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Total parenteral nutrition-induced apoptosis in mouse intestinal epithelium: modulation by keratinocyte growth factor
Barbara E Wildhaber1, Hua Yang, Daniel H Teitelbaum
1Section of Pediatric Surgery, C.S. Mott Children's Hospital, Department of Surgery, University of Michigan, Ann Arbor, Michigan, USA.
Background:
Total parenteral nutrition (TPN) induces epithelial cell (EC) apoptosis. Keratinocyte growth factor (KGF) increases EC growth; however, little is known of its effect on apoptosis. This study aimed to determine the effect of recombinant human KGF (rHuKGF) on small bowel EC apoptosis. We further determined mRNA expression of Bcl-2 family members (major mediators of epithelial cell apoptosis) with TPN and whether KGF administration influences Bcl-2 family expression in EC.
Methods:
C57BL/6J mice (n = 6/group) received oral feeding (Control); TPN; or TPN plus daily intravenous rHuKGF (TPN+KGF). After 7 days, intestines were harvested and EC isolated. Villus height was determined by microscopy and EC proliferation by immunohistochemistry using incorporation of 5-bromodeoxyuridine (BrdU). Apoptosis was identified by Annexin V as well as by TUNEL staining. EC mRNA expression of Bcl-2 family members was measured by reverse-transcriptase polymerase chain reaction and Bcl-2 protein level by immunoblot analysis.
Results:
Villus height in Controls was 310 +/- 42 microm. This decreased with TPN to 210 +/- 45 microm; however, villus height was preserved in TPN + KGF mice (273 +/- 39 microm). EC proliferation rates decreased significantly in TPN mice, and this decline was prevented by administration of rHuKGF. EC apoptotic rate in Controls was 14.4 +/- 5.1%; TPN administration resulted in doubling of largely prevented TPN-induced EC apoptosis (29.4 +/- 11.3%) rHuKGF administration largely prevented TPN-induced EC apoptosis (17.2 +/- 5.6%). Proapoptotic Bcl-2 members changed minimally with TPN or rHuKGF; however, the anti-apoptotic Bcl-2 changed significantly: Control 0.78 +/- 0.24; TPN 0.10 +/- 0.13; rHuKGF administration prevented the decline in Bcl-2 expression observed with TPN (0.76 +/- 0.36). EC Bcl-2 protein levels were: Control 0.16 +/- 0.13; TPN 0.18 +/- 0.16; and TPN+KGF 0.47 +/- 0.19.
Conclusions:
TPN-induced apoptosis was associated with decreased Bcl-2 mRNA expression. rHuKGF decreased TPN-induced EC apoptosis and increased Bcl-2 expression. rHuKGF administration may have benefit in patients on TPN.