Intravenous acid fibroblast growth factor protects intestinal mucosal cells against ischemia-reperfusion injury via regulating Bcl-2/Bax expression

Abstract

Aim: To detect the effect of acid fibroblast growth factor (aFGF) on apoptosis and gene expression of bax and bcl-2 gene in rat intestine after ischemia/reperfusion (I/R) injury, and to explore the protective mechanisms of aFGF.

Methods: One hundred and eight Wistar rats were randomly divided into sham-operated control group (C) (n = 6), intestinal ischemia group (I) (n = 6), aFGF treatment group (A) (n = 48) and intestinal ischemia-reperfusion group (R) (n = 48). In group I, the animals were killed after 45 min of superior mesenteric artery (SMA) occlusion, while in groups R and A, the rats sustained 45 min of SMA occlusion and were then treated with normal saline and aFGF, respectively, sustained 15 min, 30 min, 1, 2, 6, 12, 24, or 48 h of reperfusion, respectively. In group C, SMA was separated, but without occlusion. Apoptosis in intestinal villus was determined with terminal deoxynucleotidyl transferase mediated dUTP-biotin nick-end labeling technique (TUNEL). Intestinal tissue samples were taken not only for detection of bax and bcl-2 gene expression by RT-PCR, but also for detection of bax and bcl-2 protein expression and distribution by immunohistochemical analysis.

Results: The rat survival rates in aFGF treated group were higher than group R (P<0.05) and the improvement of intestinal histological structures was observed at 2, 6, and 12 h after the reperfusion in group A compared with group R. The apoptotic rates were (41.17+/-3.49)%, (42.83+/-5.23)% and (53.33+/-6.92)% at 2, 6 and 12 h after reperfusion, respectively in group A, apparently less than those of group R at matched time points (50.67+/-6.95, 54.17+/-7.86, 64.33+/-6.47, respectively) (P<0.05). The bax gene transcription and translation were significantly decreased in group A vs group R, while mRNA and protein contents of Bcl-2 in group A were obviously higher than those in group R during 2-12 h period after reperfusion.

Conclusion: The changes in histological structure and the increment of apoptotic rate indicated that the intestinal barrier was damaged after intestinal I/R injury, whilst intravenous aFGF could alleviate apoptosis induced by ischemia and reperfusion in rat intestinal tissues, in which genes of bax and bcl-2 might play important roles.

Figure 1

The expression of Bax protein in sham-operated control group (A), 6 h after ischemia-reperfusion in group R (B) and 6 h after ischemia-reperfusion in group A (C). Protein expression of Bax was weak in sham-operated control group. Bax was strongly expressed in villus at 6 h after reperfusion, and was mainly localized in the whole constitutive cells of villus. The positive stained cells decreased remarkably at 6 h after reperfusion in group A vs group R.

Figure 2

The expression of bcl-2 protein in sham-operated control group (A), 6 h after ischemia-reperfusion in group R (B) and 6 h after ischemia-reperfusion in group A (C) bcl-2 was strongly expressed in the sham-operated intestinal tissues and positive particles of bcl-2 were mainly localized in the lower half of the villus and in the cytoplasm and nuclei of villus cells. bcl-2 expression level decreased at 6 h after reperfusion in group R. Compared with group R, the positive cellular rates of bcl-2 were significantly higher at 6 h after reperfusion in group C.

Figure 3

Expression of bax gene in normal saline treated (A) and aFGF treated (B) rat intestinal villus. Bar indicates the size of RT-PCR cDNA products. 1: 48 h after reperfusion, 2: 24 h after reperfusion, 3: 12 h after reperfusion, 4: 6 h after reperfusion, 5: 2 h after reperfusion, 6: 1 h after reperfusion, 7: 30 min after reperfusion, 8: 15 min after reperfusion, 9: ischemia group, 10: sham-operated control group, 11: DL2000 marker.

Figure 4

Expression of bax gene in normal saline treated (A) and aFGF treated(B) rat intestinal villus. Bar indicates the size of RT-PCR cDNA products. 1: 48 h after reperfusion, 2: 24 h after reperfusion, 3: 12 h after reperfusion, 4: 6 h after reperfusion, 5: 2 h after reperfusion, 6: 1 h after reperfusion, 7: 30 min after reperfusion, 8: 15 min after reperfusion, 9: ischemia group, 10: sham-operated control group, 11: DL2000 marker.

Figure 5

Expression of β-actin gene in normal saline treated (A) and aFGF treated (B) rat intestinal villus. Bar indicates the size of RT-PCR cDNA products. 1: 48 h after reperfusion, 2: 24 h after reperfusion, 3: 12 h after reperfusion, 4: 6 h after reperfusion, 5: 2 h after reperfusion, 6: 1 h after reperfusion, 7: 30min after reperfusion, 8: 15 min after reperfusion, 9: ischemia group, 10: sham-operated control group, 11: DL2000 marker.