Dehydroepiandrosterone Resisted E.coli O157:H7-Induced Infection In Mice And Its Mechanisms

Escherichia coli O157:H7(E.coli O157:H7)exists extensively in nature.With the continuous development of intensive farming mode,E.coli O157:H7 infection in livestock and poultry is very common,which leds to a great economic losses to livestock and poultry breeding industry.E.coli O157:H7 infection has become a global public health problem due to its multi-host and multi-channel transmission.Dehydroepiandrosterone(DHEA),as the most abundant steroid in blood circulation,is considered as a multidirectional "hormone buffer" because of its unique biological function.The research on the biological function of DHEA mainly focuses on its effects on metabolism,central nervous system and oxidative stress.There are few reports on the effects of DHEA on immune regulation and its mechanism in animal with bacterial infection.In this study,the effects of DHEA on immune regulation in mice infected with E.coli O157:H7 was investigated,and then further analyzed the action of DHEA on alleviating inflammation and its related mechanisms in mice peritoneal macrophages infected with E.coli O157:H7.The aim was to reveal the anti-inflammatory role and its mechanism of DHEA in mice infected with E.coli O157:H7.These results provided substantial information for DHEA as a potential supplement to prevent infectious and inflammatory responses caused by bacteria.1 Effect of DHEA on anti-E.coli O157:H7 infection in miceIn order to reveal the effects of DHEA on the anti-E.coli O157:H7 infection ability of mice,the model of ICR mice infected with E.coli O157:H7 were established,and then the survival,spleen immune index and pathology of small intestine were analyzed.The tolerance test of mice infected with E.coli O157:H7 was anlysed by intraperitoneal injection of E.coli O157:H7.Based the results,mice were given different dose of DHEA by gavage for 3 weeks,and then the mice injected the LD50 E.coli O157:H7 by intraperitoneal administration.After 1 week,the serum,spleen and small intestine were collected for further analysis.The concentration of bacteria in peritoneal fluid of mice was determined by dilution coating plate method;histopathological changes of small intestine were observed by hematoxylin and eosin stain;lactate dehydrogenase and acid phosphatase activity were detected by a commercial kits.Results showed that the LD50 of E.coli O157:H7 in mice is 2.3×108 CFU.Intestinal villus presented breakage,shedding,bleeding and other pathological changes were observed in mice infected with LD50 E.coli O157:H7 infection,but DHEA treatment reduced the degree of damage caused by E.coli O157:H7 infection and improved the survival rate of mice in a dose-dependent manner.2 mg/kg and 4 mg/kg DHEA treatment significantly reduced the bacterial concentration in peritoneal fluid and increased the spleen immune index of mice infected with E.coli O157:H7(P<0.05).In addition,2 mg/kg and 4 mg/kg DHEA treatment significantly increased the lactate dehydrogenase and acid phosphatase activities in spleen of mice infected with LD50 E.coli O157:H7(P<0.05).These results indicated that DHEA treatment enhanced the survival rate of mice,eliminated infectious bacteria and improved the immune function of spleen in mice infected with E.coli O157:H7.2 Mechanism of DHEA against the E.coli O157:H7 infection in miceThis study aimed to investigate the effects of DHEA on inflammatory mediators and cytokines in mice infected with E.coli O157:H7,and further reveal the possible signal transduction mechanism of DHEA against E.coli O157:H7 infection.After DHEA administration and E.coli O157:H7 infection,the iNOS activity and NO content in spleen and serum were detected using a commercial kits;the mRNA levels of TNF-α,IL-1β,IL-6,IFN-γ,IL-4 and IL-10 in spleen were detected by Real-time PCR;IFN-y and IL-4 content in spleen and serum were determined by ELISA;MAPK and NF-κB protein expression level in spleen were detected by Western blot.Results showed that 0.2 mg/kg DHEA treatment reduced serum iNOS activity,and 2 mg/kg DHEA treatment significantly reduced serum NO content(P<0.05).2 mg/kg or 4 mg/kg DHEA treatment significantly reduced the activity of iNOS in spleen(P<0.01)and the mRNA levels of TNF-α,IL-1β and IFN-γin spleen(P<0.05);meanwhile,4 mg/kg DHEA treatment significantly reduced the IL-6 mRNA level in spleen(P<0.05).2 mg/kg or 4 mg/kg DHEA treatment significantly increased IL-4 mRNA level(P<0.05),and 4 mg/kg DHEA treatment significantly increased IL-10 mRNA level in spleen(P<0.05).2 mg/kg DHEA treatment significantly reduced IFN-γ content in spleen(P<0.01),while 2 mg/kg or 4 mg/kg DHEA treatment significantly increased serum IL-4 content(P<0.05).No significant differences were observed on the p-ERK1/2 and p-JNK1/2 protein levels(P>0.05),but 2 mg/kg or 4 mg/kg DHEA treatment significantly reduced p-p38 MAPK protein level in spleen(P<0.05).Importantly,2 mg/kg or 4 mg/kg DHEA treatment significantly reduced the p-IκB-α and nuclear NF-κB protein levels,and increase the IκB-α and NF-κB protein levels in cytoplasm(P<0.01).These results indicated that DHEA alleviated inflammatory response by reducing the secretion of inflammatory mediators and pro-inflammatory cytokines,and enhancing the secretion of anti-inflammatory cytokines,and these effects may be achieved by blocking the activation of p38 MAPK and NF-κB in mice infected with E.coli O157:H7.3 Regulatory effect of DHEA on primary mice peritoneal macrophages against E.coli O157:H7 infection and its mechanismsPresent study aimed to investigate the effects and its mechanism of DHEA against the inflammation in primary ICR mice peritoneal macrophages infected with E.coli O157:H7.Based on the time effect of E.coli O157:H7 caused cell damage,the treatment concentration of DHEA and the time of E.coli O157:H7 infection were determined.Macrophage phagocytosis was detected by dilution coating plate method;the content of TNF-α,IL-1β and IL-6 in supernatant of cell culture were determined by ELISA,and the protein levels of iNOS,COX-2,p38 MAPK and NF-κB were detected by western blot.Results showed that 100 μmol·L-1 DHEA significantly decreased cell viability(P<0.05).Significance cell damage occurred at 3 hours in primary mice peritoneal macrophages infected with E.coli O157:H7,and a certain concentration of DHEA pretreatment significantly alleviated the damage(P<0.05);meanwhile serious damage which were not be alleviated by DHEA in macrophages occurred after E.coli O157:H7 infection for 4 hours.No differences were observed on the macrophages phagocytosis treated with different concentrations of DHEA(P>0.05).0.1 μmol·L-1 DHEA significantly decreased the contents of TNF-α and IL-1β in cell culture supernatant(P<0.01),and 0.1 μmol·L-1 DHEA or 1μmol·L-1 DHEA significantly decreased the IL-6 content in cell culture supernatant(P<0.05).0.1 μmol·L-1 DHEA or 1 μmol·L-1 DHEA significantly decreased iNOS and COX-2 protein levels(P<0.05).Similar to the SB203580 action(inhibitor of p38 MAPK),0.1 μmol·L-1 or 10 μmol·L-1 DHEA significantly decreased the p-p38 protein level and increased IκB-α protein level in macrophage(P<0.05).In addition,0.1 μmol·L-1 or 10μmol·L-1 DHEA significantly decreased the p-IκB-α and nuclear NF-κB protein levels,and significantly inhibited the decreasing of cytoplasmic NF-κB(P<0.05).These results indicated DHEA reduced the secretion of pro-inflammatory factors by inhibiting the activation of p38 MAPK and NF-κB in primary mice peritoneal macrophages infected with E.coli O157:H7.These results suggest that DHEA increased the spleen index,decreased the concentration of peritoneal fluid,and alleviated the pathological damage of small intestine in mice infected with E.coli O157:H7.The overall performance was increased the survival rate of E.coli O157:H7 infected mice.DHEA reduced the excessive production of inflammatory mediators such as iNOS,COX-2,TNF-α,IL-1β,IL-6,and promoted the secretion of anti-inflammatory factors IL-4 and IL-10,thereby alleviating inflammation damage caused by E.coli O157:H7 infection.DHEA blocked the activation of NF-κB by inhibiting the activation of p38 MAPK protein,thereby regulating the expression of inflammatory factors to alleviate the excessive inflammatory response,and finally achieved the protective effect on E.coli O157:H7 infected mice.