Altered Gut Microbiota Associated with Hemorrhage in Chronic Radiation Proctitis

doi:10.21203/rs.3.rs-16229/v1

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Altered Gut Microbiota Associated with Hemorrhage in Chronic Radiation Proctitis

https://doi.org/10.21203/rs.3.rs-16229/v1

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Background: Pelvic cancer radiotherapy may cause chronic radiation proctitis (CRP) that adversely affects patient’s quality of life. Here, we aimed to characterize the gut microbiota of CRP patients and reveal the association of dysbiosis and hematochezia. A comparative metagenomic study of CRP with and without hematochezia was conducted by 16S rRNA gene sequencing.

Results: Different patterns of dysbiosis were observed in CRP patients with and without hematochezia. The abundance of Bacteroides was higher in CRP patients without hematochezia. The Verrucomicrobia phylum was enriched while the compositions of the Enterobacteriales family and the Porphyromonadaceae genus were relatively lower in the microbiota of CRP patients with hematochezia. PICRUSt analysis suggested that the expression of Fe-S protein, Glutathione peroxidase and Glutaredoxin-related protein were increased, indicating an exacerbated inflammation state in hematochezia patients.

Conclusions: This study provides new insight to the altered composition and function of gut microbiota in patients with hematochezia, implying the link of CRP symptom and bacterial ecosystem on rectal epithelial layer.

General Microbiology

Chronic Radiation Proctitis

Hematochezia

Gut Microbiota

Akkermansia

Cell Redox

Pelvic radiotherapy is one of the most important treatments of Gynecologic tumors. Chronic Radiation Proctitis (CRP) is a well-known complication of pelvic radiotherapy that occurs in 5–20% of patients 6 months to several years later¹. Unlike acute radiation enteropathy (RE) which presents with acute diarrhea and requires immediate termination of radiotherapy², CRP commonly manifests hematochezia which adversely affects patient’s quality of life in long term. Current treatments of CRP, such as high doses of 5-amin-osalicylic acid enemas, endoscopic thermal formalin therapy and antibiotics, are palliative^3 − 5. Prolonged, recurrent rectal inflammation and bleeding lead to weakness, anemia, and a series of mental effects including anxiety and depression, especially in female patients^6,7.

Gut microbial dysbiosis has been reported playing crucial roles in a series of gastrointestinal disorders including inflammatory bowel diseases (IBD), irritable bowel syndrome (IBS) and RE. A multi-omics study of the gut microbial ecosystem described both host and microbial activities with characteristic taxonomic, functional and biochemical shifts in IBD⁸. DNA fingerprinting and cloning-sequencing techniques revealed that acute postradiotherapy diarrhea might be linked to patient’s initial microbial composition⁹. Higher counts of Clostridium IV, Roseburia, and Phascolarctobacterium are significantly associated with RE¹⁰. Lactobacillus rhamnosus GG (LGG) protection of intestinal epithelium from radiation injury was illustrated by rodent model. The LGG-mediated radioprotection is reported to dependent on MyD88, TLR-2 and COX-2¹¹. However, little is known about the interaction between gut microbiota and prolonged proctorrhagia after irradiation.

In the present study, we set out to characterize the gut microbiota profiles of CRP patients and demonstrated dysbiosis in CRP patients with hematochezia.

Patients and sample collection

32 female participants were assigned into 2 groups: group NH, meaning chronic radiative proctitis (CRP) patients with no or mild hematochezia (hematochezia frequency < twice per week and/or no fresh blood observed under proctoscope) (n = 16); and group H, meaning CRP patients with hematochezia (hematochezia frequency > twice per week and/or fresh blood was found under proctoscope) (n = 13) (Table 1). Two patients presented with rectal ulcers (diameter >1mm) and/or bacterial infection were excluded. Age of the 30 patients ranged from 28y to 71y. Patients have received total dose of 48 to 54 Gy irradiation as for radiotherapy of uterus or cervical carcinoma but have not received antibiotic treatment for at least 3 months. 3 samples from healthy adults were collected and analyzed in parallel.

Diversity of gut microbiota in CRP patients.

To investigate the ecological complexity of the gut microbiota of CRP patients, alpha diversity analysis within each group was generated using Community richness calculators Chao1 estimator and ace estimator as well as Community diversity calculators the Simpson index and the Shannon index in MOTHUR. As shown in Figure 1, the Community richness is lower in CRP patients with hematochezia (Mean of Chao1 = 1065; Mean of ace = 1738) as compare with CRP patients without hematochezia (Mean of Chao1 = 943.0; Mean of ace = 1488) but not statistically different (p > 0.1). Nevertheless, intragroup diversities are similar between the two groups. This result suggests that the overall complexity of gut microbiota may not be a sensitive indicator for hematochezia of CRP.

Differential gut microbiota of CRP patients with hematochezia

Venn diagram was generated to illustrate the assigned OTUs in each group. About 70% of the OTUs were shared in CRP patients while 27% and 29% of the taxonomic units were unique in H group and NH group, respectively (Figure 2a). Ordination analysis including Principal Component Analysis (PCA), Principal Co-ordinates Analysis (PCoA) and Non-Metric Multi-Dimensional Scaling (NMDS) were conducted to measure the difference in bacterial community composition in two-dimension. Inter-individual variation accounted for a part of variance, which was consistent with previous studies^8,15. The ordination plots demonstrated a difference among healthy controls, CRP patients and CRP patients with hematochezia, suggesting that a proportion of bacteria population is related to the hematochezia symptom of CRP (Figure 2b, Figure S1-2).

To further study the microbiota composition, the OUT matrixes were visualized by Krona (Figure S3). As one of the most common bacterial components of the gut microbiome, Bacteroides was the most abundant bacteria in both of the CRP groups and that in healthy controls (Figure 2c, Figure S3). Composition of Bacteroides was higher in the NH group than that in H group. More Escherichia/Shigella, Fusobacterium and Prevotella were found in gut microbiota of CRP patients with hematochezia as compare to the NH group.

The linear discriminant analysis on effective size (LefSe) generated a global view of gut microbiota and revealed 8 bacteria taxa with differential relative abundance in both NH and H groups (LDA score > 2). The Verrucomicrobia phylum, Mogibacterium which is a species of the Clostridiales Incertae Sedis XIII genus, and Allisonella were identified from the H group. Akkermansia is the most abundant genus within the Verrucomicrobias (account for >99% of the Verrucomicrobias; 2% of the microbiota in the NH group; 4% in the H group) while the other two genera of Verrucomicrobias accounted for less than 0.02% of the microbiota. Our result also showed that the compositions of the Enterobacteriales family and the Porphyromonadaceae genus were lower in the H group (Figure 3, Figure S4). The linear discriminant analysis result was consistent with the Krona plots, as the Escherichia/Shigella is the major species of Enterobacteriales and the Parabacteroides belongs to the Porphyromonadaceaes.

Microbial functional dysbiosis in Hematochezia group

As the composition of microbiota community altered, functional and metabolic changes could be predicted based on previous molecular studies^16,17. The Fe-S protein (COG1600), the Glutathione peroxidase (GPx, COG0386) and the Glutaredoxin-related protein (COG4545) were significantly increased in Hematochezia group (Figure 4, Table S1). The GPx is an important enzyme for detoxin and clearage of hydrogen peroxide. Notably, the metabolism of Arachidonic Acid (ARA) was also higher in Hematochezia group according to the annotation of the KEGG database (Figure S5). ARA is not only an important composition of cell membrane but also crucial for biosynthesis of prostaglandin (PGE2 and PGI2) which are commonly known as inflammatory mediators.

Rectal bleeding is the most common symptom of Chronic Radiation Proctitis (CRP) that lays heavy medical burden to the patients. In this study, we took gut microbiota samples from CRP patients under proctoscope and revealed an altered microbial ecosystem in CRP patients with hematochezia.

CRP patients are mostly outpatients in Surgery clinic seeking for remission of rectal irritation and hematochezia. It is believed that the sign and symptom of CRP are largely contributed by interstitial fibrosis and occlusive endovascular inflammation. Proctoscope examination provides clear vision of rectal mucus in a relatively convenient and cost-effective way. It is suggested that regular proctoscope could be beneficial to patients with radiation proctitis⁶. Here we included patients with an array of criteria including patients’ observation of blood in stool, digital rectal examination and doctors’ observation under proctoscope. A systematic history collection with multiple categories would be helpful not only for research purpose but also for outpatient management.

In this population of CRP patients, hematochezia is associated with an increased proportion of Akkermansia. Akkermansia muciniphila is a probiotic for treating obesity¹⁸. Sulphated colonic mucins is known as the nutrient substrate for Akkermansia muciniphila¹⁹ as it is capable of secreting mucin-degrading enzymes to utilize mucus as carbon and nitrogen source^20,21. Mucin is important for maintaining mucosal barriers. The hydrophobic membrane-spanning domain of mucin prevent pathogens from biofilm formation and colonization^22,23. The increased proportion of Akkermansia may lead to impaired gut mucosal barriers that cause dysbacteriosis in CRP.

Diminished composition of Enterobacteriales in H group may because of the decrease of Morganella (9% of Enterobacteriales in NH group) and an unclassified Enterobacteriales species (33% of Enterobacteriales in NH group). The composition of Escherichia/Shigella was higher in H group (86% of Enterobacteriales in H group, 58% in NH group). Escherichia coli (E. coli) is one of the most common species of gut microbiota. However, metagenomic study provides information at genus level which is not able to distinguish pathogenic E. coli from the non-pathogenic one. Deeper characterization of Escherichia/Shigella should be conducted to search for hemorrhoid related pathogens (such as Enterohemorrhagic or Enteropathogenic E. coli) by qPCR typing or Matrix-Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF) Mass Spectrometry ²⁴. Further characterization of gut microbiota may provide better biomarker of hematochezia in CRP patients.

The Fe-S protein was predicted to be significantly increased in Hematochezia group. One possible explanation is that the micro-environment with continuous bleeding is favor for bacteria that utilize iron-sulfur in bacterial cell respiratory. Glutaredoxins (Grxs) ligated Fe-S cluster participates in oxidative signaling²⁵ and haem synthesis²⁶, which may be compensatory to the prolonged anemia. The Grxs system also acts as an efficient electron donor to plasma glutathione peroxidase (GPx)²⁷. GPx is known as one of the major enzymes that prevent oxidative stress by catalyzing glutathione (GSH) into glutathione disulfide (GSSG). The three proteins predicted by PICRUSt, the Grxs, FeS and GPx, are key regulators of cell redox and electron transport that involve in various processes of biosynthesis and detoxification²⁸. In addition, GPx together with Arachidonic Acid (ARA) regulate PGE2 and PGI2 which promote inflammation²⁹. Overexpression of ARA and GPx may exacerbate inflammation and result in rectal hemorrhage.

The dysbiosis could be related to the change of gene expression in glandular and/or epithelial cells induced by irradiation and inflammation³⁰. Thus, combination of metagenomic data, transcriptome profiles and proteomic profile of inflammatory factors study is expected to provide comprehensive view of gut-microbiome interaction and investigate the possible function of Akkermansia in regulating cell redox³¹. In summary, this study revealed the altered gut microbiota in CRP with hematochezia, providing a fundamental evidence for future developing and evaluating treatments for chronic radiation proctitis.

In this study, we show for the first time that the gut microbiome is associated with hematochezia in CRP. In accordance with published data, we find the differentiated microbial ecosystem in hematochezia patients where Akkermansia is dominant in rectal bacterial community. The shifted gut microbiota of CRP patients with hematochezia toward that of non-hematochezia patients is consistent with the hypothesis that gut microbiota may be responsible for CRP symptoms. Metabolites related to Akkermansia such as FeS, Grxs and GPx could further induce expression of inflammatory factor that interact with irradiated rectal epithelial cells and exacerbate inflammation. Introduction of Enterobacteriales to restore microbial balance could be a possible solution to improve hematochezia and anemia. The findings in the current study require validation in a larger cohort and the development of probiotic therapy warrant further investigation.

Ethics and patient information

This study was approved by the Ethics Board at Guangzhou Hospital of TCM. Samples were collected from 30 female outpatient under proctoscope. The patients received pelvic radiotherapy one to two years prior to the onset of chronic radiation proctitis. No antibiotic has been adopted by any patient for at least 3 months. 16 patients complained hematochezia for at least 3 times per day and bleeding was observed under proctoscope. These patients were classified as H group. 13 patients complained diarrhea with no or little blood in stool were classified as NH group (Table 1). Samples from 3 healthy volunteers were collected in parallel. Vegetarian, patients complicated with irritable bowel disease or genetic hemorrhagic disease, and patients who have received antibiotics or steroid treatment in the past 3 months were excluded from this study.

DNA extraction

Total DNA was purified from 0.25g sample by QIAamp PowerFecal DNA Kit (QIAGEN, DE). The DNA concentration and purity were determined by Multiskan™ GO (Thermo Fisher Scientific, US), and the integrity of the DNA was determined by agarose gel electrophoresis.

16S rRNA gene sequencing

PCR was performed on the V4 region (515F-806R) of the 16S rRNA gene of the sample bacteria by priming different indexes at both ends. The reaction system of PCR (20 micrometers) was performed using10 μl of KAPA HiFi HotStart ReadyMix (KAPA Biosystems, USA), 2 μl of DNA (about 30 ng/μl), and 1μl of forward and reverse primer (10 μM). The forward primer of 16S was 5 '-gtgccagcmgccgcggtaa-3' and the reverse primer was 5 '-ggactacnvgggtwtctaat-3'. The following thermal cycling program was used: an initial denaturation step at 95°C for 3 mins followed by 30 cycles of 95°C for 20s, 60°C for 30s (annealing/synthesis step), 72°C for 30s (extension) and finally 72°C for 10min extension.

The PCR products were purified with the AxyPrep™ PCR Cleanup Kit (Axygen, USA), and the concentration was determined with Qubit 3.0 (Thermo Fisher Scientific, USA). Equal amounts of each sample were mixed together to form a sequencing library. QSEP100 (Bioptic, CHN) and ABI7300 quantitative PCR (Thermo Fisher Scientific, US) were used to detect and quantify the insertion fragment and concentration of the library, respectively. The tested libraries were Paired-End 150 bp (PE150) sequenced using Illumina MiniSeq platform.

Bioinformatic analysis

Sequences generated from Illumina sequencing were analyzed with MOTHUR (version 1.39.5) for identification of operational taxonomic units (OTU), taxonomic assignment, community comparison, and data cleaning by adapting its standard operational procedure¹². Sequences were realigned with the SILVA-compatible alignment database (http://www.mothur.org/w/images/9/98/Silva.bacteria.zip). The 3% dissimilarity cut-off value was used for assigning an OTU. Shannon’s diversity, Simpson’s diversity, ACE, and Chao I richness indices were generated with the MOTHUR program. Output matrixes were further analyzed by principal coordinates analysis (PcoA) based on UniFrac distance, and linear discriminant analysis effect size (LEfSe) to identify differences in relative abundance at different taxonomic levels¹³. Gene function and metabolic pathway prediction was conducted by PICRUSt¹⁴.

Statistics and data visualization

The analysis of variance (ANOVA) test was used to assess the differences in the similarity index and the number of bands among the three groups: controls, patients with, and patients without hematochezia. The unpaired Students’ t-test was used when we analyzed between two groups using Graphpad prism 8. Krona was used for visualization of microbiome community.

Ethics approval and consent to participate

This study was approved by the Ethics Board at Guangzhou Hospital of Traditional Chinese Medicine.

Consent for publication

Not applicable.

Availability of data and material

The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests

All authors declare no competing interests.

Funding

This article is an independent research funded by the Guangzhou Hospital of Traditional Chinese Medicine.

Authors' contributions

JJL and LZL conceived the study. LZL contributed to the literature search, preparation of figures and panels, and writing of the manuscript. CYC, BCC and NNX contributed to sample collection clinical data analysis. CD contributed to 16S rRNA gene sequencing data analysis and writing of the manuscript. All authors have seen and agreed on the final submitted version of the manuscript.

Acknowledgements

We thank the Guangzhou Hospital of Traditional Chinese Medicine for supporting this study.

ARA: Arachidonic Acid

CRP: Chronic Radiation Proctitis

Grxs: Glutaredoxins

GPx: Glutathione Peroxidase

GSH: glutathione

GSSG: glutathione disulfide

LefSe: linear discriminant analysis effect size

NMDS: Nonmetric multidimensional scaling

OTUs: operational taxonomic units

PCoA: principal coordinates analysis

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Table 1. Patients information.

	Number	Age(y)	Gender	Site of cancer	Erosive mucosa and rectal bleeding	DRE: fresh blood on glove
NH	1	62	F	Cervix	-	-
	2	48	F	Uterus	-	-
	3	49	F	Cervix	-	-
	4	32	F	Cervix	-	-
	5	67	F	Cervix	-	-
	6	65	F	Cervix	-	-
	7	56	F	Cervix	-	-
	8	50	F	Cervix	-	-
	9	48	F	Cervix	-	-
	10	60	F	Cervix	-	-
	11	68	F	Cervix	-	-
	12	41	F	Uterus	-	+
	13	57	F	Cervix	-	-
	14	68	F	Cervix	-	-
	15	67	F	Cervix	-	-
	16	68	F	Cervix	-	-
H	1	59	F	Cervix	+	+
	2	56	F	Cervix	+	+
	3	71	F	Cervix	+	+
	4	61	F	Cervix	+	+
	5	56	F	Cervix	+	+
	6	32	F	Cervix	+	+
	7	37	F	Cervix	+	+
	8	28	F	Cervix	+	+
	9	58	F	Cervix	+	+
	10	58	F	Cervix	+	+
	11	57	F	Cervix	+	+
	12	61	F	Cervix	+	+
	13	34	F	Cervix	+	+

Notes: NH: Non-hematochezia group; H: hematochezia group; F: Female; DRE: Digital Rectal Examination; -: not observed; +: observed.

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Altered Gut Microbiota Associated with Hemorrhage in Chronic Radiation Proctitis

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