| In the large-scale, intensive livestock production, stress exists everywhere. With global warming, high temperature as one environmental stressor is an important factor of impacting livestock and poultry production in summer. Studies have proved that high temperature stress on animals can cause the structural damage of small intestinal villous epithelial cells, negatively affects not only the intestinal barrier function, but also nutrient digestion and absorption of intestinal epithelial cells, and leads to production performance decrease. For this reason, stress-induced intestinal structural damage and repair has always been an important research direction for domestic and foreign scholars. Heat stress Chinese herb additive, Qingliag granule, has effects of improving high-temperature-induced decline in production performance and immune function. Therefore, this doctoral thesis focused to explore high-temperature-induced structural injury of intestinal epithelial cells in pig and repair effects and mechanisms of Qingliag granule on the injury. It will lay a foundation for developing new heat-stress Chinese herb additives and searching more repair agents for intestinal structural damage.Forty eight Chinese experimental mini pigs (half male and half female), with 7.15±0.61 kg body weight at about 2 months of age, were divided into three groups according to similar principle of body weight and litter origin:Control group (CG), high temperature group (HTG) and Chinese herb group (CHG),16 animals each group. The control group was always kept at 23℃, and the high temperature group and Chinese herb group were raised in high-temperature environment which imitated Beijing summer climate with a cyclic variation from 26℃to 40℃and at 40℃for continuous four hours. The experiment lasted for continuous 10 days, basal diet fed in all groups, at the same time, Qingliang granule was given to animals in Chinese herb group. The results were as follows:1. In the high temperature group at the 3rd day, the average rectal temperature (40.22℃±0.25℃) of experimental pigs was beyond normal upper limit of reference values (38.5℃-39.5℃), the average daily gain (195 g±62 g) was obviously lower than that in the control group (238 g±80 g) and Chinese herb group (230 g±41 g). Heat-induced rapid pant, lassitude, lethargy and increased drinking water occurred in animals with decreased performance trait. Histological observation of the small intestine mucosa found that the most serious damage was seen in the duodenum and jejunum at first 3 days, the villus height, crypt depth and their ratio significantly decreased, and since then gradually returned and closed to the control group after 6 days. These results showed that animal adaptation period was more than 6 days on the cyclic-changed high temperature (26℃-40℃) stimulation. The average rectal temperature (39.52℃±0.31℃) obviously decreased (p< 0.05), average daily gain (229 g±51 g) increased (p< 0.05), the villus height, crypt depth and their ratio were somewhat improved in the Chinese herb group compared with the high temperature group. These results implied that Qingliang granule can effectively regulate rectal temperature in pigs under high temperature stress, promote the repair of small intestinal villus injury, restore intestinal villus morphological structure, and increase the absorption area of small intestinal villi to play a role in heat stress and eliminate the negative effects of high temperature on the physiological indexes and production performance in pigs.2. High temperature can cause tissue damage in intestinal mucosa. In the high temperature group, DNA and DAO contents in duodenum and jejunum decreased and were significantly different with the control group at the first three days, but six day later gradually returned and closed to the control group without obvious difference. Ultrastructural observation showed that in the high temperature group, the intestinal epithelial cell mitochondria, secondary lysosomes and transitional vesicles increased in the number, which showed the cells respond to high temperature by enhancing metabolism and promoting the repair of damage. Further studies showed that EGF and EGFR protein levels in jejunum decreased at first and then regain; EGFR was significantly reduced (p< 0.01). The trend of their mRNA expression is consistent with that of protein expression. These suggested that jejunal tissue injury and repair be closely related with the EGF and EGFR signaling down-regulation. Qingliang granule can promote not only DNA synthesis to accelerate intestinal epithelial cell turnover by maintaining the DAO content, but also the proliferation and repair of small intestinal villus epithelial cells by up-regulating the EGF and EGFR protein levels and mRNA expression, to protect the integrity of intestinal mucosal barrier against high temperature-induced epithelial cell injury. 3. Jejunal gene differential expression in high-temperature-treated pig was studied using Affymetrixz gene chip technology. The results revealed that there were 143 differential expression genes in the high temperature group compared with the control group, of which,68 genes were up-regulated and 75 genes were down-regulated (ratio> 2 or ratio<-2). Based on functional classification of Gene Ontology (GO), these differential genes are associated with endocrine, immune function, cell damage and repair, apoptosis, signal transduction, etc. Pathway analysis showed that high temperature stress can lead to differences in genome-wide expression, and 1/3 of differential gene signal pathways are related to EGF-mediated cell regeneration and repair, which maybe is an important mechanism for repair of intestinal epithelial cell injury caused by high temperature.4. IEC-6 were cultured in vitro and treated at 41℃for three hours, the study on the mechanism of IEC injury and repair showed that high-temperature-treated IEC-6 viability and proliferation index decreased by flow cytometry; EGF and EGFR mRNA level reduced significantly, and ERK1 and ERK2 signaling proteins were up-regulated by RT-PCR and Western blot (p< 0.01), respectively. These suggested that IEC-6 thermal injury and repair mechanisms be closely associated with MAPK signaling pathway. Main Chinese herb effective components, volatile oils of Atractylodes lancea and Agastache rugosa from Qingliang granule, can promote IEC-6 proliferation and increase its viability by improving the proportion of G2M and S phases in the epithelial cell cycle, realize IEC-6 viability/cell cycle, cell proliferation regulation and damaged cell repair through the regulation of P-ERK1/ERK2 signaling proteins in MAPK signaling pathway.
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