| Objective: Sepsis is a clinically significant infection with a very high mortality rate,and the excessive immune response of the body is an important pathogenic link.Xuebijing injection(XBJ)has good clinical efficacy in the treatment of sepsis,but its anti-inflammatory mechanism is not clear.The aim of this study is to investigate the relationship between XBJ and chemokine Fractalkine(FKN),as well as the relationship and role of both with NF-κB signaling pathway,from animal and cellular experimental levels,to provide new basis and ideas for the treatment of clinical sepsis and the application of XBJ.Method: After pre-injection of XBJ into the abdomen of C57BL/6 mice,the septic mouse model was obtained by Cecal Ligation Puncture(CLP).The experimental mice were randomly divided into Sham operation group(Sham),Xuebijing control group(Sham+XBJ),model group(CLP)and Xuebijing treatment group(CLP+XBJ).(1)To observe the effect of XBJ on the survival rate of mice;(2)Hematoxylin-eosin staining(HE)was used to detect lung,liver and kidney damage in mice.(3)Enzyme linked immunosorbent assay(ELISA)was used to detect the expression of serum FKN,TNF-α and IL-6 in mice.(4)The expression level of FKN and the nuclear shift of p65 in mouse lung,liver and kidney were detected by Western Blot.Human Umbilical Vein Endothelial Cells(HUVEC)were treated with XBJ,and cell damage was induced by Lipopolysaccharides(LPS).(5)The culture supernatant of cells was collected,membrane protein and plasma protein were extracted,and the expression level of FKN was detected by Western Blot.The cells were transfected with PCMV-FKN plasmid and divided into CON group,LPS group,LPS+XBJ group,p CMV-FKN group,p CMV-FKN+LPS group and p CMV-FKN+LPS+XBJ group.(6)The expression and nuclear shift of p65 were detected by Western Blot.(7)The levels of TNF-α and IL-6 in the supernatant were determined by ELISA.(8)The effect of FKN on NF-κB signaling pathway was further analyzed by immune-coprecipitation.Results:(1)There was no difference in the survival rate between Sham+XBJ group and Sham group,while the survival rate of mice in CLP+XBJ group(60%)was significantly higher than that in CLP group(40%)(P<0.01).(2)HE staining showed that the lung,liver and kidney injuries in CLP group were more serious than those in Sham group(P<0.05),while the injuries in CLP+XBJ group were significantly reduced than those in CLP group(P<0.05).(3)ELISA showed that compared with CLP group,the expressions of TNF-α,IL-6 and FKN in serum of Sham group and CLP+XBJ group were significantly decreased(P<0.05).(4)Western Blot assay showed that the expression of FKN protein in the lung,liver and kidney tissues of mice in CLP group was significantly increased compared with that in Sham group and CLP+XBJ group(P<0.05).The expression of nuclear p65 protein was also significantly increased(P<0.05).Among all the above results,the differences between Sham group and Sham+XBJ group were not statistically significant.(5)Western Blot assay showed that the expressions of FKN in culture supernatant,membrane protein and plasma protein in LPS group were significantly higher than those in CON group and LPS+XBJ group(P<0.05).After transfection of FKN plasmid,(6)the expression of p65 nuclear protein in p CMV-FKN+LPS group was significantly higher than that in CON,LPS and p CMV-FKN groups(P<0.05),while LPS+XBJ group decreased the expression of nuclear p65 protein in LPS group(P<0.05).p CMV-FKN+LPS+XBJ group also reduced the level of nuclear p65 protein in p CMV-FKN+LPS group(P<0.05).(7)After overexpression of FKN,the expressions of TNF-α and IL-6 were significantly increased by ELISA(P<0.05),and the expressions of TNF-α and IL-6 were further increased by LPS stimulation(P<0.05).The contents of TNF-α and IL-6 in p CMV-FKN+LPS+XBJ group were significantly lower than those in p CMV-FKN+LPS group(P<0.05).(8)The co-interaction of FKN and p65 was found in the immunoprecipitation experiment,and Xuebijing injection could significantly reduce their cointeraction(P<0.05).Conclusion: Xuebijing injection can effectively improve the general state and survival rate of sepsis mice,reduce the expression level of inflammatory factors(TNF-α,IL-6)and reduce lung,liver and kidney tissue injury.Xuebijing injection reduced the expression level of FKN and nuclear migration of p65 in sepsis mice.LPS can induce the increase of FKN expression in HUVEC cells,and Xuebijing injection can reduce the inflammation level of HUVEC cells induced by LPS by inhibiting the co-action of FKN and p65. |
