Polyamine Accumulation Induced Rat Enterocyte Apoptosis During Early Weaning Acute Phase, Mechanism And Modulation

To explore the inner factor for early-weaning gut impairment, according to the high similarity between two phase reaction after polyamine infusion and early-weaning gut adaptation reaction, three trails on rat pups were designed. The 1^st was to explore the effect of sharply increased polyamine supply on gut development in acute phase of early weaning. The 2^nd was to comparative study the gut polyamine metabolism difference of different early-weaning age. Meanwhile, according to the fact that polyamine infusion during suckling peroid could induces precious maturation of rat pups gut and the polyamine metabolic feature during early weaning period, the 3^rd trail investigated the possibility of artificial induced gut maturation on modifing early-weaning stress.Trial 1 Effect of polyamine accumulation on 17d rat enterocyte loss during early weaning acute phaseBoth polyamine competitive inhibitor Difluoromethyl ornithine (DFMO) and fasting could depress endogenous polyamine production, meanwhile, fasting treat had no exogenous polyamine input. In this trail, DFMO, Fasting and DFMO+Fasting were used to explore the effect of sharply increased gut polyamine supply on 17d early-weaning rat. Rat pups from same litter were assigned to one of four treatments: W, 0.1ml deionized water was infused half hour before weaning and then pups were normaly weaned; D, 0.1ml 2% DFMO solution was infused half hour before weaning, and 2% DFMO solution was provided for drinking after weaning; F, 0.1ml deionized water was infused half hour before weaning then pups were fasted only provided with normal water for drinking; M, 0.1ml 2% DFMO solution was infused and provided DFMO solution as treat D , and then be fasted. 24 litters were decapitated on 4h, 8h, 12h, 16h, 20h, 24h after weaning. Intestine weight, length, protein content, DNA content, disaccharidase specific activity, intestine morphology and intestine polyamine content were recorded. The results indicated, intestine polyamine content significantly accumulated after weaning 12h, compared with pre-weaning, the putrescine, spermidine, spermine content increased 115. 9%(P<0. 01), 75. 2%(P<0. 01), 30. 5%(P<0. 01), separately. DFMO, fasting, and DFMO+fasting could actively suppress early-weaning induced polyamine accumulation. The suppression rate of DFMO reached 83. 7%, 87.3% and 68.1%, the F treat was 89.4%, 89.3% and 48.7%, while the M treat reached 100%. Suppression polyamine accumulation had significant depressed enterocyte loss, indicated by obviously increased intestine protein content and higher villus height. The endogenous polyamine accumulation caused enterocyte loss by the time of 12-16h after early-weaning, while, the exogenous polyamine from feed would cause the enterocyte loss by the time of 4h after early-weaning. These observations indicated that increasing polyamine supply during early-weaning acute phase could cause polyamine accumulation, suppression of the accumulation can modify the entercyte loss and villus atrophy.Trail 2 The essence and molecular mechanism of polyamine accumulation induced enterocyte loss of early-weaning ratPolyamine infusion to suckling rat pups could induce intestine polyamine accumulation and enterocyte apoptosis, while had no effect on polyamine content and enterocyte survival of rats after weaning. This difference hinted the development change of intestine polyamine feedback mechanism. SD rat pups from same litter decapitated on different time, 0h after 17d weaning (1700), 12h after 17d weaning (1712), 0h after 21d weaning (2100) and 12h after 21d weaning. Intestine development related parameters, such as, intestine length, mucosa weight, mucosa protein and DNA content, lactase and sucrase specific activity, antizyme protein content in jejunum and ileum mucosa were measured, and ultrastructure of mid-intestine was observed under transmission electron microscope(TEM). The intestine maturation degree of 21d old rats was significant higher than that of 17d old rats, with higher gut length, mucosa weight and sucrase specific activity. TEM observed typical apoptosis enterocyte figure with condensed chromatin, increased nucleus-cytoplasm ratio, vacuolization cytoplasma, shrinking cellular membrane in 1712 and 2112 group. The jejunum mucosa antizyme content would increase with gut development, with 2100 group had higher content by 185.7% (P<0.05) and 46% (P>0.05) in jejunum and ileum mucosa, respectively. Early weaning had impact on mucosa antizyme content regarded with weaning age. 1712 had no significant influence on antizyme content, while 2112 increased antizyme content by 121. 7% (P<0. 05) compared with 2100. Early weaning had no impact on ileum antizyme regardless of weaning age. These observations indicated, apoptosis was important way direct enterocyte loss and villus atrophy during early-weaning acute phase. Intestine 0AZ1 protein had development change during rat pup growth, and insufficient OAZ1 protein was the main reason for polyamine accumulation during early-weaning acute phase.Trail 3 Pre-weaning infusion polyamine (spermide or spermine) to suckling rats improved early-weaning growth and gut developmentTo explore whether polyamine induced gut maturation could modify early-weaning tress, SD suckling rats of 14d old from same litter were infused 5μmol spermidine (Spd) , 5μmol spermine (Spm) or saline (control) for 3 days, respectivetly. Pups were early-weaned on 17d old, half were decapitated after weaning immediately, the residue was feeding alone and decapitated on 20d old. Body weight and intestine development parameters were examined. Spd and Spm promoted 17d pups gut maturation with higher intestine weight, length, protein content, DNA content and sucrase specific activity. Meanwhile, Spd and Spm prevent body loss of early-weaning with slight weight gain by 3.7% (P>0. 05) and 0. 7% (P>0. 05) compared with pre-weaning. However, Spd and Spm group could not improve rat body weight on 20d, and Spm group significantly decreased body weight by 9.4% (P<0. 01) compared with control group. Spd and Spm treat changed intestine goblet cell subtypes. Infusion group had much more acid goblet cell after weaning. Intestine adaptive reaction of weaning stress was dimished in infusion groups, with no change on intestine weight, length, protein and DNA content between pre-weaning and post-weaning. Spd and Spm group had decreased intestine polyamine content, especially for decreased putrescine content by 69.1% (P<0. 01) and 73. 8% (P<0.01) pre- and post-weaning in Spm group. These observations indicated that suckling infusion spermidine and spermine could promote gut maturation, this artificial maturation could diminish weaning stress, modify weaning body loss, improve mucus barrier.