Risk factors of development of gut-derived bacterial translocation in thermally injured rats

AIM: Studies have demonstrated that gut-derived bacterial translocation (BT) might play a role in the occurrence of sepsis and multiple organ dysfunction syndrome (MODS). Yet, no convincing overall analysis of risk factors for BT has been reported. The purpose of this study was to evaluate the related factors for the development of BT in burned rats. METHODS: Wistar rats were subjected to 30% third-degree burns. Then samples were taken on postburn d 1, 3, and 5. Incidence of BT and counts of mucosal bifidobacteria, fungi and E.coli mucus sIgA, degree of injury to ileal mucosa, and plasma interleukin-6 were observed. Univariate analysis and multivariate logistic regression analysis were performed. RESULTS: The overall BT rate was 53.9% (69 in 128). The result of univariate analysis showed that the levels of plasma endotoxin and interleukin-6, the counts of mucosal fungi and E. coli and the scores of ileum lesion were markedly increased in animals with BT compared with those without (P=0.000-0.005), while the levels of mucus sIgA and the counts of mucosal bifidobacteria were significantly reduced in animals with translocation compared with those without (P=0.000). There was a significant positive correlation between mucus sIgA and the counts of mucosal bifidobacteda (r=0.74, P=0.001). Moreover, there were strong negative correlations between scores of ileum-lesion and counts of bifidobacteria (r=-0.67, P=-0.001). Multivariate logistic regression revealed that ileum lesion score (odds ratio [OR] 45.52, 95% confidence interval [CI] 5.25-394.80), and counts of mucosal bifidobacteria (OR 0.039, 95% CI 0.0032-0.48) were independent predictors of BT secondary to severe burns. CONCLUSION: Ileal lesion score and counts of mucosal bifidobacteria can be chosen as independent prognosis factors of the development of BT. Specific interventions targeting these high-risk factors might be implemented to attenuate BT, including strategies for repair of damaged intestinal mucosae and restoration of the balance of gastrointestinal flora.

Enhanced anti-apoptosis and gut epithelium protection function of acidic fibroblast growth factor after cancelling of its mitogenic activity

AIM: Mitogenic and non-mitogenic activities of fibroblast growth factor (FGF) are coupled to a range of biological functions, from cell proliferation and differentiation to the onset of many diseases. Recent reports have shown that acidic fibroblast growth factor (aFGF) has a powerful antiapoptosis function, which may have potentially therapeutical effect on gut ischemia and reperfusion injuries. However,whether this function depends on its mitogenic or nonmitogenic activity remains unclear. In this study, we identified the source of its anti-apoptosis function with a mutant,aFGF28-154 and observed its effect on reducing gut ischemia and reperfusion injury.METHODS: aFGF28-154 was generated by amplification of appropriate DNA fragments followed by subcloning the products into pET-3c vectors, then they were expressed in BL21 (DE3) cells and purified on an M2 agarose affinity column.This mutant aFGF28-154 maintained its non-mitogenic activity and lost its mitogenic activity. With a dexamethasone (DEX)-induced mouse thymocyte apoptosis model in vitro and in vivo, we studied the anti-apoptotic function of aFGF28-154. Also, in vivo study was performed to further confirm whether aFGF28-154 could significantly reduce apoptosis in gut epithelium after gut ischemia-reperfusion injury in rats. Based on these studies, the possible signal transduction pathways involved were studied.RESULTS: With a dexamethasone (DEX)-induced mouse thymocyte apoptosis model in vitro and in vivo, we found that the anti-apoptotic function of aFGF28-154 was significantly enhanced when compared with the wild type aFGF. In vivo study further confirmed that aFGF28-154 significantly reduced apoptosis in gut epithelium after gut ischemiareperfusion injury in rats. The mechanisms of anti-apoptosis function of aFGF28-154 did not depend on its mitogenic activity and were mainly associated with its non-mitogenic activities, including the intracellular calcium ion balance protection, ERK1/2 activation sustaining and cell o/de balance.CONCLUSION: These findings emphasize the importance of non-mitogenic effects of aFGF, and have implications for its therapeutic use in preventing apoptosis and other injuries in tissues and internal organs triggered by ischemia-reperfusion injury.

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

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 nickend 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 groupR 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.

EXPRESSION OF HUMAN BETA-DEFENSIN 3 IN COS-7 CELL

To establish a cell line for stable expression of human beta-defensin 3 (hBD3). Methods Full length cDNA of hBD3 was isolated from previously constructed pGEM-hBD3 and then inserted into pcDNA3. The recombinant vector identified carrying hBD3 with right direction was introduced into COS-7 cells by Lipofe-ctamine. Cell clones survived in G418-rich medium and with stable expression of hBD3 in both mRNA and protein levels were identified by RT-PCR and Western blot respectively. Genomic integration of the hBD3 gene with the COS-7 cells was confirmed by Southern dot blot and primary analysis. The antimicrobial activity of the secreted hBD3 was also evaluated. Results COS-7 cells transfected with pcDNA3-hBD3 expressed hBD3 stably in mRNA and protein level. Southern dot blot analysis showed successful integration of the hBD3 gene into the genome of COS-7 cell and the hBD-3 protein secreted into the culture medium showed antimicrobial activity. Conclusion We successfully established a hBD3-expressing cell line.