HIF2 Regulates Intestinal Wnt5a Expression

Radiation therapy for abdominal tumors is challenging because the small intestine is exquisitely radiosensitive. Unfortunately, there are no FDA-approved therapies to prevent or mitigate GI radiotoxicity. The EGLN protein family are oxygen sensors that regulate cell survival and metabolism through the degradation of hypoxia-inducible factors (HIFs). Our group has previously shown that stabilization of HIF2 through genetic deletion or pharmacologic inhibition of the EGLNs mitigates and protects against GI radiotoxicity in mice by improving intestinal crypt stem cell survival. Here we aimed to elucidate the molecular mechanisms by which HIF2 confers GI radioprotection. We developed duodenal organoids from mice, transiently overexpressed non-degradable HIF2, and performed bulk RNA sequencing. Interestingly, HIF2 upregulated known radiation modulators and genes involved in GI homeostasis, including Wnt5a. Non-canonical Wnt5a signaling has been shown by other groups to improve intestinal crypt regeneration in response to injury. Here we show that HIF2 drives Wnt5a expression in multiple duodenal organoid models. Luciferase reporter assays performed in human cells showed that HIF2 directly activates the WNT5A promoter via a hypoxia response element. We then evaluated crypt regeneration using spheroid formation assays. Duodenal organoids that were pre-treated with recombinant Wnt5a had a higher cryptogenic capacity after irradiation, compared to vehicle-treated organoids. Conversely, we found that Wnt5a knockout decreased the cryptogenic potential of intestinal stem cells following irradiation. Treatment with recombinant Wnt5a prior to irradiation rescued the cryptogenic capacity of Wnt5a knockout organoids, indicating that Wnt5a is necessary and sufficient for duodenal radioprotection. Taken together, our results suggest that HIF2 radioprotects the GI tract by inducing Wnt5a expression.

Fortified Fermented Rice-Acid Can Regulate the Gut Microbiota in Mice and Improve the Antioxidant Capacity

The study aimed to explore the effects of fortified fermented rice-acid on the antioxidant capacity of mouse serum and the gut microbiota. Hair characteristics, body mass index, intestinal villus height, intestinal crypt depth, serum antioxidant capacity, and gut microbiota of mice were first measured and the correlation between the antioxidant capacity of mouse serum and the gut microbiota was then explored. The mice in the lactic acid bacteria group (L-group), the mixed bacteria group (LY-group), and the rice soup group (R-group) kept their weight well and had better digestion. The mice in the L-group had the better hair quality (dense), but the hair quality in the R-group and the yeast group (Y-group) was relatively poor (sparse). In addition, the inoculation of Lactobacillus paracasei H4-11 (L. paracasei H4-11) and Kluyveromyces marxianus L1-1 (K. marxianus L1-1) increased the villus height/crypt depth of the mice (3.043 ± 0.406) compared to the non-inoculation group (R-group) (2.258 ± 0.248). The inoculation of L. paracasei H4-11 and K. marxianus L1-1 in fermented rice-acid enhanced the blood antioxidant capacity of mouse serum (glutathione 29.503 ± 6.604 umol/L, malonaldehyde 0.687 ± 0.125 mmol/L, catalase 15.644 ± 4.618 U/mL, superoxide dismutase 2.292 ± 0.201 U/mL). In the gut microbiota of L-group and LY-group, beneficial microorganisms (Lactobacillus and Blautia) increased, but harmful microorganisms (Candidatus Arthromitus and Erysipelotrichales) decreased. L. paracasei H4-11 and K. marxianus L1-1 might have a certain synergistic effect on the improvement in antibacterial function since they reduced harmful microorganisms in the gut microbiota of mice. The study provides the basis for the development of fortified fermented rice-acid products for regulating the gut microbiota and improving the antioxidant capacity.

An Active Fraction of Trillium tschonoskii Promotes the Regeneration of Intestinal Epithelial Cells After Irradiation

Despite significant scientific advances toward the development of safe and effective radiation countermeasures, no drug has been approved for use in the clinic for prevention or treatment of radiation-induced acute gastrointestinal syndrome (AGS). Thus, there is an urgent need to develop potential drugs to accelerate the repair of injured intestinal tissue. In this study, we investigated that whether some fractions of Traditional Chinese Medicine (TCM) have the ability to regulate intestinal crypt cell proliferation and promotes crypt regeneration after radiation. By screening the different supplements from a TCM library, we found that an active fraction of the rhizomes of Trillium tschonoskii Maxim (TT), TT-2, strongly increased the colony-forming ability of irradiated rat intestinal epithelial cell line 6 (IEC-6) cells. TT-2 significantly promoted the proliferation and inhibited the apoptosis of irradiated IEC-6 cells. Furthermore, in a small intestinal organoid radiation model, TT-2 promoted irradiated intestinal organoid growth and increased Lgr5+ intestinal stem cell (ICS) numbers. More importantly, the oral administration of TT-2 remarkably enhanced intestinal crypt cell proliferation and promoted the repair of the intestinal epithelium of mice after abdominal irradiation (ABI). Mechanistically, TT-2 remarkably activated the expression of ICS-associated and proliferation-promoting genes and inhibited apoptosis-related gene expression. Our data indicate that active fraction of TT can be developed into a potential oral drug for improving the regeneration and repair of intestinal epithelia that have intestinal radiation damage.

Vitamin D Receptor Protects against Radiation-Induced Intestinal Injury in Mice via Inhibition of Intestinal Crypt Stem/Progenitor Cell Apoptosis

It is urgent to seek new potential targets for the prevention or relief of gastrointestinal syndrome in clinical radiation therapy for cancers. Vitamin D, mediated through the vitamin D receptor (VDR), has been identified as a protective nutrient against ionizing radiation (IR)-induced damage. This study investigated whether VDR could inhibit IR-induced intestinal injury and explored underlying mechanism. We first found that vitamin D induced VDR expression and inhibited IR-induced DNA damage and apoptosis in vitro. VDR was highly expressed in intestinal crypts and was critical for crypt stem/progenitor cell proliferation under physiological conditions. Next, VDR-deficient mice exposed to IR significantly increased DNA damage and crypt stem/progenitor cell apoptosis, leading to impaired intestinal regeneration as well as shorter survival time. Furthermore, VDR deficiency activated the Pmaip1-mediated apoptotic pathway of intestinal crypt stem/progenitor cells in IR-treated mice, whereas inhibition of Pmaip1 expression by siRNA transfection protected against IR-induced cell apoptosis. Therefore, VDR protects against IR-induced intestinal injury through inhibition of crypt stem/progenitor cell apoptosis via the Pmaip1-mediated pathway. Our results reveal the importance of VDR level in clinical radiation therapy, and targeting VDR may be a useful strategy for treatment of gastrointestinal syndrome.

Molecular Mechanism of Stimulation of Na-K-ATPase by Leukotriene D4 in Intestinal Epithelial Cells

Na-K-ATPase provides a favorable transcellular Na gradient required for the functioning of Na-dependent nutrient transporters in intestinal epithelial cells. The primary metabolite for enterocytes is glutamine, which is absorbed via Na-glutamine co-transporter (SN2; SLC38A5) in intestinal crypt cells. SN2 activity is stimulated during chronic intestinal inflammation, at least in part, secondarily to the stimulation of Na-K-ATPase activity. Leukotriene D4 (LTD4) is known to be elevated in the mucosa during chronic enteritis, but the way in which it may regulate Na-K-ATPase is not known. In an in vitro model of rat intestinal epithelial cells (IEC-18), Na-K-ATPase activity was significantly stimulated by LTD4. As LTD4 mediates its action via Ca-dependent protein kinase C (PKC), Ca levels were measured and were found to be increased. Phorbol 12-myristate 13-acetate (PMA), an activator of PKC, also mediated stimulation of Na-K-ATPase like LTD4, while BAPTA-AM (Ca chelator) and calphostin-C (Cal-C; PKC inhibitor) prevented the stimulation of Na-K-ATPase activity. LTD4 caused a significant increase in mRNA and plasma membrane protein expression of Na-K-ATPase α1 and β1 subunits, which was prevented by calphostin-C. These data demonstrate that LTD4 stimulates Na-K-ATPase in intestinal crypt cells secondarily to the transcriptional increase of Na-K-ATPase α1 and β1 subunits, mediated via the Ca-activated PKC pathway.