Arachidonic Acid Promotes Intestinal Regeneration by Activating WNT Signaling

Abstract

Intestinal regeneration is crucial for functional restoration after injury, and nutritional molecules can play an important role in this process. Here, we found that arachidonic acid (AA) serves as a direct proliferation promoter of intestinal epithelial cells that facilitates small intestinal regeneration in both three-dimensional cultured organoids and mouse models. As shown in the study, during post-irradiation regeneration, AA positively regulates intestinal epithelial cell proliferation by upregulating the expression of Ascl2 and activating WNT signaling, but negatively regulates intestinal epithelial cell differentiation. AA acts as a delicate regulator that efficiently facilitates epithelial tissue repair by activating radiation-resistant Msi1⁺ cells rather than Lgr5⁺ cells, which are extensively considered WNT-activated crypt base stem cells. Additionally, short-term AA treatment maintains optimal intestinal epithelial homeostasis under physiological conditions. As a result, AA treatment can be considered a potential therapy for irradiation injury repair and tissue regeneration.

Keywords: WNT signaling; arachidonic acid; differentiation; fatty acids; intestinal stem cells; irradiation injury; proliferation.

Figure 1

AA Induces Organoids to Form Spheroids and Alters the Transcriptome (A) Representative images of the dose effects of the AA treatments. Scale bars, 100 μm. (B) Quantification of the average spheroid areas after treatment with different concentrations of AA. (C) Representative images of the onset time after AA treatment (100 μM). The images at the various time points were obtained from the same well. Scale bars, 100 μm. (D–F) Heatmaps showing the significant (p_adj < 0.05) differentially expressed genes between the control and AA treatment groups identified based on FPKM (fragments per kilobase per million mapped reads) values. Three biological replicates were included in each group, as shown in the columns. The red and blue colors indicate upregulated and downregulated genes, respectively. The scale showed the row Z score. A transcriptome analysis was performed using mRNA isolated from organoids. (G) Relative mRNA expression levels of cell markers after AA treatment (100 μM) of organoids isolated from the murine small intestine. The control group was treated with 0.1% EtOH. Organoids derived from three animals served as independent biological replicates of each group, and more than 20 organoids were counted. Values are expressed as the mean ± SD. Statistical analysis was performed by one-way ANOVA and Tukey's post hoc test (in B; lowercase letters indicate significant differences, p < 0.05) and Student's t test (in G; asterisk indicates a significant difference, ^∗p < 0.05). AA, arachidonic acid; Ctrl, control; CBC, crypt base columnar; EtOH, ethanol. See also Figure S1.

Figure 2

AA Promotes Proliferation while Inducing a Low Level of Differentiation of Small Intestinal Organoids (A) Representative images of KI67 staining of organoids treated with 100 μM AA. KI67⁺ cells represent proliferative cells. (B) Quantification of the distribution of KI67⁺ cells in AA-treated organoids. (C) Relative mRNA expression level of Mki67 after treatment with different concentrations of AA. (D) Representative images of SOX9 staining of organoids treated with 100 μM AA. SOX9⁺ cells represent stem/progenitor cells. (E) Quantification of the distribution of SOX9⁺ cells in AA-treated organoids. (F) Relative mRNA expression level of Sox9 after treatment with different concentrations of AA. (G, I, K, and M) Representative images of differentiated lineage staining of organoids treated with 100 μM AA. Representative positive cells are marked by black or white arrows. (H, J, L, and N) Quantification results of the differentiated lineage staining of organoids treated with 100 μM AA. The y axes represent the positive cells per section of organoids. (O) Representative images of the AA withdrawal assay. After 3 days of AA treatment (100 μM), the organoids were integrally transferred to AA-free Matrigel and chased for 3 days. (P) Quantification of the crypt number per organoid obtained from 3 days after the removal of AA. The control group was treated with 0.1% EtOH. Organoids derived from three animals served as independent biological replicates of each group, and more than 20 organoids were counted. Values are expressed as the mean ± SD. Data were statistically analyzed using Student's t test (significant difference, ^∗p < 0.05). AA, arachidonic acid; Ctrl, control; EtOH, ethanol. Scale bars, 100 μm.

Figure 3

AA Promotes Small Intestine Epithelium Regeneration after 12 Gy IR Injury (A) Schematic of the IR injury model. (B) Representative images of IHC staining of KI67 in different parts of the small intestine on 4 days after 12 Gy IR and AA treatment (10 mg/mouse per day). Scale bars, 200 μm. (C) Quantification of KI67⁺ crypts per 1-mm tissue section from different parts of the small intestine (n = 3 biological replicates). (D) Quantification of KI67⁺ cell-distribution pattern in different parts of the small intestine (n = 3 biological replicates). (E, G, I, and K) Representative images of differentiated lineages and KI67⁺ regenerative crypts in the duodenum 3 days post 12 Gy IR injury and AA treatment (10 mg/mouse per day). The scale bars for PAS, MUC2, and CHGA represent 200 μm. The scale bars for LYZ represent 100 μm. (F, H, J, and L) Quantification of differentiated lineage staining (n = 3 biological replicates). Values are expressed as the mean ± SD. Data were statistically analyzed using Student's t test (significant difference, ^∗p < 0.05). AA, arachidonic acid; Ctrl, control; IHC, immunohistochemistry; IR, irradiation. See also Figures S2 and S3.

Figure 4

AA Promotes the Regeneration of Small Intestine Epithelial Cells by Activating the WNT Signaling Pathway (A) Cell viability of IEC-6 cells after treatments with different AA concentrations. The viability of the control group (0.1% EtOH) was considered to be 100% (n = 5 individual wells). (B) Representative western blot images of the IEC-6 cells after 24 h of AA treatment (n = 3 individual experiments). (C) TOPFlash/FOPFlash reporter assay. TWS119 is a WNT signaling activator that served as the positive control (n = 3 individual experiments). (D) Representative images of duodenal parts from Axin2-LacZ transgenic mice 3 days after 12 Gy IR injury and 10 mg AA/(mouse·day) AA treatment. Scale bars, 200 μm. (E and F) Quantification of LacZ staining 3 days after 12 Gy IR (n = 3 biological replicates and littermates). Values are expressed as the mean ± SD. Data were statistically analyzed using Student's t test (E and F; the asterisk indicates a significant difference, ^∗p < 0.05) and one-way ANOVA with Tukey's post hoc test (C; lowercase letters indicate significant differences, p < 0.05). AA, arachidonic acid; IEC-6, rat small intestinal epithelial crypt cell; EtOH, ethanol; IR, irradiation; NS, not significant.

Figure 5

AA Triggers the Expression of Ascl2 and Activates the WNT Signaling Pathway (A) Relative mRNA expression level of WNT signaling-related genes in 100 μM AA-treated organoids isolated from the small intestine (n = 3 biological replicates). (B) Representative western blot results of IEC-6 cells treated with si-Ascl2-2 and different doses of AA (n = 3 individual experiments). (C–E) qRT-PCR results of Ascl2 and WNT target genes in the IEC-6 cells after si-Ascl2-2 interference and treatment with different doses of AA (n = 3 individual experiments). Values are expressed as the mean ± SD. Data were statistically analyzed using Student's t test (A; the asterisk indicates a significant difference, ^∗p < 0.05) and one-way ANOVA with Tukey's post hoc test (C, D, and E; lowercase letters indicate significant differences, p < 0.05). AA, arachidonic acid; IEC-6, rat small intestinal epithelial crypt cell; NC, non-specific control. See also Figure S5.

Figure 6

AA Activates WNT Signaling Independent of Lgr5⁺ Cells but Might Act through Msi1⁺ Cells (A) Representative images of flow cytometry. Three days of 10 mg/(mouse·day) AA treatment after 12 Gy IR injury. (B) Quantification of flow cytometry in the radiation-induced injury experiment (n = 6 biological replicates). (C) Representative images of flow cytometry. Normal physiological conditions were administered with 7 days of 10 mg/(mouse·day) AA treatment. (D) Quantification of flow-cytometry results after AA treatment under normal physiological conditions (n = 6 biological replicates). (E) Representative images of LacZ staining of the duodenum of the Msi1-CreERT;R26RLacZ transgenic mouse model. Scale bars, 200 μm. (F) Quantification of the LacZ⁺ fully labeled crypts (Full⁺) and LacZ⁺ partially labeled crypts (Partial⁺) per 1-mm tissue section (n = 3 biological replicates; littermates). (G) Quantification of the short-term lineage-tracing labeling pattern in the duodenum. Msi1-CreERT;R26RLacZ transgenic mice were induced with tamoxifen for 15 h following 10 mg/(mouse·day) AA treatment for 3 days under physiological conditions. The right sides of the fully cut crypt-villus axes were counted. (H) Representative images of the labeling pattern in the short-term lineage-tracing experiments. The standard counting pattern for each type is shown in the dashed box. Scale bars, 50 μm. Values are expressed as the mean ± SD. Data were statistically analyzed using Student's t test (significant difference, ^∗p < 0.05). AA, arachidonic acid; Ctrl, control; IR, irradiation; NS, not significant.