Effects Of TRSDL Exttact And Its Active Ingredient On Signal Transduction Mecllanisnl Investigation By TLR4

Mongolian TRSDL, an anti-inflammatory immune compound, is prepared by our research group as an alternative for antibiotics to treat mastitis. It was reported that TRSDL had good anti-inflammatory activity as well as improve the body immunity. To research the anti-inflammatory immune mechanisms of TRSDL and its active ingredients, methods and results are as follows:①Using lipopolysaccharide LPS induced RAW264.7cells to build cell inflammation model.②Using CCK8to assay the effect of TRSDL and its active components on LPS-induced macrophage proliferation, results:show that low concentration TRSDL (1.00,5.00,10.00,25.00mg/mL) has no inhibition to murine RAW264.7cells growth, compared to the control group, there was no significant difference (p>0.05), while high concentrations TRSDL (50.00mg/mL) significantly inhibited mouse RAW264.7cell growth (p<0.05); TRSDL active ingredient monomer geniposide20mmol/L, gallic acid32μg/L and berberine40μmol/L, can inhibit RAW264.7cell growth for48h respectively (p<0.05). Three monomers in a complex at concentration gradient for48h can significantly inhibit cell growth (p<0.05).③Using fluorescence quantitative RT-PCR to assay the effect of TRSDL on IL-1, IL-6, TNF-a, TLR4and NF-κB mRNA in LPS-induced macrophages, using Western Blotting to assay the effect of TRSDL on TLR4and NF-κB proteins in LPS-induced macrophage, the results shows that TRSDL concentration-dependently inhibited the LPS-induced murine RAW264.7cells to produce inflammatory cytokines IL-1, IL-6and TNF-a mRNA and protein expression(p<0.05).5mg/ml TRSDL compared with LPS group has significantly effects to inhibite IL-1, IL-6, TNF-a mRNA and protein expression (p<0.05),10mg/ml and25mg/ml TRSDL had the same inhibition as dexamethasone (p<0.05).④WST-8method to detect the cell vaility in LPS-induced macrophage,result showthat TRSDL monomer Geniposide, gallic acid, berberine and monomer complex, NF-κB inhibitor PDTC and dexamethasone were able to significantly reverse the effect of LPS on mouse RAW264.7cell proliferation (p<0.05). Among them,⑤Using ELISA method to detect the effect of TRSDL and the main active ingredient on inflammatory cytokines IL-1, IL-6and TNF-α release in LPS-induced RAW264.7cells, the results indicatedthe single compound can non-significantly reduce the effect of PDTC on LPS-induced RAW264.7cell proliferation. In addition, Geniposide, gallic acid, berberine and monomer compound could significantly inhibit IL-1, IL-6and TNF-a expression in LPS-induced murine RAW264.7cells as TRSDL⑥Furtherly using Western Blotting and EMSA to assay the effect of TRSDL and its active ingredient on protein expression of p-IκBα, p-p65, p65and TLR4in LPS-induced RAW264.7cells and P65promoter binding activity. Results are as follows:TRSDL monomer Geniposide, gallic acid, berberine, monomer complex, NF-κB inhibitor PDTC and dexamethasone does not affect the total p65protein levels, but were able to significantly inhibit IκBα and p65phosphorylation (p-IκBα and p-p65) in LPS-induced mouse RAW264.7cells (p<0.05), in which the three single compound can promote PDTC inhibited LPS-induced mouse RAW264.7cells p-IκBα and p-p65expression, while the total p65protein levels are not affected. Each treatment group was significantly inhibited TLR4mRNA and protein levels in LPS-induced murine RAW264.7cells, which are consisten with TRSDL. TRSDL monomer Geniposide, gallic acid, berberine, monomer complex, NF-κB inhibitor PDTC and dexamethasone were able to significantly reduce p65promoter binding activity in LPS-induced murine RAW264.7cells(p<0.05). Among them, monomer complex could significantly promote the effect of PDTC to reduce p65promoter binding activity in LPS-induced murine RAW264.7cells.In summary, our research successfully constructed bacterial lipopolysaccharide LPS induced inflammatory cells model in RAW264.7cells; identified Mongolian TRSDL and its main active ingredient dosage:TRSDL concentration is less than the optional (50.00mg/mL), while three active ingredient geniposide, gallic acid and berberine were selected20mmol/L,32μg/L,40μmol/L, as commonly used in the literature; and indicated that the Mongolian TRSDL could significantly inhibit TNF-a, IL-1and IL-6mRNA levels and its protein expression in LPS-induced RAW264.7cells, and at the transcriptional level and protein level significantly inhibited TLR4and NF-κB content, three monomers and their complexes in transcription and protein levels were significantly reduced IL-1, IL-6and TNF-α expression in LPS-induced RAW264.7cells. Meanwhile, it could be speculated Mongolian TRSDL and three monomer inhibited LPS-induced murine macrophage inflammatory responses through inhibiting TLR4, thereby reducing phosphorylation of IκBα and p65subunit, inhibiting p65binding activity, then reducing the combination of p65with promoter of IL-1, IL-6and TNF-α..