P-PGBL Can Inhibit The Inflammatory Response Through TLR4/NF-κB Signaling Pathway

Ginkgo biloba is an ancient gymnosperms, which is called“pandas of flora”.Itsleaves, fruits and seed coat all have medicinal value and nourishing health function,hailed as a living fossil and all of it is treasure. In recent years, Ginkgo biloba hasbecome the hot of drug development and research in domestic and international.There are many of Ginkgo biloba products agents for the treatment of disease of heartand cerebrovascular diseases by far. PGBL (Polysaccharides of Ginkgo biloba leaf)isa kind of water-soluble ingredients which extracted From Ginkgo biloba. with avariety of biological activity, such as immune regulation, anti-inflammatory,antioxidant, anti-tumor.Lipopolysaccharide (LPS) is a major component of the cell wall ofGram-negative bacteria, Which could lead to endotoxin shock, systemic inflammatoryresponse syndrome. Toll like receptor (TLR) family is a natural receptors of the innateimmune system to recognize pathogen-associated molecular patterns, and play acrucial role in the immune process.TLR4is one of the members of TLRs receptorfamily, it is a kind of pattern recognition receptors, main identification of LPS, Whichcould regulation of inflammatory response to clear pathogen infection throμgh signaltransduction.It is reported that polysaccharides from Ginkgo biloba leaves has significantanti-inflammatory activity, but its specific anti-inflammatory mechanism needsfurther study. Based on previous studies, in this study purified PGBL(P-PGBL) wasobtained by extraction and purification. First，we investigated the cytotoxicity of theP-PGBL by CCK-8assay. The results showed that, the concentrations of P-PGBLused in this study had no significant effect on the proliferation of macrophages (P>0.05), which showed that the concentrations of P-PGBL have no toxic effects oninflammatory cells, this showed that under the condition of the concentration ofP-PGBL took place on the inflammatory cells.In order to determine the anti-inflammatory effects of P-PGBL on inflammatorycells, We use LPS stimulated RAW264.7cell to establish model of inflammatory cells. Then we Respectively detected the expression of IL-6﹑IL-1β and their genes.The results showed that P-PGBL can significantly inhibit the LPS inducedexpression of IL-6and IL-1β on RAW264.7cells, compared with the LPS group（P＜0.05）,And a certain concentration of P-PGBL also attenuated the overexpressionof IL-6mRNA and IL-1β mRNA. These results indicate that P-PGBL hasanti-inflammatory effect, and may inhibit the expression of inflammatory factors in bytranscription and translation.To study the anti-inflammatory mechanism of P-PGBL,We first detected theexpression of TLR4by immunofluorescence method、flow cytometry and westernblot method. the results of Flow cytometry and western blot showed that LPSsignificantly induced TLR4expression in RAW264.7cells. When incubated withdifferent concentrations of P-PGBL together, TLR4protein expression wassignificantly decreased, Compared with the LPS group（P＜0.05）.The results wereconsistent with the results of immunofluorescence. Secondly, we measured theexpression of TLR4mRNA, The results showed that, P-PGBL also significantlyinhibited LPS-induced TLR4mRNA expression (P <0.05).These results indicate that,P-PGBL exert anti-inflammatory activity by inhibiting the expression of TLR4receptor.In order to investigate the effect of P-PGBL on TLR4signal transductionpathways, we measured the expression of NF-κB on inflammatory cells by westernblot and RT-PCR. The western blot analysis demonstrated that, The level of NF-κBproduction induced by LPS in RAW cells decreased significantly （P＜0.05）in adose-dependent manner when treated with P-PGBL. The results of RT-PCR foundthat, P-PGBL can also significantly inhibit the overexpression of the NF-κB mRNAin inflammatory cells in a dose dependent manner（P＜0.05）.These resultsdemonstrated that p-PGBL can inhibit the expression of IL-6and IL-1β throμghregulating the NF-κB pathway.Our experimental results showed that the P-PGBL have a goodanti-inflammatory effect. The related mechanism is inhibition of the expression ofinflammatory cytokines, such as IL-6and IL-1β by regulating the TLR4/NF-κB signaling pathway. The study not only to provide valuable experimental evidence forthe development of natural polysaccharide anti-inflammatory drugs, but also toguide the new direction for the treatment of diseases which associated with TLR4.Atthe same time it paves the way not only to discover anti-inflammatory drugs withhigher efficiency and non-toxic, but also to utilize Ginkgo biloba resources.