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The Mechanism Of Inflammation Of Rumen And The Changes Of Rumen Epithelial SLC22a Transporter Proteins In Dairy Cows With SARA

Posted on:2020-05-03Degree:DoctorType:Dissertation
Country:ChinaCandidate:C X ZhaoFull Text:PDF
GTID:1363330575477825Subject:Clinical Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Subacute ruminal acidosis(SARA)is a metabolic disease that occurs in high-yielding dairy cattle herds.The main reason is that during the production process,cows were fed the excessive concentrated feed and the carbohydrates were fermented in large quantities,resulting in the accumulation of short-chain fatty acids(SCFA)in the rumen.When the concentration of SCFA in the rumen is too high which will reduce the rumen pH.SARA can be identified when the pH is lower than 5.6 for more than 3 h daily.The rumen is in an acidic environment for a long time,and the Gram-negative bacteria will dissolve and rupture,releasing its wall component,lipopolysaccharide(LPS).At the same time,some bacteria can also decarboxylate histidine to form histamine.Both LPS and histamine can induce rumen inflammation and damage the rumen epithelial tissue.When the damage reaches a certain level,it can be translocated into the bloodstream,causing inflammation in other areas such as laminitis and liver abscess,and sometimes even systemic inflammatory actions.There are two main reasons for the accumulation of SCFA in the rumen,namely,increased generation and absorption disorder.However,the causes and mechanism of the rumen SCFA absorption disorder were not clear at present.Recent studies have found that SLC22A(solute carrier family)transporter,subgroup organic anion transporters(OATs)are the uptake pathways of propionic acid and butyric acid into liver cells and play the role of SCFA transport.Another new type of organic anion transport carrier OCTNs(organic cation/carnitine transporter)transports a variety of endogenous and exogenous cations and zwitterionic ions,such as carnitine,choline and ergothione.All of them have anti-inflammatory and antioxidant effects.However,studies on the role of SLC22 A transporter in ruminant epithelium have rarely been reported.Therefore,it is of great significance to explore the occurrence mechanism of rumen inflammation and the changes of rumen epithelial SLC22 A protein expression during the period of SARA in cows,and to clarify the internal relationship between abnormal rumen metabolism of SARA,epithelial inflammatory injury and SCFA absorption disorder.It would make contribution to explaining the occurrence mechanism and prevention andtreatment of SARA in cows.In this experiment,the concentrations of various components of SCFA(including acetic acid,propionic acid and butyric acid),LPS,histamine and lactic acid in the rumen fluid of SARA cows were first detected,and found to be significantly higher than those of healthy cows.LPS,acute phase reaction proteins,including lipopolysaccharide binding protein(LBP),haptoglobin(HP)and serum amyloid loid A(SAA),tumor necrosis factor(TNF)-?,interleukin(IL)-6,and interleukin(IL)-1? in SARA cows' blood were significantly higher than those of healthy cows,which suggests that SARA cows displayed a severe inflammatory response.Protein expression changes in nuclear factor-kappa B(NF-?B)and mitogen-activated protein kinase(MAPK)inflammatory signaling pathways in the rumen epithelial of SARA cows were detected by Western blot and quantitative real-time PCR.Results showed that NF-?B and MAPK were activated in the rumen epithelial cells of SARA cows,and the phosphorylation levels of I?B,NF-?B p65,JNK and ERK1/2 were significantly increased,and the mRNA and protein expressions of cytokines TNF-?,IL-6 and IL-1? were significantly higher than those of healthy cows,which further proved the occurrence of rumen inflammation in SARA cows.Rumen epithelial cells were cultured in vitro experiment,and the best stimulation time were determined through the different time points(0,1,3,6,9,18 h).The different concentration of LPS(0,1,5,10 ?g/ml)and histamine(0,20,40,60 ?M)were added to simulate the healthy and SARA pathological state.The phosphorylation levels of key proteins in NF-?B and MAPK were tested by Western blot,and the transportation to the nucleus of p65 was detected by immunofluorescence.Quantitative real-time PCR and ELISA were used to detect the expression of cytokines.The results showed that when LPS and histamine were added to stimulate rumen epithelial cells for 6-9 h,the levels of phosphorylation of I?B,NF-?B p65,JNK and ERK1/2 were significantly increased in a dose-dependent manner,and NF-?B p65 was significantly transported into the nucleus to induce the transcription of target genes in inflammatory factors.The contents and mRNA expression of TNF-?,IL-1? and IL-6 were significantly increased which indicated the occurrence of inflammation in rumen epithelial cells.On the other hand,in vivo experiments,Western blot and immunohistochemical methods were used to detect the expression and distribution of key members of the SLC22 A family(OAT2,OAT7,OCTN1 and OCTN2)in the rumen epithelium.Theresults showed that they were expressed in the epithelium of the four lumens of the rumen of cows.The expressions of epithelial transporters OAT2 and OAT7 in the rumen of SARA cows were inhibited,with increased OCTN1 expression and slightly decreased OCTN2 expression.It indicated that the absorption and transport of SCFA in the rumen of SARA cows were influenced,and the transport of beneficial substances such as carnitine and ergothione was also impeded.In vitro,we investigated the effects of changes in rumen on SLC22 A transporter by different concentrations of SCFA,LPS,histamine and pH gradient.The results showed that stimulation with normal physiological concentrations of acetic acid,propionic acid and butyric acid had a promoting effect on the expression of transporter proteins,such as OAT2,OAT7,OCTN1 and OCTN2,while high-concentration SCFA or low-pH environment inhibited the expression.LPS and histamine served to inhibit the expressions of OAT2 and OAT7,while histamine promoted the protein expressions of OCTN1 and OCTN2.In summary,high concentrations of LPS and histamine in the rumen of SARA cows can activate the MAPK and NF-?B inflammatory signaling pathways,stimulating the transcriptional release of proinflammatory cytokines TNF-?,IL-1?and IL-6 by downstream target genes,and further induce inflammation.The expressions of rumen epithelial transporter proteins,OAT2,OAT7,and OCTN2,were inhibited,with an increased degree of SARA.
Keywords/Search Tags:SARA, short chain fatty acids, NF-?B, MAPK, solute carrier family, anion transporter
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