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Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from Salvia miltiorrhiza Bunge (Danshen)

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ABSTRACT

1–45–1844444: Four novel compounds () as well as fourteen reported compounds () were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound demonstrated the best anti-inflammatory activity and was chosen for further research. Compound greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-κB was attenuated after treatment with compound in vitro. Compound was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.

No MeSH data available.


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Compound 4 blocked LPS-induced TRL4 dimerization.(A) HEK293T cells transfected with TLR4-HA and TLR4-Flag were treated with compound 4 for 1 h. And then co-treated with LPS (1 μg/mL) for 24 h. Cells were collected and proteins were immuneprecipitated with anti-HA magnetic beads. Immunocomplexes were detected by western blotting with anti-HA and anti-Flag antibodies. (B) Quantification of TLR4-HA and TLR4-Flag in HEK293T cells. Quantification of TLR4-HA and TLR4-Flag was determined with densitometric analysis, and TLR4-Flag was compared to TLR4-HA. *p < 0.05 versus the LPS-treated group.
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f6: Compound 4 blocked LPS-induced TRL4 dimerization.(A) HEK293T cells transfected with TLR4-HA and TLR4-Flag were treated with compound 4 for 1 h. And then co-treated with LPS (1 μg/mL) for 24 h. Cells were collected and proteins were immuneprecipitated with anti-HA magnetic beads. Immunocomplexes were detected by western blotting with anti-HA and anti-Flag antibodies. (B) Quantification of TLR4-HA and TLR4-Flag in HEK293T cells. Quantification of TLR4-HA and TLR4-Flag was determined with densitometric analysis, and TLR4-Flag was compared to TLR4-HA. *p < 0.05 versus the LPS-treated group.

Mentions: Based on the promising results of compound 4 at inhibiting NF-κB translocation and JNK1/2 and ERK1/2 actinvation in vitro, we further investigated whether toll-like receptor (TLR4) would be potentially modulated by compound 4. TLR4, a transmembrane receptor located on surface of some of immune cells, is critical signaling receptor for LPS that mediates innate and acquired immunity37. Activated by LPS, TLR4 will be induced to dimer, and subsequently causes a MAPK signaling and nuclear translocation of transcription factors NF-κB through interacting between dimerized TLR4 and its downstream ‘bridging adaptor’ molecules, phosphorylated TRAM or MYD88, eventually triggering a pathogen-specific innate immune response by upregulating the proinflammatory cytokines release3839. Therefore, to suppress the formation of homodimerization of TLR4 is proposed to be a new strategy to treat inflammatory diseases40. In this study, to detect the ability of compound 4 to disrupt TLR4 dimerization induced by LPS, we employed HEK293T cells co-transfected with TLR4-HA and TLR4-Flag plasmids. As shown in Fig. 6, the decreasing of TLR4-Flag in the TLR4-HA precipitation after treatment with compound 4 showed that compound 4 suppressed the TLR4 dimerization significantly compared to that of LPS group without compound 4 treatment. Therefore, we speculated that the potential anti-inflammatory effect of compound 4 may due to its inhibition on TLR4 dimerization.


Tanshinones and diethyl blechnics with anti-inflammatory and anti-cancer activities from Salvia miltiorrhiza Bunge (Danshen)
Compound 4 blocked LPS-induced TRL4 dimerization.(A) HEK293T cells transfected with TLR4-HA and TLR4-Flag were treated with compound 4 for 1 h. And then co-treated with LPS (1 μg/mL) for 24 h. Cells were collected and proteins were immuneprecipitated with anti-HA magnetic beads. Immunocomplexes were detected by western blotting with anti-HA and anti-Flag antibodies. (B) Quantification of TLR4-HA and TLR4-Flag in HEK293T cells. Quantification of TLR4-HA and TLR4-Flag was determined with densitometric analysis, and TLR4-Flag was compared to TLR4-HA. *p < 0.05 versus the LPS-treated group.
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Related In: Results  -  Collection

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f6: Compound 4 blocked LPS-induced TRL4 dimerization.(A) HEK293T cells transfected with TLR4-HA and TLR4-Flag were treated with compound 4 for 1 h. And then co-treated with LPS (1 μg/mL) for 24 h. Cells were collected and proteins were immuneprecipitated with anti-HA magnetic beads. Immunocomplexes were detected by western blotting with anti-HA and anti-Flag antibodies. (B) Quantification of TLR4-HA and TLR4-Flag in HEK293T cells. Quantification of TLR4-HA and TLR4-Flag was determined with densitometric analysis, and TLR4-Flag was compared to TLR4-HA. *p < 0.05 versus the LPS-treated group.
Mentions: Based on the promising results of compound 4 at inhibiting NF-κB translocation and JNK1/2 and ERK1/2 actinvation in vitro, we further investigated whether toll-like receptor (TLR4) would be potentially modulated by compound 4. TLR4, a transmembrane receptor located on surface of some of immune cells, is critical signaling receptor for LPS that mediates innate and acquired immunity37. Activated by LPS, TLR4 will be induced to dimer, and subsequently causes a MAPK signaling and nuclear translocation of transcription factors NF-κB through interacting between dimerized TLR4 and its downstream ‘bridging adaptor’ molecules, phosphorylated TRAM or MYD88, eventually triggering a pathogen-specific innate immune response by upregulating the proinflammatory cytokines release3839. Therefore, to suppress the formation of homodimerization of TLR4 is proposed to be a new strategy to treat inflammatory diseases40. In this study, to detect the ability of compound 4 to disrupt TLR4 dimerization induced by LPS, we employed HEK293T cells co-transfected with TLR4-HA and TLR4-Flag plasmids. As shown in Fig. 6, the decreasing of TLR4-Flag in the TLR4-HA precipitation after treatment with compound 4 showed that compound 4 suppressed the TLR4 dimerization significantly compared to that of LPS group without compound 4 treatment. Therefore, we speculated that the potential anti-inflammatory effect of compound 4 may due to its inhibition on TLR4 dimerization.

View Article: PubMed Central - PubMed

ABSTRACT

1–45–1844444: Four novel compounds () as well as fourteen reported compounds () were isolated and purified from Salvia miltiorrhiza Bunge (Danshen). The structures of novel compounds were determined by 1D and 2D NMR, HRESIMS data, etc. The anti-inflammatory properties of all the compounds on RAW264.7 macrophages and their cytotoxicity on H1299 and Bel-7402 cell lines coupled with a structure-activity relationship (SAR) were investigated. Compound demonstrated the best anti-inflammatory activity and was chosen for further research. Compound greatly suppressed secretion of nitric oxide (NO), tumor necrosis factor (TNF)-&alpha; and interleukin-6 (IL-6) in the RAW264.7 macrophages stimulated by LPS. Additionally, the protein expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) was decreased and the nuclear translocation of NF-&kappa;B was attenuated after treatment with compound in vitro. Compound was able to dramatically inhibit LPS-induced activation of JNK1/2 and ERK1/2 and remarkably disrupted the TLR4 dimerization in LPS-induced RAW264.7 macrophages. Thus, the new compound suppressed LPS-induced inflammation partially is due to the blocking TLR4 dimerization. In addition, the anti-cancer activity investigation indicated that most of isolated compounds exhibited cytotoxicity and the SAR analysis showed that the intact D ring was indispensable and unsaturated D ring played vital role.

No MeSH data available.


Related in: MedlinePlus