The Effects Of Xuebijing Injection On The Mouse Peritoneal Macrophage In Sepsis

Objective(1) To investigate the effects of Xuebijing injection on the proliferation and phagocytic function of mouse peritoneal macrophages in the stimulation of LPS in vitro.(2) To investigate the effects of Xuebijing injection on the polarization of macrophage in sepsis and uncover the signal pathways involved in the processes.Methods(l)Mouse peritoneal macrophages were planted at a density of1×106/ml after separation and purification. The cells were inculpated with different concentrations of Xuebijing injection (Omg/ml, lmg/ml,5mg/ml,10mg/ml,50mg/ml,100mg/ml) for1h before the stimulation of LPS (100mg/ml):after adding CCK-8at12h,24h,48h and incubating for2h, read the absorbance at450nm (OD450nm); after incubation for24h, replaced the culture medium with fluorescent microsphere (1:100) and incubated for2h. Detect the fluorescence intensity of macrophage with the help of immunofluorescence microscopy and flow cytometer.(2) Mouse peritoneal macrophages were planted at a density of1×10/ml after separation and purification. The cells were inculpated with different concentrations of Xuebijing injection (Omg/ml,1mg/ml,5mg/ml,10mg/ml,50mg/ml,100mg/ml) for1h before the stimulation of LPS (100mg/ml):detecting the level of TNF-α, IL-6, IL-10in the supernatant by ELISA and testing the ratio of F4/80+CDllc+CD206-cells and F4/80+CD11c-CD206+cells by flow cytometer at6h,12h and24h; Detecting the ratio of p-JAK1-p-STAT6and JAK1-STAT6by Fast Activated Cell-based ELISA at Oh,0.5h,2h,6h and24h.(3) Mouse peritoneal macrophages were planted at a density of1×106/ml after separation and purification. JAK1or STAT6inhibitor was added for2h before the incubation of Xuebijing injection and stimulation of LPS. detecting the level of TNF-α, IL-6, IL-10in the supernatant by ELISA and testing the ratio of F4/80+CD11c+CD206-cells and F4/80+CD11c-CD206+cells by flow cytometer after24h.(4) Balb/c mice were subjected to lethal sepsis induced by CLP, and the XBJ injection (4mg/kg) was administered (i.m.) at0.5h,12h,24h,36h,48h,60h,72h,84h,96h post-CLP:counting the survival mice at12h,24h,48h,60h,72h,84h,96h,120h post-CLP, and the differences in mortality rate between groups were compared using the Kaplan-Merer method; detecting the level of TNF-α, IL-6, IL-10in the serum by ELISA and testing the ratio of F4/80+CDllc+CD206-cells and F4/80+CD11c-CD206+cells in the peritoneum of mice by flow cytometer after6h,24h and48h post-CLP.Results(1) Xuebijing injection could promote the proliferation of mouse peritoneal macrophage activated by LPS in a time and dose dependent manner with the maximum effects at a concentration of5mg/ml at48hours. Xuebijing injection could increase the phagocytic function of macrophage in a concentration of5and10mg/ml but sharply decrease the phagocytic function when the dose increased to50and100mg/ml.(2) Moderate doses (5mg/ml) of Xuebijing injection could promote the differentiation of peritoneal macrophages from Ml to M2in the stimulation of LPS especially at24h.(3) Moderate doses (5mg/ml) of Xuebijing injection could increase the phosphorylation of JAK1-STAT6at2h, and the effect of M2polarization cansed by Xuebijng injection was sharply decreased in the involvement of JAK1or STAT6inhibitor.(4) The Ml related cytokines (TNF-α、IL-6) were decreased in the serum of mice with the treatment of Xuebijng injection, while the M2related cytokine (IL-10) was increased; the percentage of M2macrophage in the peritoneum of mice post-CLP was increased and the survival rate was improved with the treatment of Xuebijng injection.Conclusion(1) Moderate doses (5mg/ml) of Xuebijing injection could increase the proliferation and phagocytic function of mouse peritoneal macrophage.(2) Moderate doses (5mg/ml) of Xuebijing injection could promote the polarization of mouse peritoneal macrophage, and the JAK1-STAT6signal pathway was related to this processes.(3) Xuebijing injection could improve the survival rate of septic mice and which was related to the promotion of macrophage polarization.