concentration ratio of DCA to its precursor primary BA cholic acid in gastric juice samples showed an incremental increase during the progression of chronic superficial gastritis into IM and GC. proteincoupled bile acid receptor 1 (GPBAR1, also called TGR5) is expressed in the gastric epithelium and activated by secondary BAs, especially DCA. Strong TGR5 staining was not present in normal gastric mucosa but was present in of IM cases Therefore, the DCA-TGR5 axis may play a pivotal role in IM initiation.
在慢性浅表性胃炎发展为IM和GC的过程中，胃液样本中DCA与其前体一级胆汁酸的浓度比呈递增趋势。 蛋白偶联胆汁酸受体1（GPBAR1，又称TGR5）在胃上皮细胞中表达，并被次级胆汁酸（尤其是DCA）激活。 正常胃黏膜中没有强烈的TGR5染色，但在 IM病例中却有 因此，DCA-TGR5轴可能在IM的启动中起着关键作用。

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that is extensively involved in proinflammatory oncogenic cellular processes by regulating the expression of target genes in numerous solid tumors. Cytokines such as interleukin-6 (IL-6) phosphorylate the STAT3 protein on tyrosine-705 and subsequently induce the nuclear translocation of STAT3, which results in DNA binding and thereby transcriptional activation of STAT3 target genes. DCA activated STAT3 signaling and its transcriptional activity and thus promoted Barrett's carcinogenesis. Wild-type mice showed more severe gastric inflammation and mucous metaplasia than gastric epithelial conditional Stat3-knockout mice. Hence, the elucidation of the mechanism underlying IM transformation from chronic gastritis based on DCA-TGR5-induced STAT3 activation is of great significance for increasing the comprehension of gastric mucosal carcinogenesis.
信号转导和激活转录因子3（STAT3）是一种转录因子，它通过调节许多实体瘤中靶基因的表达，广泛参与了促炎性致癌细胞过程。 白细胞介素-6（IL-6）等细胞因子使 STAT3 蛋白在酪氨酸-705 上磷酸化，随后诱导 STAT3 核转位，导致 DNA 结合，从而激活 STAT3 靶基因的转录。 DCA激活了STAT3信号转导及其转录活性，从而促进了巴雷特癌的发生。 野生型小鼠比条件性Stat3基因敲除的胃上皮小鼠表现出更严重的胃炎和粘液变性。 因此，阐明基于DCA-TGR5诱导STAT3激活的慢性胃炎向IM转化的机制，对于进一步了解胃黏膜癌变具有重要意义。

We aimed to fully investigate KLF5 expression during IM development in this study because substantial nuclear KLF5 staining was observed in of patients with Barrett's esophagus, and KLF5 responded positively to DCA-mediated intestinal transdifferentiation of the esophageal squamous epithelium. Moreover, the positive expression rate of KLF5 increased gradually, with significant differences in normal gastric tissues (38.5%), low-grade gastric intraepithelial neoplasia (GIN; 58.3%), highgrade GIN ( ), well-moderately differentiated adenocarcinoma ( ) and poorly differentiated adenocarcinoma (
在Barrett食管患者中观察到大量的核KLF5染色，而且KLF5对DCA介导的食管鳞状上皮肠转分化有阳性反应，因此我们在本研究中旨在全面调查IM发生过程中KLF5的表达。 此外，KLF5的阳性表达率逐渐升高，在正常胃组织（38.5%）、低级别胃上皮内瘤变（GIN；58.3%）、高级别GIN（ ）、中度分化良好的腺癌（ ）和分化不良的腺癌（ ）中均有显著差异。

Transgenic FVB/N insulin-gastrin (INS-GAS) mice initially showed an increased parietal cell number but later exhibited parietal cell loss and hypochlorhydria, which could spontaneously progress to gastric IM, dysplasia and cancer at 20 months of age, and these mice are considered to be an ideal animal model for GC research. In addition to Hp , gastric microbial dysbiosis has been determined to be involved in mucosal carcinogenesis in the stomach. The colonization densities of the bacterial community in the stomach are estimated to range from to colony forming units , and an altered community structure along with decreased microbial diversity might promote GC. -negative individuals can have better phylotype evenness and diversity on the composition of the gastric microflora than subjects positive for H. pylori, and Hp-infected INS-GAS mice with more diversified gastric commensal bacteria were observed to have less severe gastric lesions and delayed GC onset compared to Hp monoinfected mice. It is consequently necessary to explore the effects of DCA on BA metabolism and the microbial diversity and community structure in the stomachs of INS-GAS mice to validate potential microbial markers for IM occurrence and progression.
转基因FVB/N胰岛素-胃泌素（INS-GAS）小鼠最初表现为顶叶细胞数量增加，但随后出现顶叶细胞丢失和低氯血症，20月龄时可自发发展为胃IM、发育不良和胃癌，这些小鼠被认为是胃癌研究的理想动物模型。 除Hp外，胃微生物菌群失调也被确定与胃黏膜癌变有关。 据估计，胃中细菌群落的定植密度在 至 集落形成单位 之间，群落结构的改变以及微生物多样性的减少可能会促进胃癌的发生。 幽门螺杆菌阴性的个体比幽门螺杆菌阳性的个体在胃微生物区系组成上具有更好的系统型均匀性和多样性， 与Hp单一感染的小鼠相比，Hp感染的INS-GAS小鼠具有更多样化的胃共生菌，胃病变的严重程度较轻，GC的发病时间较晚。 因此，有必要探讨DCA对BA代谢以及INS-GAS小鼠胃中微生物多样性和群落结构的影响，以验证IM发生和发展的潜在微生物标记物。

In this study, we aimed to explore the involvement of TGR5-STAT3-KLF5 signaling axis on gastric IM development in response to DCA and the effects of DCA treatment on the BA metabolism and gastric microbiota of INS-GAS mice. Therefore, we investigated the roles of STAT3 signaling in KLF5 activation in response to BAs in the gastric epithelium. We found that DCA might promote the transformation of gastric mucosal inflammation to IM and carcinogenesis. The involvement of a TGR5/STAT3/KLF5 regulatory axis was observed in human IM tissues exposed to regurgitant bile, in the gastric organoids of mice treated with DCA, and in DCA-fed INS-GAS mice. Furthermore, long-term DCA supplementation in drinking water also induced gastric BA metabolism and microbiota dysbiosis during IM and dysplasia onset. These findings reveal a critical role of STAT3 signaling in regulating its target intestinal marker KLF5 in response to DCA, which is the main BA component in the stomach and the major risk factor for gastric IM.
本研究旨在探讨TGR5-STAT3-KLF5信号轴参与胃IM发育对DCA的反应，以及DCA处理对INS-GAS小鼠BA代谢和胃微生物区系的影响。因此，我们研究了 STAT3 信号在胃上皮细胞对 BA 的反应中激活 KLF5 的作用。我们发现，DCA 可能会促进胃黏膜炎症向 IM 和癌变的转化。在暴露于反流胆汁的人类 IM 组织、接受 DCA 治疗的小鼠胃有机体以及喂食 DCA 的 INS-GAS 小鼠中，都观察到了 TGR5/STAT3/KLF5 调节轴的参与。此外，在饮用水中长期补充 DCA 还会在 IM 和发育不良发病过程中诱导胃 BA 代谢和微生物群失调。这些发现揭示了 STAT3 信号在调节其靶肠道标记物 KLF5 对 DCA 的反应中的关键作用，而 DCA 是胃中的主要 BA 成分，也是胃 IM 的主要风险因素。

Sixteen subjects were enrolled from a provincial GC early detection project that screened a total of 161
16 名受试者来自一个省级 GC 早期检测项目，该项目共筛查了 161 人
volunteers without Hp infection in 2019, including 6 presenting BR (-) IM (-), 6 showing BR (+) IM (-), and 4 with BR (+) IM (+). BR represents bile reflux. IHC staining and scoring of TGR5, p-STAT3 and Kruppellike Factor 5 (KLF5) expression levels in the gastric biopsy samples were performed for the 16 enrolled individuals (Figure 1a). As shown in Figure 1a and
2019 年无肝炎感染的志愿者，其中 6 人出现 BR (-) IM (-)，6 人出现 BR (+) IM (-)，4 人出现 BR (+) IM (+)。BR 代表胆汁反流。对 16 名入选者的胃活检样本中的 TGR5、p-STAT3 和 Kruppellike Factor 5 (KLF5) 表达水平进行了 IHC 染色和评分（图 1a）。如图 1a 和
Table 1, membranal TGR5 staining and nuclear p-STAT3 staining of the gastric epithelium were both stepwise and statistically enhanced in BR (-) IM (-), BR (+) IM (-) and BR (+) IM (+) biopsies. Although nuclear KLF5 staining in BR (+) IM (-) tissues did not show a significant increase compared with that in BR (-) IM (-) tissues (-, vs., ;
表1显示，在BR（-）IM（-）、BR（+）IM（-）和BR（+）IM（+）活检组织中，胃上皮的膜TGR5染色和核p-STAT3染色均呈阶梯状增强，并具有统计学意义。虽然BR（+）IM（-）组织的核KLF5染色与BR（-）IM（-）组织相比没有显著增加（-， vs., ；

Figure 1. Enhanced TGR5, p-STAT3 and KLF5 expression was observed in the human gastric epithelium in patients with bile acid reflux. (a) IHC staining and scoring of TGR5, p-STAT3 and KLF5 in the gastric mucosal biopsy specimens of the enrolled 16 subjects. (b-c) mRNA expression levels of TGR5 and KLF5 in gastric mucosal samples from the 16 included individuals, as assessed by qRT-PCR assays. (d) Pearson correlation analysis of KLF5 and TGR5 mRNA expression levels. Scale bar, , . N.S., not significant.
图 1：胆汁酸反流患者胃上皮细胞中 TGR5、p-STAT3 和 KLF5 表达增强胆汁酸反流患者胃上皮细胞中 TGR5、p-STAT3 和 KLF5 表达增强。(a）16 例受试者胃黏膜活检标本中 TGR5、p-STAT3 和 KLF5 的 IHC 染色和评分。(b-c）通过 qRT-PCR 检测评估 16 名受试者胃黏膜样本中 TGR5 和 KLF5 的 mRNA 表达水平。(d）KLF5和TGR5 mRNA表达水平的皮尔逊相关分析。比例尺， ， 。N.S.，无意义。

Table 1. Expressions of TGR5, p-STAT3 and KLF5 in BR (-) IM (-), BR (+) IM (-) and BR (+) IM (+) human gastric epithelia based on IHC staining.
表 1.根据 IHC 染色，TGR5、p-STAT3 和 KLF5 在 BR (-) IM (-)、BR (+) IM (-) 和 BR (+) IM (+) 人胃上皮中的表达情况。

BR (-) IM (-) group vs. BR (+) IM (-) group with an unpaired parametric test. BR (-) IM (-) group vs. BR (+) IM (+) group with an unpaired parametric test. IHC, immunochemistry; -, negative staining; +, weak staining; ++ , strong staining
BR（-）IM（-）组与 BR（+）IM（-）组进行非配对参数 检验。 BR（-）IM（-）组与BR（+）IM（+）组进行非配对参数 检验。IHC，免疫化学；-，阴性染色；+，弱染色；++ ，强染色

, the substantial nuclear KLF5 staining in BR (+) IM (+) tissues was significantly different from that in BR (-) IM (-) tissues (-, vs. ++ , . The TGR5 and KLF5 mRNA expression levels further detected by qRT-PCR assays were similar to their protein expression trends determined based on IHC staining (Figure 1b-c). Interestingly, the KLF5 mRNA level had a moderate positive correlation with the TGR5 mRNA level in the gastric tissues of the 16 included subjects ( , ; Figure 1 d ).
，BR（+）IM（+）组织中KLF5的大量核染色与BR（-）IM（-）组织中KLF5的大量核染色显著不同（-， vs. ++ , ）。通过qRT-PCR检测进一步发现的TGR5和KLF5 mRNA表达水平与根据IHC染色确定的蛋白表达趋势相似（图1b-c）。有趣的是，在16名受试者的胃组织中，KLF5 mRNA水平与TGR5 mRNA水平呈中度正相关（ ， ；图1 d）。

Given that DCA is the main BAs component in DGR, we wanted to examine the phenotypic changes in immortalized GES-1 gastric epithelial cells treated with DCA. CCK-8 assays were used to detect the viability-inhibitory effects in GES-1 cells exposed to different concentrations of DCA (50 ) for , and 48 h . It was found that DCA significantly enhanced GES-1 cells viability after stimulation for only ). Low concentrations of DCA (50 or did not inhibit cell viability compared with the untreated control at each time point. However, cell viability was significantly decreased by after 48 h of treatment with DCA, while DCA showed marked cytotoxicity (Figure 2a). Hence, continuous DCA intervention showed dose-dependent cytotoxicity to GES-1 cells. Based on our subsequent experiments, DCA upregulated proinflammatory cytokines and intestinal markers and induced TGR5 expression and simultaneous STAT3 phosphorylation in a dose-dependent manner, with the greatest effects at (Figure 3a and Fig. S1E). Therefore, DCA was chosen for further exploration. Colony formation assays were conducted to observe the proliferative capacity of GES-1 cells that received short-term stimulation with DCA for 15 min . DCA was used in this study for a short time span ( 15 min ) to mimic reflux episodes. The results indicated that DCA significantly promoted the number and size of colonies formed by gastric cells ( ; Figure 2b). Afterward, the apoptosis rate of GES-1 cells exposed to DCA for 15 min and subsequently incubated in normal medium for 24 h of recovery was measured by flow cytometry. The results showed that the number of apoptotic cells in the DCA group was remarkably lower than that in the control group ( ; Figure 2c), suggesting that DCA might induce apoptotic resistance in gastric cells. To confirm these findings, we further performed western blotting to detect the expression levels of a series of apoptotic and antiapoptotic proteins in GES-1 cells under the same DCA exposure conditions. The results revealed that DCA induced resistance to apoptosis through upregulation of the protein expression levels of antiapoptotic Mcl-1 and Bcl-2 and downregulation of the protein expression levels of apoptotic cleaved caspase3 and caspase-9 in GES-1 cells (Figure 2d and Fig. . The findings demonstrated that DCA had cytotoxic effects on gastric epithelial cells, but the viable cells tended to resist apoptosis under DCA stimulation.
鉴于DCA是DGR中的主要BAs成分， 我们想研究用DCA处理的永生GES-1胃上皮细胞的表型变化。我们使用 CCK-8 试验来检测暴露于不同浓度 DCA（50 ） 和 48 小时的 GES-1 细胞的活力抑制作用。研究发现， DCA 仅在 ) 的刺激下能显著增强 GES-1 细胞的活力。与未处理的对照组相比，低浓度 DCA（50 或 在每个时间点都不会抑制细胞活力。然而，在使用 DCA 处理 48 小时后，细胞活力因 而明显降低，而 DCA 则表现出明显的细胞毒性（图 2a）。因此，持续的DCA干预对GES-1细胞具有剂量依赖性的细胞毒性。根据我们随后的实验，DCA 上调了促炎细胞因子和肠道标志物，并以剂量依赖的方式诱导了 TGR5 的表达和 STAT3 的同时磷酸化，其中 的影响最大（图 3a 和图 S1E）。因此，我们选择了 DCA进行进一步研究。进行了菌落形成试验，以观察接受 DCA 短期刺激 15 分钟的 GES-1 细胞的增殖能力。本研究中使用了短时间（15 分钟）的 DCA 来模拟回流现象。结果表明，DCA 能明显促进胃细胞形成菌落的数量和大小（ ；图 2b）。随后，用流式细胞术测量了暴露于 DCA 15分钟并在正常培养基中培养24小时恢复的GES-1细胞的凋亡率。 结果显示，DCA组的凋亡细胞数明显低于对照组（ ；图2c），表明DCA可能诱导胃细胞的凋亡抵抗。为了证实这些发现，我们进一步用 Western 印迹法检测了在相同的 DCA 暴露条件下 GES-1 细胞中一系列凋亡蛋白和抗凋亡蛋白的表达水平。结果显示，DCA通过上调GES-1细胞中抗凋亡蛋白Mcl-1和Bcl-2的蛋白表达水平，下调凋亡蛋白裂解的caspase3和caspase-9的蛋白表达水平，诱导细胞抗凋亡（图2d和图 ）。研究结果表明，DCA对胃上皮细胞有细胞毒性作用，但有活力的细胞在DCA刺激下往往能抵抗凋亡。

Since acidic bile reflux clearly contributes to the development of Barrett's esophagus and its progression to adenocarcinoma, at the end of this experiment, we aimed to clarify the different effects of neutral and acidic DCA on inflammation and IM phenotypes in normal human gastric epithelial cells. Briefly, GES-1 cells were treated with DCA in neutral and acidified media and 5.5 , respectively) for 15 min and then returned to normal medium for different times ( ; Figure 2e). The protein expression levels of the DCA receptor TGR5, phosphorylated
由于酸性胆汁反流明显有助于巴雷特食管的发展及其向腺癌的进展， 在本实验的最后，我们旨在阐明中性和酸性DCA对正常人胃上皮细胞炎症和IM表型的不同影响。简单地说，GES-1细胞在中性和酸性培养基（分别为 和5.5 ，）中用 DCA处理15分钟，然后再回到正常培养基中处理不同时间（ ；图2e）。DCA受体TGR5的蛋白表达水平、磷酸化的

Figure 2. DCA induced cytotoxicity, promoted proliferation, inhibited apoptosis, and activated STAT3 phosphorylation in gastric cells. (a) CCK-8 assays were used to examine cell proliferation/cytotoxicity in GES-1 cells treated with DCA at different doses for different times. (b) Short-term stimulation with DCA for 15 min significantly promoted colony formation in GES-1 cells. (c) Apoptosis rates in GES-1 cells exposed to DCA for 15 min and subsequently incubated in normal medium for 24 h , as measured by flow cytometry. (d) Protein expression levels of antiapoptotic Mcl-1, Bcl-2 and Bcl-xL and apoptotic cleaved caspase-3 and -9 in GES-1 cells under the same DCA exposure conditions examined by western blotting. (e) Workflow used to examine the different effects of neutral and acidic DCA ( and 5.5 ) on inflammation and IM phenotypes in GES-1 cells. ( ) Western blotting to determine the protein levels of TGR5, phosphorylated STAT3 and KLF5 in GES-1 cells treated with DCA under acidic and neutral conditions. The data are represented as the mean SEM.
图 2.DCA 在胃细胞中诱导细胞毒性、促进增殖、抑制凋亡并激活 STAT3 磷酸化。(a) 使用 CCK-8 检测不同剂量、不同时间的 DCA 处理 GES-1 细胞的细胞增殖/细胞毒性。(b) DCA 15 分钟的短期刺激可显著促进 GES-1 细胞集落的形成。(c) 用流式细胞仪测量暴露于 DCA 15 分钟并随后在正常培养基中培养 24 小时的 GES-1 细胞的凋亡率。(d) 在相同的 DCA 暴露条件下，用 Western 印迹法检测 GES-1 细胞中抗凋亡 Mcl-1、Bcl-2 和 Bcl-xL 蛋白表达水平以及凋亡裂解的 caspase-3 和 -9 蛋白表达水平。(e）用于检测中性和酸性 DCA（ 和 5.5）对 GES-1 细胞炎症和 IM 表型的不同影响的工作流程。( ) Western 印迹法测定酸性和中性条件下用 DCA 处理的 GES-1 细胞中 TGR5、磷酸化 STAT3 和 KLF5 的蛋白水平。数据以平均值 SEM表示。

STAT3 and its potential target gene KLF5, an IM marker, were detected by western blotting (figure ). The results showed that the expression of TGR5 was increased 15 minutes after the stimulation of GES-1 cells with either acidic DCA or neutral DCA, and neutral DCA had a more lasting activation effect, which lasted up to 12 hours after DCA withdrawal. Moreover, STAT3 phosphorylation (Tyr705) and KLF5 expression were significantly increased with the extension of the recovery time after acidic or neutral DCA treatment. Interestingly, there were positive correlations between p-STAT3 and KLF5 protein expression in both the acidic and neutral DCA treatment groups (Pearson's ; Pearson's , . Therefore, we observed that DCA promoted gastric epithelial inflammation and IM, regardless of the pH value, which is similar to the previously reported roles of DCA in esophageal mucosa. Given that the loss of parietal cells in the gastric body results in diminished gastric acid secretion and elevated gastric juice pH in atrophic gastritis and neutral DCA was selected for subsequent experiments studying IM development in the gastric body.
STAT3 及其潜在靶基因 KLF5（一种 IM 标记）通过 Western 印迹进行检测（图 ）。结果表明，酸性DCA或中性DCA刺激GES-1细胞15分钟后，TGR5的表达增加，中性DCA的激活作用更持久，可持续到DCA撤除后12小时。此外，STAT3 磷酸化（Tyr705）和 KLF5 表达随着酸性或中性 DCA 处理后恢复时间的延长而显著增加。有趣的是，在酸性和中性DCA处理组中，p-STAT3和KLF5蛋白表达均呈正相关（Pearson's ；Pearson's ， ）。因此，我们观察到，无论pH值如何，DCA都会促进胃上皮炎症和IM，这与之前报道的DCA在食管粘膜中的作用相似。 鉴于萎缩性胃炎和 中性DCA会导致胃体顶层细胞丧失，从而导致胃酸分泌减少和胃液pH值升高，因此我们选择了中性DCA作为随后研究胃体IM发展的实验对象。

To observe the significance of DCA in boosting gastric inflammation and IM, immortalized GES-1 gastric epithelial cells were treated with DCA at different doses for different times. The mRNA expression levels of proinflammatory cytokines and intestinal markers were determined by qRTPCR after stimulation with DCA, and the protein concentrations of proinflammatory cytokines in the supernatant were detected by ELISA. Consequently, the mRNA expression levels of IL6, CXCL8, IL-11, NFKB1, CDX2, KLF5, MUC2 and VIL1 were dose-dependently increased under DCA stimulation, and DCA had the strongest effects (Figure 3a).
为了观察DCA在促进胃炎和IM方面的意义，用不同剂量、不同时间的DCA处理永生的GES-1胃上皮细胞。用 qRTPCR 法测定受 DCA 刺激后促炎细胞因子和肠道标志物的 mRNA 表达水平，并用 ELISA 法检测上清液中促炎细胞因子的蛋白浓度。结果发现，在DCA刺激下，IL6、CXCL8、IL-11、NFKB1、CDX2、KLF5、MUC2和VIL1的mRNA表达水平呈剂量依赖性升高，其中 DCA的作用最强（图3a）。

Next, GES-1 cells were treated with DCA for 15 min and subsequently incubated in normal medium for different periods, and the results showed that the promotion effects of DCA on the mRNA expression of these genes lasted for 36 hours after DCA withdrawal (Figure 3b). In addition, since IL-6, CXCL8 and IL-11 are secretory proteins that exert initial proinflammatory signal effects, we also detected the concentrations of these proinflammatory cytokines in the cell culture medium using ELISA. The results showed that the IL-6 and CXCL-8 concentrations were significantly higher in the supernatant of GES-1 cells that received DCA stimulation than in the untreated cells vs. , vs. , ), and there was no difference in the IL-11 concentration between the two groups (Figure 3c).
接下来，用 DCA处理GES-1细胞15分钟，然后在正常培养基中培养不同时间，结果显示DCA对这些基因mRNA表达的促进作用在DCA撤除后持续了36小时（图3b）。此外，由于IL-6、CXCL8和IL-11都是分泌蛋白，具有最初的促炎信号作用，我们还利用ELISA检测了细胞培养基中这些促炎细胞因子的浓度。结果显示，接受DCA刺激的GES-1细胞上清液中IL-6和CXCL-8的浓度明显高于未处理的细胞 vs. , vs. , )，而IL-11的浓度在两组间没有差异（图3c）。

TGR5 is a predominant G-protein-coupled receptor mediated by secondary BAs. Herein, we aimed to explore the role of DCA in regulating TGR5 expression. We initially examined the baseline expression of TGR5 mRNA and protein in the gastric epithelial cell line GES-1 and GC cell lines AGS, SGC-7901, BGC-823 and MKN-45 using qRT-PCR and western blotting. As shown in Fig. S1A, TGR5 mRNA and protein expression levels were lower in GES-1 cells than in the GC cells. Moreover, TGR5 mRNA expression levels were lower in AGS and SGC-7901 cells than in BGC-823 and MKN-45 cells. Therefore, we selected a low-expressing cell line AGS and a high-expressing cell line BGC-823, respectively, for subsequent DCA intervention experiments. Next, GES-1 cells were treated with DCA for 15 min and incubated in normal medium for different times. The qRT-PCR results revealed that the expression of TGR5 mRNA reached the highest level at 3 h of recovery culture and then gradually returned to the baseline level (Fig. S1B). The immunofluorescence results also showed increased expression of TGR5 in the cytoplasm and membrane of GES-1 cells and AGS and BGC-823 GC cells stimulated by DCA with a 3-hour recovery (Fig. S1C-D). Furthermore, we treated GES-1 cells with DCA at different doses with a 3 -hour recovery culture. The qRT-PCR and western blotting results showed that compared with untreated cells, DCA induced TGR5 expression and simultaneous STAT3 phosphorylation in a dose-dependent manner, with the greatest effects at (Fig. S1E). Based on these results
TGR5是一种由次级BA介导的主要G蛋白偶联受体。 在此，我们旨在探索DCA在调节TGR5表达中的作用。我们首先用qRT-PCR和Western印迹法检测了胃上皮细胞系GES-1和GC细胞系AGS、SGC-7901、BGC-823和MKN-45中TGR5 mRNA和蛋白质的基线表达。如图 S1A 所示，GES-1 细胞的 TGR5 mRNA 和蛋白表达水平均低于 GC 细胞。此外，TGR5 mRNA 在 AGS 和 SGC-7901 细胞中的表达水平也低于 BGC-823 和 MKN-45 细胞。因此，我们分别选择了低表达细胞株 AGS 和高表达细胞株 BGC-823 进行后续的 DCA 干预实验。接下来，用 DCA处理GES-1细胞15分钟，并在正常培养基中培养不同时间。qRT-PCR结果显示，TGR5 mRNA的表达在恢复培养3 h时达到最高水平，随后逐渐恢复到基线水平（图S1B）。免疫荧光结果也显示，受 DCA 刺激的 GES-1 细胞、AGS 和 BGC-823 GC 细胞在恢复培养 3 小时后，其细胞质和细胞膜中 TGR5 的表达均有所增加（图 S1C-D）。此外，我们用不同剂量的 DCA 处理 GES-1 细胞，并进行 3 小时恢复培养。qRT-PCR和Western印迹结果显示，与未处理的细胞相比，DCA以剂量依赖的方式诱导TGR5的表达并同时诱导STAT3磷酸化，在 时效果最好（图S1E）。基于这些结果

Figure 3. DCA upregulated proinflammatory cytokines and intestinal markers in gastric cells. (a) DCA dosages: 50, 100 and in 24 h interventions. The mRNA expression levels of IL-6, CXCL8, IL-11, NFKB1, CDX2, KLF5, MUC2 and VIL1 were then detected by qRTPCR assays. (b) DCA dosage: in a 15 min intervention; recovery times: , and 24 h . The mRNA expression levels of the abovementioned genes were then detected with qRT-PCR. (c) DCA dosage: in a 15 min intervention; recovery time: 24 h . The concentrations of IL-6, CXCL8 and IL-11 in the culture medium of GES-1 cells after DCA stimulation were determined by ELISA. , . N.S., not significant.
图 3.DCA 上调胃细胞中的促炎细胞因子和肠道标志物。(a) DCA 剂量：50、100 和 24 小时干预。然后通过 qRTPCR 检测 IL-6、CXCL8、IL-11、NFKB1、CDX2、KLF5、MUC2 和 VIL1 的 mRNA 表达水平。(b) DCA 剂量： ，15 分钟干预；恢复时间： 和 24 小时。然后用 qRT-PCR 检测上述基因的 mRNA 表达水平。(c) DCA 剂量： ，干预 15 分钟；恢复时间：24 小时。用 ELISA 法测定 DCA 刺激后 GES-1 细胞培养液中 IL-6、CXCL8 和 IL-11 的浓度。 ， 。N.S.，无意义。

together, DCA mediated the expression of the secondary BA receptor TGR5 on the membrane of gastric epithelial cells.
同时，DCA 在胃上皮细胞膜上介导二级 BA 受体 TGR5 的表达。

STAT3 is a transcription factor involved in gastric tumorigenesis. Detection based on clinical specimens found that tyrosine-phosphorylated STAT3 (p-STAT3) expression was elevated in GC and precancerous lesions, including IM and dysplasia, the underlying molecular mechanism of which requires further investigation. We first investigated the phosphorylation and localization of STAT3 in DCA-treated GES-1 cells. Transient stimulation with
STAT3 是一种参与胃肿瘤发生的转录因子。基于临床标本的检测发现，酪氨酸磷酸化STAT3（p-STAT3）在胃癌和癌前病变（包括IM和发育不良）中表达升高， 其潜在的分子机制有待进一步研究。我们首先研究了经DCA处理的GES-1细胞中STAT3的磷酸化和定位。瞬时刺激
DCA mimicking a reflux episode resulted in remarkable increases in p-STAT3 and KLF5 levels in the nuclear fraction (Figure 4a and Fig. S4B). We further observed that DCA treatment drove the nuclear accumulation of p-STAT3 and KLF5 in GES-1 cells compared to untreated cells (Figure 4b). Similar to the effects in gastric normal epithelial cells, in two gastric cancer cell lines, AGS and BGC-823, the secondary bile acid receptor TGR5 was significantly activated after transient DCA stimulation, the expression of p-STAT3 and KLF5 increased stepwise with prolonged recovery
模拟回流事件的 DCA 导致核部分中 p-STAT3 和 KLF5 水平显著增加（图 4a 和图 S4B）。我们进一步观察到，与未处理的细胞相比，DCA 处理促进了 GES-1 细胞中 p-STAT3 和 KLF5 的核积累（图 4b）。与对胃正常上皮细胞的影响相似，在 AGS 和 BGC-823 两种胃癌细胞系中，二级胆汁酸受体 TGR5 在短暂的 DCA 刺激后被显著激活，p-STAT3 和 KLF5 的表达随着恢复时间的延长而逐步增加。
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Figure 4. DCA-TGR5 axis facilitated STAT3 phosphorylation, nuclear accumulation and transcriptional activation. After 15 min of exposure to DCA followed by 24 h of recovery in fresh complete media, the cytoplasmic and nuclear proteins in GES-1 cells (a) and AGS and BGC-823 cells (d) were completely separated and then detected by western blotting. Immunofluorescence images of GES1 cells (b) and AGS and BGC-823 cells (e) treated with and without DCA under the same conditions mentioned above. Scale bars, . (c) Gastric cancer cells AGS and BGC-823 were treated with DCA for 15 min , followed by different recovery times in complete media. TGR5, p-STAT3 and KLF5 expression in the total cell fractions was detected by immunoblot analysis. (f) Luciferase reporter assays examining STAT3 transcriptional activity were performed in GES-1, AGS and BGC-823 cells after transfection with the p -Stat3Luciferase reporter followed by DCA treatment for 15 min . The reporter activity was measured post recovery in complete media for 24 h . UT, untreated. .
图 4.DCA-TGR5 轴促进了 STAT3 磷酸化、核积累和转录激活。暴露于 DCA 15 分钟后，在新鲜的完全培养基中恢复 24 小时，GES-1 细胞（a）和 AGS 及 BGC-823 细胞（d）的胞质蛋白和核蛋白完全分离，然后用 Western 印迹法检测。在上述相同条件下，用或不用DCA处理GES1细胞（b）和AGS及BGC-823细胞（e）的免疫荧光图像。比例尺， 。(c) 胃癌细胞 AGS 和 BGC-823 用 DCA 处理 15 分钟，然后在完全培养基中进行不同时间的恢复。通过免疫印迹分析检测总细胞组分中 TGR5、p-STAT3 和 KLF5 的表达。(f）检测 STAT3 转录活性的荧光素酶报告实验是在 GES-1、AGS 和 BGC-823 细胞中转染 p -Stat3Luciferase 报告基因，然后用 DCA 处理 15 分钟后进行的。在完全培养基中恢复 24 小时后测量报告活性。UT，未处理。 。

time (0-24 h), and activated p-STAT3 mainly accumulated in the nucleus (Figure 4 c -e). To investigate whether DCA-mediated nuclear p-STAT3 accumulation influenced STAT3 transcriptional activity, the validity of the p -Stat3-Luciferase reporter was first confirmed. The reporter was successfully transfected into GES-1 cells and indeed enhanced the luciferase signal ( ). Then, on this basis, a significant increase in STAT3 transcriptional activity was found in normal and cancerous gastric cells after
图 4 c -e）。为了研究DCA介导的核内p-STAT3积累是否影响STAT3的转录活性，首先确认了p-STAT3-荧光素酶报告物的有效性。该报告物被成功转染到 GES-1 细胞中，并确实增强了荧光素酶信号（ ）。在此基础上，研究人员发现正常胃细胞和癌变胃细胞的 STAT3 转录活性在转录后都有显著增加。

DCA stimulation (figure 4f). Taken together, the DCA-TGR5 axis facilitated nuclear p-STAT3 accumulation and STAT3 transcriptional activation, which may be related to KLF5 overexpression.
图 4f）。综上所述，DCA-TGR5 轴促进了核 p-STAT3 的积累和 STAT3 的转录激活，这可能与 KLF5 的过表达有关。

To confirm that KLF5 expression is regulated by STAT3, especially the active form of STAT3 (p-STAT3), we first silenced STAT3 using siRNAs in GES-1 cells. It was observed that si-STAT3 transfection with each of the three oligonucleotide sequences could significantly inhibit STAT3 mRNA expression (Figure 5a), and we selected siSTAT3, which had the strongest inhibitory effect, for use in the following experiment. In cells not treated with DCA, si-STAT3 transfection did not change the KLF5 mRNA levels. In cells that received DCA stimulation for 15 min , pretransfection with si-STAT3 resulted in a significant decrease in KLF5 mRNA levels (Figure 5b). The inhibition of KLF5 at the protein level by siSTAT3 transfection was also analyzed, and equivalent results were obtained (Figure 5c and Fig. S4C). To further assess the implication of STAT3 phosphorylation in regulating KLF5 expression, GES-1 cells were treated with a selective STAT3 inhibitor, Stattic, which inhibits the phosphorylation, dimerization and nuclear translocation of STAT3 by interacting with the SH 2 domain. The addition of Stattic dose-dependently decreased the expression of KLF5 (Figure 5d and Fig. S4D).
为了证实KLF5的表达受STAT3，尤其是STAT3的活性形式（p-STAT3）调控，我们首先在GES-1细胞中使用siRNA沉默STAT3。我们选择了抑制作用最强的 siSTAT3 用于接下来的实验。在未经 DCA 处理的细胞中，si-STAT3 转染不会改变 KLF5 mRNA 水平。在接受 DCA 刺激 15 分钟的细胞中，预转染 si-STAT3 导致 KLF5 mRNA 水平显著下降（图 5b）。我们还分析了 siSTAT3 转染在蛋白水平上对 KLF5 的抑制作用，也得到了相同的结果（图 5c 和图 S4C）。为了进一步评估 STAT3 磷酸化在调控 KLF5 表达中的作用，用选择性 STAT3 抑制剂 Stattic 处理 GES-1 细胞，该抑制剂通过与 STAT3 的 SH 2 结构域相互作用来抑制 STAT3 的磷酸化、二聚化和核转运。加入 Stattic 后，KLF5 的表达呈剂量依赖性下降（图 5d 和图 S4D）。

In vivo binding of the active form of STAT3 (p-STAT3) to the KLF5 promoter was further confirmed by ChIP assays after two putative binding sites for STAT3 were located within 2 kb of the KLF5 proximal promoter through the JASPAR website (https://jaspar.genereg.net/; Figure 5e). Two specific primers, one including two STAT3-binding sites in the KLF5 promoter used for the ChIP experiments, were designed and are shown in Figure 5e. Consequently, no binding was detected when a negative control primer pair was used for q-PCR, whereas visible binding of p -STAT3 to the KLF5 promoter was observed in untreated GES-1 cells. Furthermore, a significant increase in binding was detected in GES-1 cells treated with DCA when using each of the primers (figure 5fandg). Together, DCA might promote STAT3 phosphorylation and subsequently activate KLF5 transcription through the direct binding of p-STAT3 to the KLF5 promoter.
通过JASPAR网站（https://jaspar.genereg.net/;图5e），在KLF5近端启动子的2 kb范围内找到了两个STAT3的推定结合位点，通过ChIP实验进一步证实了STAT3的活性形式（p-STAT3）与KLF5启动子的体内结合。设计了两个特异引物，其中一个包括用于 ChIP 实验的 KLF5 启动子中的两个 STAT3 结合位点，如图 5e 所示。因此，当使用阴性对照引物对进行 q-PCR 时未检测到结合，而在未经处理的 GES-1 细胞中观察到 p -STAT3 与 KLF5 启动子的明显结合。此外，当使用每种引物时，在使用 DCA 处理的 GES-1 细胞中检测到的结合明显增加（图 5fandg）。总之，DCA 可能会促进 STAT3 磷酸化，随后通过 p-STAT3 与 KLF5 启动子的直接结合激活 KLF5 的转录。

Given that gastric organoids better mimic the genetic, phenotypic and behavioral traits of normal stomach tissues and tumors than frequently used cell lines, we further conducted in vitro and in vivo mouse organoid experiments to explore the regulatory effects of DCA on the TGR5/ p-STAT3/KLF5 axis of the gastric epithelium. The workflow used to examine the phenotypic changes in gastric organoids treated with DCA in vitro is shown in Figure 6a. Stomach organoids were extracted from three 8 -week-old male FVB/N mice and treated with DCA in vitro. Representative bright-field microscopic and -stained images of the stomach organoids in the two groups are shown in Figure 6b. Immunofluorescence staining analysis showed that the basal levels of TGR5 membrane staining (Figure 6c-d) and p-STAT3 and KLF5 nuclear costaining (Figure ) were low in gastric organoids, and these levels could be increased by DCA in vitro vs. (18.11 1.14)% vs. (75.74 , both . Western blot analysis was also performed, and a similar trend to that shown in the IF assays was observed (Figure 6g and Fig. S4E).
鉴于胃有机体比常用细胞系更好地模拟了正常胃组织和肿瘤的遗传、表型和行为特征， 我们进一步进行了小鼠有机体的体外和体内实验，以探索DCA对胃上皮细胞TGR5/ p-STAT3/KLF5轴的调控作用。图 6a 显示了用于研究体外用 DCA 处理的胃有机体表型变化的工作流程。从三只 8 周大的雄性 FVB/N 小鼠体内提取胃器官组织并用 DCA 在体外处理。图 6b 中显示了两组胃有机体的代表性明视野显微镜图像和 染色图像。免疫荧光染色分析表明，胃有机体中TGR5膜染色（图6c-d）、p-STAT3和KLF5核染色（图 ）的基础水平较低，体外DCA可提高这些水平 对 （18.11 1.14）%对（75.74 ，均为 。还进行了 Western 印迹分析，观察到与 IF 试验中显示的类似趋势（图 6g 和图 S4E）。

We next verified our findings regarding DCAmediated gastric IM using three mouse stomach organoid lines in vivo. As shown in Figure 7a, a total of three C57BL/6 mice, three FVB/N wildtype mice and six FVB/N INS-GAS mice were prepared, all of which were male mice aged 8 weeks. Among them, three INS-GAS transgenic mice were randomly selected to be given free access to DCA drinking water, as previously reported, and all remaining mice were given NS in their drinking water. In this part of the in vivo experiments, we used NS as the solvent for DCA instead of DMEM to eliminate the bias caused by the toxicity of DMEM. Three months after the drinking water intervention, gastric organoids were extracted and
接下来，我们利用体内的三个小鼠胃器官系验证了我们关于DCA介导的胃IM的发现。如图7a所示，我们共制备了3只C57BL/6小鼠、3只FVB/N野生型小鼠和6只FVB/N INS-GAS小鼠，均为8周龄的雄性小鼠。其中，3只INS-GAS转基因小鼠被随机选中，按照之前的报道，让它们自由饮用 DCA饮用水， 其余所有小鼠的饮用水中都添加了NS。在这部分体内实验中，我们用NS代替DMEM作为DCA的溶剂，以消除DMEM毒性造成的偏差。在饮水干预三个月后，提取胃有机体并将其与DCA混合。

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Figure 5. After DCA treatment, p-STAT3 activates KLF5 expression by binding to the KLF5 promoter in gastric cells. (a) STAT3 mRNA levels detected by qRT-PCR in GES-1 cells 24 h after si-STAT3 transfection with different oligonucleotide sequences. (b-c) GES-1 cells were transfected with si-STAT3 (398) for 24 h and stimulated with DCA for 15 min followed by 24 h of recovery. KLF5 mRNA expression was detected by qRT-PCR, and p-STAT3, STAT3 and KLF5 protein expression was examined by western blotting. (d) After the inhibition of STAT3 phosphorylation by Stattic (Cons. 10-150 M) for 2 hours, as recommended in the manufacturer's instructions, KLF5 protein expression was detected by western blotting. (e) Schematic representation of the KLF5 promoter, in which two putative STAT3 binding sites and the primers used for ChIP assays are shown. (f) p-STAT3 bound to the KLF5 promoter in untreated GES-1 cells and GES-1 cells treated with of DCA for 15 min with subsequent recovery in normal medium for 24 h was detected by ChIP assays. (g) Enrichment levels of p-STAT3 binding to the KLF5 promoter were significantly increased according to q-PCR detection with each of the two primers. . N.S., not significant.
图 5.DCA 处理后，p-STAT3 通过与胃细胞中的 KLF5 启动子结合激活 KLF5 的表达。(a）不同寡核苷酸序列的 si-STAT3 转染 GES-1 细胞 24 小时后，通过 qRT-PCR 检测 STAT3 mRNA 水平。(b-c）用 si-STAT3 (398) 转染 GES-1 细胞 24 小时后，用 DCA 刺激 15 分钟，然后恢复 24 小时。qRT-PCR 检测 KLF5 mRNA 的表达，Western 印迹检测 p-STAT3、STAT3 和 KLF5 蛋白的表达。(d）按照生产商说明书的建议，用 Stattic（Cons. 10-150 M）抑制 STAT3 磷酸化 2 小时后，用 Western 印迹法检测 KLF5 蛋白表达。(e）KLF5 启动子示意图，其中显示了两个推定的 STAT3 结合位点和用于 ChIP 检测的引物。(f）通过 ChIP 检测未处理的 GES-1 细胞和经 DCA 处理 15 分钟后在正常培养基中恢复 24 小时的 GES-1 细胞中与 KLF5 启动子结合的 p-STAT3。(g）根据两种引物中每一种的 q-PCR 检测，p-STAT3 与 KLF5 启动子结合的富集水平显著增加。 。N.S.，不显著。

cultured for 7 days, and then the sizes of the organoids in the four groups were observed by brightfield microscopy and H&E staining (Figure 7b). A previous study found that INS-GAS mice showed decreased maximal gastric acid secretion and decreased parietal cell number in later stage (five months and older), which suggests the beginning of gastric atrophy. Hence, we selected the three
培养7天后，用明视野显微镜和H&E染色观察四组有机体的大小（图7b）。先前的研究发现，INS-GAS小鼠在后期（5个月以上）出现最大胃酸分泌减少和顶细胞数量减少， 这表明胃开始萎缩。因此，我们选择了三种
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Figure 6. DCA promoted STAT3 phosphorylation and the expression of the IM marker KLF5 in gastric organoids derived from FVB/N mice in vitro. (a) Workflow used to examine the phenotypic changes in gastric organoids treated with DCA in vitro. Dosage: for 15 min ; recovery time: 24 h in normal medium. (b) Bright-field microscopy and hematoxylin and eosin staining of representative stomach organoids of FVB/N mice treated with NS or DCA. Scale bars, (top) and (middle and bottom). (c-f) Immunofluorescence staining for TGR5, p-STAT3 and KLF5 in gastric organoids from FVB/N mice stimulated with DCA in vitro. Scale bars, (top) and (bottom). (g) Western blot analysis of gastric organoids from FVB/N mice stimulated with DCA in vitro. NS, normal saline. .
图 6.DCA 促进了 STAT3 磷酸化和体外 FVB/N 小鼠胃有机体中 IM 标记 KLF5 的表达。 (a) 用于检测体外用 DCA 处理的胃有机体表型变化的工作流程。剂量： 15分钟；恢复时间：正常培养基中24小时。(b）NS或DCA处理的FVB/N小鼠代表性胃有机体的明视野显微镜及苏木精和伊红染色。比例尺， （上）和 （中和下）。(c-f）体外用DCA刺激的FVB/N小鼠胃有机体中TGR5、p-STAT3和KLF5的免疫荧光染色。比例尺， （上）和 （下）。(g）体外用DCA刺激FVB/N小鼠胃器官组织的Western印迹分析。NS：生理盐水。 。

Figure 7. STAT3 phosphorylation and the IM phenotype were observed in gastric organoids derived from INS-GAS transgenic mice after drinking DCA for 3 months. (a) Schematic diagram of the in vivo experimental procedure. (b) Stomach organoids derived from the four groups of mice were extracted and cultured for 7 days for bright-field microscopy examination and H&E staining. Scale bar, . (c) For the statistical analyses of the average maximum diameters of the four groups of organoids, three microscopic fields (under 100× magnification) were randomly selected from each sample, and the three largest organoids in each field were selected for diameter measurement. (d) Immunofluorescence staining for nuclear p-STAT3 and KLF5 coexpression in gastric organoids from FVB/N (NS), INS-GAS (NS) and INS-GAS (DCA) mice. Scale bar, . (e) Quantification of nuclear p-STAT3 and KLF5 costaining in the three groups. (f) Western blot analysis of gastric organoids from FVB/N (NS), INS-GAS (NS) and INS-GAS (DCA) mice. ; N.S., not significant.
图 7.饮用 DCA 3 个月后，在 INS-GAS 转基因小鼠的胃器官组织中观察到 STAT3 磷酸化和 IM 表型。(a）体内实验过程示意图。(b) 提取四组小鼠的胃器官组织，培养 7 天后进行明视野显微镜检查和 H&E 染色。比例尺， 。(c）为了统计分析四组有机体的平均最大直径，从每个样本中随机选取三个显微镜视野（放大 100 倍），每个视野中选取三个最大的有机体进行直径测量。(d）免疫荧光染色显示FVB/N（NS）、INS-GAS（NS）和INS-GAS（DCA）小鼠胃组织细胞核p-STAT3和KLF5共表达。比例尺， 。(e) 三组小鼠核 p-STAT3 和 KLF5 染色定量。(f）FVB/N（NS）、INS-GAS（NS）和INS-GAS（DCA）小鼠胃器官组织的Western印迹分析。 ；N.S.，不显著。
months of DCA administration for the organoids in vivo experiments. The average maximum diameter of the organoids was in the C57BL/6 mouse group, in the FVB/N mouse group, in the INS-GAS mouse group, and in the INS-GAS (DCA) mouse group. Compared with FVB/N wild-type mice, INS-GAS transgenic mice showed significant glandular atrophy regardless of whether they received DCA (both ; Figure 7c). Immunofluorescence staining images and the results of quantification of the nuclear coexpression of p-STAT3 and KLF5 are shown in Figure 7d and e, indicating obvious enhancement in organoids extracted from INS-GAS mice that received DCA administration compared to those from untreated INS-GAS mice . figure 7 f and Fig. S4F show the western blot results, which were consistent with the immunofluorescence photomicrographs. Moreover, DCA ingestion did not affect gastric mucosal CDX2 expression, although it was more highly expressed in INS-GAS mice.
C57BL/6小鼠组的平均最大直径为 ，FVB/N小鼠组为 ，FVB/N小鼠组为 。C57BL/6小鼠组的器官组织平均最大直径为 ，FVB/N小鼠组为 ，INS-GAS小鼠组为 ，INS-GAS（DCA）小鼠组为 。与 FVB/N 野生型小鼠相比，INS-GAS 转基因小鼠无论是否接受 DCA 都表现出明显的腺体萎缩（均为 ；图 7c）。图7d和e显示了免疫荧光染色图像以及p-STAT3和KLF5核共表达的定量结果，表明与未接受治疗的INS-GAS小鼠相比，接受DCA治疗的INS-GAS小鼠提取的器官组织明显增强 。图7f和图S4F显示了Western印迹结果，与免疫荧光显微照片一致。此外，摄入DCA并不影响胃粘膜CDX2的表达，尽管它在INS-GAS小鼠中的表达量更高。

We also collected and mixed the gastric contents of each group of mice ( ) to prepare gastric content supernatants used to stimulate GES-1 cells, aiming to observe the effects of long-term DCA ingestion by INS-GAS mice on the TGR5/STAT3/ KLF5 axis of normal human gastric epithelial cells. Promisingly, the results showed that the gastric extracts of the INS-GAS (DCA)-group mice also promoted inflammation and IM transformation in GES-1 cells (Fig. S2). Therefore, in the next experiment, we further explored the effects of DCA on the gastric environment of INS-GAS mice from the perspectives of gastric bile acid metabolism profiling and gastric bacterial alteration. Taken together, the data from the gastric epithelial cell and organoid experiments indicate that DCA might play a vital role in multistep precancerous processes involving inflammation and IM and the transformation from chronic inflammation to IM.
我们还收集并混合了各组小鼠（ ）的胃内容物，制备了用于刺激GES-1细胞的胃内容物上清液，旨在观察INS-GAS小鼠长期摄入DCA对正常人胃上皮细胞TGR5/STAT3/ KLF5轴的影响。结果表明，INS-GAS（DCA）组小鼠的胃提取物也促进了 GES-1 细胞的炎症和 IM 转化（图 S2）。因此，在接下来的实验中，我们从胃胆汁酸代谢谱分析和胃细菌改变的角度进一步探讨了DCA对INS-GAS小鼠胃环境的影响。总之，胃上皮细胞和类器官实验的数据表明，DCA可能在涉及炎症和IM的多步骤癌前病变过程以及从慢性炎症到IM的转化过程中发挥重要作用。

To understand the pathogenesis of gastric IM induced by BAs, we utilized a model of intestinaltype GC called transgenic INS-GAS mice, as previously reported. DCA in drinking water was given to mice aged 2 months, and the treatment was maintained for 6 months (Figure 8a). We found that beginning in the fourth month, DCA induced a significant body weight decrease in INS-GAS mice (Figure 8 b ). We further detected the serum TBA concentration in the two groups by the enzymatic cycling method and observed a remarkable accumulation of serum TBA in the DCA group compared to the NS group ( vs. ; Figure 8c). For the subsequent analysis, a macroscopic-level schematic drawing of mouse stomach anatomy is shown in Figure 8d, which depicts the sampling sites used in each experiment. More specifically, after incision along the greater curvature of the stomach, the left portion of the stomach was used for western blotting and qPCR assays, while the right portion was used for histopathological assessments. As shown in Figure 8e and Fig. S4G, DCA treatment led to markedly increased expression of TGR5 and the IM markers CDX2 and KLF5 and to the phosphorylation of STAT3 in corpus tissues compared to the slight expression detected in NS-treated samples. We also observed significantly higher levels of IL-6, CDX2, KLF5 and MUC2 mRNA expression in both corpus and antrum tissues in the DCA group than in the control group (figure 8f,g), which were indicative of activated mucosal chronic inflammation and IM progression caused by DCA in INS-GAS mice, and these findings were consistent with the aforementioned gastric epithelial cell and organoid results.
为了了解BA诱发胃IM的发病机制，我们利用了一种肠型GC模型--转基因INS-GAS小鼠，如前所述。 给2个月大的小鼠喂食饮用水中的DCA，并维持6个月（图8a）。我们发现，从第四个月开始，DCA 引起 INS-GAS 小鼠体重显著下降（图 8 b）。我们进一步用酶循环法检测了两组小鼠的血清 TBA 浓度，观察到 DCA 组与 NS 组相比，血清 TBA 显著积累（ vs. ; 图 8c）。为了进行后续分析，图 8d 显示了小鼠胃部的宏观解剖示意图，其中描述了每次实验中使用的取样部位。更具体地说，沿胃大弯切开后，胃的左侧部分用于 Western 印迹和 qPCR 检测，右侧部分用于组织病理学评估。如图 8e 和图 S4G 所示，与 NS 处理样本中检测到的轻微表达相比，DCA 处理导致胃体组织中 TGR5、IM 标记 CDX2 和 KLF5 的表达以及 STAT3 的磷酸化明显增加。我们还观察到，与对照组相比，DCA组胃体和胃窦组织中IL-6、CDX2、KLF5和MUC2 mRNA的表达水平均明显升高（图8f,g），这表明INS-GAS小鼠DCA引起了活化的粘膜慢性炎症和IM进展，这些发现与前述胃上皮细胞和类器官的结果一致。

We next histologically assessed the effects of DCA on gastric carcinogenesis in INS-GAS mice by H&E, Alcian blue and periodic acid-Schiff (AB/ PAS) and IHC staining. First, H&E staining of the gastric mucosa of INS-GAS mice revealed accelerated gastric IM and low-grade dysplasia (LGD) after DCA treatment. The red arrow in the representative image indicates a typical goblet cell in an irregular gastric gland in the DCA group (Figure 9a). Second, since of cases of IM columnar cells that secreted intestinal mucus showed AB -positive staining due to the presence of acidic mucin, staining was further performed to identify the type of mucin. Consequently, mucosal columnar cells in the control mice were stained magenta with PAS, indicating the presence of neutral mucin, whereas
接下来，我们通过H&E、Alcian蓝和定期酸-Schiff（AB/ PAS）以及IHC染色对DCA对INS-GAS小鼠胃癌发生的影响进行了组织学评估。首先，INS-GAS小鼠胃粘膜的H&E染色显示，DCA治疗后胃IM和低度发育不良（LGD）加速。代表图像中的红色箭头表示 DCA 组不规则胃腺中的一个典型小腺细胞（图 9a）。其次，由于 分泌肠粘液的IM柱状细胞因存在酸性粘蛋白而显示AB阳性染色，因此进一步进行 染色以确定粘蛋白的类型。结果，对照组小鼠的粘膜柱状细胞被 PAS 染成洋红色，表明存在中性粘蛋白，而非对照组小鼠的粘膜柱状细胞被 PAS 染成红色，表明存在酸性粘蛋白。

Figure 8. DCA resulted in the accumulation of serum TBA and enhanced STAT3 phosphorylation and IM marker expression in the gastric mucosae of INS-GAS mice. (a) Treatment schemata used for DCA administration in the drinking water of INS-GAS mice. INS-GAS (NS), INS-GAS (DCA). (b) Body weight changes in the two groups of INS-GAS mice during the intervention period. (c) Concentrations of serum TBA in the two groups determined by the enzymatic cycling method. (d) Macroscopic-level schematic drawing of mouse stomach anatomy depicting the sampling sites used in each experiment. (e) Western blotting for TGR5, p-STAT3 and IM markers CDX2 and KLF5 in the corpus tissues of the two groups. ( f and g ) mRNA expression detection by qRT-PCR in the corpus and antrum tissues of the two groups of INS-GAS mice.
图 8.DCA 导致 INS-GAS 小鼠血清 TBA 的积累，并增强 STAT3 磷酸化和 IM 标志物的表达。(a) 在 INS-GAS 小鼠饮用水中施用 DCA 的处理示意图。 INS-GAS（NS）， INS-GAS（DCA）。(b) 两组 INS-GAS 小鼠在干预期间的体重变化。(c) 用酶循环法测定的两组小鼠血清 TBA 浓度。(d) 小鼠胃部解剖的宏观示意图，描绘了每个实验中使用的取样部位。(e) 两组胃体组织中 TGR5、p-STAT3 和 IM 标记 CDX2 和 KLF5 的 Western 印迹。( f 和 g ) 通过 qRT-PCR 检测两组 INS-GAS 小鼠胃体和胃窦组织中的 mRNA 表达。
columnar cells in the bottoms of glands and the fovea of gastric mucosa in all cases in the DCA group reacted with AB and were stained blueviolet (Figure 9b). Considering both the presence of goblet cells and/or the presence of acidic mucin, all INS-GAS mice that received DCA treatment developed gastric IM at 6 months (9/9, 100%), while only 2 of mice in the control group were identified as having gastric IM ( ; Figure 9c). Furthermore, we tested p-STAT3 and KLF5 expression levels by IHC staining and further scored the corresponding nuclear staining intensities (Figure 9d-f). Compared with those of the control mice, the nuclear staining intensities of p-STAT3 and KLF5 in the gastric tissues of the DCA group were remarkably higher (
DCA组所有病例胃黏膜腺体底部和胃黏膜窝中的柱状细胞均与AB反应并被染成蓝紫色（图9b）。考虑到鹅口疮细胞的存在和/或酸性粘蛋白的存在，所有接受DCA治疗的INS-GAS小鼠在6个月时都出现了胃IM（9/9，100%），而对照组的 小鼠中只有2只被鉴定为胃IM（ ；图9c）。此外，我们还通过IHC染色检测了p-STAT3和KLF5的表达水平，并进一步对相应的核染色强度进行了评分（图9d-f）。与对照组相比，DCA 组小鼠胃组织中 p-STAT3 和 KLF5 的核染色强度明显更高 ( ) 。

d

C

Figure 9. Effects of DCA on gastric carcinogenesis in INS-GAS mice assessed by and staining. (a) Representative images of H&E staining from the two groups of INS-GAS mice. The red arrow shows a goblet cell in a dysplastic gastric gland. (b) Representative images of AB/PAS staining in the gastric and duodenal mucosae of the two groups. Duodenal sections containing intestinal mucus were stained blue as positive references for reaction with . The red arrows indicate positive staining. (c) Comparison of the number of gastric IM cases between the two groups (Fisher's exact test). (d) IHC staining for p-STAT3 and KLF5 in the stomach sections of the two groups. The right boxes depict enlarged regions of the left images. Scale bars represent (left) and (right). (e-f) IHC staining scores of p-STAT3 and KLF5 between the two groups.
图 9.通过 和 染色评估 DCA 对 INS-GAS 小鼠胃癌发生的影响。(a) 两组 INS-GAS 小鼠 H&E 染色的代表性图像。红色箭头显示的是发育不良胃腺中的一个腺细胞。(b）两组小鼠胃和十二指肠粘膜 AB/PAS 染色的代表性图像。含有肠粘液的十二指肠切片被染成蓝色，作为与 反应的阳性参照物。红色箭头表示 阳性染色。(c）两组胃 IM 病例数比较（费雪精确检验）。(d）两组胃切片中 p-STAT3 和 KLF5 的 IHC 染色结果。右侧方框描述了左侧图像的放大区域。比例条代表 （左）和 （右）。(e-f）两组间 p-STAT3 和 KLF5 的 IHC 染色评分。
vs. vs. . In summary, DCA resulted in the systemic accumulation of TBA and enhanced STAT3 phosphorylation and IM marker expression in the gastric tissues of INS-GAS mice. Next, we explored DCA-mediated alterations in the gastric microenvironment in INS-GAS mice, including changes in gastric BA metabolism and microbial structure.
vs. vs. 。总之，DCA导致TBA的系统性积累，并增强了INS-GAS小鼠胃组织中STAT3磷酸化和IM标记物的表达。接下来，我们探讨了 DCA 介导的 INS-GAS 小鼠胃微环境的改变，包括胃 BA 代谢和微生物结构的改变。

To identify whether BA metabolites other than DCA promote IM occurrence, we performed a BAtargeted metabolomics approach on gastric content samples from DCA-supplemented mice and control mice, with both groups possessing a transgenic INS-GAS background. The gastric BA concentrations in the two groups are shown in Figure 10(a,b), and those with significant differences between the two groups are listed in Table S1. The concentrations of twelve BAs (UCA, ursocholic acid; GCA, sodium glycocholate hydrate; 12-ketoLCA, 12ketolithocholic acid; GDCA, glycodeoxycholic acid; -MCA, -muricholic acid; CA, cholic acid; T- -MCA, tauro- -muricholic acid; GUDCA, glycoursodeoxycholic acid; NorCA, norcholic acid; GHDCA, glycohyodeoxycholic acid; DCA, deoxycholic acid; and TCA, taurocholic acid) were greater in gastric content samples from the DCA group compared with the controls. A partial least squares-discriminate analysis (PLS-DA) plot showed distinct clustering patterns between the DCA-supplemented mice and control mice (Figure 10C). Additionally, correlation analyses revealed that the concentrations of all BA metabolites were positively correlated except for GCDCA and TDCA (Figure 10D).
为了确定除DCA以外的BA代谢物是否会促进IM的发生，我们对补充DCA的小鼠和对照组小鼠的胃内容物样本进行了BA靶向代谢组学研究，两组小鼠均具有转基因INS-GAS背景。图 10（a,b）显示了两组小鼠胃中 BA 的浓度，表 S1 列出了两组之间存在显著差异的 BA。12 种 BA 的浓度（UCA，乌苏胆酸；GCA，甘氨胆酸钠水合物；12-ketoLCA，12-ketolitho 胆酸；GDCA，甘去氧胆酸； -MCA， -甲基胆酸；CA，胆酸；T- -MCA，tauro- -甲基胆酸；与对照组相比，DCA组胃内容物样本中的GUDCA（糖脱氧胆酸）、NorCA（诺胆酸）、GHDCA（糖羟基脱氧胆酸）、DCA（脱氧胆酸）和TCA（牛磺酸胆酸）含量更高。偏最小二乘判别分析（PLS-DA）图显示，补充 DCA 的小鼠与对照组小鼠之间存在明显的聚类模式（图 10C）。此外，相关性分析表明，除 GCDCA 和 TDCA 外，所有 BA 代谢物的浓度均呈正相关（图 10D）。

Deep sequencing of the 16 S rRNA genes in the gastric contents was used to determine the microbial profiles in the stomachs of INS-GAS mice treated with DCA and NS. The top three bacterial genera in terms of relative abundance were Lactobacillus, unidentified_Chloroplast, and Alloprevotella in both groups (Figure 11A). Microbial a-diversity analysis revealed that the microbial richness and evenness in the DCAtreated group was significantly lower than that in the NS control group (Figure 11(B,C), Simpson index and Shannon index, both with ). NMDS and ANOSIM analyses were used to compare the -diversity and showed a significant difference in microbial community structure between the two groups (Figure 11D, Stress ; Figure 11E, ). We further screened potential DCA-induced cancer-specific microbial candidates in INS-GAS mice. At the genus level, significantly increased abundances of Gemmobacter and Lactobacillus and significantly decreased abundances of Alloprevotella and Anaerovorax were observed in the DCA group compared to the control group (Figure 11 (F,G). Interestingly, at the species level, we found that the relative abundance of Lactobacillus johnsonii was significantly higher in the DCA group than in the control group (Figure 11 G and H). GC-associated genus Lactobacillus was enriched in the stomachs of H. Pylori-infected INSGAS mice, while the beneficial short-chain fatty acids-producing bacteria including Alloprevotella were more abundant in H. Pylori-infected mice with subsequent probiotics supplementation, which was consistent with our findings. However, gastric wash samples of patients with GC and superficial gastritis were detected by shotgun metagenomic sequencing, and it was found that the genus Alloprevotella that usually colonizes the oral cavity was highly abundant in GC. Therefore, unlike genus Lactobacillus, the pathophysiological roles of genus Alloprevotella in the mouse and human stomachs are inconsistent. In summary, DCA mediated decreased bacterial diversity and increased microbial dysbiosis in INS-GAS mice, especially enriching the Lactobacillus genus.
对胃内容物中的 16 S rRNA 基因进行深度测序，以确定接受 DCA 和 NS 治疗的 INS-GAS 小鼠胃中的微生物概况。就相对丰度而言，两组中排名前三位的细菌属分别是乳酸杆菌、未鉴定的叶绿体和异尖孢霉（Alloprevotella）（图 11A）。微生物a-多样性分析表明，DCA处理组的微生物丰富度和均匀度明显低于NS对照组（图11（B，C），Simpson指数和Shannon指数，均为 ）。用NMDS和ANOSIM分析比较了 多样性，结果显示两组微生物群落结构有明显差异（图11D，压力 ；图11E， ）。我们进一步筛选了INS-GAS小鼠中潜在的DCA诱导癌症特异性微生物候选群。在属的水平上，与对照组相比，DCA组中鬼臼菌和乳酸杆菌的丰度明显增加，而Alloprevotella和Anaerovorax的丰度则明显下降（图11（F,G））。有趣的是，在物种水平上，我们发现 DCA 组约翰逊乳杆菌的相对丰度明显高于对照组（图 11 G 和 H）。幽门螺杆菌感染的INSGAS小鼠胃中富含与GC相关的乳酸杆菌属，而幽门螺杆菌感染的小鼠在随后补充益生菌后，胃中包括Alloprevotella在内的产生短链脂肪酸的有益菌更为丰富， 这与我们的研究结果一致。 然而，通过霰弹枪元基因组测序对胃癌和浅表性胃炎患者的洗胃样本进行检测，发现通常定植于口腔的异型普氏菌属在胃癌中含量很高。 因此，与乳酸杆菌属不同，Alloprevotella属在小鼠和人类胃中的病理生理作用并不一致。总之，DCA介导了INS-GAS小鼠细菌多样性的减少和微生物菌群失调的增加，尤其是乳酸杆菌属的富集。

Previous studies have depicted bile acid-microbiota crosstalk in the gastrointestinal tract. On the one hand, BAs regulate the microbial structure, while on the other hand, gut microbiota modulate the size and composition of the BA pool, which consequently contributes to digestive diseases and even cancer. In this study, we explored the interactions between BA profiles and gastric microbes during
以往的研究描述了胃肠道中胆汁酸与微生物群之间的相互关系。一方面，胆汁酸调节微生物结构，另一方面，肠道微生物群调节胆汁酸池的大小和组成，从而导致消化系统疾病甚至癌症。 在这项研究中，我们探讨了BA与胃肠微生物在胃酸分泌过程中的相互作用。

Figure 10. Alterations in gastric BA metabolites in INS-GAS mice treated with DCA detected by a targeted metabolomics approach. (a) Heatmap of the relative concentrations of BAs in the two groups determined by agglomerate hierarchical clustering analyses. (b) Z score plot based on BA concentrations in the stomachs of the two groups. (c) PLS-DA score plot of gastric BA metabolites. PLS-DA model validation parameters: Pre 2, R2X (cum) 0.622, R2Y (cum) 0.834 and Q2 (cum) 0.674. (d) Correlation heatmap of gastric BA metabolites based on Pearson correlation analyses. , .
图 10.通过靶向代谢组学方法检测INS-GAS小鼠接受DCA治疗后胃中BA代谢物的变化。(a）通过聚类分层聚类分析确定的两组中 BAs 相对浓度的热图。(b) 基于两组胃中 BA 浓度的 Z 评分图。(c) 胃中 BA 代谢物的 PLS-DA 评分图。PLS-DA 模型验证参数：Pre 2，R2X（cum）0.622，R2Y（cum）0.834 和 Q2（cum）0.674。(d) 基于皮尔逊相关分析的胃 BA 代谢物相关热图。 ， 。

the process of IM and carcinogenesis in the gastric mucosa caused by DCA in INS-GAS mice. The results of multiomics correlation analyses of the BA profiles and gastric microbes at the genus level are presented in Figure 12A. Since the abundance of the Lactobacillus genus and the aforementioned twelve BA species levels were upregulated in the DCA group compared with the controls, we further performed correlation analyses of the Lactobacillus genus and the levels of these BA species and revealed that the Lactobacillus genus was significantly positively correlated with GCA, CA, T- MCA, TCA and -MCA in the stomachs of INSGAS mice administrated DCA (Figure 12B-F). Among them, CA and T- -MCA had the highest correlation with DCA ( and 0.7385). Furthermore, as shown in Figure 10A, CA and T- MCA were clustered closely with each other. More specifically, eleven of the twelve BA species had significant positive correlations with Lactobacillus
图 12A 显示了多组学分析的结果。图 12A 展示了 BA 图谱与胃微生物属水平的多组学相关性分析结果。由于与对照组相比，DCA 组乳酸杆菌属的丰度和上述 12 种 BA 的水平都有所升高、我们进一步对乳酸杆菌属和这些BA物种的水平进行了相关性分析，结果发现乳酸杆菌属与INSGAS小鼠胃中的GCA、CA、T- MCA、TCA和 -MCA呈显著正相关（图12B-F）。其中，CA 和 T- -MCA 与 DCA 的相关性最高（ 和 0.7385）。此外，如图 10A 所示，CA 和 T- MCA 的聚类关系也很密切。更具体地说，在 12 个 BA 物种中，有 11 个与乳酸杆菌有显著的正相关关系。

h

Figure 11. 16S rRNA gene sequencing results of the gastric contents of INS-GAS mice treated with DCA. (A) Top ten gastric bacterial genera with the highest relative abundance in both groups. ( and ) Simpson index and Shannon index comparison of the a-diversity of gastric microbes between the two groups. ( and ) -diversity comparison between the two groups based on NMDS and ANOSIM analyses. ( and ) Significantly different taxa enriched in the gastric microbiota of the two groups. (H) Comparison of the relative abundance of Lactobacillus johnsonii in the two groups.
图 11.用 DCA 处理 INS-GAS 小鼠胃内容物的 16S rRNA 基因测序结果。(A) 两组相对丰度最高的前十个胃细菌属。( 和 ) 两组胃微生物 a-多样性的辛普森指数和香农指数比较。( 和 ) 基于 NMDS 和 ANOSIM 分析的两组 多样性比较。( 和 ) 两组胃微生物群中富集的类群显著不同。(H）两组约翰逊乳杆菌相对丰度的比较。
johnsonii enrichment (Fig. S3A-K). These data indicate that the Lactobacillus genus might be a potential IM microbial marker that interacts with gastric BA metabolites and is involved in BAinduced gastric IM occurrence and development. Besides Lactobacillus genus, Alloprevotella genus was only significantly positively correlated with
图 S3A-K）。这些数据表明，乳酸杆菌属可能是一种潜在的 IM 微生物标记物，它与胃 BA 代谢产物相互作用，并参与 BA 诱导的胃 IM 的发生和发展。除乳酸杆菌属外，Alloprevotella 菌属也只与胃中的 BA 代谢产物呈显著正相关。
GLCA (Figure 12G). There was no significant correlation between Gemmobacter and Anaerovorax genera and BAs in the stomach. The mechanisms underlying gastric IM caused by DCA are shown in Figure 13. In conclusion, DCA induces gastric IM by activating the TGR5-STAT3-KLF5 axis and disturbing gastric BA metabolism and microbiota.
GLCA（图 12G）。胃中的鬼门氏菌属和厌氧菌属与 BA 之间没有明显的相关性。图 13 显示了 DCA 引起胃 IM 的机制。总之，DCA 通过激活 TGR5-STAT3-KLF5 轴和扰乱胃 BA 代谢及微生物群诱导胃 IM。

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Figure 12. Multiomics analyses showed that Lactobacillus genus enrichment was positively correlated with increased levels of GCA, CA, T-a-MCA, TCA and -MCA in DCA-treated INS-GAS mice. (a) Heatmap of the multiomics correlation analyses of BA profiles and gastric microbes at the genus level. Pearson correlation analyses of the Lactobacillus genus and the levels of GCA (b), CA (c), T-a-MCA (d), TCA (e) and -MCA (f) in the stomachs of INS-GAS mice. (g) Pearson correlation analysis of the Alloprevotella genus and the level of GLCA in the stomachs of INS-GAS mice. , .
图 12.多组学分析表明，在 DCA 处理的 INS-GAS 小鼠中，乳酸杆菌属富集与 GCA、CA、T-a-MCA、TCA 和 -MCA 水平的增加呈正相关。(a) BA 特征与胃微生物属水平的多组学相关性分析热图。INS-GAS小鼠胃中乳酸杆菌属与GCA（b）、CA（c）、T-a-MCA（d）、TCA（e）和 -MCA（f）水平的皮尔逊相关性分析。(g）INS-GAS 小鼠胃中 Alloprevotella 属与 GLCA 水平的皮尔逊相关性分析。 、 。

Chronic DGR, wherein BAs abnormally reflux from the duodenum into the stomach, is another main risk factor in addition to Hp infection for the development of gastric IM and its progression to intestinal-type gastric adenocarcinoma. In this study, we found that exposure to DCA, as the predominant secondary BA in the stomach, activates a novel signaling axis comprising TGR5-STAT3-KLF5 in the gastric epithelium. We also revealed that in addition to altered molecular signaling, altered gastric conditions are also essential for IM development. The long-term increase of the DCA concentration dramatically influenced the metabolism of BAs and dominant bacteria in the stomach. Therefore, bile reflux leads to IM and gastric carcinogenesis in a complex and comprehensive manner, at least evolving abnormal molecular pathways, bile acid metabolism, and microbial structures.
慢性十二指肠溃疡（DGR）是指十二指肠中的胆碱酯酶异常反流到胃中，它是除Hp感染之外导致胃IM及其发展为肠型胃腺癌的另一个主要危险因素。 在这项研究中，我们发现，作为胃中最主要的次级BA，暴露于DCA会激活胃上皮细胞中由TGR5-STAT3-KLF5组成的新型信号轴。我们还发现，除了分子信号转导的改变，胃部条件的改变也对 IM 的发生至关重要。DCA浓度的长期增加极大地影响了胃中BA和优势菌的代谢。因此，胆汁反流导致IM和胃癌发生的方式是复杂而全面的，至少会进化出异常的分子通路、胆汁酸代谢和微生物结构。

Since dysplastic and subsequent intestinal-type cancerous lesions arise within regions of preexisting IM, the identification of IM markers indicating the risk for the transition from IM to dysplasia is valuable for clinical diagnoses and prognoses. Spasmolytic polypeptide-expressing metaplasia (SPEM) is the earliest metaplasia form derived from atrophy, which then evolves into complete and incomplete IM under chronic inflammation. Although incomplete IM is a major risk factor that ultimately leads to predisposure to adenocarcinoma compared with complete IM, biopsies with H&E staining can only detect a mixture of SPEM lesions
由于发育不良和随后的肠癌病变都发生在已存在的 IM 区域内，因此鉴定 IM 标记物以显示从 IM 过渡到发育不良的风险对临床诊断和预后很有价值。表达痉化多肽的移行细胞（SPEM）是最早的移行细胞形态，源于萎缩，然后在慢性炎症的作用下演变为完全和不完全IM。 虽然与完全性IM相比，不完全性IM是最终导致腺癌的主要风险因素，但用H&E染色进行活检只能发现SPEM病变的混合物。

Figure 13. Mechanisms underlying gastric intestinal metaplasia caused by secondary bile acids. DCA activates the TGR5-STAT3-KLF5 axis in gastric tissues and disturbs gastric BA metabolism and microbiota in INS-GAS mice.
图 13.次级胆汁酸导致胃肠化生的机制。DCA 激活了 INS-GAS 小鼠胃组织中的 TGR5-STAT3-KLF5 轴，并扰乱了胃 BA 代谢和微生物群。
and complete and incomplete IM. The INS-GAS mouse model demonstrates the full spectrum of progressive metaplastic lineages from SPEM to IM, dysplasia and finally tumorigenesis.
以及完全和不完全IM。INS-GAS小鼠模型展示了从SPEM到IM、发育不良以及最终肿瘤发生的全过程。

The interaction between Hp infection and bile reflux in driving gastric IM is complex and controversial. From one perspective, a clinical study showed that premalignant lesions (atrophic gastritis and IM) were more common in patients with both DGR reflux and Hp infection than in those with only DGR reflux ( vs. ), but the association was not statistically significant. Mechanistically, chronic Hp infection contributes to antroduodenal motility disorder, which indirectly induces the retrograde passage of alkaline duodenal content into the stomach. Additionally, the presence of DGR does not interfere with the presence and severity of H. pylori. Therefore, together with our findings presented herein, the eradication of Hp without the treatment of DGR might not be sufficient to prevent gastric epithelial carcinogenesis.
Hp 感染和胆汁反流在胃 IM 的驱动力方面的相互作用既复杂又有争议。一项临床研究显示，同时患有DGR反流和Hp感染的患者比仅患有DGR反流的患者更容易发生恶性前病变（萎缩性胃炎和IM）（ vs. ），但两者之间的关系并无统计学意义。 从机理上讲，慢性 Hp 感染会导致反十二指肠运动障碍，从而间接诱导碱性十二指肠内容物逆行进入胃部。 此外，DGR的存在并不影响幽门螺杆菌的存在和严重程度。 因此，结合我们在本文中的发现，不治疗 DGR 而根除幽门螺杆菌可能不足以预防胃上皮癌变。

STAT3, as a DNA-binding transcription factor, is critical for mediating normal cellular processes upon tyrosine phosphorylation. The importance of STAT3 signaling in inducing chronic gastritis is now widely accepted. However, the molecular mechanisms underlying how STAT3 signaling drives the transformation of chronic mucosal inflammation to IM have not been clarified. Our study provided evidence that STAT3 was continuously phosphorylated and underwent nuclear accumulation during DCA-induced inflammation and IM and directly bound to the two sites on the promoter of KLF5 to activate its transcription in gastric cells. Furthermore, we also demonstrated that DCA induced the upregulation of other STAT3 target genes, including Mcl-1, Bcl-2, IL-6 and CXCL8; thus, DCA-treated cells showed inflammatory and apoptosis-resistant phenotypes different from those of normal cells. Hence, we present data showing that STAT3 may be a key mediator of carcinogenesis in BA-exposed IM tissues. The secondary BA receptor TGR5 may be the link between BAs and STAT3 activation. Nevertheless, other downstream molecules mediating STAT3-induced IM need to be further verified in future studies.
STAT3 作为一种 DNA 结合型转录因子，在酪氨酸磷酸化后对介导正常的细胞过程至关重要。 STAT3信号在诱导慢性胃炎中的重要性现已被广泛接受。然而，STAT3信号如何驱动慢性粘膜炎症转变为IM的分子机制尚未明确。我们的研究提供的证据表明，STAT3在DCA诱导的炎症和IM过程中持续磷酸化并发生核聚集，直接与KLF5启动子上的两个位点结合，激活其在胃细胞中的转录。此外，我们还证明了 DCA 诱导了其他 STAT3 靶基因的上调，包括 Mcl-1、Bcl-2、IL-6 和 CXCL8；因此，DCA 处理的细胞表现出不同于正常细胞的炎症和抗凋亡表型。因此，我们提供的数据表明，STAT3 可能是暴露于 BA 的 IM 组织发生癌变的关键介质。二级 BA 受体 TGR5 可能是 BA 与 STAT3 激活之间的纽带。然而，其他介导 STAT3 诱导 IM 的下游分子还需要在今后的研究中进一步验证。

We found that Gemmobacter and Lactobacillus were DCA-induced IM-associated genera in the stomachs of INS-GAS mice. In addition to Hp , microbial factors may contribute to the progression of gastric carcinogenesis. Several studies have revealed that Lactobacillus widely colonizes the human gastric mucosa. It can metabolize lactose into lactic acid, thus acidifying the gastric mucous layer and subsequently inhibiting gastrin secretion by antral G cells and gastric acid secretion. Therefore, a higher relative abundance of the Lactobacillus genus in the gastric microbiota might accelerate atrophy, IM and tumorigenesis in the gastric mucosae in INS-GAS mice, which have a natural loss of parietal cells and hypochlorhydria. Moreover, the prevalence of Lactobacillus in patient stomachs was found to increase gradually from gastritis to gastric metaplasia and cancer by 16 S rRNA gene sequencing analysis. In healthy stomachs, Lactobacillus is a commensal bacterial genus with a relative abundance of up to Noteworthily, a case-control study reported that Lactobacillus was the dominant genus in GC patients without Hp infection, and its relative abundance was These results are in accordance with our study, which showed that DCA induced gastric IM and dysplasia along with Lactobacillus genus enrichment in INS-GAS mice. However, the mechanism by which DGR leads to increased Lactobacillus abundance in the stomach is still poorly understood. Additionally, our study is the first to depict the increased abundance of the Gemmobacter genus in the tumorigenic stomachs of INS-GAS mice exposed to DCA for a prolonged period.
我们发现，在 INS-GAS 小鼠的胃中，鬼臼菌和乳酸杆菌是 DCA 诱导的 IM 相关菌属。除 Hp 外，微生物因素也可能导致胃癌的发生。多项研究表明，乳酸杆菌广泛定植于人类胃粘膜。 它能将乳糖代谢成乳酸，从而酸化胃黏膜层 ，进而抑制前胃G细胞分泌胃泌素和胃酸分泌。 因此，胃微生物群中乳酸杆菌属的相对丰度较高，可能会加速INS-GAS小鼠胃黏膜的萎缩、IM和肿瘤发生，而INS-GAS小鼠的顶叶细胞天然缺失，且胃酸分泌过少。此外，通过16 S rRNA基因测序分析发现，从胃炎到胃移行症和胃癌，患者胃中乳酸杆菌的数量逐渐增加。 在健康的胃中，乳酸杆菌是一种共生菌属，其相对丰度高达 值得注意的是，一项病例对照研究报道，乳酸杆菌是未感染Hp的胃癌患者的优势菌属、这些结果与我们的研究相符，我们的研究表明，DCA诱导INS-GAS小鼠胃IM和发育不良，同时乳酸杆菌属富集。然而，DGR 导致胃中乳酸杆菌丰度增加的机制仍不甚明了。此外，我们的研究首次描述了长期暴露于DCA的INS-GAS小鼠肿瘤性胃中鬼臼菌属丰度的增加。

To the best of our knowledge, this is the first study that used several gastric organoid lines derived from C57BL/6, FVB/N wild-type and FVB/N INS-GAS mice with DCA treatment, and the results suggest that secondary BAs may be tightly linked to the etiology of IM. Moreover, we first revealed that DCA altered the BA metabolism profile and disrupted microbiome homeostasis in the stomachs of INS-GAS mice, which is a mouse model with complete metaplastic lineages for dysplasia and adenocarcinoma. Notably, these findings remain murinespecific, and the administration of only DCA simplified the BA mixture that regurgitates into the stomach during DGR. In animal studies,
据我们所知，这是第一项使用来自C57BL/6、FVB/N野生型和FVB/N INS-GAS小鼠的多个胃器质系进行DCA处理的研究，结果表明继发性BA可能与IM的病因密切相关。此外，我们首次发现DCA改变了INS-GAS小鼠胃中BA的代谢谱并破坏了微生物组的平衡，而INS-GAS小鼠是一种具有发育不良和腺癌完整移行谱系的小鼠模型。值得注意的是，这些发现仍然是针对小鼠的，而且只施用 DCA 简化了 DGR 期间反流到胃中的 BA 混合物。在动物研究中、