The Study On Manipulation Of Host Macrophage Apoptosis By Toxoplasma Gondii Isolates Of TgCtwh3and RH

Background Toxoplasma gondii is an obligate and opportunistic apicomplexan parasitewhich can invade and replicate in almost all nucleated cells of warm-blooded animal.Feline animals including domestic cats are definitive hosts,whereas manywarm-blooded animals including human are intermediate hosts.In Europe and NorthAmerica, the population structure of T.gondii is dominated by three genotypes,designated as strains types I, II and III, according to the virulence to mice and humans.The genotype which is dominantly prevalent in China is Chinese1.Many studies haveconfirmed that T.gondii can establish extensive and effective infection in the hostthrough regulating the host cell apoptosis(inhibiting or promoting) for survival andproliferation. At present, studies on mechanisms underlying the manipulation of hostcells apoptosis by toxoplasma are mainly focused on the death receptor pathway andmitochondrial pathway, rare approach, however, has been reported on the modulation of endoplasmic reticulum stress-mediated apoptosis by Toxoplasma. In addition, studies inthis field are mainly focused on the strain of Toxoplasma with archetypical type such asRH(virulent strain), and the characteristics of modulating host cell apoptosis by Chinese1(TgCtwh3,virulent) remain unknown.Objective To observe the apoptosis of macrophage Raw264.7infected with TgCtwh3and RH strains, to investigate the transcription and expression profile ofapoptosis-related factors in macrophage, to explore the difference in modulatingapoptosis between TgCtwh3and RH strain, and thus may provide insights into thedevelopment of prevention and treatment of toxoplasmosis in China.Methods1) The cells were divided into three groups: control, TgCtwh3infection, andRH infection, and each group was treated with apoptosis inducer(Staurosporine)respectively. Apoptosis of macrophages was detected by flow cytometry;2) The mRNAlevels of anti-apoptotic family members(Bcl-2,Bcl-xl), pro-apoptotic familymembers(Bax,Bad) and endoplasmic reticulum stress marker(CHOP) were detected ineach group by qRT-PCR.3) Using Western blotting to analyse the protein expression ofanti-apoptotic family members(Bcl-2,Bcl-xl), pro-apoptotic members(Bax,Bad), CHOPand a key protease Caspase9.Results1) The early apoptosis of infected cells significantly decreased;2) The mRNAlevels of Bcl-2, Bcl-xl, and CHOP in infection group significantly increased, and themRNA levels of Bax, Bad had no obvious change in each group. In addition, therewere no significant differences in mRNA levels of Bcl-2, CHOP among infectiongroups, but the mRNA levels of Bcl-xl in TgCtwh3infection group was much higherthan that of RH infection group;3) The results of Western blotting showed that theprotein expression levels of Bcl-2, Bcl-xl, and CHOP rather than Bax, Bad increased obviously in all infection groups. Moreover, the protein levels of Bcl-xl in TgCtwh3group was remarkablely high in infection groups,but there was no difference in proteinlevels of Bcl-2.Conclusion In the early phase of Toxoplasma infection, both TgCtwh3and RH isolatescan effectively inhibit the apoptosis of host macrophages,but the effect of TgCtwh3inhibiting host cell apoptosis is stronger than RH.