ABSTRACT
ObjectiveTo explore the effects of phillygenin （PHI） on the inflammation in L02 cells induced by lipopolysaccharide （LPS） and adenosine triphosphate （ATP） and the expression of purinergic 2X7 receptor （P2X7R）， NOD-like receptor family pyrin domain containing 3 （NLRP3）， and nuclear factor kappa B （NF-κB） expression. MethodIn this study， the inflammation model was induced in L02 cells by 100 μg·L-1 LPS treatment for 24 h and 5 mmoL·L-1 ATP treatment for 5 h. The cells in the PHI groups were cultured with PHI （100， 50， 25 mg·L-1） for 6 h in the LPS treatment period， followed by LPS treatment for another 18 h. After ATP treatment for 5 h， the mRNA and protein expression of interleukin-1β （IL-1β）， interleukin-18 （IL-18）， P2X7R， NLRP3， Caspase-1 precursor （pro-Caspase-1）， cleaved Caspase-1， NF-κB， and NF-κB inhibitor protein α （IκBα） in L02 cells was detected by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. Molecular docking was used to predict whether P2X7R could bind to PHI， and DCFH-DA was employed to detect the accumulation of reactive oxygen species （ROS） in cells. P2X7R was silenced by small interfering ribonucleic acid （siRNA）， and then the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was detected by Real-time PCR. ResultReal-time PCR and Western blot showed that compared with the normal group， the model group showed increased expression of IL-1β and IL-18 （P<0.05）， and compared with the model group， the PHI groups showed down-regulated IL-1β， IL-18 mRNA and protein expression （P<0.05）. Molecular docking suggested a good binding effect of PHI to P2X7R. Real-time PCR and Western blot analysis showed that the expression of P2X7R in the model group was significantly up-regulated compared with that in the normal group （P<0.05）， and compared with the model group， the PHI groups showed down-regulated mRNA and protein expression of P2X7R （P<0.05）. DCFH-DA results showed that compared with the normal group， the model group showed increased content of ROS （P<0.05）， and compared with the model group， the PHI groups decreased the accumulation of ROS （P<0.05）. As demonstrated by Real-time PCR and Western blot， compared with the normal group， the model group showed increased expression of NLRP3 inflammasome and NF-κB （P<0.05）， and compared with the model group， the PHI groups significantly decreased the mRNA and protein expression of NLRP3 and cleaved Caspase-1， and up-regulated the mRNA and protein expression of NF-κB and IκBα （P<0.05）. Real-time PCR analysis showed that compared with the results in the model group， after silencing P2X7R by siRNA， the mRNA expression of IL-1β， IL-18， P2X7R， NLRP3， Caspase-1， NF-κB， and IκBα was decreased （P<0.05）. PHI exerted the same effect， and the mRNA expression was further reduced after the combination of them. ConclusionPHI is presumed to suppress the expression of the NLRP3/NF-κB signaling pathway by down-regulating upstream P2X7R to alleviate the LPS/ATP-induced inflammation in L02 cells， suggesting that P2X7R may be the target of PHI against inflammation.
ABSTRACT
Objective:To investigate the relationship between NOD-like receptor pyrin domain containing 3(NLRP3)/cysteine aspartate-specific protease(Caspase)-1 signaling pathway and esophageal inflammation by observing the effect of Xuanfu Daizhe Tang on the composition of inflammatory body and the expression of relevant inflammatory factors in rats with reflux esophagitis (RE), so as to explain the mechanism of Xuanfu Daizhe Tang in treating RE. Method:Sixty healthy male Wistar rats were randomly divided into four groups:the normal control group, the model control group, the Xuanfu Daizhe Tang group (9.89 g·kg-1) and the positive control group (omeprazole enteric-coated tablets+mosapride, 2.58 mg·kg-1), with 15 rats in each group. Except for the blank control group, the remaining rats were operated by " 4.2 mm pyloric clip+2/3 gastric fundus ligation" to establish models. Since the 8th day after the operation, the rats were given corresponding drugs twice a day for 14 days. The arterial blood and esophageal tissues were taken out at the 15th day after the intervention. The pathological morphology of esophagus was observed by naked eyes and under light microscopy. The secretion of cytokines Caspase-1 and interleukin(IL)-1β in serum was detected by enzyme linked immunosorbent assay(ELISA). The expressions of NLRP3, Caspase-1 and IL-1β in esophagus were detected by Western blot. Result:Compared with the normal group, the injury of esophageal mucosa in the model group was the most serious. Compared with the normal group, the levels of Caspase-1 and IL-1β in serum and the expression of NLRP3 protein in esophageal tissue of the model group were significantly increased (PPβ in serum of rats, and down-regulate the expressions of NLRP3, Caspase-1 and IL-1β protein in esophageal tissue (P0.05, PConclusion:Xuanfu Daizhe Tang can regulate the expressions of NLRP3 and Caspase-1, and reduce the content of IL-1β, suggesting that it may antagonize esophageal inflammatory response, reduce esophageal inflammatory injury and treat RE by inhibiting the activation of NLRP3/Caspase-1 signaling pathway.