Background：
Brain-gut-microbiota axis (BGMA) disorder is an important pathophysiology of irritable bowel syndrome (IBS). There are still unclear how brain affects gut microbiota. Colon belongs to hypoxic environment, thus forming gut microbiota with obligate anaerobic bacteria as dominant strain. Oxygen hypothesis raised that decrease of obligate anaerobic bacteria and increase of facultative anaerobic bacteria in intestinal are caused by oxygen concentration changes. Therefore, we speculate that central stress may affect gut microbiota by damage colonocyte mitochondria.
Method：
1) C57BL/6 male mice aged 5-6 weeks were randomly divided into acute water avoidance stress group (WAS) and Sham group. ELISA tested corticosterone and CRF concentration of serum. Quantitative Real-time-PCR (qPCR) revealed mRNA expression of corticotropin releasing hormone receptor (CRHR), and mitochondria related genes. Mitochondria function was performed by O2K-tecnology and ELISA. Mitochondria ultrastructure was observed by transmission electron microscopy (TEM). Epithelium hypoxia was tested by Non-invasive Micro-test Technology (NMT) and Hypoxia stain. Metagenomic sequencing revealed microbiota composition.
2) C57BL/6 male mice aged 5-6 weeks were randomly divided into four groups [Control, CRF, CRF+NBI30775 (CRHR1 antagonist) and CRF+5-amino salicylic (5-ASA) group]. We used stereotactic injection to inject CRF or vehicle in PVA of mice, and then gave NBI3077 or 5-ASA to CRF intervention mice by intraperitoneal injection immediately. All aspects of indicators tested by above methods.
Results：
1) WAS group mice displayed increased corticosterone and CRF concentration of serum and elevated colonocyte CRHR1 expression. O2K-tecnology and ELISA revealed reduced mitochondria respiration and decreased activity of pyruvate dehydrogenase (PDH), citrate synthetase (CS), level of ATP in WAS group. TEM exhibited damaged mitochondria ultrastructure in WAS group. qPCR revealed that mitochondria related genes were differentially expressed in WAS and Sham group. WAS group mice showed reduced net flux of oxygen absorption and diminished hypoxia in colon epithelial. Finally, WAS group mice showed decreased obligate anaerobic bacteria in microbiota (Figure 1).
2) CRF group mice displayed increased corticosterone and CRF concentration of serum and elevated colonocyte CRHR1/2 expression. CRF treatment decreased oxygen consumption and damaged ultrastructure of mitochondria. Epithelium hypoxia in CRF group mice has dramatically impaired. NBI30775 treatment blocked the expression of CRHR1 and 5-ASA treatment has barely influenced these indicators, both of them improved mitochondria and restored epithelium hypoxia (Figure 2).
Conclusion：
Acute central stress release CRF elevate colonocyte CRHR1 expression damage mitochondria and induce microbiota dysbiosis.

<b>Figure 1. Acute water avoidance stress damage colonocyte mitochondria and alter microbiota composition.</b> (A) Acute water avoidance model. (B) Corticosterone, CRF level of serum, <i>N</i> = 10. (C) CRHR1/2 genes expression level, <i>N</i> = 10. (D) H&E images of the colon (scale bar = 50 um), <i>N</i> = 10. (E) TEM images of mitochondria (scale bar = 500 nm). (F) Quantitative indicators of mitochondria ultrastructure, <i>N</i> = 5. (G) Mitochondria dynamic genes, <i>N</i> = 10. (H) O2K technology. OXPHOS, ETS, Complex IV, <i>N</i> = 10. (I) Cytosolic level of ATP, lactate and activity of PDH and CS, <i>N</i> = 7. (J) Mitochondria biogenesis and DNA translation genes, <i>N</i>= 10. (K) Oxygen absorption flux in colon epithelial, <i>N</i> = 5. (L) Hypoxia stain images and hypoxia scoring of colon epithelium, <i>N</i> = 5. (M) Different taxa composition. Purple, elevated in WAS; Green, reduced in WAS (N) Different microbiota in top family and genus level, <i>N</i> = 10. Data: Mean ± SEM.

Figure 1. Acute water avoidance stress damage colonocyte mitochondria and alter microbiota composition. (A) Acute water avoidance model. (B) Corticosterone, CRF level of serum, N = 10. (C) CRHR1/2 genes expression level, N = 10. (D) H&E images of the colon (scale bar = 50 um), N = 10. (E) TEM images of mitochondria (scale bar = 500 nm). (F) Quantitative indicators of mitochondria ultrastructure, N = 5. (G) Mitochondria dynamic genes, N = 10. (H) O2K technology. OXPHOS, ETS, Complex IV, N = 10. (I) Cytosolic level of ATP, lactate and activity of PDH and CS, N = 7. (J) Mitochondria biogenesis and DNA translation genes, N= 10. (K) Oxygen absorption flux in colon epithelial, N = 5. (L) Hypoxia stain images and hypoxia scoring of colon epithelium, N = 5. (M) Different taxa composition. Purple, elevated in WAS; Green, reduced in WAS (N) Different microbiota in top family and genus level, N = 10. Data: Mean ± SEM.

<b>Figure 2.</b> <b>CRF of PVA damage colonocyte mitochondria and increases colon oxygen bioavailability via CRHR1. </b>(A) Mice stereotactic injection. (B)Corticosterone, CRF level of serum. (C) CRHR1/2 genes expression level. (D) OXPHOS, ETS, Complex IV. (E) Cytosolic level of ATP, lactate and activity of PDH and CS. (F) Mitochondria biogenesis, mt-DNA translation genes, <i>N</i> = 7-10. (G) H&E images of colon (scale bar = 50 um), <i>N</i> = 10. (H) TEM images of mitochondria (scale bar = 500 nm). (I) Quantitative indicators of mitochondria ultrastructure, <i>N</i> = 5. (J) Mitochondria dynamic genes, <i>N</i> = 10. (K) Hypoxia stain images and hypoxia scoring of colon epithelium, <i>N</i> = 5. Data: Mean ± SEM.

Figure 2. CRF of PVA damage colonocyte mitochondria and increases colon oxygen bioavailability via CRHR1. (A) Mice stereotactic injection. (B)Corticosterone, CRF level of serum. (C) CRHR1/2 genes expression level. (D) OXPHOS, ETS, Complex IV. (E) Cytosolic level of ATP, lactate and activity of PDH and CS. (F) Mitochondria biogenesis, mt-DNA translation genes, N = 7-10. (G) H&E images of colon (scale bar = 50 um), N = 10. (H) TEM images of mitochondria (scale bar = 500 nm). (I) Quantitative indicators of mitochondria ultrastructure, N = 5. (J) Mitochondria dynamic genes, N = 10. (K) Hypoxia stain images and hypoxia scoring of colon epithelium, N = 5. Data: Mean ± SEM.

XIAOANG LI

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