells, as evidenced by decreased expression of AQP5, Ki67, and GIF. Furthermore, DOP suppresses M2 macrophage polarization both in vivo and in vitro via modulation of the IL-13/STAT6 signaling pathway.

Nonetheless, our study has certain limitations. Recent lineage tracing studies indicate that the active depletion of chief cells, along with the involvement of isthmus stem cells, also contribute to the development of SPEM[36]. Such as the cholecystokinin-2/gastrin receptor, expressed in corpus isthmus progenitors and PCs, regulates acid secretion and cellular turnover[37]. Simultaneously, multiple studies have established that gastric PCs are crucial for the maintenance of gastric health and the regulation of acid secretion. For instance, Estrogen-related receptor gamma serves as a pivotal factor in the differentiation of gastric PCs from progenitor cells. Furthermore, the co-regulation of sirtuin 1 and Estrogen-related receptor gamma may facilitate the differentiation process of gastric PCs[38]. Therefore, a comprehensive understanding of how these mechanisms relate to the loss and progression of SPEM will provide valuable insights and guide the direction of future experimental investigations.

CONCLUSION

Our experimental findings demonstrate that DOP effectively inhibits the initiation and progression of SPEM. The primary mechanism involves DOP upregulating the expression of APT4α in PCs, reducing the levels of the SPEM marker TFF2 and the mucous neck cell marker GS II, and concurrently inhibiting the differentiation of chief cells, as evidenced by decreased expression of AQP5, Ki67, and GIF (Figure 7). Furthermore, DOP suppresses M2 macrophage polarization both in vivo and in vitro via modulation of the IL-13/STAT6 signaling pathway.

Figure 7 Molecular mechanism of Dendrobium officinale polysaccharides in inhibiting spasmolytic polypeptide-expressing metaplasia. DOP: Dendrobium officinale polysaccharides; IL: Interleukin; BMDMs: Bone marrow-derived macrophages; SPEM: Spasmolytic polypeptide-expressing metaplasia; TFF2: Trefoil factor 2; AQP5: Aquaporin 5.

ACKNOWLEDGEMENTS

We thank all those who provided excellent technical support and assistance during the study.