Bacteroides fragilis strain ZY-312 promotes intestinal barrier integrity via upregulating the STAT3 signaling pathway in a radiation-induced intestinal injury mouse model

1.1. FIGURE 1 | B. fragilis alleviated total body radiation-induced intestinal injury in mice. (A) (B) The model of radiation-induced intestinal injury in mice. (C)The weight loss. (D)The statistical analysis of SI and colon length. (E) The histopathology morphology of SI. (F) The statistical analysis of histopathology-associated index (HAI) between groups. (G) Periodic acid-Schiff stain (PAS) staining of SI. Control group (N=6), TBI group (N=12), TBI+ZY-312 group (N=12). N, the number of mice. The data are presented as mean ± SEM, and *p < 0.05, **p < 0.01, **p < 0.001, **p < 0.0001.
1.2. FIGURE 2 | B. fragilis alleviated abdominal radiation-induced intestinal injury in mice through B. fragilis-derived PSA. (A) The weight loss. (B) The statistical analysis of small intestinal(SI) length. (C) The statistical analysis of colon length. (D) The histopathology of small intestinal at different magnifications. (E) The HAI between groups. (F) The length of SI crypts between groups. Control group (N=6), TAI group (N=9), TAI+ZY-312 group (N=10), TAI+PSA group (N=8). The data are presented as mean ± SEM, and *p < 0.05, **p < 0.01, **p < 0.001, **p < 0.0001.
1.3. FIGURE 3 | B. fragilis promoted intestinal epithelial cell proliferation, stem cell regeneration, goblet cell secretion, and tight junction repair. (A)The immunohistochemical staining of Ki-67 in SI between groups. (B)The immunofluorescence staining of Ki-67 and Lgr5. (C) The immunofluorescence and Alcian blue-periodic acid-Schiff staining(AB-PAS) of SI goblet cells. (D) The immunofluorescence staining of Claudin-1 and ZO-1.
1.4. FIGURE 4 | B. fragilis did not significantly alter intestinal microbiota to relieve radiation-induced intestinal injury in mice. (A) (B)The phylum level of microbiota composition and comparison of microbiota in mice between samples and groups. (C) (D) The genus level of microbiota composition and comparison of microbiota in mice between samples and groups. (E) The abundance and diversity of microbiota ( Shannon index) in mice. (F) The principal coordinate analysis of microbiota. (G) The bug base analysis of microbiota. (H) The LEfSe analysis of differential bacteria in SI meeting a significant LDA threshold value of >3.5 between groups with different levels. (I) The cladogram based on LEfSe shows differential bacteria of the gut microbiota between groups with different levels. Nd30, Control group; xd30 and TAI, TAI group; xzd30 and TAIZ, TAI+ZY-312 group.
1.5. FIGURE 5 | B. fragilis up-regulated the STAT3 signaling pathway in intestinal epithelial cells. (A)The western blot analysis of pJAK2, pNF-KB, p38, pmTOR, mTOR, pSTAT3, STAT3, MUC2, Claudin-1, Lgr5, and PCNA in SI tissue between groups. GAPDH was regarded as an internal reference. (B)The immunohistochemical staining of pSTAT3 in SI between groups. (C) The western blot analysis of pSTAT3, STAT3 in SI IECs. (D) The western blot analysis of pSTAT3, STAT3 in Non-SI epithelial cells.
1.6. FIGURE 6 | B. fragilis relieved radiation-induced intestinal injury through the STAT3 signaling pathway in intestinal epithelial cells. (A)The weight loss. (B)The statistical analysis of SI and colon length. (C) The histopathology morphology of SI. (D) The HAI of SI between groups. (E) The length of SI crypts between groups.Control group/Stat3△IEC (N=3), TAI/Stat3△IECgroup (N=3), TAI+ZY-312/Stat3△IEC group (N=4), TAI+ZY-312/WT group (N=5). The data are presented as mean ± SEM, and *p < 0.05, **p < 0.01, **p < 0.001, **p < 0.0001.
1.7. FIGURE 7 | B. fragilis promoted intestinal epithelial cell proliferation, stem cell regeneration, goblet cell secretion, and tight junction repair through the STAT3 signaling pathway. (A) The histological staining of pSTAT3, Ki-67, MUC2, PAS, and ZO-1 in SI IECs. (B) The western blot analysis of pSTAT3, STAT3, MUC2, Claudin-1, Lgr5, and PCNA in SI IECs between groups. GAPDH was regarded as an internal reference.
1.8. FIGURE S1 | WT mice received relieved radiation-induced intestinal injury. (A)The weight loss. (B)The statistical analysis of SI and colon length. (C) The statistical analysis of HAI. (D) The PAS staining of SI. Control group/WT (N=3), TAI/WT group (N=5). The data are presented as mean ± SEM, and *p < 0.05, **p < 0.01, **p < 0.001, **p < 0.0001.

1.1 图1 | 脆弱拟杆菌（Bacteroides fragilis, B. fragilis）可缓解小鼠全身照射（total body radiation, TBI）诱导的肠损伤。(A)(B) 小鼠照射诱导肠损伤模型。(C) 小鼠体重变化。(D) 小肠（small intestine, SI）与结肠长度的统计分析。(E) 小肠组织病理学形态。(F) 各组间组织病理学损伤评分（histopathology-associated index, HAI）的统计分析。(G) 小肠过碘酸-希夫染色（Periodic acid-Schiff stain, PAS）结果。对照组（N=6）、TBI组（N=12）、TBI+ZY-312组（N=12）。N为小鼠数量。数据以平均值±标准误（standard error of the mean, SEM）表示，*p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001。
1.2 图2 | 脆弱拟杆菌（B. fragilis）可通过其分泌的多糖A（polysaccharide A, PSA）缓解小鼠腹部照射（total abdominal irradiation, TAI）诱导的肠损伤。(A) 小鼠体重变化。(B) 小肠（SI）长度统计分析。(C) 结肠长度统计分析。(D) 不同放大倍数下的小肠组织病理学图像。(E) 各组间HAI评分。(F) 各组间小肠隐窝长度统计分析。对照组（N=6）、TAI组（N=9）、TAI+ZY-312组（N=10）、TAI+PSA组（N=8）。数据以平均值±SEM表示，*p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001。
1.3 图3 | 脆弱拟杆菌可促进肠上皮细胞增殖、干细胞再生、杯状细胞分泌及紧密连接修复。(A) 各组小肠组织Ki-67免疫组化染色结果。(B) Ki-67与Lgr5免疫荧光双染结果。(C) 小肠杯状细胞免疫荧光及阿尔辛蓝-过碘酸-希夫双染色（Alcian blue-periodic acid-Schiff, AB-PAS）结果。(D) 紧密连接蛋白1（claudin-1, Claudin-1）与紧密连接蛋白ZO-1（zonula occludens-1, ZO-1）免疫荧光双染结果。
1.4 图4 | 脆弱拟杆菌未显著改变肠道菌群组成，从而缓解小鼠照射诱导的肠损伤。(A)(B) 各组小鼠肠道菌群门水平组成及组间菌群差异比较。(C)(D) 各组小鼠肠道菌群属水平组成及组间菌群差异比较。(E) 各组小鼠肠道菌群丰度与多样性（香农指数，Shannon index）。(F) 肠道菌群主坐标分析（principal coordinate analysis, PCoA）。(G) 肠道菌群bugbase分析。(H) 各组间差异菌的线性判别分析效应大小（linear discriminant analysis effect size, LEfSe）分析，阈值为线性判别分析（linear discriminant analysis, LDA）得分>3.5。(I) 基于LEfSe的进化分支图，展示各组间肠道菌群的差异物种。分组标记：Nd30为对照组；xd30与TAI为TAI组；xzd30与TAIZ为TAI+ZY-312组。
1.5 图5 | 脆弱拟杆菌可上调肠上皮细胞中的信号转导与转录激活因子3（signal transducer and activator of transcription 3, STAT3）信号通路。(A) 各组小肠组织中磷酸化Janus激酶2（phosphorylated Janus kinase 2, pJAK2）、磷酸化核因子κB（phosphorylated nuclear factor kappa B, pNF-κB）、p38丝裂原活化蛋白激酶（p38 mitogen-activated protein kinase, p38）、磷酸化雷帕霉素靶蛋白（phosphorylated mammalian target of rapamycin, pmTOR）、雷帕霉素靶蛋白（mammalian target of rapamycin, mTOR）、pSTAT3、STAT3、黏蛋白2（mucin 2, MUC2）、Claudin-1、Lgr5及增殖细胞核抗原（proliferating cell nuclear antigen, PCNA）的蛋白质印迹（western blot）分析，以甘油醛-3-磷酸脱氢酶（glyceraldehyde-3-phosphate dehydrogenase, GAPDH）作为内参。(B) 各组小肠组织pSTAT3免疫组化染色结果。(C) 小肠肠上皮细胞（intestinal epithelial cells, IECs）中pSTAT3与STAT3的蛋白质印迹分析。(D) 非小肠上皮细胞中pSTAT3与STAT3的蛋白质印迹分析。
1.6 图6 | 脆弱拟杆菌可通过肠上皮细胞中的STAT3信号通路缓解照射诱导的肠损伤。(A) 小鼠体重变化。(B) 小肠与结肠长度的统计分析。(C) 小肠组织病理学形态。(D) 各组间小肠HAI评分。(E) 各组间小肠隐窝长度统计分析。对照组/Stat3△IEC（N=3）、TAI/Stat3△IEC组（N=3）、TAI+ZY-312/Stat3△IEC组（N=4）、TAI+ZY-312/野生型（wild type, WT）组（N=5）。数据以平均值±SEM表示，*p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001。
1.7 图7 | 脆弱拟杆菌可通过STAT3信号通路促进肠上皮细胞增殖、干细胞再生、杯状细胞分泌及紧密连接修复。(A) 小肠肠上皮细胞中pSTAT3、Ki-67、MUC2、PAS及ZO-1的组织化学染色结果。(B) 各组小肠肠上皮细胞中pSTAT3、STAT3、MUC2、Claudin-1、Lgr5及PCNA的蛋白质印迹分析，以GAPDH作为内参。
1.8 补充图S1 | 野生型（wild type, WT）小鼠的照射诱导肠损伤得以缓解。(A) 小鼠体重变化。(B) 小肠与结肠长度的统计分析。(C) HAI评分统计分析。(D) 小肠PAS染色结果。对照组/WT（N=3）、TAI/WT组（N=5）。数据以平均值±SEM表示，*p < 0.05，**p < 0.01，***p < 0.001，****p < 0.0001。