Effects of Shenling Baizhu Powder on lipopolysaccharide-induced inflammatory response of GES-1 cells through TLR4/NFκB/Nrf2 pathway and autophagy
Objective To study the anti-inflammatory effect of Shenling Baizhu Power on human gastric mucosal epithelial cell (GES-1) induced by Helicobacter pylori lipopolysaccharide,and its mechanism. Methods The lipopolysaccharide-induced GES-1 cell inflammation model was established. NO production was assessed by Griess reagent. Cell viability was determined by CCK8 assay. The levels of TNF-α,IL-1β,and IL-6 in cell culture supernatant were measured by ELISA. ROS production were detected through fluorescent probe assay. Caspase 3/7 activation was evaluated using Caspase 3/7 Green Ready Probe. JC-1 was used for the detection of the mitochondrial membrane potential. NF-κB was detected by immunofluorescence. TLR4 and Nrf2 mRNA expression were detected by qRT-PCR. Transmission electron microscopy was applied to observe the number of autophagosomes in GES-1 cells. The expression levels of TLR4,Nrf2,LC3Ⅱ and p62 were determined by Western blotting. Results Compared with model groups,Shenling Baizhu Powder 6.25 and 12.5 μg/mL groups could significantly reduce the content of NO in cell supernatant (P<0.01). The contents of IL-6 and IL-1β in Shenling Baizhu Powder 6.25 and 12.5 μg/mL groups were significantly decreased,and the contents of TNF-α in Shenling Baizhu Powder 12.5 μg/mL groups were significantly decreased (P<0.05,0.01). Compared with model group,ROS production in Shenling Baizhu Powder 6.25 and 12.5 μg/mL groups was significantly decreased (P<0.05,0.01). The level of mitochondrial membrane potential in Shenling Baizhu Powder 12.5 μg/mL group was significantly increased (P<0.05),Shenling Baizhu Powder could significantly inhibit NF-κB p65 nuclear shift (P<0.05). Compared with model group,the green fluorescence intensity of Caspase 3/7 in Shenling Baizhu Powder 12.5 μg/mL group was significantly decreased (P<0.05). The relative expression levels of TLR4 in Shenling Baizhu Powder 12.5 μg/mL group were significantly decreased,while Nrf2 expression levels significantly increased (P<0.05),and the relative expression levels of TLR4 mRNA in each dose group were significantly decreased (P<0.01). The relative expression of Nrf2 mRNA in Shenling Baizhu Powder groups 6.25 and 12.5 μg/mL were significantly increased (P<0.05,0.01). Shenling Baizhu Powder can increase the number of autophagosomes in GES-1 cells in lipopolysaccharide environment. Compared with model group,LC3Ⅱ expression in Shenling Baizhu Powder 12.5 μg/mL group was significantly increased,and p62 expression was significantly decreased (P<0.05). Conclusion Shenling Baizhu Power mitigated lipopolysaccharide-induced inflammatory response in GES-1 cells,which may be associated with the TLR4/NFκB/Nrf2 signaling pathway and activating autophagy.
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