| There is fundamental knowledge of the consequences of subacute ruminal acidosis (SARA) on ruminal epithelial barrier function, remarkably little information is currently available about the underlying molecular changes in ruminal epithelial barrier function during SARA. The objective of this thesis was to investigate the effects of SARA on ruminal epithelial barrier function at the tissue, molecular and cellular level and was to explore the regulational roles of cellular signal pathway in SARA induced tight junction erosion.1. Experimental SARA induced by high-grain feeding in goatsTen ruminally fistulated, castrated male goats were randomly assigned to either a hay diet (0% grain; n=5) or high-grain (HG) diet group (65% grain; n=5) with continuous feeding of 7 weeks. Weekly 0,2,3,4,6,8 and 12 h after morning feeding, rumen fluid was collected to monitor the changes in ruminal pH. The rumen fluid collected at 0、3、6 and 12 h after morning feeding was used to determine volatile fatty acid (VFA) concentration. The rumen fluid collected at 6 h after morning feeding was used to determine lipopolysaccharide (LPS) concentration. At 6 h after morning feeding, the jugular vein blood was collected to measure the free LPS in peripheral blood. The results showed that, at 1st,2nd,3rd,6th and 7th weeks, the rumen pH below 5.8 lasted for more than 4 h within 12 h in HG-fed goats. The results suggested that, at 1st,2nd,3rd,6th and 7th weeks, SARA has been successfully induced. Throughout the feeding period, compared with the control group, HG feeding reduced rumen pH, acetate concentration and acetate-propionate ratio significantly (P<0.05) and increased concentrations of ruminal propionate, butyrate and free LPS significantly (P<0.05). Principal response curve (PRC) analysis of the overall trend of changes in rumen physiological parameters with feeding weeks. The results showed that there was a dramatic change in physiological parameters of HG-fed goats at the 1st week of feeding, then there is a process of adaptation and recovery, but at 6th week, the SARA occurs again. The free LPS in the peripheral blood was not detectable in all hay-fed goats. At the 1st and 2nd weeks, the free LPS in the blood of HG-fed goats was not detectable. At the 3nd and 4th weeks, the free LPS in the blood was detectable in some HG-fed individuals. At 5th,6th and 7th weeks, the free LPS in the peripheral blood were detectable in all HG-fed goats. These results indicate that long-term HG diet feeding has induced SARA successfully and the incidence of SARA experienced the process of occurrence, adaptation and repeated occurrence.2. The effect of SARA on ruminal epithelial barrier functionOn day 50, goats were slaughtered at 4-5 h after last feeding and samples were collected. As expected, the ruminal pH of HG-fed goats was lower than that of hay-fed goats (P<0.00I). HG feeding increased the concentration of propionate, butyrate, valerate, isovalerate, total VFA and lactic acid (P<0.05), but decreased the acetate concentration (P=0.009) when compared with the hay diet. Ruminal free LPS in HG group increased significantly (P<0.001) compared with the hay group and free LPS in the peripheral blood was detectable with concentrations of 0.80±0.20 EU/ml, while not detectable in the control, suggesting a leakage of LPS into the blood in the HG group. Correspondingly, the HG-fed goats showed profound alterations in ruminal epithelial structure. Cross sections of the rumen papillae revealed that HG-fed goats had a higher thickness of ruminal epithelial stratum corneum (P<0.001), but a less granulosum stratum thickness (P<0.001) than hay-fed goats. Scanning electron micrographs of rumen papillae showed the rumen papillae surface cells exhibited profound cellular damage and parakeratosis in the HG-fed goats. Transmission electron micrographs of rumen papillae cross sections showed that HG diet feeding caused profound impairment of ruminal epithelial ultrastructure structure, depicted by intercellular junctions erosion and marked epithelial cellular damage.The quantitive realtime PCR revealed that, compared with the hay-fed goats, HG-fed goats had a decline in the levels of ruminal epithelial claudin-4 (P= 0.003), occludin (P <0.001)and ZO-1 (P<0.001) mRNA expression, while, there was an increase in the levels of claudin-1 (P=0.001) mRNA expression. Correspondingly, western blot analysis showed that the HG diet caused a decrease in the ruminal epithelial claudin-4 (P= 0.042), occludin (P= 0.008), and ZO-1 (P= 0.001) proteins expression levels, while, there was an increase in the levels of claudin-1 proteins expression in the HG group (P= 0.022). The immunofluorescence results showed that, in ruminal epithelial tissues from hay-fed goats, claudin-1, claudin-4, and occludin were precisely localized to the surface of epithelial cells and were present in a continuous band at the cell borders. However, HG diet caused a marked change in claudin-1, claudin-4 and occludin localization from the usual circumferential fencework pattern to one with substantial fragmentation. Furthermore, these changes in TJ proteins in HG group were coupled with the up-regulation of mRNA levels for the cytokines TNF-a and IFN-y in the ruminal epithelia.In conclusion, the long-term high-grain diet feeding lead to rumen environmental disorder, causing profound alterations in ruminal epithelial structure and TJ proteins, depicted by marked epithelial cellular damage and intercellular junctions erosion, down-regulation of TJ proteins claudin-4, occludin and ZO-1 mRNA and proteins expression, as well as redistribution of claudin-1, claudin-4 and occludin. These changes potentially increased the ruminal epithelial permeability, providing opportunity for the ruminal LPS translocation into the blood, thus activating the local immune response in rumen epithelium, ultimately affecting animal performance.3. The underlying mechanism of SARA-induced ruminal epithelial tight junction erosionThe objective of this chapter was to investigate the effects of low pH and LPS on the expression of tight junction protein in ruminal epithelial cells and was to explore the regulatory role for extracellular signal regulated kinases (ERK)1/2 signal pathway in ruminal epithelial tight junction proteins expression. The primary ruminal epithelial cells were isolated from three hay-fed goats. After 24 h growing, cells from subconfluent dishes were divided into 8 group (pH7.4 group; pH5.5 group; pH 7.4+10 μg/mL LPS group; pH5.5+10 μg/mL LPS group; pH7.4+20 μM PD98059 group; pH5.5+20 μM PD98059 group; pH7.4+10 μg/mL LPS+20 μM PD98059 group; pH 5.5+10 μg/mL LPS+20 μM PD98059 group). The results showed that, compared with the control group, the combination of low pH and LPS treatment increased the protein expression of claudin-1 and claudin-4 significantly (P<0.05), while reduced the protein expression of occludin and ZO-1 significantly (P<0.05); Compared with the control group, SARA elevated the phophorylation level of ERK1/2 of ruminal epithelia in goats in vivo (P <0.05); Correspondingly, the combination of low pH and LPS treatment increased the phophorylation level of ERK1/2 of ruminal epithelial cells in vitro (P<0.05). Inhibition of MAPK activity by the ERK1/2 inhibitor PD98059 alleviated the depression of occludin and ZO-1 induced by the low pH and LPS. In conclusion, low pH and LPS induced alteration in tight junction expression involves ERK1/2 signal pathway. |
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