Exquisite visualization of mitophagy and monitoring the increase of lysosomal micro-viscosity in mitophagy with an unusual pH-independent lysosomal rotor.

BACKGROUND: Mitophagy plays indispensable roles in maintaining intracellular homeostasis in most eukaryotic cells by selectively eliminating superfluous components or damaged organelles. Thus, the co-operation of mitochondrial probes and lysosomal probes was presented to directly monitor mitophagy in dual colors. Nowadays, most of the lysosomal probes are composed of groups sensitive to pH, such as morpholine, amine and other weak bases. However, the pH in lysosomes would fluctuate in the process of mitophagy, leading to the optical interference. Thus, it is crucial to develop a pH-insensitive probe to overcome this tough problem to achieve exquisite visualization of mitophagy. RESULTS: In this study, we rationally prepared a pH-independent lysosome probe to reduce the optical interference in mitophagy, and thus the process of mitophagy could be directly monitored in dual color through cooperation between IVDI and MTR, depending on Förster resonance energy transfer mechanism. IVDI shows remarkable fluorescence enhancement toward the increase of viscosity, and the fluorescence barely changes when pH varies. Due to the sensitivity to viscosity, the probe can visualize micro-viscosity alterations in lysosomes without washing procedures, and it showed better imaging properties than LTR. Thanks to the inertia of IVDI to pH, IVDI can exquisitely monitor mitophagy with MTR by FRET mechanism despite the changes of lysosomal pH in mitophagy, and the reduced fluorescence intensity ratio of green and red channels can indicate the occurrence of mitophagy. Based on the properties mentioned above, the real-time increase of micro-viscosity in lysosomes during mitophagy was exquisitely monitored through employing IVDI. SIGNIFICANCE AND NOVELTY: Compared with the lysosomal fluorescent probes sensitive to pH, the pH-inert probe could reduce the influence of pH variation during mitophagy to achieve exquisite visualization of mitophagy in real-time. Besides, the probe could monitor the increase of lysosomal micro-viscosity in mitophagy. So, the probe possesses tremendous potential in the visualization of dynamic changes related to lysosomes in various physiological processes.

Fecal Microbiota Transplant via Endoscopic Delivering Through Small Intestine and Colon: No Difference for Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease (CD) is a chronic inflammatory bowel disorder associated with intestinal dysbiosis. This study aimed to determine the efficacy and safety of different methods of fecal microbiota transplantation (FMT), a potential therapy for CD. METHODS: Patients with CD were randomized to receive FMT by gastroscopy or colonoscopy; a second transplantation was performed 1 week later. Patients were assessed by clinical evaluation and serum testing (at weeks 1, 2, 4, 6, and 8) and endoscopy (8 weeks after transplantation). Fecal DNA was extracted and analyzed using the Illuminal sequencing platform. RESULTS: Of the 27 patients included in the study, clinical remission was achieved in 18 (66.7%); no significant difference was seen between the two methods. 76.9% of gastroscopy group patients and 64.3% of colonoscopy group patients experienced mild adverse events during or shortly after treatment. Microbiota diversity analyses showed that, in comparison with the donors, patients had lower operational taxonomic units (OTU; 117 vs. 258, p < 0.05) and Shannon diversity index (2.05 vs. 3.46, p < 0.05). The CD patients showed a significant increase in OTU and Shannon diversity index 2 weeks after FMT. In comparison with the donors, CD patients had lower levels of Bacteroides, Eubacterium, faecalibacterium, and Roseburia, and higher levels of Clostridium, Cronobacter, Fusobacterium, and Streptococcus. CONCLUSIONS: FMT was seen to be safe and effective in this cohort of patients with CD. No significant differences in clinical remission rate and adverse events were seen between the gastroscopy and colonoscopy groups. FMT was seen to increase the species richness in CD patients.

Roseburia intestinalis supernatant ameliorates colitis induced in mice by regulating the immune response.

Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), has a complex etiology that may be associated with dysbiosis of the microbiota. Previously, our study revealed significant loss of Roseburia intestinalis from the gut of untreated patients with CD, and that R. intestinalis exerted anti­inflammatory functions in TNBS­induced colitis; however, the function of R. intestinalis supernatant is unknown. Therefore, LPS­induced macrophages, including RAW264.7 macrophages and bone marrow­derived macrophages were treated with R. intestinalis supernatant. The results indicated that R. intestinalis supernatant suppressed expression of interleukin (IL)­6 and signal transducer and activator of transcription 3 (STAT3) by macrophages. Additionally, these findings were further verified in vivo in DSS­ and TNBS­induced mouse models of colitis. It was observed that R. intestinalis supernatant ameliorated IBD colitis by reducing the number of inflammatory macrophages and Th17 cells in the colon, and by downregulating the expression of IL­6 and STAT3. Finally, the non­protein components of R. intestinalis supernatant were examined using gas chromatography­mass spectrometry analysis and identified the presence of short­chain fatty acids. In conclusion, the results of the present study indicated that R. intestinalis supernatant may regulate immune responses and ameliorate colitis.

Roseburia intestinalis-derived flagellin is a negative regulator of intestinal inflammation.

Our previous study showed that the Roseburia intestinalis (R. intestinalis), one of the dominant intestinal bacterial microbiota, was significantly decreased in Crohn's disease patients and protected colon epithelial cells from inflammatory damage. However, the roles of lncRNAs in R. intestinalis flagellin-mediated anti-inflammation remain unclear. In this study, we investigate global lncRNA expression profiles using microarray analysis of ulcerative colitis samples from DSS/Flagellin-challenged mice and identified a Flagellin-induced upregulated lncRNA (HIF1A-AS2). Flagellin induced HIF1A-AS2 expression in a dose- and time-dependent manner via p38-stat1 activation. Selective pharmacological inhibitors of Stat1 and p38, and genetic knockdown of these genes abolished Flagellin-induced HIF1A-AS2 expression. In addition, luciferase reporter assay showed that Flagellin activated HIF1A-AS2 promotor via increasing stat1 phosphorylation. Silencing of HIF1A-AS2 abolished Flagellin-mediated anti-inflammatory effects, evaluating by upregulation of cytokines expression, including TNF-α, IL-1ß, IL-6 and IL-12, but not TNFß. In addition, knockdown of HIF1A-AS2 significantly increased p65 and Jnk phosphorylation, and sufficiently abolished Flagellin-mediated anti-inflammatory affects in vivo. Our study provides new insights into the mechanisms that lncRNAs regulate flagellin-mediated alleviation of colonic inflammation. It is indicated that HIF1A-AS2 may be a modulator of intestinal inflammation and represent a novel target for future therapeutics.

Roseburia intestinalis inhibits interleukin­17 excretion and promotes regulatory T cells differentiation in colitis.

Roseburia intestinalis (R. intestinalis) is one of the dominant intestinal bacterial microbiota and is decreased in patients with inflammatory bowel disease (IBD). It helps protect colonic mucosa against the development of inflammation and subsequent IBD, however its underlying mechanisms are unclear. The aim of the present study was to evaluate the anti­inflammatory properties of R. intestinalis in vitro and in an animal model of IBD. The effects of R. intestinalis on disease activity index (DAI) scores, intestinal pathology, the expression of interleukin (IL)­17 and the frequency of CD4+CD25+Foxp3+ regulatory T cells (Treg) were evaluated in vivo in a model of 2,4,6­trinitrobenzenesulfonic acid solution (TNBS)­induced colitis. Compared with the control group, TNBS­treated mice had significantly higher secretion of IL­17, higher DAI scores, a lower ratio of Treg, reduced colon lengths and higher histological scores for colon inflammation. The administration of R. intestinalis significantly downregulated the expression of IL­17, increased the ratio of Treg and ameliorated the high DAI scores and the pathological signs of inflammation in the colon compared with mice treated with TNBS alone. Gene expression profiling was also used to detect the expression of IL­17 in human IBD and healthy control specimens. To extend these findings to an in vitro model of inflammation the human colon epithelial cell line NCM460 was stimulated with lipopolysaccharide (LPS) to induce inflammation and co­cultured with R. intestinalis and changes in IL­17 expression were evaluated. R. intestinalis inhibited the LPS­induced secretion of IL­17 by NCM460 cells. In conclusion, these results demonstrate that R. intestinalis inhibits IL­17 secretion and promotes Treg differentiation in colitis, suggesting that R. intestinalis could be of potential use in the treatment of IBD.

Insights into Roseburia intestinalis which alleviates experimental colitis pathology by inducing anti-inflammatory responses.

BACKGROUND AND AIM: The study aims to elucidate the anti-inflammatory effect and mechanism of Roseburia intestinalis (R. intestinalis) in Crohn's disease (CD). METHODS: 16S-rRNA genome sequencing technique is used to detect the characteristics of intestinal microbiota in untreated CD patients and healthy controls. Then the study investigates the effects of R. intestinalis on disease activity index score, intestinal pathology, the differentiation of Treg cells, and the expressions of Thymic stromal lymphopoietin (TSLP), TGF-ß and IL-10 by using TNBS colitis models. At the cellular level, the study uses LPS to stimulate Caco-2 cells to conduct inflammation models and then co-culture with R. intestinalis and detect changes of TSLP and TGF-ß. The study then uses R. intestinalis to stimulate peripheral blood mononuclear cells, and the change of Treg cells was detected. RESULTS: Genome sequencing of fecal samples from untreated CD patients (n = 10) revealed decreases in the abundance and diversity of intestinal microbiota, including R. intestinalis. Moreover, R. intestinalis reduced disease activity index scores, colon shortening, intestinal mucosal epithelial injury, and mucosal lymphocyte infiltration in a colitis mice model. It suppressed intestinal inflammation by increasing Treg cell numbers and expression of the anti-inflammatory cytokines TSLP, TGF-ß, and interleukin-10 (P < 0.05). R. intestinalis also increased secretion of TSLP and TGF-ß in lipopolysaccharide-treated Caco-2 cells. CONCLUSION: These findings suggest that R. intestinalis suppresses CD pathogenesis by inducing anti-inflammatory responses.

Knockdown of long non-coding RNA MAP3K20 antisense RNA 1 inhibits gastric cancer growth through epigenetically regulating miR-375.

Emerging evidence has demonstrated that long noncoding RNAs (lncRNAs) play a critical role in tumorigenesis of gastric cancer. LncRNA MAP3K20 antisense RNA 1 (MLK7-AS1) has been identified as one of gastric cancer-specific lncRNAs. However, its precise role in gastric cancer remains unknown. In this study, we found that lncRNA MLK7-AS1 was significantly increased in gastric cancer tissues compared with in adjacent tissues. Gastric cancer patients with high MLK7-AS1 expression had a shorter survival and poorer prognosis. By loss-function assay, we demonstrated that knockdown of MLK7-AS1 inhibited cell proliferation and induced apoptosis in HGC27and MKN-45 cells. Furthermore, we identified miR-375 as a target of MLK7-AS1. MLK7-AS1 interacted with Dnmt1 and recruited it to miR-375 promotor, hyper-methylating miR-375 promotor and repressing miR-375 expression. Taken together, our findings demonstrate that knockdown of MLK7-AS1 by siRNA inhibits gastric cancer growth by epigenetically regulating miR-375. Thus, MLK7-AS1 may be a useful prognostic marker and therapeutic target for gastric cancer patients.

Relationship between intestinal microbiota and ulcerative colitis: Mechanisms and clinical application of probiotics and fecal microbiota transplantation.

Ulcerative colitis (UC) is an inflammatory disease that mainly affects the colon and rectum. It is believed that genetic factors, host immune system disorders, intestinal microbiota dysbiosis, and environmental factors contribute to the pathogenesis of UC. However, studies on the role of intestinal microbiota in the pathogenesis of UC have been inconclusive. Studies have shown that probiotics improve intestinal mucosa barrier function and immune system function and promote secretion of anti-inflammatory factors, thereby inhibiting the growth of harmful bacteria in the intestine. Fecal microbiota transplantation (FMT) can reduce bowel permeability and thus the severity of disease by increasing the production of short-chain fatty acids, especially butyrate, which help maintain the integrity of the epithelial barrier. FMT can also restore immune dysbiosis by inhibiting Th1 differentiation, activity of T cells, leukocyte adhesion, and production of inflammatory factors. Probiotics and FMT are being increasingly used to treat UC, but their use is controversial because of uncertain efficacy. Here, we briefly review the role of intestinal microbiota in the pathogenesis and treatment of UC.

Update on intestinal microbiota in Crohn's disease 2017: Mechanisms, clinical application, adverse reactions, and outlook.

The pathogenesis of Crohn's disease (CD) is complex, and it is thought to be associated with the environment, immune, hereditary, microbe, and other factors. If the balance between the host and the intestinal microbes in CD patients was broken, immune-inflammatory response of susceptible individuals might be triggered. Probiotics could improve the intestinal microbial flora balance and treat human effectively. There are several new mechanisms that might explain the role of probiotics. Fecal microbiota transplantation (FMT) is becoming more and more attractive in treating a large amount of digestive system diseases that are related to the dysbiosis of intestinal microbiota. FMT has been widely used in recurrent Clostridium difficile infection. More and more attention has been paid on the clinical application of FMT in CD, while the exact mechanism is still a mystery. So in this review, we explore the mechanism, clinical application, and adverse reactions of intestinal microbiota in CD so that we can use the tool to cure more diseases. Enteric microbiota leads to new therapeutic strategies for CD.