草苁蓉多糖对THP-1巨噬细胞炎症反应的抑制作用及其机制OA北大核心CSTPCD

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.