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

View Article: PubMed Central - PubMed

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.

No MeSH data available.


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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.
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f5: 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.

Mentions: To determine if an increase in stem cell number and proliferation was responsible for the increased villus height observed with AA-ORS, we studied the effect of AA-ORS on markers for stem cells and proliferation. Irradiation resulted in a significant decrease in Lgr5 protein levels (Fig. 5a and Supplementary Fig. 1a). AA-ORS increased Lgr5 protein levels in 0 Gy and 5 Gy irradiated mice when compared to saline-treated control groups. However, intestinal tissues from 5 Gy irradiated mice showed no significant change in Bmi1 protein levels when compared to 0 Gy. Similarly, AA-ORS did not change Bmi1 protein levels in 0 Gy and 5 Gy irradiated mice (Fig. 5a and Supplementary Fig. 1b). Lgr5 transcript levels, but not Bmi1 levels, significantly increased in AA-ORS-treated 0 Gy and 5 Gy mice (Fig. 5c,d).


An amino acid-based oral rehydration solution (AA-ORS) enhanced intestinal epithelial proliferation in mice exposed to radiation
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.
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f5: 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.
Mentions: To determine if an increase in stem cell number and proliferation was responsible for the increased villus height observed with AA-ORS, we studied the effect of AA-ORS on markers for stem cells and proliferation. Irradiation resulted in a significant decrease in Lgr5 protein levels (Fig. 5a and Supplementary Fig. 1a). AA-ORS increased Lgr5 protein levels in 0 Gy and 5 Gy irradiated mice when compared to saline-treated control groups. However, intestinal tissues from 5 Gy irradiated mice showed no significant change in Bmi1 protein levels when compared to 0 Gy. Similarly, AA-ORS did not change Bmi1 protein levels in 0 Gy and 5 Gy irradiated mice (Fig. 5a and Supplementary Fig. 1b). Lgr5 transcript levels, but not Bmi1 levels, significantly increased in AA-ORS-treated 0 Gy and 5 Gy mice (Fig. 5c,d).

View Article: PubMed Central - PubMed

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&thinsp;&plusmn;&thinsp;0.4) compared to the saline-treated group (6.1&thinsp;&plusmn;&thinsp;0.3; P&thinsp;&lt;&thinsp;0.001) without a change in D0 (4.8&thinsp;&plusmn;&thinsp;0.1&thinsp;Gy). The Dq values increased from 8.8&thinsp;&plusmn;&thinsp;0.4&thinsp;Gy to 10.5&thinsp;&plusmn;&thinsp;0.5&thinsp;Gy with AA-ORS treatment (P&thinsp;&lt;&thinsp;0.01), indicating an increased radiation tolerance of 1.7&thinsp;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.

No MeSH data available.


Related in: MedlinePlus