An amino acid-based oral rehydration solution (AA-ORS) enhanced intestinal epithelial proliferation in mice exposed to radiation

Liangjie Yin, Reshu Gupta, Lauren Vaught, Astrid Grosche, Paul Okunieff, Sadasivan Vidyasagar, Liangjie Yin, Reshu Gupta, Lauren Vaught, Astrid Grosche, Paul Okunieff, Sadasivan Vidyasagar

Abstract

Destruction of clonogenic cells in the crypt following irradiation are thought to cause altered gastrointestinal function. Previously, we found that an amino acid-based oral rehydration solution (AA-ORS) improved gastrointestinal function in irradiated mice. However, the exact mechanisms were unknown. Electrophysiology, immunohistochemistry, qPCR, and Western blot analysis were used to determine that AA-ORS increased proliferation, maturation, and differentiation and improved electrolyte and nutrient absorption in irradiated mice. A single-hit, multi-target crypt survival curve showed a significant increase in crypt progenitors in irradiated mice treated with AA-ORS for six days (8.8 ± 0.4) compared to the saline-treated group (6.1 ± 0.3; P < 0.001) without a change in D0 (4.8 ± 0.1 Gy). The Dq values increased from 8.8 ± 0.4 Gy to 10.5 ± 0.5 Gy with AA-ORS treatment (P < 0.01), indicating an increased radiation tolerance of 1.7 Gy. We also found that AA-ORS treatment (1) increased Lgr5+, without altering Bmi1 positive cells; (2) increased levels of proliferation markers (Ki-67, p-Erk, p-Akt and PCNA); (3) decreased apoptosis markers, such as cleaved caspase-3 and Bcl-2; and (4) increased expression and protein levels of NHE3 and SGLT1 in the brush border membrane. This study shows that AA-ORS increased villus height and improved electrolyte and nutrient absorption.

Conflict of interest statement

Drs. Vidyasagar and Paul Okunieff have shares in Entrinsic Health Solutions, Inc.

Figures

Figure 1. AA-ORS increased crypt count &…
Figure 1. AA-ORS increased crypt count & villus length following irradiation.
Normal saline (saline) was used as a control; saline and AA-ORS were given by gastric gavage. 6 mice per radiation group (0, 1, 3, 5, 6, 7, 9, 13 and 15 Gy) with and without treatment. (a) Semi-log survival curve showing the effect of AA-ORS on crypt count. AA-ORS shifted the graph to the left. The crypt survival curve was modeled using a single-hit, multi-target cell survival model to assess the biological effect. The probability of survival of the mitotic cells in the crypt following radiation was calculated using the equation [S = 1-(1-e^-D/D0)n. S represents the fraction of mitotic cells in the crypts that survived in each of the radiation dose, D represents radiation dose; D0, a measure of the intrinsic radiation resistance of the crypt reproductive units. Dq values for saline treated mice and AA-ORS treated mice are represented by black arrow and red arrow respectively. Dq is calculated from the formula Dq = D0 In n. Without constraining constant cell sensitivity, the N values were 10.4 ± 0.2 and 5.3 ± 0.1 (P < 0.001), indicating a near doubling of progenitor units per circumference from a control. When a constant D0 (4.8 ± 0.1 Gy) was constrained, the difference remained significant at 8.8 ± 0.4 to 6.1 ± 0.3 (P < 0.001). (b) Shows the height of villus following treatment using saline and AA-ORS in irradiated mice. Significant increase in villus height with AA-ORS treated mice compared to mice receiving saline as treatment. Crypts per circumference were counted, and villus length was measured from 10 sections obtained from the ileum. Data are shown as the mean ± S.E.M. for 6 mice per group. *Indicates statistically significant difference (P < 0.01). Normal saline (saline) was used as control and both saline and AA-ORS was given by gastric gavage.
Figure 2
Figure 2
(a) Confococal microscopy of longitudinal ileal section with prominently stained epithelial cells along the villus length. Paraffin embedded tissues at 5-μm thickness were used. Cell nuclei were stained with DAPI (blue) and EdU+ epithelial cells stained red. Image Pro Plus software was used for measurements of distance migrated by the EdU+ cells along the villus height. Bar – 100 μm. Cells were scored per entire crypt and villus unit. At least 60 crypts and corresponding villi were analyzed per mouse. EdU-labeled cells were normalized to the total cell number per crypt or villus. Minimum of five well-oriented villi were counted per tissue section and the results were averaged. EdU+ cells were seen all the way to the tip of the villus in 5 Gy irradiated tissues but not in AA-ORS-treated mice. (b) EdU+ cell migration distance measured at 72 hours. The 5 Gy irradiated saline-treated mice showed significant decrease in cell migration distance (black bar) compared to 0 Gy. AA-ORS had increased migration distance when compared to irradiated saline treated mice. Values are means ± S.E.M. for 6 mice per group.
Figure 3
Figure 3
(a,b) Ussing chamber flux studies using 22Na and 36Cl showing the effect of AA-ORS on Na+ and Cl− absorption. AA-ORS increased net Na+ (JnetNa) and Cl− (JnetCl) absorption in 0 Gy and 5 Gy irradiated tissues (n = 8). (c) Immunohistochemistry showing a magnified view of NHE3 expression (red) along the brush border membrane (BBM) of villus epithelial cells (white arrows). Paraffin embedded tissues at 5-μm thickness were used. Cell nuclei were stained with DAPI (blue). A minimum of five well-oriented villi were used. (d) Western blot analysis for NHE3 protein, (e) graphical representation of NHE3 protein density in intestinal tissues from mice treated with saline (black bars) or AA-ORS (hatched bars) following 0 or 5 Gy irradiation. Immunoblots were repeated four times. Values are means ± S.E.M. from n = 4, *indicates statistically significant difference (P < 0.05) from saline treated animals. (f) NHE3 transcript levels in intestinal tissues from mice treated with saline (black bars) or AA-ORS (hatched bars) following 0 or 5 Gy irradiation. Values are means ± S.E.M. from n = 6, *indicates statistically significant difference (P < 0.05) from saline treated animals. Saline or AA-ORS was given for 6 days.
Figure 4. Glucose-stimulated sodium absorption and SGLT1…
Figure 4. Glucose-stimulated sodium absorption and SGLT1 protein levels.
(a) Ussing chamber flux studies using 22Na showing the effect of AA-ORS on glucose coupled Na absorption. AA-ORS treatment increased JnetNa absorption in 5 Gy irradiated tissues (n = 8). (b) Western blot analysis for SGLT1 protein and beta-galactosidase, showed increased protein levels with AA-ORS treatment in villus cells from 0 Gy and 5 Gy mice. Immunoblots were repeated four times. (c) Normalized SGLT1 protein levels for western analysis. Significant difference in SGLT1 protein levels was observed in 5 Gy irradiated mice treated with AA-ORS when compared to 5 Gy mice. (d) SGLT1 transcript levels in intestinal tissues from mice treated with saline (black bars) or AA-ORS (hatched bars) following 0 or 5 Gy irradiation. Values are means ± S.E.M. from n = 6, *indicates statistically significant difference (P < 0.05) from saline treated animals. Saline or AA-ORS was given for 6 days.
Figure 5. Protein levels and mRNA expression…
Figure 5. Protein levels and mRNA expression of Lgr5, Bmi1, p-Akt, Akt, p-Erk, Erk in villus epithelial cells from mice treated with saline and AA-ORS following 0 and 5 Gy irradiation.
Immunoblots were repeated four times and q-PCR were repeated 6 times. (a) Western blot analysis for stem cell and proliferation markers (Lgr5, Bmi1, p-Akt, Akt, p-Erk, Erk and PCNA). The protein band of interest was normalized to the total amount of protein in each lane using Coomassie blue stain. (b) Western blot analysis for apoptotic proteins (Bcl2, Bax, cleaved caspase-3, caspase-3 and p53). (c) Lgr5 mRNA levels in mice treated with saline or AA-ORS and 0 Gy or 5 Gy radiation. (d) Changes in Bmi1 mRNA levels in mice treated with saline or AA-ORS and 0 Gy or 5 Gy. (e) Changes in Erk mRNA levels in mice treated with saline or AA-ORS treatment and 0 Gy or 5 Gy. (f) Changes in Akt mRNA levels in mice treated with saline or AA-ORS treatment and 0 Gy or 5 Gy. (g) mRNA expression for caspase-3. Values are means ± S.E.M from n = 6 different mice repeated in triplicates. #P < 0.05 and *P < 0.001 compared with saline control.
Figure 6. Representative microphotographs of the distribution…
Figure 6. Representative microphotographs of the distribution of Lgr5+, Ki-67+ and PCNA+ cells within ileal mucosa of 0 Gy (left) and 5 Gy (right) after treatment with saline (top) or AA-ORS (bottom).
(a) Immunostaining for Lgr5: Lgr5+ cells were seen in the lower 1/3rd of the crypt. Mice irradiated with 5 Gy resulted in a significant decline of Lgr5+ stem cells in ileal crypts, and AA-ORS increased Lgr5+ stem cells. Scale bars represent 25 μm (b) Mean number of Lgr5+ cells expressed in crypt. Error bars indicate S.E.M. (c) Immunostaining for Ki-67: The number of Ki-67-expressing cells, a proliferation marker, showed no significant difference in 0 Gy radiated mice treated with AA-ORS when compared to saline-treated groups. 5 Gy irradiated mice showed significant increase in Ki-67+ cells with AA-ORS treatment. Scale bars represent 100 μm. (d) Mean number of Ki-67 expressing cells in crypt and/or villus cells. Error bars indicate S.E.M. (e) Immunostaining for PCNA: The number and distribution of PCNA+ cells. PCNA+ cells were reduced in mice after 5 Gy radiation, but increased with AA-ORS treatment. Scale bars represent 100 μm. (f) Mean number of PCNA expressing cells in crypt and/or villus cells. Error bars indicate S.E.M.
Figure 7. Schematic figure of small intestinal…
Figure 7. Schematic figure of small intestinal villus and enterocytes: AA-ORS treatment increases rapidly dividing stem cells that are Lgr5+, as well as proliferation markers p-Erk, p-Akt and PCNA.
The treatment also increases cleaved caspase-3, p53 and Bcl-2. The AA-ORS treatment increases villus height, increased expression of NHE3, SGLT1 and β-galactosidases thereby increasing electrolyte absorption, sodium-coupled glucose absorption, and breakdown of disaccharides at the brush border membrane, respectively. A cartoon of the enterocyte on the top right shows the functional improvement in NHE3 mediated Na+ absorption and glucose-coupled sodium transport with AA-ORS.

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