The gut microbiota participates in the effect of linaclotide in patients with irritable bowel syndrome with constipation (IBS-C): a multicenter, prospective, pre-post study
肠道菌群参与利那洛肽对便秘型肠易激综合征（IBS-C）患者疗效的影响：一项多中心、前瞻性、前后对照研究

Subjects  受试者

This multicenter, pre-post clinical trial, which spanned a treatment period of six weeks, was conducted at six Chinese hospitals between January 2020 and June 2021 (Additional file 7: Table S1). The inclusion criteria for IBS-C patients were the following: ① age > 18 years, ② diagnosis of IBS-C (Rome IV diagnostic criteria), ③ Bristol type 1 or 2 > 25% [22], and ④ < 3 bowel movements per week. The exclusion criteria were as follows: ① pregnant or lactating women; ② patients with mental illness; ③ patients who consumed probiotics or antibiotics one month prior to the study; ④ patients who had undergone intestinal cleansing in the past two weeks; ⑤ patients allergic to the drugs used in the study; ⑥ patients with a history of digestive system tumors or gastrointestinal surgery, intestinal obstruction or gastrointestinal bleeding; and ⑦ patients with severe heart or lung diseases.
这项为期六周的、多中心的、前瞻性临床试验于 2020 年 1 月至 2021 年 6 月在中国六家医院进行（附加文件 7 : 表 S1）。IBS-C 患者的纳入标准如下：①年龄 > 18 岁，②符合 IBS-C 诊断标准（罗马 IV 诊断标准），③布里斯托类型 1 或 2 > 25% [ 22 ]，④每周排便次数 < 3 次。排除标准如下：①孕妇或哺乳期妇女；②精神疾病患者；③研究前一个月内服用过益生菌或抗生素的患者；④过去两周内进行过肠道清洁的患者；⑤对研究中使用的药物过敏的患者；⑥有消化系统肿瘤或胃肠道手术、肠梗阻或胃肠道出血病史的患者；⑦患有严重心脏或肺部疾病的患者。

The exclusion criteria for the healthy volunteers were as follows: ① age < 18 years; ② use of antibiotics, probiotics, bowel cleansing products or proton-pump inhibitors within 1 month prior to the study; and ③ diagnosis of diseases such as IBS, inflammatory bowel disease, coeliac disease, or digestive system tumors or history of gastrointestinal surgery, intestinal obstruction or gastrointestinal bleeding, cardiac, renal or hepatic diseases, metabolic diseases, lactose intolerance, or active infection with pathogenic microorganisms.
健康志愿者的排除标准如下：①年龄<18 岁；②研究前 1 个月内使用抗生素、益生菌、肠道清洁产品或质子泵抑制剂；③患有 IBS、炎症性肠病、乳糜泻或消化系统肿瘤，或既往有胃肠道手术、肠梗阻或胃肠道出血病史，或患有心脏、肾脏或肝脏疾病、代谢性疾病、乳糖不耐受，或存在病原微生物的急性感染。

Treatment and follow-up  治疗和随访

All patients received the same dosage of linaclotide (290 μg orally once a day, half an hour before breakfast) for 6 weeks. The medication was supplied by AstraZeneca. All patients were followed up twice a week for 10 weeks post enrollment. The patients were not allowed to take any other constipation-related drugs, including probiotics and laxatives, during the treatment period. For patients with other diseases, treatment could be administered according to the established guidelines, with the requirement of keeping objective records.
所有患者均接受相同剂量的利那洛肽治疗（290 μg，每日早餐前半小时口服），持续 6 周。该药物由阿斯利康公司提供。所有患者在入组后均接受 10 周的每周两次随访。治疗期间，患者不得服用任何其他与便秘相关的药物，包括益生菌和泻药。对于患有其他疾病的患者，可根据既定指南进行治疗，并要求保持客观记录。

Collection of fecal samples
粪便样本收集

Fecal samples were collected before and after 6 weeks of linaclotide treatment. All samples were collected using a special stool collection tube in the hospital, and samples that did not come into any obvious contact with the external environment were collected with a stool shovel. Three stool samples (each consisting of at least 1 g) were collected from each patient, placed in a special refrigerator for specimen collection, stored at – 20 ℃, and rapidly transferred to − 80 ℃ by laboratory personnel within 4 h [23].
粪便样本在利那洛肽治疗 6 周前后进行收集。所有样本均使用医院特制的粪便收集管进行收集，未与外界环境发生明显接触的样本则使用粪便铲收集。每位患者收集三份粪便样本（每份至少 1 克），置于特制样本收集冰箱中，储存于–20℃，并由实验室人员在 4 小时内迅速转移至–80℃[ 23 ]。

Indicator collection  指标收集

The changes in abdominal pain (numeric rating scale (NRS), gastrointestinal symptom rating scale (GSRS), symptom severity score of irritable bowel syndrome (IBS-SSS), and quality of life scale of irritable bowel syndrome (IBS-QoLS)) were evaluated biweekly for 10 weeks, and the Bristol stool form scale (BSFS) and spontaneous bowel movements (SBMs) were assessed daily during treatment based on dietary conditions [24]. Drug-related side effects were also monitored. Fecal samples were collected before and 6 weeks after linaclotide administration. All these indicators were collected by trained personnel responsible for data collection at each center, and the organizing unit inspected and reassessed the data weekly. After treatment, the patients were categorized into relief and no relief groups. The patients in the relief group were further divided into responders and nonresponders based on the FDA response criteria [25].
腹部疼痛的变化（数字评分量表（NRS）、胃肠道症状评分量表（GSRS）、肠易激综合征症状严重程度评分（IBS-SSS）和肠易激综合征生活质量量表（IBS-QoLS））在 10 周内每两周评估一次，并在治疗期间根据饮食情况每日评估布里斯托大便形态量表（BSFS）和自发性排便（SBMs）[ 24 ]。同时监测药物相关副作用。粪便样本在给予利那洛肽前和 6 周后收集。所有这些指标均由各中心的负责数据收集的培训人员收集，组织单位每周检查并重新评估数据。治疗后，患者被分为缓解组和无缓解组。根据 FDA 反应标准[ 25 ]，缓解组中的患者进一步分为应答者和非应答者。

The FDA response endpoint criteria for IBS-C were as follows: ① ≥ 30% reduction from baseline in the weekly mean of the daily scores for abdominal pain; ② ≥ 1 increase in the CSBM per week from baseline; and ③ improvement in abdominal pain and CSBM in the same week during at least 50% of the treatment period.
FDA 对 IBS-C 的响应终点标准如下：①腹痛每日评分的每周平均值较基线减少≥30%；②每周 CSBM 较基线增加≥1 次；③治疗期间至少 50%的周数腹痛和 CSBM 均有改善。

The relief criterion for IBS-C was the alleviation of abdominal pain or constipation during at least 50% of the treatment period.
IBS-C 的缓解标准是在治疗期间至少 50%的时间内缓解腹部疼痛或便秘。

16S rRNA sequencing  16S rRNA 测序

DNA extraction, PCR amplification, fluorescence quantification, MiSeq library construction and MiSeq sequencing were subsequently performed. PE reads obtained by MiSeq sequencing were first spliced according to overlap, and the sequence quality was then controlled and filtered. Operational taxonomic unit (OTU) clustering analysis and species taxonomy analysis were performed after sample segmentation. Multiple diversity indices could be analyzed based on the OTU clustering analysis results and the detected sequencing depth. Using taxonomic information, the community structure was statistically analyzed at each taxonomic level. Based on the abovementioned analysis, a series of in-depth statistical and visual analyses, including multivariate analysis and significance tests, were conducted to analyze the community composition and phylogenetic information of diverse species.
DNA 提取、PCR 扩增、荧光定量、MiSeq 文库构建和 MiSeq 测序随后进行。通过 MiSeq 测序获得的 PE 读段首先根据重叠进行拼接，然后进行序列质量控制和过滤。样本分割后进行操作分类单元（OTU）聚类分析和物种分类分析。基于 OTU 聚类分析结果和检测测序深度，可以分析多种多样性指数。利用分类信息，在各个分类水平上对群落结构进行统计分析。基于上述分析，进行一系列深入统计和可视化分析，包括多元分析和显著性检验，以分析不同物种的群落组成和系统发育信息。

Metabolomics testing  代谢组学检测

Appropriate amounts of pure standards of short-chain fatty acids, including acetic acid, propionic acid, butyric acid, isobutyric acid, valeric acid, isovaleric acid and hexanoic acid, were used. Ten standard concentration gradients (0.02, 0.1, 0.5, 2, 10, 25, 50, 100, 250, and 500 μg/ml) were formulated with ether. An appropriate amount of sample was placed into a 2-ml centrifuge tube, and 50 μl of 15% phosphoric acid, 100 μl of 125 μg/ml internal standard (isohexic acid) solution, and 400 μl of ether were then added. After homogenization for 1 min, centrifugation was performed at 12,000 RPM at 4 ℃ for 10 min, and the supernatant was collected for testing. Appropriate chromatographic and mass spectrometry conditions were used. The precision, repeatability, and recovery were within a reasonable range.
使用了适量的短链脂肪酸纯标准品，包括乙酸、丙酸、丁酸、异丁酸、戊酸、异戊酸和己酸。使用乙醚配制了十个标准浓度梯度（0.02、0.1、0.5、2、10、25、50、100、250 和 500 μg/ml）。将适量的样品放入 2-ml 离心管中，然后加入 50 μl 15%磷酸、100 μl 125 μg/ml 内标（异己酸）溶液和 400 μl 乙醚。混合均匀 1 分钟后，在 4℃下以 12,000 RPM 离心 10 分钟，收集上清液进行检测。使用了适当的色谱和质谱条件。精密度、重复性和回收率均在合理范围内。

Statistical methods  统计方法

The appropriate statistical analysis for comparisons between groups was selected based on the data distribution and patient characteristics, and the analyses were performed using GraphPad Prism 8.0 and IBM SPSS Statistics 26 software. The alpha diversity was determined by sampling-based OTU analysis, and the beta diversity was visualized and tested by principal coordinate analysis (PCoA) plots and analysis of similarities (ANOSIM). Linear discriminant analysis (LDA) was employed to analyze the differences in the bacterial community predominance between groups. Correlation analyses were performed using Spearman’s correlation. The data are presented as the means ± SDs, medians (P25-P75 values), medians (mix-max values), and n (%) values, and the significance was marked as follows: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and NS for no significance.
根据数据分布和患者特征选择了适当的统计分析方法，并使用 GraphPad Prism 8.0 和 IBM SPSS Statistics 26 软件进行数据分析。Alpha 多样性通过基于样本的 OTU 分析确定，Beta 多样性通过主坐标分析（PCoA）图和相似性分析（ANOSIM）进行可视化和检验。采用线性判别分析（LDA）分析组间细菌群落优势的差异。相关性分析采用 Spearman 相关性分析。数据以均值±标准差、中位数（P25-P75 值）、中位数（混合-最大值）和 n（%）表示，显著性标记如下：*p<0.05，**p<0.01，***p<0.001，****p<0.0001，无显著性用 NS 表示。

Patient demographics  患者人口统计学特征

A total of sixty-two patients diagnosed with IBS-C were recruited for the study between January 2020 and June 2021. Two patients (3.2%) discontinued participation due to severe diarrhea (Fig. 1). Among the recruited IBS-C patients, 86.7% were female, and the average age, IBS-SSS score, and body mass index (BMI) of the patients were 45.2 ± 10.97 years, 225.17 ± 92.296, and 22.62 ± 2.76, respectively (Table 1). No significant differences in age, BMI, education, underlying diseases, or drug-related side effects were observed between the patients who experienced relief and those who did not (P > 0.05). Prior to linaclotide therapy, no significant differences in the IBS-SSS scores were found between the two groups (Table 1). Concurrently, a cohort of thirty healthy volunteers was enrolled, and no notable differences in baseline characteristics were observed between the group of volunteers and the IBS-C patient group (Additional file 8: Table S2). To mitigate the potential confounding effects of dietary factors on the gut microbiota, we assessed the nutrient intake of in IBS-C patients using a food frequency questionnaire (FFQ) [26], and no significant differences were found in the total calorie, protein, fat, fiber, or carbohydrate intake from baseline to week 10 (Additional file 9: Table S3).
研究于 2020 年 1 月至 2021 年 6 月期间招募了 62 名诊断为 IBS-C 的患者。其中两名患者（3.2%）因严重腹泻而退出研究（图 1 ）。在招募的 IBS-C 患者中，86.7%为女性，患者的平均年龄、IBS-SSS 评分和体重指数（BMI）分别为 45.2 ± 10.97 岁、225.17 ± 92.296 和 22.62 ± 2.76（表 1 ）。在缓解组和未缓解组之间，年龄、BMI、教育程度、基础疾病或药物相关副作用无显著差异（P > 0.05）。在利那洛肽治疗之前，两组的 IBS-SSS 评分无显著差异（表 1 ）。同时，还招募了 30 名健康志愿者，志愿者组与 IBS-C 患者组在基线特征方面无显著差异（附加文件 8 ：表 S2）。 为减轻饮食因素对肠道菌群可能产生的混杂效应，我们使用食物频率问卷（FFQ）[ 26 ]评估了 IBS-C 患者的营养摄入情况，从基线到第 10 周，总热量、蛋白质、脂肪、纤维或碳水化合物摄入量均未发现显著差异（附加文件 9 ：表 S3）。

Clinical efficacy of linaclotide
利那洛肽的临床疗效

To evaluate the impact of linaclotide, we collected relevant clinical data (see the supplementary data and patient information table for detailed information). The IBS-SSS, GSRS, IBS-QoLS, and abdominal pain scores gradually decreased over six weeks compared with the baseline scores (Fig. 2A-D). The BSFS scores and number of SBMs progressively increased during the six weeks of treatment (Fig. 2E, F. After the six-week medication period, we conducted a four-week follow-up. During this period, the IBS-SSS, GSRS, IBS-QoLS, abdominal pain, BSFS, and SBM scores of the patients remained stable. By applying the FDA response endpoint criteria, we determined that 43.3% (26/60) of patients achieved the FDA response endpoint. Eighty-five percent (51/60) of the patients reported some relief. Among the patients who did not meet the FDA response criteria, 73.5% (25/34) reported at least some alleviation of abdominal pain and constipation with clinical relief, whereas 26.5% (9/34) reported no relief over six-week treatment course. These findings highlight the interindividual variation in the therapeutic efficacy of linaclotide.
为评估利那洛肽的影响，我们收集了相关的临床数据（详见补充数据和患者信息表以获取详细信息）。与基线评分相比，IBS-SSS、GSRS、IBS-QoLS 和腹痛评分在六周内逐渐下降（图 2 A-D）。BSFS 评分和 SBM 数量在六周的治疗期间逐渐增加（图 2 E, F）。在六周的用药期结束后，我们进行了四周的随访。在此期间，患者的 IBS-SSS、GSRS、IBS-QoLS、腹痛、BSFS 和 SBM 评分保持稳定。通过应用 FDA 响应终点标准，我们确定 43.3%（26/60）的患者达到了 FDA 响应终点。85%（51/60）的患者报告了某种程度的缓解。在未达到 FDA 响应标准的患者中，73.5%（25/34）报告了腹痛和便秘至少有所减轻，并出现临床缓解，而 26.5%（9/34）报告在六周的治疗过程中未得到缓解。这些发现突出了利那洛肽治疗效力的个体差异。

Linaclotide altered the intestinal flora of IBS-C patients
利那洛肽改变了 IBS-C 患者的肠道菌群

To investigate whether the effect of linaclotide treatment was associated with changes in the gut microbiota, we collected initial (0-week) and final (6-week) fecal samples from 60 patients and performed high-throughput gene sequencing analysis of 16S rRNA to analyze the gut microbial composition. We assessed the alpha diversity of the gut microbiota using various methodologies through a generalized linear model, and consistent results across different indices (ACE and Chao1) revealed that the 6-week group exhibited significantly higher alpha diversity than the 0-week group (p < 0.0001; Fig. 3A, Additional file 10: Table S4). To further elucidate the differences in the gut microbial composition, we evaluated the beta diversity through principal coordinate analysis (PCoA) using the Jaccard distance algorithm. Clear clustering separation of operational taxonomic units (OTUs) indicated distinct community structures between the 0-week and 6-week groups, indicating significant differences in the structures (Fig. 3B).
为探究利那洛肽治疗的效果是否与肠道菌群的变化相关，我们从 60 名患者中收集了初始（0 周）和最终（6 周）的粪便样本，并进行了 16S rRNA 高通量基因测序分析，以分析肠道微生物组成。我们通过广义线性模型，采用多种方法评估了肠道菌群的α多样性，不同指数（ACE 和 Chao1）的一致结果表明，6 周组的α多样性显著高于 0 周组（p < 0.0001；图 3 A，附加文件 10 ：表 S4）。为进一步阐明肠道微生物组成的差异，我们使用 Jaccard 距离算法进行主坐标分析（PCoA）评估了β多样性。操作分类单元（OTU）的清晰聚类分离表明 0 周组和 6 周组之间存在不同的群落结构，表明结构存在显著差异（图 3 B）。

Subsequently, we conducted a comprehensive examination of the gut microbiota landscape in all available samples to further explore the potential composition differences between the 0-week and 6-week groups. At the phylum level, Firmicutes constituted the most abundant phylum, accounting for 63.81% and 77.93% of the gut microbial community in the 0-week and 6-week groups, respectively (p < 0.0001) (Fig. 3C, Additional file 11: Table S5). Notably, at the genus level, distinct differences in the biological composition were observed between the two groups. We analyzed bacterial genera with a relative abundance exceeding 1%. Among them, the genera Blautia (18.57% vs. 7.77%, respectively; p < 0.001) (Fig. 3D, Additional file 12: Table S6) and Fusicatenibacter (1.23% vs. 1.69%, respectively; p < 0.001) (Additional file 12: Table S6) exhibited relatively higher abundances in the 6-week group. We also compared the taxonomic compositions at the class/order/family level between the two groups (Additional file 1: Figure S1B-D, Additional file 13: Table S7, Additional file 14: Table S8, Additional file 15: Table S9) and detected significant differences in the abundances of Lachnospiraceae, Clostridiales and Clostridia, to which Blautia belong, between before and after treatment.
随后，我们对所有可用样本的肠道菌群景观进行了全面分析，以进一步探索 0 周组和 6 周组之间潜在的组成差异。在门水平上，厚壁菌门是含量最丰富的门，分别占 0 周组和 6 周组肠道微生物群落的 63.81%和 77.93%（p < 0.0001）（图 3 C，附加文件 11 ：表 S5）。值得注意的是，在属水平上，两组在生物学组成上存在明显差异。我们分析了相对丰度超过 1%的细菌属。其中，布劳蒂亚属（18.57% vs. 7.77%，分别；p < 0.001）（图 3 D，附加文件 12 ：表 S6）和纤维弧菌属（1.23% vs. 1.69%，分别；p < 0.001）（附加文件 12 ：表 S6）在 6 周组中相对丰度较高。 我们还比较了两组在科/目/科水平上的分类组成（附加文件 1 ：图 S1B-D，附加文件 1 3 ：表 S7，附加文件 1 4 ：表 S8，附加文件 1 5 ：表 S9），并检测到在治疗后，与普拉梭菌属（Blautia）所属的毛螺菌科、梭菌目和梭菌纲的丰度存在显著差异。

To confirm the specific bacterial taxa affected by linaclotide treatment, linear discriminant analysis of effect size (LEfSe) was used for high-dimensional class comparisons, revealing significant differences in the predominance of the bacterial communities between the two groups (Fig. 3E, Additional file 2: Figure S2A). According to the results, the genus Blautia (from the phylum Firmicutes and the family Lachnospiraceae) emerged as a key bacterial type contributing to gut microbiota dysbiosis in the 6-week group. Additionally, a heatmap comparing the gut microbiota between the two groups based on the OTU abundance was generated at the genus level, further demonstrating the relatively higher abundance of the genus Blautia in the 6-week group, which aligned with the findings from the LEfSe analysis (Fig. 3F).
为确认利那洛肽治疗影响的具体细菌类群，采用高维分类比较的线性判别分析效应大小（LEfSe）方法，揭示了两组间细菌群落组成存在显著差异（图 3 E，附加文件 2 ：图 S2A）。根据结果，厚壁菌门毛螺菌科中的属 Blautia 成为导致 6 周组肠道菌群失调的关键细菌类型。此外，基于 OTU 丰度在属水平上比较两组肠道菌群的差异热图显示，6 周组中 Blautia 属的相对丰度较高，这与 LEfSe 分析结果一致（图 3 F）。

Moreover, we analyzed the differences in the gut microflora between healthy volunteers and IBS-C patients. The LEfSe results indicated that Firmicutes at the phylum level and Blautia at the genus level were predominant in the healthy volunteers (Fig. 4A). In contrast, IBS-C patients exhibited a lower abundance of Blautia than did healthy volunteers, whereas the abundance of Blautia after linaclotide treatment was higher than that of the healthy volunteers (Fig. 4B). In addition, in the continuous observation (4 weeks after linaclotide withdrawal), we also found that Blautia remained higher than pretherapy, but lower than the period of treatment (Additional file 3: Figure S3). These findings further support the notion that linaclotide may exert its effects by modulating the intestinal flora.
此外，我们分析了健康志愿者和 IBS-C 患者肠道菌群的差异。LEfSe 结果指出，在门水平上厚壁菌门（Firmicutes）和在属水平上布劳蒂亚菌属（Blautia）在健康志愿者中占主导地位（图 4 A）。相比之下，IBS-C 患者的布劳蒂亚菌属丰度低于健康志愿者，而接受利那洛肽治疗后，布劳蒂亚菌属的丰度高于健康志愿者（图 4 B）。此外，在持续观察（利那洛肽停药后 4 周）期间，我们发现布劳蒂亚菌属的丰度仍高于治疗前水平，但低于治疗期间（附加文件 3 ：图 S3）。这些发现进一步支持利那洛肽可能通过调节肠道菌群发挥其作用的观点。

The abundance of Blautia was related to the efficacy of linaclotide
布劳蒂亚的丰度与利那洛肽的疗效相关

A relationship between the abundance of Blautia and the effectiveness of linaclotide treatment was observed in this study. To control for confounding factors such as sex, age, and BMI, a ratio of 1:4 was used to match 9 patients who did not experience relief (NR) and 36 out of 51 patients who reported relief [27]. Stool specimens collected at 0 and 6 weeks were also analyzed (Additional file 4: Figure S4A-E), which demonstrated that the overall structure and alpha diversity of the gut microbiota did not differ between the relief and no relief patients (Additional file 5: Figure S5A, B). Interestingly, more pronounced symptom relief was observed if the Blautia abundance after treatment was markedly higher than that before treatment (Fig. 4C, D. We also investigated the predictive ability of the intestinal flora by receiver operating characteristic (ROC) analysis and found that the baseline abundance at the genus level could be used to predict the efficacy of the IBS-C treatment with an area under the curve (AUC) of 0.819, and Blautia (AUC of 0.708) was among the top 21 genera (Fig. 4E). These findings suggested that some specific gut microbes, such as Blautia, exhibited a significant (p < 0.01) difference in their impact on the effect of linaclotide between patients who experienced relief and those who did not.
本研究观察到普拉梭菌丰度与利那洛肽治疗效果之间存在关联。为控制性别、年龄和 BMI 等混杂因素，采用 1:4 的比例匹配了 9 例未缓解（NR）患者和 51 例中报告缓解的 36 例患者[ 27 ]。同时分析了 0 周和 6 周收集的粪便标本（附加文件 4 ：图 S4A-E），结果显示缓解组和非缓解组的肠道菌群整体结构和α多样性无差异（附加文件 5 ：图 S5A, B）。有趣的是，若治疗后普拉梭菌丰度显著高于治疗前，则症状缓解更为明显（图 4 C, D）。我们还通过受试者工作特征（ROC）分析研究了肠道菌群预测能力，发现属水平上的基线丰度可用于预测 IBS-C 治疗效果，曲线下面积（AUC）为 0.819，其中普拉梭菌（AUC 为 0.708）位列前 21 个属之中（图 4 E）。 这些发现表明，某些特定的肠道微生物，如布劳蒂亚，在缓解症状的患者和未缓解症状的患者之间，对利那洛肽疗效的影响存在显著差异（p < 0.01）。

The Blautia and SCFA levels were positively correlated with the alleviation of clinical symptoms
布劳蒂亚和短链脂肪酸水平与临床症状的缓解呈正相关

According to relevant studies, Blautia species are SCFA-producing bacteria. A metabolomic analysis of fecal samples revealed that the concentrations of acetic acid (p < 0.01), propionic acid (p < 0.05), butyric acid (p < 0.01) and isobutyric acid (p < 0.05) were significantly increased in the feces of IBS-C patients treated with linaclotide for 6 weeks (Fig. 5A). A comparison between the relief and no relief groups demonstrated significantly higher levels of acetic acid (p < 0.0001), propionic acid (p < 0.0001), and isobutyric acid (p < 0.001) in the relief group after the treatment (Fig. 5B). We then performed a correlation analysis to investigate the relationships among the abundance of Blautia, the content of SCFAs, and clinical symptoms. Interestingly, we found a positive relationship between the abundance of Blautia and the contents of acetic acid, propionic acid, and butyric acid. Increased IBS-QoLS and decreased IBS-SSS and GSRS scores were positively correlated with the Blautia abundance. Additionally, increased IBS-QoLS and decreased IBS-SSS scores were positively correlated with the contents of acetic acid and butyric acid (Fig. 5C). These findings suggest that Blautia may play a crucial role in the efficacy of linaclotide treatment.
根据相关研究，布劳蒂亚属是产生短链脂肪酸的细菌。对粪便样本进行的代谢组学分析显示，接受利那洛肽治疗 6 周的 IBS-C 患者粪便中乙酸（p < 0.01）、丙酸（p < 0.05）、丁酸（p < 0.01）和异丁酸（p < 0.05）的浓度显著升高（图 5 A）。缓解组与未缓解组的比较显示，治疗后缓解组的乙酸（p < 0.0001）、丙酸（p < 0.0001）和异丁酸（p < 0.001）水平显著更高（图 5 B）。我们随后进行了相关性分析，以研究布劳蒂亚丰度、短链脂肪酸含量与临床症状之间的关系。有趣的是，我们发现布劳蒂亚丰度与乙酸、丙酸和丁酸含量之间存在正相关关系。IBS-QoLS 评分升高和 IBS-SSS、GSRS 评分降低与布劳蒂亚丰度呈正相关。此外，IBS-QoLS 评分升高和 IBS-SSS 评分降低与乙酸和丁酸含量呈正相关（图 5 C）。 这些发现表明，布劳蒂亚可能在利那洛肽治疗的疗效中发挥关键作用。

Ethics approval and consent to participate
伦理批准和参与同意

The trial adhered to ethical guidelines and received approval from the Ethics Committee of Xinqiao Hospital, Third Military Medical University (Ethical Review No. 2019-125-01).
该试验遵循伦理指南，并获得了第三军医大学新桥医院伦理委员会的批准（伦理审查编号 2019-125-01）。

Consent for publication  发表同意

Not applicable.  不适用。

Competing interests  竞争利益

All the authors declare that they have no competing interests.
所有作者声明他们没有利益冲突。