The Effects Of RhUTI On The Splenic Dendritic Cells In Mice With Sepsis

Objective(1)To investigate whether rh UTI has beneficial effects on immune function(the status of DC maturation and apoptosis)of splenic dendritic cells in CLP mice,a septic model in mice.(2)To investigate the effects of rh UTI on the function of DC in the stimulation of LPS in vitro and uncover the influences of ERS involved in this processes.Methods(1)Balb/c mice were subjected to lethal sepsis induced by CLP,and the rh UTI,UTI were administered(i.m.)at 0.5h,12h,24h post-CLP:counting the sur-vival mice at 12h,24h,48h,60h,72h,84h,96h,120h post-CLP,and the differences in mortality rate between groups were compared using the Kaplan-Merer method;de-tecting the level of TNF-α,IL-12,IL-4,INF-γin the serum by ELISA and testing the ratio of CD80,CD86,MHC-II and apoptosis in splenic DCs of mice by flow cy-tometer after 24h,48h and 72h post-CLP.(2)Mice splenic DC were planted at a density of 1×10~6/ml after separation and puri-fication.The cells were inculpated with different concentrations of rh UTI and UTI injection(0U/ml,100U/ml,300U/ml,900U/ml,2700U/ml,8100U/ml)for 2h before the stimulation of LPS(100ng/ml):after 6h,24h,48h,72h,detecting of the level of TNF-α,IL-12,IL-4,INF-γin the serum by ELISA and testing the ratio of CD80,CD86,MHC-II and apoptosis by flow cytometer;(3)Mouse splenic DC were planted at a density of 5×10~5/ml after separation and pu-rification.The cells were inculpated with different concentrations of of rh UTI and UTI(0U/ml,100U/ml,300U/ml,900U/ml,2700U/ml,8100U/ml)for 2h before the stimulation of LPS(100ng/ml)for 12h;Mice splenic CD 4~+T cells were planted at a density of 1×10~6/ml after separation and purification.The cells were inculpated with different concentrations of of rh UTI and UTI injection(0U/ml,100U/ml,300U/ml,900U/ml,2700U/ml,8100U/ml)for 2h before the stimulation of Con A(5μg/ml)for 12h,then at the rate of 1:200 for72h,detecting the level of the level of IL-4,INF-γin the serum by ELISA,adding CCK-8 incubating for 3h,read the absorbance at 450nm(OD450nm)(4)Mouse splenic DC were planted at a density of 2×10~6/ml after separation and purification.The cells were inculpated with different concentrations of of rh UTI and UTI injection(8100U/ml)for 2h before the stimulation of LPS(100ng/ml)for24h.Expressions of GRP-78,caspase12,chop in DC both three groups were detected by Western blotting.Results(1)The survival rate of septic mice was improved with the treatment of rh UTI,without difference from UTI,the expression of costimulatory molecules induding CD80,CD86and MHC-II was improved by rh UTI at a concentr tion of 250 000U/kg at 24 hours in vivo;(2)rh UTI can reduce the rate of apoptosis of DC at a concentr tion of 250000U/kg at 24 hours,in vivo;(3)Moderate-intensive doses(2700～8100U/ml)of rh UTI could promote the he expression of costimulatory molecules by LPS in a time and dose dependent manner with the maximum effects at 48 hours;(4)rh UTI can re-duce the rate of apoptosis of DCat a concentr tion of 250 000U/kg at 24 hours;(5)The Th1 related cytokines(INF-γ)were increased decreased in the supernatants of DC with the treatment of rh UTI,while the Th2 related cytokine(IL-4)was decreased;rh UTI could promote the proliferation of mouse T cell;(6)The cytokines(IL-12)related to maturationrelated of DC increased,TNF-αdecreased in the serum of DC with the treatment of rh UTI;(7)The expression of GRP-78 related to ERS were im-proved,chop and caspase-12 were both reduced.Conclusion(1)rh UTI could improve the survival rate of septic mice and which was related to the promotion of the immune function and the reduction of apoptosis of DC.(2)Moderate-intensive doses(2700～8100U/ml)of rh UTI could promotion of the im-mune function and the reduction of apoptosis of DC,and the ERS was related to this processes.