The inhibition of enterocyte proliferation by lithocholic acid exacerbates necrotizing enterocolitis through downregulating the Wnt/β‐catenin signalling pathway

Abstract Objectives Necrotizing enterocolitis (NEC) is a catastrophic gastrointestinal emergency in preterm infants, whose exact aetiology remains unknown. The role of lithocholic acid (LCA), a key component of secondary bile acids (BAs), in NEC is unclear. Methods Clinical data were collected to analyse the changes of BAs in NEC patients. In vitro studies, the cell proliferation and cell death were assessed. In vivo experiments, the newborn rats were administered with low or high dose of LCA and further induced NEC. Results Clinically, compared with control group, total BAs in the NEC patients were significantly higher when NEC occurred. In vitro, LCA treatment significantly inhibited the cell proliferation through arresting cell cycle at G1/S phase without inducing apoptosis or necroptosis. Mechanistically, the Wnt/β‐catenin pathway was involved. In vivo, LCA inhibited intestinal cell proliferation leading to disruption of intestinal barrier, and thereby increased the severity of NEC. Specifically, LCA supplementation caused higher levels of FITC‐labelled dextran in serum, reduced PCNA expression and inhibited the activity of Wnt/β‐catenin pathway in enterocytes. The LC–MS/MS test found that LCA was significantly higher in intestinal tissue of NEC group, and more obviously in the NEC‐L and NEC‐H group compared with the DM group. Conclusion LCA exacerbates NEC by inhibiting intestinal cell proliferation through downregulating the Wnt/β‐catenin pathway.

perforation, sepsis, shock and even death. 4 Survivors of severe episodes of NEC frequently suffer serious sequelae such as short bowel syndrome, cholestasis and neurodevelopmental retardation. 5,6 Although prematurity, 7 enteral feeding, 8,9 intestinal bacterial colonization, 10,11 and immune imbalance, 12 are considered as major risk factors, the pathophysiology of NEC is still poorly understood. The intestinal epithelial cells (IECs), immune system and microbiome, coordinate with each other to maintain normal intestinal homeostasis and barrier integrity. 13 In response to microbial invasion, the death of IECs increases, but more importantly, there is an enhanced proliferation and renewal to retain the intestinal barrier and tissue homeostasis. However, an impairment of IEC proliferation leads to increased intestinal permeability and barrier dysfunction.
The Wnt/β-catenin pathway, a multitasking and evolutionary conserved pathway, plays essential roles in embryonic development, tissue homeostasis and regeneration. 14,15 β-Catenin is a key component of the Wnt signalling cascade; the levels and activity of this protein are tightly regulated by a destruction complex consisting of the enzyme glycogen synthase kinase-3β (GSK-3β), the adenomatosis polyposis coli (APC) protein and the scaffolding protein AXIN. 16 β-Catenin can be phosphorylated by the destruction complex through GSK-3β and degraded by the ubiquitination-proteasome pathway in the cytoplasm, thereby failing to enter the nucleus to promote the transcription of target genes, including cyclin D1, c-jun and c-myc. 17 These target genes are well known to play important roles in the proliferation of intestinal cells. [18][19][20] In addition, it has been reported that impairment of the Wnt/β-catenin pathway can lead to the dysfunction of intestinal regeneration during NEC. 21  where BAs are reabsorbed. The accumulation of secondary BAs in the intestine can cause damage to the intestinal epithelium. [24][25][26] It has been reported that DCA is closely related to NEC by regulating cell proliferation or the BA transporter. 27,28 Similarly, LCA has been found to be elevated in intestinal bile salts from patients with NEC. 29 However, the role and mechanisms of LCA in NEC remains clear. In this study, we aimed to unravel the impact of LCA in the pathogenesis of NEC.

| Clinical data collection
Clinical data were obtained between April 2021 and October 2021 in accordance with the clinical research and applied ethics committee of the Third Affiliated Hospital of Guangzhou Medical University. Written informed consent was obtained from the patient's parents. The following data were collected, including gestational age (GA), birth weight (BW), gender, Apgar scores at 5 min, total BAs within 24 h after birth, as well as total BAs, total bilirubin and direct bilirubin at the time of NEC occurrence. In addition, treatment with red blood cell (RBC) or nonsteroidal anti-inflammatory drugs (NASIDs), including ibuprofen or indomethacin, was also collected, which are risk or protective factors for NEC. 30,31 The diagnosis of NEC was defined as Bell's stage ≥II with radiographic evidence of NEC. 32 In this study, infants with NEC were matched by GA, BW and days of age at collection of total BAs, with a maximum allowable deviation of 10% to two controls. 33   All assays were performed in triplicate.

| RNA sequencing
RNA sequencing was performed by Novogene (Suzhou, China). In brief, RNA quantification and identification were determined by an RNA Nano 6000 assay kit and a Bioanalyzer 2100 system (Agilent Technologies, CA). Then, cDNA library construction and quality assessment of the library were performed with an AMPure XP system (Beckman Coulter, Beverly, MA). Qualified libraries were sequenced on an Illumina Novaseq platform and 150 bp paired-end reads were generated. Differentially expressed genes (DEGs) were identified using the edger R package with a log2 jfold changej >1 and adjust pvalue (padj) ≤0.05. KEGG pathway enrichment analysis of DEGs was performed using the clusterprofiler R package. Gene set enrichment analysis (GSEA), a computational method used to determine whether a pre-defined gene set can show significant concordant differences between two biological genes, was also carried out.

| Immunofluorescence
IEC-6 cells were seeded on coverslips, fixed in 4% paraformaldehyde for 15 min and permeabilized in 0.15% Triton-100X for 10 min. Then, cells were blocked with 5% goat serum for 1 hour. Anti-Ki67 or antiβ-catenin (CST, Danvers) antibody was then added at a dilution of 1:100 in 1% BSA and incubated overnight at 4 C. Then, cells were washed three times with PBS and incubated with FITC-labelled secondary antibodies at a dilution of 1:100 in 1% BSA for 2 h in the dark.

| Western blotting
Preparation of whole cell lysates and western blotting analysis was performed as previously described. 34

| Experimental NEC induction and evaluation
Newborn rats were randomly divided into four groups immediately after birth: DM, NEC, NEC-L and NEC-H (seven rats in each group).
The DM group was left with their mothers and fed breast milk as normal control. The NEC, NEC-L and NEC-H groups were fed with cow's milk-based rat milk substitute formula and were subjected to asphyxia (breathing 100% nitrogen for 60 s) followed by cold stress (4 C for 10 min) twice a day for 4 days to induce experimental NEC. [36][37][38] The substitute formula for the NEC-L and NEC-H groups were mixed with LCA at final concentrations of 10 and 20 mM, respectively. After 4 days, all surviving rats were euthanized. The intestines were carefully removed, formalin-fixed, paraffin-embedded, microtome-sectioned, stained with haematoxylin-eosin (H&E) and histologically evaluated by two blinded pathologists independently based on an established histology damage scoring system. [39][40][41] Damage scores ≥2 were considered to have developed experimental NEC.

| BAs composition assay
In rats, the composition of BAs in terminal ileum tissue were detected using a liquid chromatography tandem mass spectrometry (LC-MS/ MS) system (BioNovoGene, Suzhou, China). In brief, approximately 50 mg of lyophilized homogenized tissue was weighed, ground and methanol was added to precipitate protein. After vertexing for 1 min and centrifugation at 4 C, the supernatant was concentrated and dried in a vacuum; the residue was then redissolved with 100 μl methanol. Finally, the supernatant was analysed with a LC-MS/MS system.

| Immunohistochemistry
Immunohistochemical analysis was completed as described previously. 42 In brief, after deparaffinization, antigen retrieval and background blocking, sections were incubated with anti-PCNA at 4 C overnight. The next morning, the sections were washed with PBS and then incubated with a HRP conjugated secondary antibody at room temperature for 1 h. Immunoreactivity was then observed by staining with 3,3 0 -diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA). Finally, the slides were counterstained with H&E. To quantify immunoreactivity, 15 consecutive non-overlapping fields at Â400 magnification were scored blindly.

| Measurement of intestinal barrier permeability
Four thousand dalton fluorescent dextran-FITC (DX-4000-FITC; Sigma-Aldrich) was used to measure intestinal barrier permeability as described previously. 43,44 In brief, DX-4000-FITC (80 mg/100 g body weight, 40 mg/ml) was administered after the rats were fasted for 8 h. Four hours later, blood was taken from the heart and the upper plasma was collected by centrifugation. The level of DX-4000-FITC in the plasma was then measured using a Synergy H1 microplate reader (BioTek, Winooski, VT) (excitation, 480 nm; emission, 520 nm). The concentration of DX-4000-FITC was then calculated based on a standard curve.

| Statistical analysis
Statistical analysis was performed using the SPSS 22 software (SPSS, Chicago, IL) and the GraphPad Prism 7 software (La Jolla, CA). Unless otherwise stated, results are expressed as mean ± standard deviation (SD). Statistical differences between two groups were analysed using the Student's t-test or Wilcoxon test, while multiple groups were evaluated using one-way analysis of variance (ANOVA) or the Kruskal-Wallis test, and rates were compared and analysed using chi square or Fisher's exact tests. p <0.05 was considered statistically significant.     Figure S2A). Of these, 48 DEGs related to the cell cycle were identified ( Figure 2A). KEGG pathway analysis indicated that LCA was associated with cell cycle (Figures 2B and S2B). GSEA also indicated that genes related to cell cycle progression were significantly repressed by LCA treatment ( Figure 2C).
Next, we investigated key regulatory proteins involved with the cell cycle. We found that treatment of IEC-6 cells with LCA (60-180 μM) for 24 h led to a dose-dependent reduction in the expression of cyclin D1, cyclin D3, cyclin A2, cyclin B1, cyclin E1, CDC2, CDK2, CDK4 and CDK6 ( Figure 2D, Figure S3A-J). Similar results were obtained after both 12 and 6 h of treatment ( Figure S2C,D). Furthermore, we used flow cytometry to determine which phase of the cell cycle was arrested. As shown in Figure 2E

| The Wnt/β-catenin pathway was involved in the inhibition of cell proliferation
Mechanistically, it has been reported that the MAPK and Wnt/β-  S4E-K,N). These similar trends were observed after both 12 and 6 h of treatment ( Figure S5C,D). Moreover, the immunofluorescence staining of β-catenin also decreased in a dose-dependent manner ( Figure 4D). Wnt-3A is the major cytokine responsible for β-catenin activation. 53,54 IEC-6 was treated with LCA (80, 120 and 160 μM), following by treatment with Wnt-3A (20 ng/ml). 55 The cell viability was tested using CCK-8 assay. And Wnt-3A can alleviate the inhibition of the cell proliferation at low concentrations of LCA, but not at high concentrations ( Figure S6).
Next, we compared LCA with DCA or CA, and found that only LCA had an inhibitory effect on the Wnt/β-catenin pathway ( Figures 4E and S7A-J). DCA was more likely to act through the MAPK pathway while CA had no functional role in the Wnt/β-catenin or MAPK pathway ( Figure 4E,F, Figure S7A-N). Similar results were obtained after 12 and 6 h of treatment ( Figure S5E-H). Collectively, our data suggest that LCA inhibited IEC-6 cell proliferation in vitro by arresting cell cycle at the G1/S phase via the Wnt/β-catenin pathway.

| LCA triggered intestinal injury by inhibiting cell proliferation
The reduction of IEC cell proliferation leads to increased intestinal permeability and barrier dysfunction and is associated with a variety of intestinal diseases. Next, we investigated the effect of LCA on the rat intestine in vivo. Newborn rats were administered with low or high doses of LCA (namely the CON-L or CON-H groups) or fed with rat milk (namely DM group). Compared with the DM group, body weight gain was slower in the CON-L and CON-H groups ( Figure 5A). Histological examination of the distal ileum showed more serious injury and higher damage scores in the CON-L or CON-H group than in the DM group ( Figure

| DISCUSSION
In this study, we confirmed that LCA aggravated NEC by inhibiting the proliferation of enterocytes through arresting at G1/S phase in a β-catenin-dependent manner (Figure 7). To our knowledge, our study is the first to illustrate the effect of LCA on Wnt/β-catenin signalling in NEC. We also found that LCA had a more obvious inhibitory role in enterocyte proliferation than DCA or CA.
BAs are produced by the liver, secreted into the duodenum and then undergo the conversion of primary BAs (CDCA, CA) to secondary BAs (DCA, LCA) by the gut microbiota; they are then recycled back to the liver in the terminal ileum via the enterohepatic circulation. [56][57][58] When NEC occurs, the distal ileum is damaged; this can lead to the obstruction of the enterohepatic circulation, thus causing difficulties in the recycling of BAs and the consequent accumulation of BAs. 25,59 In turn, the accumulation of BAs can aggravate intestinal damage. 28,60 The chemical structure and properties of BAs are diverse; therefore, the role of BAs differs across many diseases. It has been reported that CDCA can stimulate the release of inflammatory factors and lead to increased intestinal permeability 61 while UDCA has been shown to inhibit apoptosis and alleviate NEC-induced injury. 62 Other research has shown that tauroursodeoxycholic acid alleviates intestinal injury by inhibiting endoplasmic reticulum stress in NEC. 63 In our  The intestinal epithelium undergoes renewal every 3-5 days in humans and every 2-3 days in mice. 68,69 When the epithelial layer is damaged, epithelial regeneration and cell proliferation occur rapidly to repair the defective cell barrier. 70 Thus, the inhibition of proliferation can result in disruption of this renewal process, thus leading to the F I G U R E 7 Model indicated that LCA exacerbates necrotizing enterocolitis (NEC) by inhibiting enterocyte proliferation through arresting at G1/S phase in a β-catenin-dependent manner initiation or exacerbation of disease. Notably, most inflammatory bowel diseases, including NEC, Crohn's disease and ulcerative colitis, are closely associated with impaired enterocyte proliferation and disruption of the intestinal barrier. [71][72][73] Our findings confirmed the possibility that LCA resulted in severe disruption of the intestinal barrier due to inhibition of intestinal epithelial renewal and regeneration.
The Wnt/β-catenin pathway is involved in a wide range of physiological and pathological mechanisms. [74][75][76] It is well known that the Wnt/β-catenin signalling pathway plays a key role in embryonic intestinal development, homeostasis of the adult intestine and pathogenesis of intestinal cells. 77,78 Signalling molecules associated with the Wnt/βcatenin pathway are expressed along the villi axis and regulate epithelial homeostasis between cell proliferation and differentiation in both a spatial and temporal manner. 79 Previous studies have shown that Wnt/βcatenin-dependent mechanisms stimulate epithelial cell proliferation. 80,81 Therefore, inhibition of the Wnt/β-catenin pathway leads to the blockade of enterocyte proliferation, and subsequently, the aggravation of NEC. 17,21,82,83 Our data are consistent with these previous findings.
However, we also discovered that LCA, a natural metabolite of the intestinal flora, can specifically regulate the Wnt/β-catenin pathway. However, the detailed underlying mechanisms responsible for how LCA inhibits the Wnt/β-catenin pathway have yet to be elucidated.
Some limitations exist in the current study that need to be considered. LCA caused a deterioration of NEC by inhibiting intestinal cell proliferation through the inhibition of the Wnt/β-catenin pathway. Accordingly, this study indicates a possible role for LCA as a predictors of NEC.