Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery methods

[
  {
    "@context": "https://schema.org",
    "@type": "HowTo",
    "name": "Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery methods",
    "description": "Evidence-backed execution summary for Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery methods from Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery.",
    "totalTime": "PT34080M",
    "step": [
      {
        "@type": "HowToStep",
        "position": 1,
        "name": "Therapeutic efficacy of ECN-pE(C/A) 2 against IBD",
        "text": "We first detected the ROS scavenging effect of ECN-pE(C/A) 2 in a DSS-induced murine IBD model by DCFH-DA (2,7-dichlorodihydrofluorescein diacetate) staining. To verify the ROS eliminating capability of ECN-pE(C/A) 2, colon tissues collected from mice with different treatments were stained with DCFH-DA. As shown in Supplementary Fig., compared to the control group (PBS + water), the level of ROS in the colon tissue of mice treated with DSS (PBS + 3% DSS) was obviously increased. Interestingly, the ROS signal in the colon tissue collected from mice in the ECN-pE(C/A) 2 + 3% DSS group was obviously reduced, demonstrating that ECN-pE(C/A) 2 could indeed effectively eliminate ROS. We next assessed the therapeutic efficacy of ECN-pE(C/A) 2 in DSS-induced IBD mice. Compared with the control group (PBS + water), the mice in the DSS-tre..."
      },
      {
        "@type": "HowToStep",
        "position": 2,
        "name": "Therapeutic efficacy of ECN-pE(C/A) 2 against IBD",
        "text": "Then, we investigated the effect of ECN-pE(C/A) 2 on colonic epithelial cells and the impaired colonic epithelial barrier, which are crucial characteristics of DSS-induced acute IBD. According to the immunofluorescence staining results (Fig. a, b, d, ), DSS treatment markedly reduced the expression of ZO-1 and Occludin in colon tissue, which are the main tight junction-associated proteins and play a crucial role in maintaining the structure and function of the intestinal epithelium and the integrity of the intestinal barrier. Compared to ECN-pE and ECN(C/A) 2 treatment, ECN-pE(C/A) 2 treatment effectively upregulated the expression of ZO-1 and Occludin in colon tissue, which further indicated the key role of antioxidases (CAT and SOD) and chitosan/sodium alginate coating in the restoration of the colonic epithelium. Furthermore, a FITC-dextran perfusion test was also carried o..."
      },
      {
        "@type": "HowToStep",
        "position": 3,
        "name": "Regulatory effect of ECN-pE(C/A) 2 on the gut microbiome",
        "text": "The gut microbiome is one of the most investigated components of the GI tract and has been demonstrated to be an important factor during the development of intestinal inflammation,. To explore the role of the gut microbiome during treatment, we investigated the therapeutic efficacy of ECN-pE(C/A) 2 in DSS-induced acute IBD in mice pretreated with antibiotics. The therapeutic efficacy of ECN-pE(C/A) 2 was obviously reduced with the pretreatment of antibiotics, indicating that the intestinal microbiota may play an important role in regulating acute IBD (Fig. and Supplementary Fig. ). Therefore, we further investigated whether ECN-pE(C/A) 2 could regulate the abundance of intestinal microbiota in a DSS-induced murine IBD model. Then, the 16S rRNA gene sequencing assay was carried out to evaluate the abundance of intestinal microbiota, and the results indicated that oral adm..."
      },
      {
        "@type": "HowToStep",
        "position": 4,
        "name": "Bacterial viability assay",
        "text": "To analyze the viability of ECN-pE with different chitosan/sodium alginate coatings, 190 µL ECN-pE, ECN-pE(C/A), ECN-pE(C/A) 2, and ECN-pE(C/A) 3 were added to the 96-well plate, 10 µL CCK-8 solution was added, and the mixture was cultured at 37 °C for 4 h. The viability of bacteria was assessed by the OD value of the mixture solution at 450 nm."
      },
      {
        "@type": "HowToStep",
        "position": 5,
        "name": "Therapeutic efficacy of ECN-pE(C/A) 2 against IBD with delayed treatment",
        "text": "Finally, to further explore the therapeutic efficacy of ECN-pE(C/A) 2, we investigated the therapeutic effects of ECN-pE(C/A) 2 against IBD with delayed treatment. C57BL/6 mice were also administered drinking water containing 3% DSS for 6 days to induce an acute IBD model and then orally delivered ECN(C/A) 2, ECN-pE(C/A) 2 or ECN-pE on days 5, 6, 7, and 9 (Fig. ). Excitingly, ECN-pE(C/A) 2 treatment effectively protected the mice against DSS-induced acute IBD, including colon length shortening, body weight loss, colon epithelial tissue damage and increased MPO activity (Fig. and Supplementary Fig. ). Moreover, ECN-pE(C/A) 2 treatment also suppressed the levels of pro-inflammatory cytokines, including IL-1β, TNF-α, and IL-6, and increased the levels of anti-inflammatory cytokines, such as IL-10 and TGF-β (Fig. ). All these results verified that..."
      },
      {
        "@type": "HowToStep",
        "position": 6,
        "name": "Methods",
        "text": "ECN was a gift from Jinyao Liu's group (Shanghai Jiao Tong University) and was cultured at 37 °C in LB broth (Sangon Biotech, Shanghai) with vigorous shaking. Then, the ECNs were stored within bacterial cryopreservation fluid (50% LB broth, 25% water, and 25% glycerol) at -80 °C for further experiments. The pET28a-T5-luxCDABE plasmid was prepared by adding the luxCDABE sequence, which was copied from pGEN-luxCDABE (a gift from Harry Mobley; Addgene plasmid # 44918; http://n2t.net/addgene:44918; RRID:Addgene_44918) by PCR, into pET28a-T5 through polylinker. The ECN-lux was prepared by transfecting plasmid pET28a-T5-luxCDABE. Female C57BL/6 mice (6-8 weeks) were purchased from Nanjing Pengsheng Biological Technology Co. Mice were housed in individually ventilated cages with five mice per cage and kept on a regular 12-h:12-h light:dark cycle (8:00..."
      },
      {
        "@type": "HowToStep",
        "position": 7,
        "name": "Activity of superoxide dismutase",
        "text": "To analyze the activity of recombinant SOD, ECN(C/A) 2 or ECN-pE(C/A) 2 (10 7 CFU/mL) with or without 1 mM IPTG incubation was collected by centrifugation, dispersed in 4 mL of bacterial cell lysis buffer (50 mM Tris-HCl, 2 mM EDTA, 100 mM NaCl, 100 µg/mL lysozyme, 0.1% Triton X-100), and then lysed by an ultrasonic cell crusher with a power of 400 W. Then, the hybrid solution was centrifuged to remove the precipitate. Then, the supernatant was added to the test solution, including 2.95 mL Tris-HCl buffer and 0.05 mL pyrogallol solution (60 mM), and the absorbance at 325 nm was recorded every 30 s for 5 min ( A sample ). For the blank group, the lysate was replaced with deionized water, and the absorbance at 325 nm was recorded in the same way ( A 0 ). The ·O 2 - inhibition rate of reco..."
      },
      {
        "@type": "HowToStep",
        "position": 8,
        "name": "Resistance analysis of ECN-pE in vitro and in vivo",
        "text": "ECN-pE with or without chitosan/sodium alginate coating was resuspended in PBS (Control), 4% bile salt solution or SGF at 37 °C with gentle shaking for 2 h. The starting number of different bacteria (ECN-pE, ECN-pE(C), ECN-pE(C/A) and ECN-pE(C/A) 2 ) in PBS, 4% bile salt solution or SGF was fixed at 1 × 10 6 CFU. Then, the bacteria in each sample were collected at different time points by centrifugation, washed and diluted with PBS, and 100 µL of each diluted sample was spread on solid agar plates containing ampicillin. The number of bacteria was determined by the number of colonies after 48 h of incubation in microbiological incubators. The morphology of bacteria after different treatments was observed by TEM. After different treatments, the bacterial samples were washed with PBS and deposited on formvar/carbon-supported copper grids..."
      }
    ],
    "tool": [
      {
        "@type": "HowToTool",
        "name": "Protection effect of probiotics with chitosan/sodium alginate coating against the GI tract"
      },
      {
        "@type": "HowToTool",
        "name": "Regulatory effect of ECN-pE(C/A) 2 on the gut microbiome"
      },
      {
        "@type": "HowToTool",
        "name": "Preparation and characterization of ECN-pE(L100)"
      },
      {
        "@type": "HowToTool",
        "name": "Resistance analysis of ECN-pE in vitro and in vivo"
      },
      {
        "@type": "HowToTool",
        "name": "In vivo immunofluorescence staining analysis"
      },
      {
        "@type": "HowToTool",
        "name": "Gut microbiota 16S sequencing assay"
      },
      {
        "@type": "HowToTool",
        "name": "Gut microbiota 16S sequencing assay"
      },
      {
        "@type": "HowToTool",
        "name": "Statistical analysis"
      }
    ],
    "supply": [
      {
        "@type": "HowToSupply",
        "name": "Protection effect of probiotics with chitosan/sodium alginate coating against the GI tract"
      },
      {
        "@type": "HowToSupply",
        "name": "Therapeutic efficacy of ECN-pE(C/A) 2 against IBD"
      },
      {
        "@type": "HowToSupply",
        "name": "Therapeutic efficacy of ECN-pE(C/A) 2 against IBD"
      },
      {
        "@type": "HowToSupply",
        "name": "Biosafety evaluation of ECN-pE(C/A) 2"
      },
      {
        "@type": "HowToSupply",
        "name": "Regulatory effect of ECN-pE(C/A) 2 on the gut microbiome"
      },
      {
        "@type": "HowToSupply",
        "name": "Bacterial viability assay"
      },
      {
        "@type": "HowToSupply",
        "name": "Methods"
      },
      {
        "@type": "HowToSupply",
        "name": "Preparation and characterization of ECN-pE(C/A) 2"
      }
    ],
    "isBasedOn": {
      "@type": "ScholarlyArticle",
      "headline": "Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery",
      "datePublished": "2022",
      "author": [
        {
          "@type": "Person",
          "name": "Jun Zhou"
        },
        {
          "@type": "Person",
          "name": "Maoyi Li"
        },
        {
          "@type": "Person",
          "name": "Qiufang Chen"
        },
        {
          "@type": "Person",
          "name": "Xinjie Li"
        },
        {
          "@type": "Person",
          "name": "Linfu Chen"
        },
        {
          "@type": "Person",
          "name": "Ziliang Dong"
        },
        {
          "@type": "Person",
          "name": "Wenjun Zhu"
        },
        {
          "@type": "Person",
          "name": "Yang Yang"
        },
        {
          "@type": "Person",
          "name": "Zhuang Liu"
        },
        {
          "@type": "Person",
          "name": "Qian Chen"
        }
      ],
      "identifier": "10.1038/s41467-022-31171-0"
    }
  },
  {
    "@context": "https://schema.org",
    "@type": "BreadcrumbList",
    "itemListElement": [
      {
        "@type": "ListItem",
        "position": 1,
        "name": "Experiments",
        "item": "https://replicatescience.com/experiments"
      },
      {
        "@type": "ListItem",
        "position": 2,
        "name": "Programmable probiotics modulate inflammation and gut microbiota for inflammatory bowel disease treatment after effective oral delivery methods",
        "item": "https://replicatescience.com/experiments/programmable-probiotics-modulate-inflammation-and-gut-microbiota-for-inflammatory-bowel-disease-treatment-after-effective-oral-delivery-methods-jun-zhou-pmc9198027/programmable-probiotics-modulate-inflammation-and-gut-microbiota-for-inflammatory-bowel-disease-trea-mlph7qos"
      }
    ]
  }
]