Abstract：
BACKGROUND: Long-term chronic inflammation often leads to chronic diseases. Although Sophora flavescens has been shown to have anti-inflammatory properties, its detailed molecular mechanism is still unknown.
OBJECTIVE: This study investigated the effect of Radix Sophorae Flavescentis on the LPS-induced inflammatory response in macrophages.
METHODS: LPS was used to induce the peritoneal macrophages to simulate the inflammatory environment in vitro. Different concentrations of Radix Sophorae Flavescentis-containing (medicated) serum were used for intervention. The peritoneal macrophages were identified by using hematoxylin-eosin and immunofluorescence staining. ELISA was used to measure the TNF-α and IL-6 expression to determine the concentration of LPS. ELISA and Western blot (WB) were used to detect the PGE2 and CFHR2 expression in each group, respectively. The lentiviral vector for interference and overexpression of the CFHR2 gene was constructed, packaged, and transfected into LPS-induced macrophages. The transfection efficiency was verified by WB. Then, ELISA was used to detect the TNF-α, PGE2, and IL-6 expression. WB was used to detect the CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression.
RESULTS: The primary isolated cells were identified as macrophages. The LPS-treated macrophages exhibited significantly higher expression of PGE2 and CFHR2, and the inflammatory factors TNF-α and IL-6, as well as iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression compared with the control group (P < 0.05). The TNF-α, PGE2, and IL-6 levels, as well as CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression were considerably lower in the LPS-induced+10% medicated-serum group, LPS-induced+20% medicated-serum group, and shCFHR interference group compared with the LPS group (P < 0.05).
CONCLUSIONS: Radix Sophorae Flavescentis might mediate CFHR2 expression and play an important role in inhibiting the LPS-induced pro-inflammatory response of macrophages. Radix Sophorae Flavescentis could be a potential treatment for LPS-induced related inflammatory diseases.
OBJECTIVE: This study investigated the effect of Radix Sophorae Flavescentis on the LPS-induced inflammatory response in macrophages.
METHODS: LPS was used to induce the peritoneal macrophages to simulate the inflammatory environment in vitro. Different concentrations of Radix Sophorae Flavescentis-containing (medicated) serum were used for intervention. The peritoneal macrophages were identified by using hematoxylin-eosin and immunofluorescence staining. ELISA was used to measure the TNF-α and IL-6 expression to determine the concentration of LPS. ELISA and Western blot (WB) were used to detect the PGE2 and CFHR2 expression in each group, respectively. The lentiviral vector for interference and overexpression of the CFHR2 gene was constructed, packaged, and transfected into LPS-induced macrophages. The transfection efficiency was verified by WB. Then, ELISA was used to detect the TNF-α, PGE2, and IL-6 expression. WB was used to detect the CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression.
RESULTS: The primary isolated cells were identified as macrophages. The LPS-treated macrophages exhibited significantly higher expression of PGE2 and CFHR2, and the inflammatory factors TNF-α and IL-6, as well as iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression compared with the control group (P < 0.05). The TNF-α, PGE2, and IL-6 levels, as well as CFHR2, iNOS, COX-2, TLR2, TLR4, IFN-γ, STAT1, and p-STAT1 expression were considerably lower in the LPS-induced+10% medicated-serum group, LPS-induced+20% medicated-serum group, and shCFHR interference group compared with the LPS group (P < 0.05).
CONCLUSIONS: Radix Sophorae Flavescentis might mediate CFHR2 expression and play an important role in inhibiting the LPS-induced pro-inflammatory response of macrophages. Radix Sophorae Flavescentis could be a potential treatment for LPS-induced related inflammatory diseases.
摘要：
背景：长期慢性炎症常导致慢性疾病。虽然苦参已被证明具有抗炎特性，其详细的分子机制尚不清楚。
目的：本研究探讨苦参对LPS诱导的巨噬细胞炎症反应的影响。
方法：用LPS诱导腹腔巨噬细胞体外模拟炎症环境。采用不同浓度的苦参含药血清进行干预。通过使用苏木精-伊红和免疫荧光染色鉴定腹膜巨噬细胞。采用ELISA法检测TNF-α和IL-6的表达,以确定LPS的浓度。ELISA和Westernblot(WB)检测各组PGE2和CFHR2的表达,分别。构建了干扰和过表达CFHR2基因的慢病毒载体,已包装,并转染到LPS诱导的巨噬细胞中。通过WB验证转染效率。然后，ELISA法检测TNF-α,PGE2和IL-6表达。WB用于检测CFHR2，iNOS，COX-2、TLR2、TLR4、IFN-γ、STAT1和p-STAT1表达。
结果:原代分离的细胞被鉴定为巨噬细胞。LPS处理后的巨噬细胞中PGE2和CFHR2、炎症因子TNF-α和IL-6以及iNOS的表达明显增高,COX-2、TLR2、TLR4、IFN-γ、STAT1和p-STAT1表达与对比组比拟(P<0.05)。TNF-α,PGE2和IL-6水平，以及CFHR2，iNOS，COX-2、TLR2、TLR4、IFN-γ、STAT1和p-STAT1表达在LPS诱导的+10%含药血清组中显著降低，LPS诱导+20%含药血清组，而shCFHR干涉组与LPS组比拟(P<0.05)。
结论：苦参可能介导CFHR2的表达，在抑制LPS诱导的巨噬细胞促炎反应中起重要作用。苦参可能是治疗LPS诱导的相关炎性疾病的潜在药物。
目的：本研究探讨苦参对LPS诱导的巨噬细胞炎症反应的影响。
方法：用LPS诱导腹腔巨噬细胞体外模拟炎症环境。采用不同浓度的苦参含药血清进行干预。通过使用苏木精-伊红和免疫荧光染色鉴定腹膜巨噬细胞。采用ELISA法检测TNF-α和IL-6的表达,以确定LPS的浓度。ELISA和Westernblot(WB)检测各组PGE2和CFHR2的表达,分别。构建了干扰和过表达CFHR2基因的慢病毒载体,已包装,并转染到LPS诱导的巨噬细胞中。通过WB验证转染效率。然后，ELISA法检测TNF-α,PGE2和IL-6表达。WB用于检测CFHR2，iNOS，COX-2、TLR2、TLR4、IFN-γ、STAT1和p-STAT1表达。
结果:原代分离的细胞被鉴定为巨噬细胞。LPS处理后的巨噬细胞中PGE2和CFHR2、炎症因子TNF-α和IL-6以及iNOS的表达明显增高,COX-2、TLR2、TLR4、IFN-γ、STAT1和p-STAT1表达与对比组比拟(P<0.05)。TNF-α,PGE2和IL-6水平，以及CFHR2，iNOS，COX-2、TLR2、TLR4、IFN-γ、STAT1和p-STAT1表达在LPS诱导的+10%含药血清组中显著降低，LPS诱导+20%含药血清组，而shCFHR干涉组与LPS组比拟(P<0.05)。
结论：苦参可能介导CFHR2的表达，在抑制LPS诱导的巨噬细胞促炎反应中起重要作用。苦参可能是治疗LPS诱导的相关炎性疾病的潜在药物。
