| Toxoplasmosis is a parasitic disease caused by the intracellular infection of Toxoplasma gondii.It is a global zoonosis affecting one third of the population around the world.T.gondii has a serious negative effect on health and development of human and livestock worldwide because of its strong transmissibility and high infection rate.The defense mechanism by which host response to T.gondii is relatively complex,and the specific immune response and defense methods are yet to be completely understood.Usually,apoptosis,autophagy and other programmed cell death process are important defense mechanisms employed by host in response to bacteria,parasites and other pathogens.By this "suicide" process,the innate immune system of multicellular organisms can independently deal with infection of pathogens and remove bacteria,parasites and other pathogens from host cells.However,studies have found that bacteria,parasites and other pathogens can interfere with the recognition of proteins related to apoptosis and autophagy,and influence the formation of autophagosome and apoptotic body to inhibit the host cell death.Hence,there is a competitive relation between the intiation of host cell death as a host defence mechanism and the inhibition of host cell death intiation by pathogens,and the strength of this competitive relationship affect host response to pathogens.However,this competitive relation between host cells and the pathogens is not completely known.Therefore,the further understanding to mechanism of T.gondii infection and defense response in host is an important foundation for effective prevention and treatment of toxoplasmosis.CCAAT/enhancer binding protein beta(C/EBP ?)is an important transcription factors,belonging to the transcription factor family of CCAAT/enhancer binding protein(C/EBPs).C/EBP ? is highly homologous,and expressed in various tissues and organs of human,cattle,pigs,rats,and many other species.By regulating the transcription of target genes,C/EBP ? plays roles in cell growth,differentiation,apoptosis,metabolism and immune response.Researches show that the abnormal expression of C/EBP ? is correlated with inflammation,endoplasmic reticulum stress and other pathological changes,and participates in the process of human disease.C/EBP ? is also involved in the regulation of cell autophagy.Under normal physiological conditions,C/EBP ? can adjust the autophagy process to maintain the nutritional and metabolic balance.Whereas,C/EBP ? can mediate autophagy to affect the defense response to pathogens and are involved in inflammation,fibrosis and cancer related pathological events.In conclusion,as an important transcription factor,the abnormal expression of C/EBP ? has an important regulatory role in cell growth,death,inflammation and stress reaction.To have a better/further understanding of T.gondii infection and the host defence evasion mechanism there are few questionsthat needs to be addressed like whether C/EBP ? is abnormally expressed in host infected by T.gondii? Whether C/EBP ? is involved in the defense response of host to T.gondii infection? Is the molecular mechanism related to the programmed cell death of infected host cells? To address these questions,we constructed the T.gondii infection mouse model and cell model.We analyzed the expression of C/EBP ? in presence of T.gondii infection in vivo and in vitro with an aim to study the effect of C/EBP ? expression level on the ability of T.gondii's infection and host's defense response to T.gondii.In the meanwhile,we want to know if C/EBP ?'s function is related to cell autophagy and apoptosis during T.gondii infection.To further analyze whether C / EBP ? regulates p38 MAPK,m TOR and other apoptotic and autophagy-related signaling pathways,and uncover the possible molecular mechanisms to provide fundamental basis for the understanding of host defence against the infection of Toxoplasma gondii.Chapter 1: T.gondii infection on the expression of C/EBP ?1.Expression of C/EBP ? in cells infected by T.gondiiBy co-culturing of T.gondii RH or ME49 strain with brain microvascular endothelial cells,umbilical vein endothelial cells or retinal pigment epithelial cells,T.gondii infected cell models were constructed.q RT-PCR and Western blot were conducted to measure the expression of C/EBP ? in cells infected by T.gondii after 24 h.The results showed that m RNA and protein levels of C/EBP ? were significantly increased in cells infected by T.gondii compared to control,and the infection efficiency of T.gondii ME49 strain is much better.2.Expression of C/EBP ? in mice infected by T.gondiiBy infecting mice with T.gondii ME49 strain,T.gondii infected mice models were constructed.q RT-PCR and Western blot were conducted to measure the expression of C/EBP ? in celiac fluid,brain and eye tissues of model mice after 2,4,6 days of T.gondii infection.The correlation between C/EBP ? expression and time of T.gondii infected mice were analyzed.The results indicated that m RNA and protein levels of C/EBP ? were significantly increased in celiac fluid,brain and eye tissues of model mice compared to control.And there was a positive correlation between C/EBP ? expression and time of T.gondii infected mice.Chapter 2: C/EBP ?'s effect on T.gondii infection1.Effects of C/EBP ? on mice cells infected by T.gondiiRecombinant Ad-C/EBP ? adenovirus and C/EBP ? si RNA were constructed,and C/EBP ? was overexpressed or inhibited in cells infected by T.gondii to analyze its effects on T.gondii infection and tachyzoite in cells.Results showed that,expression of C/EBP ? was significantly up-regulated or inhibited in T.gondii infected mice after treated by Ad-C/EBP ? or C/EBP ? si RNA.After 24 h of T.gondii infection,the proportion of infected cells and number of tachyzoite were significantly reduced in cells treated by Ad-C/EBP ? compared to the control.On the contrary,the proportion of infected cells and number of tachyzoite were enhanced in cells treated by C/EBP ? si RNA.2.Effects of C/EBP ? on mice infected by T.gondiiC / EBP ?-specific si RNA was constructed and the expression of C / EBP ? in the mice infected with T.gondii was inhibited by intraperitoneal injection of C / EBP ?-specific si RNA.After the inhibition of C / EBP ?,microscopy was used to check the changes of pseudocyst and tachyzoites in peritoneal fluid.The results showed that the expression of C / EBP ? in the peritoneal fluid of mice was significantly inhibited by C / EBP ?-specific si RNA in mice infected with T.gondii.After the infection of T.gondii,C / EBP ? silencing group showed increased vertical hairy and the action ability,mental state and diarrhea symptoms were more serious;Meanwhile,rupture of false cyst and number of tachyzoite in peritoneal fluid were sharply increased in T.gondii infected mice treated by C/EBP ? si RNA.Chapter 3: C/EBP ?'s role in anti-T.gondii infection mediated by apoptosis and autophagyC/EBP ? was overexpressed or inhibited in cells infected by T.gondii to analyze its effects on proliferation,cell cycle progression,apoptosis and autophagy of cells infected by T.gondii.By treating with autophagy inhibitor or inducer,the effect of autophagy on T.gondii infection and tachyzoite was analyzed.Compared to the control,overexpression or inhibition of C/EBP ? has no significant effect on proliferation and cell cycle progression of T.gondii infected cells.The apoptosis and autophagy levels of T.gondii infected cells were significantly increased by C/EBP ? overexpression but reduced by C/EBP ? inhibition.In addition,the reducing effect of C/EBP ? overexpression on proportion of infected cells and number of tachyzoite were affected by autophagy inhibitor 3-MA,and the inducing effect of C/EBP ? inhibition on proportion of infected cells and number of tachyzoite were repressed by autophagy inducer rapamycin.Chapter 4: The role of C/EBP ?-dependent signaling pathways in the regulation of cell apoptosis and autophagy during T.gondii infectionC/EBP ? was overexpressed or inhibited,and the activation of m TOR,ERK,p38 MAPK and JNK signaling molecules were analyzed in cells infected by T.gondii.In the meantime,the effect of these signaling on C/EBP ? mediated killing of T.gondii was analyzed by treating cells with m TOR and p38 MAPK signaling inhibitor or activator.Compared to control,the phosphorylation level of p38 MAPK was significantly increased and m TOR phosphorylation level was decreased in T.gondii infected cells treated by Ad-C/EBP ?.On the contrary,p38 MAPK phosphorylation level was decreased and m TOR phosphorylation level was increased in T.gondii infected cells treated by C/EBP ? inhibition.And there was no significant change in the phosphorylation level of ERK and JNK.Additionally,the reducing effect of C/EBP ? overexpression on proportion of infected cells and number of tachyzoite were enhanced by m TOR inhibitor but inhibited by m TOR activator and p38 MAPK inhibitor.In summary,C/EBP ? was up-regulated in the main organisms of host when infected by T.gondii.The change of C/EBP ?'s expression level can active p38 MAPK signaling pathway and inhibit the phosphorylation level of m TOR,both of which play positive roles in apoptosis and autophagy of host cells.These processes may be related to C/EBP ? mediated killing of T.gondii.By regulating the downstream signaling pathways,C/EBP ? could promote apoptosis and autophagy of host cells to inhibit T.gondii infection and accelerate the clear of tachyzoite. |
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