| Dairy cows are often fed high concentrate(HC)diets to meet the energy demand of high milk production that may lead to subacute ruminal acidosis(SARA).It is well known that induction of SARA declines the ruminal pH and increases the accumulation of endotoxins such as y-D-glutamyl-meso-diaminopimelic acid(iE-DAP)by the lysis of Gram-negative bacteria.Consiquently,iE-DAP translocated into blood circulation and tirggers the systemic inflammation.Previously,studies have been focused on the role of nucleotide-binding oligomerization domain-containing protein 1(Nod1)involved in the activation of inflammatory pathways through the recognition of iE-DAP.However,very little is known about the contribution of Nod1 in apoptosis within the mammary gland of dairy cows during SARA.Therefore,the present research was designed to explore the effects of long term high concentrate diet feeding on Nod1 mediated injury in the mammary gland of dairy cows and activation of Nod1/Caspase8 apoptotic response.Furthermore,our in vitro study confirmed the role of Nod1/Caspase8 to cause apoptosis in response to iE-DAP treatment in bovine mammary epithelial cells.Additionally,for the first time,protective effects of sodium butyrate(SB)were observed against the iE-DAP induced Nod1/Caspase8 apoptotic response and enhance the Bcl2 production in bovine mammary epithelial cells.EXPERIMENT 1:Long term high-concentrate feeding induces apoptosis via the Nodl/Caspase8 activation in mammary gland of dairy cowsThe aim of the present study was to evaluate how long term high-concentrate diet feeding affects mammary glands of dairy cows.Twelve Holstein Frisian cows at midlactation were selected to conduct this research.The animals were randomly allocated into two groups(n=6),and both groups received one of two diets:a low-concentrate(LC)(forage:concentrate 6:4)or a high-concentrate(HC)(forage:concentrate 4:6)for 20 weeks.Remarkable upregulation of y-D-glutamyl-meso-diaminopimelic acid(iE-DAP)level and decreased pH in rumen fluid were induced by HC as compared with LC group,which indicated that SARA model was successfully generated.The tunnel cell assay revealed a significant number of apoptotic cells in HC group,and the concentration of Caspase3,and Caspase8 was also higher in HC group.Nod1,Rip2,Caspase3,Caspase8,Caspase9,and Bax mRNA expressions,and Nod1,Caspase3,Caspase8,and Bax protein expressions,in the HC group were markedly higher as compared to LC group.Furthermore,Bcl2 mRNA and protein expressions were markedly decreased in HC compared to those in LC group.Nod1 and Caspase8 abundance showed similar trends as expressed by their protein blots.A HC diet fed to dairy cows for longer period induces SARA,elevates the rumen iE-DAP concentration that induces apoptosis by activation of Nod1/Caspase8 and modulation of Bcl2 in mammary gland of dairy cows.EXPERIMENT 2:Effects of iE-DAP stimulation in BMECs,induction of apoptosis by regulating Nod1 mediated Caspase8 and modulation of Bcl2Nod1,a cytoplasm located receptor which senses iE-DAP is before related to the inflammatory diseases in different type of cells in vivo and in vitro.Very poorly known about how bovine mammary epithelial cells(BMECs)respond to iE-DAP and its efficacy in inducing cellular apoptosis by regulating Nod1 mediated Caspase8 apoptotic response.Thus,the main purpose of this research was to investigate the effects of iE-DAP treatments in BMECs to initiate Nod1 mediated Caspase8 apoptosis.BMECs were treated with different doses of iE-DAP(1,10,20,50 and 100 μg/mL)for 48 h to conduct cell viability test and Lactate dehydrogenase(LDH)activity.Only 100 μg/mL iE-DAP has a significant toxic effect resulted 60%viability.Time and dose dependent mRNA expresions of Nod1 shows significantly higher levels with 20 μg/mL dose of iE-DAP for 12 h.BMECs were divided into four groups:Control as a non-treated,and three treated groups with(iE-DAP 10 μg/mL for 12 h)and(iE-DAP 20μg/mL for 12 h and 24 h)to conduct the further experiments.20 μg/mL iE-DAP showed a significant number of apoptotic cells for 12 and 24 h.The mRNA expressions of Rip2,Caspase3,Caspase8,Caspase9,Bax and protein intensity of Nod1 and Caspase8 were significantly increased with 20μg/mL iE-DAP dose for 12 h and 24 h.However,the mRNA and protein expressions of Bcl2 were totally reversed in the 20 μg/mL iE-DAP for 12 h.Moreover,confocal microscopy revealed the protein abundance of Caspase8 and Bcl2 which showed similar results to western blotting reports.Briefly,these results suggested that stimulation of 20μg/mL iE-DAP for 12 h remarkably induces apoptosis in BMECs by the activation of Nod1/Caspase8 and modulation of Bcl2.EXPERIMENT 3:Sodium butyrate mitigates iE-DAP induced cellular injury and suppression of Nod1 dependent Caspase8 apoptotic response in BMECsThe aim of present study was to evaluate the protective role of sodium butyrate(SB)against iE-DAP induced apoptotic response in BMECs.First of all,to adjust the doses and time points of SB,BMECs were pre-treated with different doses of SB(0.5,1,5,10 and 100 mM)for 48h to conduct cell viability test and LDH activity.Bovine mammary epithelial cells pretreated with 1mM SB were utilized for the SB group,and those exposed to 20 μg/mL iE-DAP for 12 h were used for the iE-DAP and pre-treated with SB(iEDAP+SB)totally four groups created.The iE-DAP stimulated BMECs exposed increase in Nod1 mRNA and protein production,that increase was markedly reduced by the pretreatment of SB in the iE-DAP+SB group.In iE-DAP treated cells the Nod1 and Caspase8 protein expressions and fluorescence intensity were repressed with the addition of SB.mRNA expressions of Rip2,Caspase3,Caspase8,Caspase9,and Bax and protein expressions of Caspase3,Caspase3C,and Bax were down regulated in iE-DAP+SB group as compared to iE-DAP treated cells.1mM SB pretreatment significantly increases the Bcl2 mRNA as well as protein expressions and fluorescence intensity that was decreased by iEDAP stimulation in BMECs.Conclusively,SB reduces apoptosis caused by iE-DAP stimulation.This is the first report showing that SB protects against the cellular injury caused by iE-DAP stimulation and also modulate Nod1/Caspase8 and enhance the Bcl2 production in bovine mammary epithelial cells.Thus,SB plays a dynamic role in neutralizing the negative effects of infection and keep the mammary glands healthy. |
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