MS ｜ Xie Liwei group found that mixed probiotics and their metabolites could alleviate colitis

     at present , Inflammatory bowel disease （Inflammatory Bowel Disease, IBD） Because the incidence rate is high and the cure rate is low , It has become a global public health problem . Although the pathogenesis of the disease is not clear , However, a potential association between gut microbiota and inflammatory signals has been established , It can be the starting point for the research and prevention of the disease . probiotics , Especially Lactobacillus and Bifidobacterium , Because of its safety, effectiveness and strong ability to regulate flora , Recently became IBD Hot spots for treatment . However, most studies focus on the combination of multiple strains and their metabolites IBD Evaluation of the effects has rarely been reported .

    2022 year 6 month 22 Japan , Researcher Xie Liwei （ South China State Key Laboratory of Applied Microbiology 、 Institute of Microbiology, Guangdong Academy of Sciences , Intestinal microecology and health team PI） The team , stay Microbiology Spectrum Periodical （IF：7.17） Published on “Probiotic Consortia and Their Metabolites Ameliorate the Symptoms of Inflammatory Bowel Diseases in a Colitis Mouse Model” The article , This paper reports the efficacy of indigenous probiotic strains isolated independently in the laboratory in inhibiting intestinal inflammation and restoring intestinal microenvironment , among , Compared with the control group and the single strain treatment group , Lactobacillus reuteri , Lactobacillus grignai , The combination of probiotics of Lactobacillus acidophilus and B. lactis or their fermentation metabolites has a unique effect in inhibiting intestinal inflammation, accelerating inflammation and restoring intestinal microenvironment in colitis model . The findings of this study support the combination of the patented strain of mixed probiotics and its metabolites , Adjuvant therapy IBD New methods and new strategies .

    IBD It is a chronic nonspecific inflammatory disease occurring in the gastrointestinal tract , It mainly includes ulcerative colitis and Crohn's disease [1], The clinical manifestation of most patients is abdominal pain , diarrhea , Bloody stool , Perianal lesions , With weight loss , Malnutrition, etc [2,3].IBD Prone to recurrent attacks , It is difficult to cure and has a high risk of developing colorectal cancer （Colorectal cancer, CRC）, Although it is generally considered to be a common intestinal inflammatory disease in western countries , But in recent years , In developing countries , The incidence rate of the disease is accelerating , Seriously increase the disease burden of patients and damage the quality of life [4–6].IBD The exact cause of the disease is still unclear , But there is more evidence that , The disease is genetic , The result of the joint action of various complex factors in the environment , meanwhile , Persistent intestinal infection 、 Abnormal immune regulation of intestinal mucosa and imbalance of intestinal microbiota are important to IBD The occurrence and development of . among , Intestinal microbiota has been reported to be one of the key factors in disease progression , Intestinal microflora composition of individuals with colitis （ Type and quantity ） Different from healthy individuals , Will activate the host immune response , This will increase intestinal permeability and destroy the barrier structure of intestinal mucosa , And then destroy the symbiotic microorganisms and activate the serious immune response , Cause the disease to recur [7–9].

     as everyone knows , Probiotics are living microorganisms that colonize the human gut to have beneficial effects on the gut , Two main genera of probiotics , Bifidobacterium and Lactobacillus have also been reported to mediate the formation of short chain fatty acids IBD and CRC The improvement of symptoms has potential benefits [10]. besides , Lactobacillus and Bifidobacterium can also increase the adhesion of healthy probiotics to intestinal mucosa 、 Inhibition of pathogen adhesion to mucosal surface 、 Competitive elimination of pathogenic microorganisms 、 Produce antibacterial substances and regulate immune function to rebuild intestinal symbiosis and protect intestinal mucosa [11]. The patented strains of probiotics used in this study include Lactobacillus reuteri , Lactobacillus grignai , Lactobacillus acidophilus and Bifidobacterium lactis , They belong to Lactobacillus and Bifidobacterium respectively , The formula of the bacteria mixed preparation meets the national food addition standard . Although it has been proved that these four bacteria have positive effects in the host . However , The bacterium and its metabolites are found in dextran sodium sulfate （Dextran Sulfate Sodium, DSS） The combined effect in inducing colitis has not been clearly evaluated , The underlying mechanisms also need to be further clarified .

     This study adopts 14 Weeks old mice were established DSS Induced colitis model , The experimental period is 14 God . According to the experimental conditions, the mice were divided into 7 Group ：CONTROL, control group; MIX, probiotic consortia; LR1, Lactobacillus reuteri PLBK1 group; LR2, Lactobacillus reuteri PLBK2 group; LG, Lactobacillus gasseri PLBK3 group; LA, Lactobacillus acidophilus PLBK4 group; BL, Bifidobacterium lactis PLBK5 group. among ,6 The mice in the experimental group were given the corresponding single strain or probiotic mixture by gavage every day , During the whole probiotic intervention experiment , Record your weight every day 、 Stool hardness changes 、 Blood stool condition and other experimental indicators , in addition , In the 8 Day and day 14 It is necessary to collect feces for 16S rRNA Sequence , After the mice were killed, the colon tissues were collected for subsequent hematoxylin and eosin （Hematoxylin and eosin, H&E） Staining and immunohistochemistry （Immunohistochemistry, IHC） experiment （ chart 1A）. Through analysis, it was found that probiotics could alleviate the pathological symptoms of colitis in mice . Compared with the control group ,MIX Group can improve DSS The resulting shortening of the colon （ chart 1B）, Reduce the fluctuation of weight change （ chart 1C） And significantly reduced the disease activity index （Disease Activity Index, DAI） The score （ chart 1D）, in addition , The effect of single strain group on body weight change and DAI The score reduction effect is not as good as MIX Group significant （ chart 1）.

chart 1 Probiotics can alleviate DSS Induce pathological symptoms of experimental colitis

     Colon sections were H&E Staining to assess colonic mucosal damage , From the colon section of the control group, it was found that the intestinal structure and barrier were damaged . by comparison , The treatment of probiotics has a protective effect on colon , The histological score was also significantly lower . in addition , Although inflammation can still be observed in the single strain group , But most crypt structures are normal , Especially in LA Group , Low colon pathological score （ chart 2A）. Further use of immunohistochemistry , To detect macrophage markers associated with inflammation -F4/80, The processing result is similar to H&E The staining results are consistent , Compared with the control group ,MIX Of the group F4/80 The level decreased significantly （ chart 2B）, It indicates that the infiltration of innate immune cells into the colon is reduced .

chart 2 Probiotic protection DSS Induce the intestinal structure of colitis model and inhibit the infiltration of macrophages

     The fecal samples were tested 16S rRNA Sequencing to explore the regulatory effect of probiotic strains on the intestinal flora of colitis . Results found , In different experimental periods ,7 The composition of intestinal microbiota in group a differs at the species level （ chart 3A-B）, And compared with the control group ,MIX The group is in the 8 Days of Simpson The index is significantly different , There was no significant difference in other single strain groups （ chart 3C）, The results showed that the intervention of probiotics could affect the composition and diversity of the community , Thus further accelerating the recovery of inflammation . Besides , Whether on the eighth day or 14 God ,β Diversity shows , The control group and MIX The intestinal microbiota of the group was clearly divided into two clusters , On the eighth day, the difference of single strain group was not significant （ chart 3E-H）, These results suggest that , Compared with single strain , Probiotics treatment has a regulatory effect on the intestinal microflora during the duration and recovery of intestinal inflammation . And the control group and MIX The group also showed significantly different bacterial composition in the two stages （ chart 3I-J）.

chart 3 Probiotics reconstitute the composition of the intestinal microbiota

     besides , To further identify the microbial markers that play a role during intestinal inflammation , This research mainly focuses on MIX The control group and the control group were divided into two groups 8 Day and day 14 Days to take random forest combination 5 Multiple cross validation algorithm （Random forest analysis and 10 trials of 5-fold cross-validation, RFCV） To further identify characteristic bacteria （ chart 4A, C）. After analysis, it was found that the bacteria with significant difference were 11 plant , stay 8 There is 7 Strains （ chart 4B）, In the 14 There is 4 Strains （ chart 4D）, among , Yes 8 There was no significant difference among strains in the single strain group （ Supplementary drawing ）, These data show that , Probiotics are likely to play a comprehensive role through specific regulation of the intestinal microenvironment . To explore the control group and MIX The interaction between flora and phenotype in the group , The co occurrence network diagram is also analyzed , It turns out that it passed RFCV Screened out 4 Strains of key bacteria can be found in the three network diagrams , And the first 8 Days show more complex interaction network relationships （ chart 4E-G）, Hint this 4 The importance of key bacteria in the regulation of intestinal microecology and disease phenotype by probiotics .

chart 4 Identification of the marked intestinal microbiota in a mouse model of colitis by random forest

     Considering that probiotic preparations, in addition to balancing the intestinal flora , It can also produce a large number of metabolites and be absorbed by intestinal epithelial cells , In order to further explore the effect of the metabolites of the probiotic preparation on colitis , In this study, the fermentation metabolites of unpurified probiotics were applied to the colitis model . Because the effect of probiotic preparation is better than that of single strain , Therefore, only the control and mixed metabolite groups were intervened by metabolites ： Control and mixed metabolites MIX Group . And in the first place 14 Days to collect small intestine （Small intestine, SI） Contents （ chart 5A）. Analysis found , Control group due to DSS Handle , Causes colon shortening （ chart 5B）, The weight changes a lot （ chart 5C）, However, the mixed metabolites decreased significantly DAI score （ chart 5D）, Improved survival （ chart 5E）. meanwhile , In the control group ,H&E Dyeing and IHC The results show that DSS Processing causes crypt loss 、 Mucosal damage and F480 The expression of （ chart 5F-G）, These conditions were significantly relieved after the intervention of mixed metabolites . in addition , By comparing the phenotypic results of probiotic intervention experiments , Probiotics can be found to be more effective than mixed metabolites in reducing weight loss and mucosal damage .

chart 5 Mixed metabolites attenuate colon injury in a mouse model of colitis

     To explore the regulatory effect of metabolite treatment on microbiota , The study also examined faeces collected during mixed metabolite interventions 16S rRNA Sequence . Results found , In the 14 God , There was no significant difference in the diversity of colon stool samples and the key bacteria screened in the probiotic intervention between the two groups （ chart 6A-D）, in addition , The co occurrence network diagram also shows that the key bacteria are mostly isolated points in the network structure （ chart 6E-G）. It is suggested that the mixed metabolites have little ability to regulate the colonic flora , At the same time, combined with the literature research findings , The proximal small intestine is the main site for microbial metabolite absorption [12,13], Therefore, it is assumed that mixed metabolites may affect bacteria in the small intestine more effectively . To test this conjecture , The contents of small intestine were studied 16S rRNA Sequence , Find out MIX Group and control group β There are significant differences in diversity （ chart 6H）. The thermogram analysis of the core flora showed that there were obvious differences between the two groups （ chart 6I）. These results support the hypothesis , That is, the mixed metabolites significantly changed the bacterial composition and structure of the small intestine .

chart 6 stay DSS In a mouse model of induced colitis , Mixed metabolite intervention is inferior to probiotics in the specific regulation of intestinal microorganisms

     Overall speaking , Current research has proved that , Lactobacillus reuteri PLBK1, Lactobacillus reuteri PLBK2, Lactobacillus grignai PLBK3, Lactobacillus acidophilus PLBK4 And B. lactis PLBK5 The intervention of mixed probiotics can reduce the intestinal inflammation of colitis , By regulating the level of intestinal bacteria, the composition and function of dysfunctional microbiota in colitis mice can be improved , It is proved that there is a complex correlation between intestinal flora and colitis phenotype . Provide new insights into the role of probiotics in maintaining intestinal health , And will promote IBD And other inflammatory diseases . Microecological products of five patented strains （ The "three high companion" microecological preparation jointly produced by Baiyun Mountain and Huang pharmaceutical ）, At present, it is being carried out in the digestive department of the First Affiliated Hospital of Xinxiang Medical College IBD and UC Study on direct clinical intervention of patients' microecology （ Ethics number ：[2001] Lenko quick review word (23) Number ）.

    The main authors of this study , The first author xulimin is a graduate student of the school of public health of Xinxiang Medical College ; The co first author liubingdong is a doctoral student jointly trained by Professor Pan Jiyang of the Department of psychiatry of the First Affiliated Hospital of Jinan University and researcher Xie Liwei of the State Key Laboratory of Applied Microbiology in South China ; Huangliujing is a postgraduate student jointly trained by Professor Ma Ying of Department of Obstetrics and Gynecology of Zhujiang Hospital of Southern Medical University and researcher Xie Liwei of State Key Laboratory of Applied Microbiology in South China ; Li Ze is a postgraduate student in the school of public health, Xinxiang Medical College ; The corresponding author of this article is a distinguished professor of School of public health, Xinxiang Medical College 、 Master Tutor , Distinguished professor, Department of Endocrinology and metabolism, Zhujiang Hospital, Southern Medical University 、 Doctoral supervisor , Institute of Microbiology, Guangdong Academy of Sciences 、 South China State Key Laboratory of Applied Microbiology 、 Intestinal microecology and health team PI Researcher Xie Liwei .

Full text link ：DOI:https://doi.org/10.1128/spectrum.00657-22

reference

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[6]      KUIPERS E J, GRADY W M, LIEBERMAN D, et al. Colorectal cancer[J/OL]. Nature Reviews Disease Primers, 2015, 1(1): 15065. DOI:10.1038/nrdp.2015.65.

[7]      SCHIRMER M, GARNER A, VLAMAKIS H, et al. Microbial genes and pathways in inflammatory bowel disease[J/OL]. Nature Reviews Microbiology, 2019, 17(8): 497-511. http://www.nature.com/articles/s41579-019-0213-6. DOI:10.1038/s41579-019-0213-6.

[8]      ODENWALD M A, TURNER J R. The intestinal epithelial barrier: a therapeutic target?[J/OL]. Nature Reviews Gastroenterology & Hepatology, 2017, 14(1): 9-21. DOI:10.1038/nrgastro.2016.169.

[9]      XAVIER R J, PODOLSKY D K. Unravelling the pathogenesis of inflammatory bowel disease[J/OL]. Nature, 2007, 448(7152): 427-434. DOI:10.1038/nature06005.

[10]    PARADA VENEGAS D, DE LA FUENTE M K, LANDSKRON G, et al. Short Chain Fatty Acids (SCFAs)-Mediated Gut Epithelial and Immune Regulation and Its Relevance for Inflammatory Bowel Diseases[J/OL]. Frontiers in Immunology, 2019, 10. DOI:10.3389/fimmu.2019.00277.

[11]     MISHRA J, STUBBS M, KUANG L, et al. Inflammatory Bowel Disease Therapeutics: A Focus on Probiotic Engineering[J/OL]. Mediators of Inflammation, 2022, 2022: 1-15. DOI:10.1155/2022/9621668.

[12]    HERREMA H, NIESS J H. Intestinal microbial metabolites in human metabolism and type 2 diabetes[M/OL]//Diabetologia. Springer Science and Business Media Deutschland GmbH, 2020: 2533-2547. DOI:10.1007/s00125-020-05268-4.

[13]    HE Y, FU L, LI Y, et al. Gut microbial metabolites facilitate anticancer therapy efficacy by modulating cytotoxic CD8+ T cell immunity[J/OL]. Cell Metabolism, 2021, 33(5): 988-1000.e7. DOI:10.1016/j.cmet.2021.03.002.

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