Study of regulatory effect of Astragalus injection on inflammatory response of sepsis / 中国中西医结合急救杂志

ABSTRACT

Objective To observe the effect of Astragalus injection on the expressions of inflammatory cytokines in human primary macrophages stimulated by lipoteichoic acid (LTA) and lipopolysaccharide (LPS),and investigate its effects on inflammatory reactions of Gram-positive (G+) and Gram-negative (G-) bacteria sepsis and its mechanisms.Methods Percoll density gradient centrifugation was used to isolate the human peripheral blood mononuclear cells,then they were purified by immune Anti-Biotin Microbeads with magnetic character and under the induction of recombinant human granulocyte-macrophage colony stimulating factor (rhGM-CSF),the cells were cultivated for 12 days in vitro,eventually the human monocyte-derived macrophage was formed.The cultured human macrophages were inoculated in 96-well plates (each group 3 wells) and 6-well plates (each group 3 wells).The cells were divided into control group (200 μL DMEM added in each well),LTA 1 mg/L group,LPS 0.1 mg/L group and low astragalus injection (0.1 mg/L) and high astragalus injection (0.2 mg/L) dose groups.After the incubator plates were put in an incubator for 24 hours,the protein content of IL-8 and IL-10 in supernatant were detected by enzymelinked immunosorbent assay (ELISA),and the mRNA expression levels of IL-8 and IL-10 were detected by real time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR).Results LTA and LPS all can obviously up-regulate the expression levels of pro-inflammatory factor IL-8 and anti-inflammatory factor IL-10 of macrophage.The expressions of IL-8 and IL-10 protein and mRNA in LTA group and LPS group were significantly higher than those in control group after cuhure for 8 hours and 24 hours,the degrees of increment were more significantly at 24 hours [LTA stimute groupIL-8 protein (ng/L,× 103)41.57± 1.90 vs.1.58 ±0.24,IL-8 mRNA (A value)21.49±8.05 vs.1.00±0.16;IL-10 protein (ng/L)5.90±3.02 vs.2.91 ± 1.54,IL-10 mRNA (A value)1.35±0.34 vs.0.95±0.14;LPS stimute groupIL-8 protein (ng/L,× 103)345.00±22.80 vs.5.60±0.31,IL-8 mRNA (A value)29.84 ± 8.93 vs.1.00 ± 0.16,IL-10 protein (ng/L)122.37 ± 39.26 vs.44.79 ± 3.67,IL-10 mRNA (A value)7.38 ± 1.58 vs.1.35 ± 0.34,all P ＜ 0.05].The Astragalus injection could regulate LTA and LPS to stimulate the macrophage to decrease the expression levels of pro-inflammatory factor IL-8 protein and mRNA and increase the expression levels of anti-inflammatory factor IL-10 protein and mRNA in the macrophage;the changes of regulatory effect in the 24 hour-culture of Astragalus injection high dose group was the most significant [LTA stimute groupIL-8 protein (ng/L,×103)22.63±1.91 vs.41.57±1.90,IL-8 mRNA (A value)12.10±1.93 vs.21.49±8.05,IL-10 protein (ng/L)14.03±2.22 vs.5.90±3.02,IL-10 mRNA (A value)10.37±6.08 vs.1.35±0.34;LPS stimute groupIL-8 protein (ng/L,× 103)167.75 ± 19.90 vs.345.01 ±22.80,IL-8 mRNA (A value)15.61 ± 3.63 vs.29.84±8.93;IL-10 protein (ng/L)243.22±14.41 vs.122.37±39.26,IL-10 mRNA (A value)16.14±4.10 vs.7.38± 1.58,all P ＜ 0.05].Conclusions In the process of inflammatory response,the pro-inflammatory and anti-inflammatory factors co-exist simultaneously.Astragalus injection can inhibit the expression levels of pro-inflammatory factor gene and protein in the inflammatory response of G+ and G-bacteria sepsis and in the mean time,it can promote the expression levels of anti-inflammatory factor gene and protein,thus the immune mechanism of sepsis is affected,achieving the balance between pro-inflammation and anti-inflammation.