Abstract:

Objective To discuss the effect of Boschnikia rossica polysaccharides rapa polysaccharides （BRPS） on lipopolysaccharide （LPS）-induced inflammatory responses in the THP-1 macrophages， and to clarify its mechanism. Methods The THP-1 monocytes were differentiated into the macrophages， and the inflammation model was established using LPS to induce the THP-1 macrophages. CCK-8 method was used to detect the survial rates of the THP-1 macrophages after treated with different concentrations （0， 100， 200， 500， 1 000， and 2 000 μg·L^-1） of LPS and different concentrations （0， 12.5， 25.0， 50.0， 100.0， and 200.0 mg·L^-1） of BRPS to select the concentrations for the subsequent experiments. The THP-1 macrophages were divided into blank group， model group， low dose of BRPS group （25.0 mg·L^-1 BRPS）， medium dose of BRPS group （50.0 mg·L^-1 BRPS）， and high dose of BRPS group （100.0 mg·L^-1 BRPS）. P38 inhibitor SB203580， ERK inhibitor U0126， c-Jun N-terminal kinase（JNK） inhibitor SP600125， and nuclear factor of kappa B（NF-κB） inhibitor BAY11-7082 were used to verify the effects on THP-1 cells. The THP-1 cells were divided into control group， LPS group， inhibitor group， 100.0 mg·L^-1 BRPS group， and inhibitor+100.0 mg·L^-1 BRPS group. ELISA method was used to detect the levels of tumor necrosis factor α （TNF-α）， interleukin （IL）-6， and IL-1β in culture fluid of the THP-1 macrophages in various groups； DCFH-DA fluorescence probe method was used to detect the reactive oxygen species （ROS） levels in the THP-1 macrophages in various groups； Hoechst33342/PI fluorescence staining method was used to detect the membrane damage in the THP-1 macrophages in various groups； JC-1 fluorescence staining was used to observe mitochondrial membrane potential in the THP-1 macrophages in various groups； Western blotting method was used to detect the expression levels of cyclooxygenase-2 （COX-2）， high mobility group protein B1 （HMGB1）， NOD-like receptor thermal protein domain assciated protein 3 （NLRP3）， cysteinyl aspartate specific protease （Caspase）-1， gasdermin D （GSDMD）-N， IL-1β， mitogen-activated protein kinase （MAPK）， and nuclear factor-kappa B （NF-κB） related proteins in the THP-1 macrophages in various groups. Results The CCK-8 method results showed that when the LPS concentration was 100-2 000 μg·L^-1， the survival rates of the THP-1 macrophages were over 90%. Compared with 0 μg·L^-1 LPS group， the IL-6 levels in culture fluid of the THP-1 macrophages in 100， 200， 500， 1 000， and 2 000 μg·L^-1 LPS group were increased （P<0.05）， indicating a significant enhancement of the inflammatory response in the macrophages， so 100 μg·L^-1 LPS was used to construct the inflammation model.After treated with 12.5， 25.0， 50.0， 100.0， and 200.0 mg·L^-1 BRPS， the survival rates of the THP-1 macrophage were 91.2%， 93.8%， 91.4%， 90.6%， and 91.8%， respectively， so 25.0， 50.0， and 100.0 mg·L^-1 BRPS were selected as the drug concentrations for low， medium， and high doses of BRPS groups in the subsequent experiments.The ELISA results showed that compared with blank group， the levels of IL-6， TNF-α， and IL-1β in culture fluid of the THP-1 macrophages in model group were increased （P<0.05）； compared with model group， the levels of IL-6， TNF-α， and IL-1β in low， medium， and high doses of BRPS groups were decreased （P<0.05）. The DCFH-DA fluorescence probe method results showed that compared with blank group， the ROS level in the THP-1 macrophages in model group was increased （P<0.05）； compared with model group，the ROS levels in low， medium， and high doses of BRPS groups were decreased （P<0.05）. The Hoechst33342/PI fluorescence staining results showed that compared with blank group， the degree of membrane damage in the THP-1 macrophages in model group was increased； compared with model group， the degrees of membrane damage in low， medium， and high doses of BRPS groups were decreased. The JC-1 fluorescence staining results showed that compared with blank group， the mitochondrial membrane potential in the THP-1 macrophages in model group was decreased significantly； compared with model group， the mitochondrial membrane potential in low， medium， and high doses of BRPS groups were increased gradually.The Western blotting results showed that compared with blank group， the expression levels of COX-2， HMGB1， NLRP3， Caspase 1， GSDMD-N， and IL-1β proteins and the ratios of p-P38/P38， p-ERK/ERK， p-JNK/JNK， and p-NF-κB/NF-κB in the THP-1 macrophages in model group were increased （P<0.05）； compared with model group， the expression levels of HMGB1， NLRP3， Caspase-1， GSDMD-N， and IL-1β proteins and the ratios of p-P38/P38， p-ERK/ERK， p-JNK/JNK， and p-NF-κB/NF-κB in the THP-1 macrophages in medium and high doses of BRPS groups were decreased （P<0.05）， the expression levels of NLRP3， Caspase-1， and IL-1β proteins in the cells in low dose of BRPS group were decreased （P<0.05）， the expression level of COX-2 protein in the cells in high dose of BRPS group was decreased （P<0.05）. Compared with control group， the ratios of p-P38/P38， p-ERK/ERK， p-JNK/JNK， and p-NF-κB/NF-κB， and the expression level of IL-1β protein in the THP-1 macrophages in LPS group were increased （P<0.05）； compared with LPS group， the ratios of p-P38/P38， p-ERK/ERK， p-JNK/JNK， and p-NF-κB/NF-κB， and the expression level of IL-1β protein in the THP-1 macrophages in inhibitor group， 100 mg·L^-1 BRPS group， and inhibitor+100 mg·L^-1 BRPS group were decreased （P<0.05）； compared with inhibitor group， the ratios of p-P38/P38， p-ERK/ERK， p-JNK/JNK， and p-NF-κB/NF-κB in the THP-1 macrophages in inhibitor+100 mg·L^-1 BRPS group were decreased （P<0.05）. Conclusion BRPS inhibits the inflammatory response of the THP-1 macrophages， which may be related to the MAPK and NF-κB signaling pathways regulated by BRPS.

Key words: Boschnikia rossica polysaccharides, NOD-like receptor family pyrin domain-containing protein 3, Mitogen-activated protein kinase, Nuclear factor-κB, Pyroptosis