| Transport stress can cause metabolic disorders,suppression of immune function and reduction of body resistance in animals,and even cause diseases(such as transport stress syndrome in beef cattle),bringing serious economic losses to the farming industry.When stress occurs in animals,it excites the nerve center and hypothalamus to release glucocorticoids,catecholamines and other neuroendocrine hormones,which in turn affects the function of immune organs and causes disruption of Th1/Th2 type cytokine secretion,leading to tissue oxidative imbalance and cell apoptosis.It was shown that signal transducer and activator of transcription 3(STAT3)is involved in signaling of cytokines(e.g.IL-6,TNF-α,etc.),affecting cell proliferation,differentiation and apoptosis,and its expression was significantly increased in the spleen of chronically restrained stress rats.A number of inflammatory cytokines(IL-6 and TNF-α,etc.)were significantly changed in the thymus and spleen of fertile pigs after 4 h of transport,and it remains to be verified whether the mechanism is related to the activation of STAT3.Powerfeel is an alkaline cationic compound additive,mainly used in anti-heat stress of dairy cows and anti-weaning stress of piglets,etc.It also has the effect of stimulating the proliferation of immune cells and regulating the secretion of Th1/Th2 type cytokines,but whether it affects the apoptosis of spleen and thymus cells and the differentiation of T lymphocytes due to transport stress is not clear.In this study,we investigated the effects of pre-transport and post-transport administration of Powerfeel on apoptosis and T-lymphocyte fractionation in thymus and spleen of rats under transport stress,and investigated the molecular regulation mechanism of Powerfeel on anti-transport stress in rats based on JAK2/STAT3 signaling pathway.There were five time points in this experiment,namely three days before transport(D-3),before transport(D0),after transport(D0+),the third day after transport(D3)and the tenth day after transport(D10).According to the experimental design,240 25-day-old male SD rats were randomly divided into 6 groups according to the principle of similar total body weight per cage(4 rats/cage):blank control group(I,60 rats,tested at the time points of D-3,D0,D0+,D3,D10),transport stress group(II,36 rats,tested at the time points of D0+,D3,D10),Powerfeel prevention group(III,48 rats,tested at the time points of D0,D0+,D3,D10),vitamin C prevention group(Ⅳ,48 rats,detection time point as groupⅢ),Powerfeel post-transport addition group(V,24 rats,detection time point as D3,D10),vitamin C post-transport addition group(VI,24 rats,detection time point as group V).Rats in group I were not treated for transport,while rats in groups II,III,IV,V and VI were treated for 3.5 h of transport stress on the day of transport(D0+)(road transport,average speed 42 km/h,total distance 116 km).According to the experimental design,12 rats were randomly selected and sampled from different test groups at each time point.The following experiments were conducted:Experiment Ⅰ.Effects of Powerfeel on the tissue structure and antioxidant indexes of thymus and spleen of rats under transport stress:The effects of Powerfeel on the indices,histomorphology and antioxidant indexes of thymus and spleen of rats before and after transport stress.Results:1.The addition of Powerfeel and vitamin C promoted the increase of body mass and thymus mass,but had no significant effect on the organ mass and index of spleen;it reduced the weight loss of body mass and organ(thymus and spleen)mass in rats caused by transport stress;and promoted the recovery of body mass and organ(thymus and spleen)mass in 3 d and 10 d after transport stress.2.The addition of Powerfeel enhanced the thymus The addition of Powerfeel mainly enhanced the CAT activity of thymus(P<0.05)and enhanced the SOD activity of thymus(P<0.05)before transportation.3.The addition of vitamin C had the effect of enhancing thymic antioxidant capacity,improving T-AOC capacity and increasing MDA content(P<0.05);pre-and post-transport addition mainly enhanced thymic CAT activity and decreased MDA content(P<0.05).5.The addition of vitamin C enhanced the antioxidant capacity of spleen,increased CAT and SOD activities and increased MDA content(P<0.05);pre-and post-transport addition mainly enhanced CAT activity and decreased MDA content(P<0.05).6.Pre-transport and post-transport addition of Powerfeel or vitamin C had the effect of reducing a small number of lymphocytes in the thymus caused by transport stress.ExperimentⅡ Effect of Powerfeel on apoptosis of thymus and spleen cells in rats under transport stress:The effects of Powerfeel on apoptosis in thymus and spleen tissues were investigated using Annexin V/PI assay,RT-q PCR and WB assay for apoptosis-related factors(Bax,Bcl-2,Caspase-3 and Caspase-9).Results:1.The addition of Powerfeel and vitamin C had enhanced anti-thymocyte apoptotic effect,and both pre-and post-transport addition of Powerfeel and vitamin C could alleviate the effect of transport stress-induced thymocyte apoptosis rate(P<0.05).2.The addition of Powerfeel and vitamin C had anti-apoptotic effect on spleen cells,and both pre-and post-transport addition of Powerfeel and pre-transport addition of vitamin C could alleviate the effect of transport stress-induced spleen cell apoptosis rate.Powerfeel had the effect of down-regulating Bax,Caspase-9 and up-regulating Bcl-2 gene and protein expression levels in thymus tissue(P<0.05),and both pre-and post-transport addition of Powerfeel alleviated the increase in expression levels of pro-apoptotic factors and decrease in expression levels of anti-apoptotic factors caused by transport stress.4.Powerfeel had the effect of down-regulating Powerfeel had the effect of down-regulating Bax gene and protein expression level,down-regulating Caspase-3 and Caspase-9 protein expression level in spleen(P<0.05),and both pre-and post-transportation administration could alleviate the increase of pro-and anti-apoptotic factor expression levels caused by transport stress.5.Vitamin C had the effect of down-regulating Caspase-3,Caspase-9 and Vitamin C had the effect of down-regulating Caspase-3,Caspase-9 and up-regulating Bcl-2 gene and protein expression levels in thymus tissue(P<0.05),and its addition both before and after transportation could alleviate the increase of pro-apoptotic factor expression level and decrease of anti-apoptotic factor expression level caused by transportation stress.6.Vitamin C had the effect of down-regulating Caspase-3 and up-regulating Bcl-2 gene and protein expression levels(P<0.05),and its addition both before and after transportation could alleviate the increase of pro-apoptotic factor expression level and decrease of anti-apoptotic factor expression level caused by transportation stress.The addition of vitamin C before and after transportation could alleviate the increase in the expression level of pro-apoptotic factors and decrease in the expression level of anti-apoptotic factors caused by transportation stress.ExperimentⅢ Effect of Powerfeel on T-lymphocyte subsets in peripheral blood,thymus and spleen of transport-stressed rats:The effect of Powerfeel on lymphocyte subsets was investigated by flow cytometry in peripheral blood,thymus and spleen.Results:1.Powerfeel had the effect of promoting the emigration of thymic CD3~+cells,which in turn increased the ratio of CD3~+cells in peripheral blood(P>0.05)and spleen(P<0.05),and the pre-transport addition of Powerfeel could alleviate the decrease of CD3~+cells in peripheral blood due to transport stress(P<0.05).2.ratio(P>0.05),and pre-shipment addition alleviated the decrease of CD4~+/CD8~+cell ratio in thymus and spleen due to transport stress(P<0.05),and the effect was better than post-shipment addition(P>0.05).3.Vitamin C had the effect of promoting the ratio of CD3+cells in peripheral blood,thymus and spleen(P>0.05),and post-shipment addition of vitamin C decreased the 4.Vitamin C had the effect of increasing the ratio of CD4~+/CD8~+cells in peripheral blood and spleen(P>0.05),and pre-transport addition alleviated the decrease of CD4~+/CD8~+cell ratio in peripheral blood(P>0.05)and thymus(P<0.05)caused by transport stress.Experiment Ⅳ.Effect of Powerfeel on JAK2/STAT3 signaling pathway in transport-stressed rats:The effects of Powerfeel on JAK2/STAT3 signaling pathway in thymus and spleen tissues of transport-stressed rats were detected by RT-q PCR for Th1/Th2 cytokines,and RT-q PCR and WB for Powerfeel.RESULTS:1.Addition of Powerfeel caused Th1/Th2 to Th2drift in thymus and had the effect of increasing IL-4/IFN-γratio in thymus(P<0.05).2.Addition of Powerfeel caused Th1/Th2 to Th1 drift in spleen and had the effect of decreasing IL-4/IFN-γratio in thymus(P<0.05).had the effect of decreasing IL-4/IFN-γratio(P<0.05),but both pre-and post-transport addition of Powerfeel alleviated the decrease in splenic IL-4/IFN-γratio(P<0.05).3.Addition of vitamin C caused thymic Th1/Th2 to drift toward Th2 and had the effect of increasing thymic IL-4/IFN-γratio(P<0.05),and both pre-and post-transport The addition of vitamin C effectively alleviated the decrease of IL-4/IFN-γratio caused by transport stress(P<0.05).4.The addition of vitamin C caused the drift of spleen Th1/Th2 to Th1,which had the effect of decreasing IL-4/IFN-γratio(P<0.05),and the addition of vitamin C before and after transport aggravated the decrease of IL-4/IFN-γratio caused by transport stress(P<0.05).5.The addition of Powerfeel had the effect of increasing the thymic p-STAT3/STAT3 ratio(P>0.05)and JAK2 protein expression level(P<0.05),and the addition of Powerfeel before shipment could alleviate the decrease of p-STAT3/STAT3 ratio and JAK2 protein expression level caused by transport stress(P<0.05);after recovery from stress,the addition of Powerfeel after shipment could alleviate the decrease of p-STAT3/STAT3 ratio and JAK2 protein expression level(P<0.05).Powerfeel was effective in reducing the p-STAT3/STAT3 ratio and JAK2 protein expression level after recovery from stress(P<0.05).6.The addition of vitamin C increased the p-STAT3/STAT3ratio and JAK2 protein expression level in thymus(P<0.05),and the addition of vitamin C before and after shipment alleviated the decrease of p-STAT3/STAT3 ratio and JAK2 protein expression level due to transport stress.After recovery from stress,post-transport addition of vitamin C alleviated the high p-STAT3/STAT3 ratio and JAK2 protein expression levels(P<0.05).8.Vitamin C had the effect of lowering spleen p-STAT3/STAT3 ratio(P>0.05)and lowering JAK2 protein expression levels(P>0.05),and pre-transport addition of vitamin C before shipment effectively alleviated the decrease in p-STAT3/STAT3 ratio caused by transport stress(P<0.05),and the addition of vitamin C after shipment effectively alleviated the increase in JAK2 protein expression level caused by transport stress(P<0.05)Comprehensive analysis of the results showed that transport stress led to a small amount of apoptotic lymphocyte nuclei consolidation in rat thymus tissues and an increased rate of apoptosis in thymus and spleen tissues;pre-and post-transport addition of Powerfeel upregulated Bcl-2 expression and downregulated Bax and Caspase-9 expression in rat thymus and spleen to alleviate the rate of apoptosis;these processes were mediated by activation of the JAK2/STAT3 pathway. |
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