Study On The Histology Structure Of Spleen And The Changes Of Related Molecular Expression In Weaned Piglet' Stress Model Induced By Lipopolysaccharide

A new model that newly weaned piglets were treated repeatedly with LPS was established to analysis the immune stress process of piglet.Moreover,the process of inflammation,cell proliferation and apotosis in weaned piglets would be further studied and explored from different levels including anatomy,histology,morphogy,cytology,mRNA and protein.In our study,it was very helpful for us to better understand immune regulation mechanism of piglets.In our study,fifty weaned piglets were divided randomly into two groups with equal number each group,LPS group received a single dose LPS?100?g/kg bw?for consecutive 13 days,and control group were treated with equal volume saline.Respectively in the 1st,4th,7th,10 th and 13 th day of the experiment,five inviduals from each group firstly were weighed and undergone precaval vein collection.And then automatic hamocyte analyzer was used to examine routine blood parameters and Elisa techonolgy was applied to detect the concentration of pro-inflammatory cytokines IL-1?,IL-6 and TNF-? in different serums.Compelte spleen tissue in each piglet was collected and weighed,and spleen index was calculated accordingly after pigs were sacrificed.Some spleen tissues were fixed with 4% paraformaldehyde solution,and finally were made into five-micro-thick slices through many procedures including water-washing,dehydration,transparency,embedding and section.Then,sections were stained with HE?hematoxylin-Eosin?to observe the histology and morpgology changes of spleen tissues.At the same moment,sections were treated with immunohistochemistry techonology to analyse the distribution and expression of IL-1?,IL-6,TNF-?,Skp2,P27^Kip1 and Casapase-3.In addition,sections were handled with TUNOLPOD method to observre the distribution of the apoptotic cells.Other spleen tissues were tranfered into ultra deep-freeze equipment after liquid nitrogen freezing.Subsequently,realtime fluorescent quantitative PCR was utilized to examine the mRNA levels of cell cycle regulators Skp2 and P27^Kip1.The major findings in weaned piglets' stress model induced by LPS showed that:?1?LPS-treated piglets suffered from vomiting,diarrhea,mental fatigue,shortness of breath,poor appetite and fever in the early stage of the experiment,became better at the metaphase experiment,and finally restored normal;?2?Spleen weight and index of LPS-treated piglets increased evidently,especially in the 10 th day;?3?Multiple LPS treatment disturbed the balance system of blood cells in piglets,the number and proportion of leukocyte presented a significant change;?4?A marked expansion of germinal center,increased amount of splenic nodules and thickened PALS were formed in spleen parenchyma after piglets were treated by LPS on day 4,7 and 10;?5?In the process of LPS stimulation,the levels of IL-1? and IL-6 increased significantly in serum and spleen tissue on day 1 but IL-1?,IL-6 and TNF-? decreased remarkablely on day 4,7;?6?Skp2-positive cells distributed mainly in splenic nodules,marginal zone and PALS were more in LPS-treated piglets than in saline-treated piglets but fewer P27^Kip1-positive cells on day 4,7 and 10,and consisted of lymphocytes;?7?Transcription and translation level of Skp2 increased signinficantly and accumulation of P27^Kip1 decreased obviously on day 7 and 10,the changes were negative correlative between Skp2 and P27^Kip1 mRNA;?8?Apoptotic protein Caspase-3 reactivity was inhibited and apoptotic cells in spleen tissues was difficult to be found during the processing of LPS induction,which resulted in arrestment of apoptotic pathway.Our study demonstrated that spleen of piglets could resist LPS attack and strength immunity to maintain homeostasis and normal physical activity by itself.This can be embodied three aspects: Synthesis and secretion of pro-inflammatory cytokines IL-1?,IL-6 and TNF-? was surpressed to prevent spleen from inflammatory damage;Incresased level of Skp2 transcription and translation could promote proliferation and differentiation of lymphocyte and improve the immune function of the weaned piglets;Repeated LPS stimulation would cause extensive gene reprogram in the apoptotic pathway,resulting in inhibition of Caspase-3 activity and arrestment of apoptotic pathway.