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Influence of the chemical constituents of Salvia miltiorrhiza Bunge on lipopolysaccharide-induced activation of Kupffer cells
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摘要:
目的探讨丹参化学成分对脂多糖激活Kupffer细胞（KCs）作用的影响及机制。方法 60 ng/ml脂多糖（LPS）作用原代培养的KCs 12 h后,加入丹参提取物及分离纯化的丹参化学成分——丹参新醌乙、丹参新酮、丹参醇A、丹参酮I、异丹参酮I、丹参新醌甲、隐丹参酮、丹参酮II A、丹参酚酸B各100μg/ml。培养12 h后,以MTT法检测KCs的增殖情况,ELISA检测培养上清中TNFα、IL-6和IL-8的含量,Western Blot检测KCs内CD14、TLR2和TLR4蛋白的表达。结果除丹参新醌乙外,其余受试的丹参化学成份和丹参提取物均能不同程度地抑制LPS刺激后的KCs增殖,但以丹参酮I、丹参新醌甲和丹参新酮的抑制率最高。LPS刺激后,KCs分泌TNFα、IL-6、IL-8的能力显著升高;丹参新醌乙和丹参酚酸B对三种细胞因子的分泌无明显抑制作用,丹参酮II A对IL-8的分泌无影响,其余受试成分对KCs的分泌能力有不同程度的抑制作用。Western Blot结果表明,LPS刺激KCs后CD14、TLR2、TLR4的蛋白表达量显著增加,加入受试的各丹参成分后,丹参新醌乙、丹...
Abstract:Objective To survey the chemical constituents of Salvia miltiorrhiza bunge and characterize their abilities to influence lipopolysaccharide (LPS) -induced activation of Kupffer cells (KCs) .Methods After culturing KCs with 60 ng/ml of LPS for 12 h, the supernatant medium was collected and divided for respective mixing with 100 μg/ml each of the isolated constituents of Salvia miltiorrhiza bunge: neotanshinone B, miltionone, tanshinol A, tanshinone I, isotanshinone I, neotanshinone A, cryptotanshinone, tanshinone IIA, and salvianolie acid B.Untreated supernatant was used as control.The mixtures and control were added to fresh cultures of KCs, respectively, and incubated for 12 h.Effects on cellular proliferation were assessed by MTT assay, on secreted levels of cytokines (TNF α, IL-6, and IL-8) were measured by enzyme linked immunosorbent assay (ELISA) , and on protein expressions of immunity-related receptors (CD14, TLR2, and TLR4) were detected by Western blot.Results Except for neotanshinone B, the Salvia miltiorrhiza bunge chemical constituents inhibited proliferation of KCs.The extent of inhibition varied for each constituent, but tanshinone I, neotanshinone A, and miltionone produced the highest inhibitory rates.LPS stimulation of KCs led to enhanced TNFα, IL-6, and IL-8 expression.Subsequent treatment with neotanshinone B, salvianolie acid B, or tanshinone IIA had no detectable effect on the LPS-induced expression of IL-8.The other constituents, however, inhibited the ability of KCs to secret cytokines, to various degrees.In addition, LPS stimulation of KCs led to increased expression of CD14, TLR2 and TLR4 proteins.Subsequent treatment with neotanshinone B and salvianolie acid B had no effect on LPS-induced CD14;neotanshinone B and miltionone had no effect on LPS-induced TLR2;and neotanshinone B, tanshinone IIA, and salvianolie acid B had no effect on LPS-induced TLR4.The other constituents, however, significantly down-regulated the expression levels of these three receptors.Conclusion With the exception of neotanshinone B, the full panel of Salvia miltiorrhiza burge's chemical constituents can inhibit LPS-induced activation, proliferation, and cytokine secretion of KCs.These effects may be related to the ability of the chemical constituents to decrease LPS-induced protein expression of CD14, TLR2, and TLR4.
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Key words:
- salvia miltiorrhiza /
- kupffer cells /
- lipopolysaccharides /
- cytokine /
- CD14 /
- receptors
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