A biomarker capable of reflecting oxidative stress levels in the gut and uses thereof

Abstract

The application discloses a biomarker capable of reflecting oxidative stress level in intestines and use thereof, belongs to the technical field of biotechnology, and relates to a peroX operon in a Bacteroides genome from an endogenous intestinal flora, wherein the peroX operon has four genes, namely, BT_3735, BT_3736, BT_3737 and sRNA0246, the sequences of the four genes are arranged adjacently and the transcription directions of the four genes are consistent. The application takes the expression level of the peroX operon in feces as an early-stage diagnosis index, a disease severity evaluation index or a treatment effect monitoring index of inflammatory bowel disease, and can realize continuous dynamic evaluation of oxidative stress state.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a dynamic biomarker based on the Bacteroides peroX operon and its application in monitoring the redox state of enteritis. Background Technology

[0002] Many intestinal diseases, such as IBD, irritable bowel syndrome (IBS), dysbiosis, and colon cancer, are closely related to oxidative stress. Elevated levels of reactive oxygen species (ROS) in the chronic inflammatory microenvironment are a hallmark of these diseases. Physiologically, ROS participate in cell signaling and immune defense mechanisms; however, excessive ROS can lead to biomolecular damage, increase intestinal mucosal permeability, and accelerate the inflammatory process, thus creating a vicious cycle of inflammation and oxidative stress. Simultaneously, during inflammatory attacks or in the early stages of disease, intestinal oxygen levels significantly increase. Excessive ROS production and persistently elevated oxygen concentrations not only exacerbate existing inflammatory responses but may also activate oncogenes and downregulate tumor suppressor gene expression, thereby promoting tumor formation and progression.

[0003] Therefore, the state of oxidative stress in the gut can serve as a key health indicator, especially for patients with intestinal diseases (particularly asymptomatic patients). Persistent oxidative stress during the quiescent phase of disease may lead to further deterioration. Tracking and monitoring the oxidative status of the gut in these inactive patients helps to identify risk factors associated with disease exacerbation early, providing important evidence for clinical decision-making.

[0004] However, despite the widespread recognition of the importance of intestinal oxidation levels, effective monitoring methods for assessing this indicator are still lacking. Existing detection strategies primarily diagnose the presence of intestinal oxidative stress by measuring the activity or concentration of stress-related substances in the patient's blood, thereby inferring the occurrence of disease. These methods share a common drawback: the low and dynamic levels of these components in the blood often lead to inaccurate results reflecting the oxidative state of the local intestinal environment. In recent years, analyzing oxidative stress levels in fecal samples has provided a non-invasive method for assessing intestinal inflammation, particularly suitable for patients with inactive IBD. However, our understanding of the mechanisms of fecal oxidative stress remains limited. Identifying suitable biomarkers in fecal samples and developing non-invasive monitoring technologies with high specificity and stability to continuously and dynamically track changes in the oxidative state of the intestinal environment will provide important support for the diagnosis and treatment of IBD and colorectal cancer. Summary of the Invention

[0005] Based on the above technical problems, the objective of this invention is:

[0006] 1. To provide a biomarker derived from endogenous gut microbiota that can reflect the level of oxidative stress in the gut.

[0007] 2. To provide a method for non-invasive, quantitative assessment of intestinal oxidative stress levels using this biomarker.

[0008] 3. Provide the application of this method in the diagnosis, disease monitoring, or efficacy evaluation of inflammatory bowel disease.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] A biomarker reflecting the level of oxidative stress in the gut is proposed. This biomarker is the peroX operon derived from the genome of Bacteroides, an endogenous gut microbiota. The peroX operon contains four genes: BT_3735, BT_3736, BT_3737, and sRNA0246. The sequences of BT_3735, BT_3736, BT_3737, and sRNA0246 are as follows:

[0011] The forward primer sequence for BT_3737 is shown in SEQ ID NO.1;

[0012] The reverse primer sequence for BT_3737 is shown in SEQ ID NO.2;

[0013] The forward primer sequence for sRNA0246 is shown in SEQ ID NO.3;

[0014] The reverse primer sequence for sRNA0246 is shown in SEQ ID NO.4;

[0015] The forward primer sequence for BT_3736 is shown in SEQ ID NO.5;

[0016] The reverse primer sequence for BT_3736 is shown in SEQ ID NO.6;

[0017] The forward primer sequence for BT_3735 is shown in SEQ ID NO.7;

[0018] The forward primer sequence of BT_3735 is shown in SEQ ID NO.8.

[0019] Furthermore, the BT_3735, BT_3736, and BT_3737 sequences of this invention are arranged adjacently and have the same transcription direction.

[0020] This invention provides a method for screening the peroX operon, which obtains the peroX operon through transcriptomic analysis.

[0021] Furthermore, the peroX operator selection method includes the following steps:

[0022] 1.1 Strains culture and sample collection

[0023] Bacteroides multiforme VPI-5482 strain was scraped with a sterile inoculation loop and incubated upside down at 37°C for 48 hours.

[0024] A single colony with typical morphology was inoculated into 3 mL of BHI liquid medium and anaerobic cultured at 37°C for 16-18 h to obtain a seed culture. The seed culture was then anaerobically cultured at 37°C until the OD600 reached 0.2-0.3.

[0025] The culture was transferred to a sterile centrifuge tube, inverted twice in the air, and cultured with shaking at 37°C and 200 rpm for 120 min. The cells were then collected by centrifugation at 8,000 rpm for 5 min.

[0026] The collected bacterial pellet was washed once with PBS buffer pre-cooled at 4°C, and immediately flash-frozen in liquid nitrogen and stored at -80°C.

[0027] 1.2 RNA extraction and transcriptome sequencing

[0028] The collected bacterial cells were deribosomal RNA removed, and then the mRNA was randomly fragmented to approximately 200 bp.

[0029] Using fragmented mRNA as a template, cDNA was synthesized using random primers and the SuperScript double-stranded cDNA synthesis kit. When synthesizing the second strand of cDNA, dUTP was used instead of dTTP for strand-specific labeling.

[0030] End repair of double-stranded cDNA was performed, including 5' phosphorylation and 3' "A" tail addition, followed by ligation of Y-shaped sequencing adapters, and selective digestion of the second strand of cDNA using uracil DNA glycosylation enzyme.

[0031] 1.3 Differentially expressed gene analysis

[0032] Gene expression was quantified using RSEM software with TPM as the quantitative indicator, and differential expression analysis was performed using DESeq2 software package. The screening criteria were |log2FC|≥1 and p-adjust<0.05.

[0033] The four genes with the most significant differential expression were BT_3735, BT_3736, BT_3737 and sRNA0246. Genomic mapping showed that BT_3735, BT_3736 and BT_3737 were arranged adjacently and had the same transcription direction, and were named peroX.

[0034] This invention provides the application of the aforementioned biomarkers in the non-invasive, quantitative assessment of intestinal oxidative stress levels.

[0035] Furthermore, the biomarkers described herein are used in the diagnosis, disease monitoring, or efficacy evaluation of inflammatory bowel disease.

[0036] This invention provides a drug for the diagnosis, disease monitoring, or efficacy evaluation of inflammatory bowel disease, the drug comprising the biomarkers described in claim 1 or 2.

[0037] The present invention has the following beneficial effects:

[0038] The peroX operon is highly conserved in Bacteroides bacteria but absent in other genera or host cells. This specificity opens up the possibility of using it as a biomarker. Given the uniqueness of this operon, peroX could become a tool for identifying specific gut microbes.

[0039] The expression of the peroX gene can serve as an early warning signal reflecting the host's gut health, as its expression level fluctuates with changes in inflammatory response and oxidative stress. Validation using an inflamed gut mouse model revealed a temporal correlation between peroX gene expression and the deterioration of oxidative state during inflammation, further confirming its sensitivity in bacterial responses to changes in the gut microenvironment.

[0040] The low levels and significant dynamic changes of relevant components in blood often lead to inaccurate results reflecting the oxidative state of the local intestinal environment. Compared to using indirect markers in blood for detection, this method has the advantages of high specificity and stability. It is also simple to operate, can utilize mature molecular biology techniques (RT-qPCR or digital PCR), and does not require the introduction of exogenous engineered bacteria. It directly utilizes endogenous flora markers and has the advantages of being non-invasive, simple, dynamic, and accurately reflecting the state of the microenvironment. Attached Figure Description

[0041] Figure 1 This is a schematic diagram of the "OxyR-sRNA0246-peroX" cascade regulatory network of the present invention. Wherein:

[0042] (a) Volcano plot analysis showing differential gene expression in B. theta before and after oxidative exposure. Locations marked a, b, c, and d correspond to the four genes most significantly upregulated in the detection, i.e., the four genes within the peroX operon. (b) Heatmap showing the top thirty genes with the most significant expression differences before and after oxidative exposure. The right side -1 to 2 (corresponding to a color change from green to red) represent transcriptional levels from low to high; the two clusters represent the anaerobic group and the oxidative exposure group, respectively. (c) Schematic diagram of the peroX polycistronic operon structure.

[0043] Figure 2 peroX can be used as a biomarker to detect fluctuations in oxidative levels in the inflamed gut of mice. Specifically:

[0044] (a) Phylogenetic analysis of the peroX operon showed that the gene is conserved in Bacteroides. (b) After applying different concentrations of hydrogen peroxide to B. theta under anaerobic conditions to induce oxidative stress, the expression level of the peroX gene significantly increased, indicating its sensitivity to stress response. (c) Changes in mouse body weight and colon length during the construction of the DSS-induced colitis model. (d) H&E staining sections of colon tissue from healthy and experimental mice. (e) Quantitative analysis of peroX operon expression levels in total fecal RNA from healthy and colitis mice: After random hexamer reverse transcription, the cDNA from total fecal RNA was distributed into millions of chip droplets for digital PCR amplification, with primers corresponding to the BT3737 plus sRNA sequence.

[0045] Figure 3 This is a schematic diagram of the assessment of oxidative kinetics in colitis mice based on the peroX gene. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0047] This invention provides a biomarker that can reflect the level of oxidative stress in the gut, wherein the biomarker is the peroX operon from the genome of Bacteroides, which is derived from the endogenous gut microbiota.

[0048] The principle behind this invention is:

[0049] Bacteroides is one of the dominant anaerobic bacteria in the gastrointestinal tract. Its survival is closely related to the intestinal redox environment, playing a crucial role in maintaining intestinal homeostasis. However, it is also sensitive to oxygen, easily generating reactive oxygen species (ROS) and triggering complex defense mechanisms upon oxygen exposure. Existing research mainly focuses on traditional H2O2 scavenging systems in Bacteroides, such as Kat, Ahp, Tpx, and Gpx. The existence of novel oxidative stress response units that can reflect the local redox state of the intestine remains insufficiently understood. This invention discovers a previously unreported, naturally occurring endogenous operon, peroX, in Bacteroides polymorpha that significantly responds to oxygen exposure and hydrogen peroxide stimulation. This operon is composed of genes BT3735, BT3736, sRNA0246, and BT3737, which are adjacent in genomic position and have the same transcriptional direction. These genes all exhibit enhanced expression during cellular oxidative stress and participate in the regulation of bacterial hydrogen peroxide scavenging and oxidative stress tolerance.

[0050] Therefore, in the gut environment, peroX gene expression can serve as an early warning signal reflecting the host's gut health, as its expression level fluctuates with changes in inflammatory response and oxidative stress. By monitoring peroX gene expression levels, the state of oxidative stress in the gut can be tracked more sensitively, thus providing novel biomarkers for the early diagnosis and treatment of inflammatory bowel disease (IBD) and colorectal cancer. Four genes in the peroX operon are highly sensitive to external oxidative stress conditions, and their expression levels are significantly upregulated. Upstream genes within this operon influence the transcription of downstream genes through hierarchical regulatory mechanisms, forming a fine cascade effect from top to bottom. BT3737 and sRNA0246 are regulated by the transcription factor OxyR, but their upregulation sensitivity to oxidative stress is significantly higher than that of OxyR. Primers designed for these genes were used for absolute quantitative PCR (digital PCR) analysis of total RNA in mouse fecal microbiota. The results showed that the expression pattern of the peroX operon is closely related to changes in oxidative stress in the gut, providing empirical support for the potential value of this gene as a biomarker. Based on the results of a DSS-induced colitis model experiment, this study found a time correlation between peroX gene expression and the deterioration of oxidative state during inflammation, further confirming its sensitivity in bacterial responses to changes in the gut microenvironment. Non-invasive biosensor technology developed based on the peroX regulatory network holds promise for real-time monitoring of intestinal oxidation levels. By analyzing specific biomarkers in patient excrement, this technology can achieve continuous dynamic assessment of oxidative stress, opening up new avenues for clinical application.

[0051] In the selection of biomarkers in this invention, the peroX operon from Bacteroides intestinalis is explicitly identified for the first time as a biomarker for assessing the level of intestinal oxidative stress. This operon is almost not expressed in a normal anaerobic intestinal environment, but is significantly upregulated under oxidative stress (upregulation can reach more than 200-fold), exhibiting extremely high sensitivity and specificity.

[0052] In the target and primer design of this invention, specific amplification primers were designed for the BT_3737 gene and / or sRNA0246 in the peroX operon for the quantitative detection of target RNA in fecal samples. BT_3737 encodes a protein containing a YtxH domain and participates in oxidative stress signal transduction; sRNA0246, as a core hub in a cascade regulatory network, exhibits more sensitive expression changes.

[0053] This invention uses the fecal peroX operon expression level as an early diagnostic indicator, a disease severity assessment indicator, or a treatment efficacy monitoring indicator for inflammatory bowel disease, enabling continuous dynamic assessment of oxidative stress.

[0054] Example 1: Screening of the peroX operon using transcriptomics analysis

[0055] 1.1 Strains culture and sample collection

[0056] (1) Culture medium: BHI liquid culture medium (after cooling, add 10% NaHCO3 solution after filtration and sterilization, 2mL per 100mL); BHI solid culture medium (add 1.5g of agar powder per 100mL).

[0057] (2) Culture conditions: Bacteroides thetaiotaomicron VPI-5482 strain was taken out of the -80°C glycerol tube, and the bacterial solution was scraped with a sterile inoculation loop in the anaerobic workstation and streaked on the surface of BHI solid medium. It was then incubated upside down at 37°C for 48 hours.

[0058] (3) Select a single colony with typical morphology and inoculate it into 3 mL of BHI liquid medium. Incubate anaerobically at 37°C for 16-18 h to obtain seed culture. Transfer the seed culture to fresh BHI liquid medium at an inoculation rate of 1% (v / v) and incubate anaerobically at 37°C until the OD600 is 0.2-0.3.

[0059] (4) Divide the culture into two equal parts: one part is directly centrifuged at 8,000 rpm for 5 min to collect the bacterial cells, which serves as the control group (anaerobic group); the other part is transferred to a sterile centrifuge tube, inverted twice in air, and cultured with shaking at 37°C and 200 rpm for 120 min, and then centrifuged at 8,000 rpm for 5 min to collect the bacterial cells, which serves as the experimental group (aerobic exposure group). Three biological replicates are set up for each group.

[0060] (5) The collected bacterial pellet was washed once with PBS buffer pre-cooled at 4°C, and immediately frozen in liquid nitrogen and stored at -80°C.

[0061] 1.2 RNA extraction and transcriptome sequencing

[0062] (1) Total RNA was extracted using TRIzol reagent (Invitrogen, USA), strictly following the manufacturer's experimental protocol. DNase I (TaKaRa, Japan) was used to remove genomic DNA contamination.

[0063] (2) Ribosomal RNA was removed using the Ribo-Zero Magnetic Kit (Epicenter, USA), and then the mRNA was randomly fragmented to approximately 200 bp.

[0064] (3) Using fragmented mRNA as a template, cDNA was synthesized using random primers (Illumina) and the SuperScript double-stranded cDNA synthesis kit (Invitrogen, CA, USA). dUTP was used instead of dTTP for strand-specific labeling during the synthesis of the second strand of cDNA.

[0065] (4) End repair of double-stranded cDNA was performed using End Repair Mix, including 5' phosphorylation and 3' A-tailing, followed by ligation of Y-shaped sequencing adapters. The second strand of cDNA was selectively digested using uracil DNA glycosylation enzyme (UNG).

[0066] (5) The library was enriched by PCR amplification and quantitatively analyzed using the TBS380 (Picogreen) real-time fluorescence method. Sequencing was performed on the Illumina HiSeq X Ten platform using paired-end sequencing mode with a read length of 2×150bp.

[0067] 1.3 Differentially expressed gene analysis

[0068] (1) The raw sequencing data underwent quality control: reads containing adapter sequences were removed, low-quality bases (Phred quality value <20) were removed, reads with more than 5% N bases were removed, and high-quality sequences with a length greater than 50 bp were retained.

[0069] (2) The clean data of each sample were aligned with the reference genome using the BLAST algorithm, and the alignment efficiency was optimized using the Burrows-Wheeler transform (BWT) algorithm.

[0070] (3) Gene expression was quantified using RSEM software with TPM (Transcripts Per Million) as the quantitative indicator. Differential expression analysis was performed using the DESeq2 software package, with the screening criteria being |log2FC|≥1 and p-adjust<0.05.

[0071] (4) The results showed that a total of 1,699 differentially expressed genes were identified, of which 1,120 were upregulated and 579 were downregulated.

[0072] (5) The four genes with the most significant differential expression were BT_3735 (FC=242.06), BT_3736 (FC=197.27), BT_3737 (FC=88.49), and sRNA0246 (FC=209.09) (e.g. Figure 1 (As shown in a). Genomic mapping showed that BT_3735, BT_3736, and BT_3737 were arranged adjacently and had the same transcription direction, suggesting that they constituted a single operon, named peroX ( Figure 1 c).

[0073] (6) Volcano plots of differentially expressed genes related to the peroX operon were constructed (Figure 1a), and hierarchical cluster analysis was performed (Figure 1b). Volcano plot analysis showed that under oxygen stress, the expression levels of three genes of the peroX operon (BT_3735, BT_3736, BT_3737) and their associated sRNA0246 were significantly upregulated, with changes far exceeding those of other differentially expressed genes. The gene expression profiles (horizontal axis) of the aerobic treatment group and the anaerobic control group formed clear clusters, confirming that oxygen stress can induce global reprogramming of the B. theta transcriptome.

[0074] Example 2: Phylogenetic Analysis of the peroX Operator

[0075] 2.1 Collection of Homologous Sequences

[0076] (1) Homologous sequences of the peroX operon from different species were collected from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), covering major bacterial groups such as Bacteroidetes, Firmicutes, and Proteobacteria.

[0077] (2) Using the amino acid sequences encoded by the genes of Bacteroides thetaiotaomicron BT_3737, BT_3736, and BT_3735 as query sequences, we performed homology search using BLASTp, setting E-value < 1e-5 and sequence similarity > 30%.

[0078] 2.2 Multiple sequence alignment

[0079] (1) Multiple sequence alignment of the collected amino acid sequences was performed using the ClustalW algorithm in MEGA-X software.

[0080] (2) Parameter settings: Gap Opening Penalty is 10, Gap Extension Penalty is 0.2, and BLOSUM62 matrix is ​​used.

[0081] (3) The comparison results were manually corrected to remove areas with poor alignment at both ends.

[0082] 2.3 Phylogenetic Tree Construction

[0083] (1) A phylogenetic tree was constructed using the maximum likelihood method. The best alternative model was selected from MEGA-X, and the LG+G+F model was selected after model testing.

[0084] (2) The Bootstrap iteration count was set to 1000 times to evaluate the reliability of each branch. Partial deletion was used to handle missing data, with a cutoff of 95%.

[0085] (3) Results are displayed (e.g.) Figure 2 As shown in a), the peroX operon is found only in *Bacteroides polymorpha* and its closely related *Bacteroides enterica*, exhibiting a specific distribution within the Bacteroidetes phylum. The phylogenetic tree shows the peroX operon located at the base of the evolutionary tree, suggesting its possible origin in the early stages of life evolution. The three genes BT_3737, BT_3736, and BT_3735 show strict collinearity within the Bacteroidetes phylum, indicating that their function was subjected to strong selective pressure during evolution.

[0086] Example 3: Expression of the peroX operon in B. theta under different concentrations of hydrogen peroxide induction

[0087] 3.1 Strain culture and hydrogen peroxide induction treatment

[0088] (1) Culture medium: BHI liquid medium

[0089] (2) Remove Bacteroides polymorpha from the -80°C glycerol tube, scrape the bacterial solution with a sterile inoculation loop in the anaerobic workstation, streak it on the surface of BHI solid medium, and incubate it upside down at 37°C for 48 hours.

[0090] (3) Select a single colony with typical morphology and inoculate it into 3 mL of BHI liquid medium. Incubate anaerobically at 37°C for 16-18 h to obtain seed culture. Transfer the seed culture to fresh BHI liquid medium at an inoculation rate of 1% (v / v) and incubate anaerobically at 37°C until the OD600 is 0.2-0.3.

[0091] (4) Divide the culture into 4 equal parts, each 5 mL. Set up hydrogen peroxide (H2O2) treatment groups with final concentrations of 0 μM (control group), 1 μM, 5 μM, and 10 μM respectively. Set up 3 biological replicates for each group.

[0092] (5) Induction treatment: H2O2 of the corresponding concentration was added to each treatment group every 20 minutes (to maintain the final concentration at the above-set value), for a total of 5 additions, with a total treatment time of 1.5 hours. An equal volume of sterile water was added to the control group. The entire process was carried out under anaerobic conditions at 37°C.

[0093] (6) After induction, centrifuge at 8,000 rpm for 5 min to collect the cells, discard the supernatant, immediately add liquid nitrogen for quick freezing, and store at -80°C.

[0094] 3.2 RNA extraction and reverse transcription

[0095] (1) Total RNA extraction: Total RNA was extracted using a cell / tissue isolation kit (Vazyme, RC112). 200 μL of lysozyme solution (10 mg / mL) was added to the bacterial pellet, and the pellet was vortexed for resuspending. 500 μL of Buffer RL was added, and the pellet was vortexed until no obvious cell clumps were observed. The lysis buffer was transferred to FastPure gDNA-Filter Columns III, centrifuged at 12,000 rpm for 30 seconds, and the filtrate was collected. 350 μL of anhydrous ethanol was added to the filtrate, and the mixture was mixed. The filtrate was transferred to FastPure RNA Columns III, centrifuged at 12,000 rpm for 30 seconds, and washed sequentially with 700 μL of Buffer RW1, 700 μL of Buffer RW2 (containing anhydrous ethanol), and 500 μL of Buffer RW2. The pellet was centrifuged at 12,000 rpm for 1 min to remove residual ethanol. 50 μL of enzyme-free water was added to elute the RNA.

[0096] (2) Use NanoDrop to determine RNA concentration and purity, requiring the A260 / A280 ratio to be between 1.8 and 2.0.

[0097] (3) Reverse transcription was performed using a one-step reverse transcription and genome removal kit (Yeasen). Reaction volume (20 μL): 5 μL of 4×Hifair® AdvanceFast One-Step RT SuperMix, 1 μL of gDNA Remover Mix, 1 μg of total RNA, and enzyme-free water to a final volume of 20 μL. Reaction program: 37°C for 5 min; 85°C for 30 sec; store at 4°C. cDNA was stored at -20°C for later use.

[0098] 3.3 qRT-PCR detection of gene expression levels in the peroX operon

[0099] (1) Primer design: BT_3735, BT_3736, BT_3737 and sRNA0246 in the peroX operon were used as targets, and 16S rRNA was used as an internal reference gene. Primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd., and their sequences are as follows:

[0100] BT_3737-F: 5'-GCGCCCTCGTTCTATCGTTTT-3' (SEQ ID NO.1)

[0101] BT_3737-R: 5'-GGCAGCACCCGTATCCAATA-3' (SEQ ID NO.2)

[0102] sRNA0246-F: 5'-CACTAGAGTGTTTCTGAAATAAACCTAA-3' (SEQ ID NO.3)

[0103] sRNA0246-R: 5'-ACTCTTTAGCTTGCGGGTC-3' (SEQ ID NO.4)

[0104] BT_3736-F: 5'-ATCCTTCACGTGCTGTA-3' (SEQ ID NO.5)

[0105] BT_3736-R: 5'-CTGCGCTGTTTTGAACGTCT-3' (SEQ ID NO.6)

[0106] BT_3735-F: 5'-TGGCGTATCCATGCACCAAT-3' (SEQ ID NO.7)

[0107] BT_3735-R: 5'-TCGTGCCTTGCGGTAGAAAT-3' (SEQ ID NO.8)

[0108] 16S rRNA-F: 5'-ACAGCGAGCGTGGCTCTCATTC-3' (SEQ ID NO.9)

[0109] 16S rRNA-R: 5'-GTTGACGATGTCGGCGGCAGTT-3' (SEQ ID NO.10)

[0110] (2) qRT-PCR reaction system (20μL): 10μL SYBR qPCR mix (TOYOBO), 0.5μL each of upstream and downstream primers (10μM), 2μL cDNA template, and 7μL sterile deionized water.

[0111] (3) Reaction program: 95°C pre-denaturation for 30s; 95°C denaturation for 5s, 58.6°C annealing for 30s, 72°C extension for 15s, for a total of 40 cycles; then melting curve analysis was performed (95°C for 15s, 60°C for 1min, 95°C for 15s).

[0112] (4) The fold increase of each target gene relative to 16S rRNA was calculated using the 2^(-ΔΔCt) method. The 0 μM H2O2 treatment group was used as a control. Three technical replicates were set up for each sample.

[0113] 3.4 Experimental Results

[0114] The results showed that ( Figure 2(b) The expression levels of four genes (BT_3735, BT_3736, BT_3737, and sRNA0246) in the peroX operon were significantly upregulated in a dose-dependent manner with increasing hydrogen peroxide concentration, with sRNA0246 and BT_3737 showing the most sensitive responses. This result validates the high sensitivity of the peroX operon to oxidative stress and suggests it could serve as a more sensitive indicator molecule for oxidative stress.

[0115] Example 4: Assessing intestinal oxidative stress in colitis mice using fecal peroX expression levels

[0116] 4.1 Establishment of a DSS-induced mouse colitis model

[0117] (1) Experimental animals: Ten female SPF grade C57BL / 6J mice, aged 6-8 weeks and weighing 18-22g, were selected and acclimatized for 1 week. Husbandry environment: temperature 22±2°C, humidity 50±10%, 12h light / 12h dark cycle.

[0118] (2) Grouping: The animals were randomly divided into two groups (6 animals in each group): the control group (Control) drank sterile water throughout the process; the model group (DSS) drank sterile water containing 2.5% (w / v) sodium dextran sulfate (DSS) from day 1 to day 7, and then switched back to sterile water on day 8.

[0119] (3) Record the mouse's weight daily ( Figure 2 c).

[0120] 4.2 Fecal sample collection and RNA extraction

[0121] (1) Feces were collected at three time points: on the 3rd day, the 10th day after modeling, and the 4th day after modeling. Fresh feces from each mouse were collected, immediately placed in liquid nitrogen and frozen, and then transferred to -80°C for storage.

[0122] (2) Total RNA extraction: Total RNA was extracted from feces using the TRIzol method combined with a centrifugation column.

[0123] (3) Use NanoDrop to determine RNA concentration and purity, requiring the A260 / A280 ratio to be between 1.8 and 2.0.

[0124] 4.3 Reverse transcription

[0125] (1) Reverse transcription was performed using a one-step reverse transcription and genome removal kit (Yeasen).

[0126] (2) Reaction system (20μL): 4×Hifair® AdvanceFast One-Step RT SuperMix 5μL, gDNA Remover Mix 1μL, total RNA 1μg, enzyme-free water to 20μL.

[0127] (3) Reaction procedure: 37°C for 5 min (removal of genomic DNA); 85°C for 30 sec (reverse transcription inactivation); store at 4°C. The obtained cDNA is stored at -20°C for later use.

[0128] 4.4 qRT-PCR detection of peroX operon expression level

[0129] (1) Primer design: The BT_3737 gene and sRNA0246 in the peroX operon were used as targets, and Bacteroides 16S rRNA was used as an internal reference gene.

[0130] (2) Absolute quantification was performed using the same cDNA sample and primer pair as in Example 4, and a digital PCR system was used.

[0131] (3) Prepare the dPCR reaction system: 10 μL of 2× ddPCR Supermix, 1 μL each of upstream and downstream primers (10 μM), 2 μL of cDNA template, and ddH2O to 20 μL.

[0132] (4) Use a droplet generator to generate droplets, and then perform PCR amplification: 95°C for 10 min; 94°C for 30 s, 60°C for 1 min, for a total of 40 cycles; terminate the reaction at 98°C for 10 min.

[0133] (5) The signal was read using a droplet reader, and the absolute copy number of the target molecule per μL of cDNA was calculated using QuantaSoft software. Figure 2 e)

[0134] 4.4 Colon tissue sampling and pathological examination

[0135] On day 14 (experimental endpoint), all mice were euthanized by cervical dislocation. After dissection, the colon was completely removed from the ileocecal junction to the anus. The colon was gently rinsed with pre-cooled PBS to remove intestinal contents, and its length was measured. Figure 2 c).

[0136] 4.5 HE staining and histological scoring

[0137] Colon tissue was fixed, embedded, sectioned, and stained with hematoxylin and eosin (HE) to observe the morphology of the intestinal mucosa. HE histological scoring was performed based on villus integrity, degree of inflammatory cell infiltration, and degree of mucosal damage.

[0138] The above results indicate that ( Figure 2 The expression level of peroX in the feces of model mice showed a time correlation with the deterioration of oxidative state during inflammation, further confirming its sensitivity in bacterial responses to changes in the intestinal microenvironment. The results of the DSS colitis modeling experiment showed that the model mice exhibited significant inflammatory symptoms, consistent with findings from studies using this method to induce colitis, and also led to increased oxidative stress levels in colitis tissues.

[0139] Appendix Figure 3 A schematic diagram illustrating the assessment of oxidative kinetics in colitis mice based on the peroX gene is shown. A method for tracking oxidative changes in the colon of colitis mice using the Bacteroides peroX operon is outlined. This platform provides a sensitive strategy for assessing redox fluctuations in intestinal tissue and studying colitis-related reactive oxygen species (ROS)-mediated processes.

[0140] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A biomarker capable of reflecting the level of oxidative stress in the gut, characterized in that: The biomarker is the peroX operon from the genome of Bacteroides, an endogenous gut microbiota. This peroX operon contains four genes: BT_3735, BT_3736, BT_3737, and sRNA0246. The sequences of BT_3735, BT_3736, BT_3737, and sRNA0246 are as follows: The forward primer sequence for BT_3737 is shown in SEQ ID NO.1; The reverse primer sequence for BT_3737 is shown in SEQ ID NO.2; The forward primer sequence for sRNA0246 is shown in SEQ ID NO.3; The reverse primer sequence for sRNA0246 is shown in SEQ ID NO.4; The forward primer sequence for BT_3736 is shown in SEQ ID NO.5; The reverse primer sequence for BT_3736 is shown in SEQ ID NO.6; The forward primer sequence for BT_3735 is shown in SEQ ID NO.7; The forward primer sequence of BT_3735 is shown in SEQ ID NO.

8.

2. The biomarker capable of reflecting the level of oxidative stress in the intestine according to claim 1, characterized in that: The BT_3735, BT_3736, and BT_3737 sequences are arranged adjacently and have the same transcription direction.

3. The method for screening peroX operators according to claim 1, characterized in that: The peroX operon was obtained through transcriptomics analysis.

4. The method for screening peroX operators according to claim 3, characterized in that, Includes the following steps: 1.1 Strains culture and sample collection Bacteroides multiforme VPI-5482 strain was scraped with a sterile inoculation loop and incubated upside down at 37°C for 48 hours. A single colony with typical morphology was inoculated into 3 mL of BHI liquid medium and anaerobic cultured at 37°C for 16-18 h to obtain a seed culture. The seed culture was then anaerobically cultured at 37°C until the OD600 reached 0.2-0.

3. The culture was transferred to a sterile centrifuge tube, inverted twice in the air, and cultured with shaking at 37°C and 200 rpm for 120 min. The cells were then collected by centrifugation at 8,000 rpm for 5 min. The collected bacterial pellet was washed once with PBS buffer pre-cooled at 4°C, and immediately flash-frozen in liquid nitrogen and stored at -80°C. 1.2 RNA extraction and transcriptome sequencing The collected bacterial cells were deribosomal RNA removed, and then the mRNA was randomly fragmented to approximately 200 bp. Using fragmented mRNA as a template, cDNA was synthesized using random primers and the SuperScript double-stranded cDNA synthesis kit. When synthesizing the second strand of cDNA, dUTP was used instead of dTTP for strand-specific labeling. End repair of double-stranded cDNA was performed, including 5' phosphorylation and 3' "A" tail addition, followed by ligation of Y-shaped sequencing adapters, and selective digestion of the second strand of cDNA using uracil DNA glycosylation enzyme. 1.3 Differentially expressed gene analysis Gene expression was quantified using RSEM software with TPM as the quantitative indicator, and differential expression analysis was performed using DESeq2 software package. The screening criteria were |log2FC|≥1 and p-adjust<0.

05. The four genes with the most significant differential expression were BT_3735, BT_3736, BT_3737 and sRNA0246. Genomic mapping showed that BT_3735, BT_3736 and BT_3737 were arranged adjacently and had the same transcription direction, and were named peroX.

5. The application of the biomarker of claim 1 in the non-invasive, quantitative assessment of intestinal oxidative stress levels.

6. The application according to claim 5, characterized in that: Applications in the diagnosis, disease monitoring, or efficacy evaluation of inflammatory bowel disease.

7. A drug for the diagnosis, disease monitoring, or efficacy evaluation of inflammatory bowel disease, characterized in that: The drug includes the biomarker described in claim 1 or 2.

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