Inhibitory Effect and Mechanism of Jingulian Extract on LPS-induced RAW264.7 Cell Inflammatory Response Based on PI3K/Akt Signaling Pathway / 中国实验方剂学杂志

ABSTRACT

Objective:To explore the inhibitory effect and mechanism of Jingulian extract （JGL） on inflammation. Method:The following groups were set up in this study： a control group （10% fetal bovine serum）， a lipopolysaccharide （LPS） model group （0.5 mg·L^-1）， and JGL groups （10， 20， 40， 60， 80， 120， 160， 200， 250， 300 mg·L^-1 + 0.5 mg·L^-1LPS）. The RAW264.7 cells were cultured for 24 hours. Cell proliferation was detected by cell counting kit-8 （CCK-8） assay. Nitric oxide （NO） release was detected by Griess assay. The release of cytokines interleukin （IL）-1<italic>β</italic>， IL-6， IL-10， and tumor necrosis factor （TNF）-<italic>α</italic> was determined by enzyme linked immunosorbent assay （ELISA）. The expression of inducible nitric oxide synthase （iNOS） and intraprostaglandin peroxidase synthase 2 （PTGS2）/cyclooxygenase-2 （COX-2） was measured by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR） and the activation of key proteins in the phosphatidylinositol 3-kinase （PI3K）/protein kinase B （Akt） signaling pathway by Western blot. Result:Compared with the control group， LPS （0.5 mg·L^-1）could promote the proliferation of RAW264.7 cells after stimulation for 24 hours （<italic>P</italic><0.01）. Compared with the model group， JGL had no significant effect on cell proliferation. Compared with the control group， LPS （0.5 mg·L^-1）increased the release of NO， IL-1<italic>β</italic>， IL-6， IL-10， and TNF-<italic>α</italic> （<italic>P</italic><0.01）. Compared with the model group， JGL （20-300 mg·L^-1）inhibited the release of NO in a dose-dependent manner after stimulation for 24 hours （<italic>P</italic><0.05） and reduced IL-1<italic>β</italic>， IL-6， and IL-10 （<italic>P</italic><0.05， <italic>P</italic><0.01）， but no obvious inhibition on the release of TNF-<italic>α</italic> was observed. LPS （0.5 mg·L^-1） could induce the expression of iNOS and PTGS2/COX-2 genes as compared with the control group （<italic>P</italic><0.05， <italic>P</italic><0.01）. JGL could down-regulate the mRNA expression of iNOS and PTGS2/COX-2 genes as compared with the model group （<italic>P</italic><0.05， <italic>P</italic><0.01）. LPS （0.5 mg·L^-1） could activate the PI3K/Akt pathway （<italic>P</italic><0.01） as compared with the control group， while JGL （10， 20， 40， and 80 mg·L^-1） decreased the expression of PI3K-p110， p-p85， and p-Akt （<italic>P</italic><0.01）， and inhibited the activation of PI3K/Akt pathway. Conclusion:JGL extract could significantly inhibit the inflammatory response and activation of the PI3K/Akt pathway induced by LPS in RAW264.7 cells. The anti-inflammatory effect was related to the inhibition of the PI3K/Akt pathway.