Pinellia pedatisecta lectin exerts a proinflammatory activity correlated with ROS-MAPKs/NF-κB pathways and the NLRP3 inflammasome in RAW264.7 cells accompanied by cell pyroptosis.

Pinellia pedatisecta, a widely used herb in Chinese medicine, has proinflammatory toxicity related to its Pinellia pedatisecta lectin (PPL), but the mechanism is still unknown. However, for safer use, it is necessary to clarify its proinflammatory mechanism. Herein, we studied the mechanism in RAW264.7 cells. PPL decreased the mitochondrial membrane potential (MMP) and increased the outflow of calcium, accompanied by the overproduction of reactive oxygen species (ROS), which resulted in the activation of the MAPK and NF-κB pathways and the release of IL-1ß. The maturation of IL-1ß relied on caspase-1 p20, the active caspase-1, as demonstrated by adding caspase-1 inhibitor. While caspase-1 was associated with the activation of the NLRP3 inflammasome, we further found that the stimulation of PPL also contributed to the activation. In addition, TXNIP was downregulated, whereas NLRP3/caspase-1 p20/ASC was upregulated, and there was binding of TXNIP with NLRP3. There was also binding of NLRP3 with ASC and caspase-1. Further, we found that N-acetylcysteine (NAC), an ROS scavenger, could inhibit the PPL-stimulated activation of these pathways and the release of IL-1ß. Moreover, PPL led to cell pyroptosis with pyknotic nuclei and plasma membrane rupture, which could be inhibited by NAC. All of these findings demonstrated an important role of ROS in the inflammation caused by PPL. Taken together, our data provide new mechanistic insights into the possible endogenous signaling pathways involved in the inflammation of RAW264.7 cells, stimulated by PPL.

Upregulation of aquaporin 3 expression by diterpenoids in Euphorbia pekinensis is associated with activation of the NF-κB signaling pathway in the co-culture system of HT-29 and RAW 264.7 cells.

This study was designed to evaluate the toxic effects of diterpenoids separated from the roots of Euphorbia pekinensis, a type of widely used traditional Chinese medicine. This herb has intestinal toxicity associated with its complex diterpenoids. In this study, the diterpenoids (pekinenin A, pekinenin C, pekinenin F, pekinenin G, yuexiandajisu A, (-)-(1S)-15-hydroxy-18-carboxycembrene) elevated the expression of interleukin 1 beta and tumor necrosis factor alpha in a dose-dependent manner at doses of 6.25, 12.5, and 25 µM in RAW264.7 monocultures. Pekinenin C increased the expression of phosphorylated IκB and phosphorylated p65 in RAW264.7 monocultures, indicating that it stimulated a substantial inflammatory response and activated the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway. A co-culture model of RAW 264.7 mouse macrophage cells and HT-29 human intestinal epithelial cells was established to study the correlation between inflammation and aquaporin (AQP) expression and to evaluate the toxicity of different diterpenoids from E. pekinensis. Pekinenin C (6.25, 12.5, and 25 µM) increased AQP3 mRNA and protein expression of HT-29 cells in the co-culture system in a dose-dependent manner but not in HT-29 monocultures. AQP3 mRNA and protein expression peaked at 2 and 3 h of HT-29 cells in the co-culture system, respectively. In contrast, their expression peaked more slowly in the monoculture system. After the specific NF-κB inhibitor BAY11-7082 (5, 10, and 20 µM) was added to the co-culture system, the release of cytokines and increased AQP3 expression caused by pekinenin C were inhibited. Comparisons of the representative monomeric compound pekinenin C, diterpenoid monomer mixtures, and total diterpenoids from E. pekinensis showed that the monomer mixtures had the most toxicity. In conclusion, this study demonstrated that E. pekinensis induces inflammation and increases the expression of AQP3, causing disorders of water metabolism, which may lead to gastrointestinal side effects such as diarrhea.

Circular RNAs promote TRPM3 expression by inhibiting hsa-miR-130a-3p in coronary artery disease patients.

We investigated the differential expression of circular RNAs (circRNAs) in plasma samples from three coronary artery disease (CAD) patients to identify putative therapeutic targets. We identified 24 differentially expressed circRNAs (18 up-regulated and 6 down-regulated) and 7 differentially expressed mRNAs (6 up-regulated and 1 down-regulated) in CAD patients based on competing endogenous RNA (ceRNA) microarray analysis. MiR-221(p = 0.001), miR-155(p = 0.049), and miR-130a (p = 0.001) were downregulated in CAD patients based on qRT-PCR analysis of another independent population of 932 study subjects (648 CAD subjects and 284 controls). We constructed a hsa-miR-130a-3p-mediated circRNA-mRNA ceRNA network using the miRanda database. This included 9 circRNAs (hsa_circ_0089378, hsa_circ_0083357, hsa_circ_0082824, hsa_circ_0068942, hsa_circ_0057576, hsa_circ_0054537, hsa_circ_0051172, hsa_circ_0032970, and hsa_circ_0006323) and 1 mRNA (transient receptor potential cation channel subfamily M member 3 [TRPM3]). We have shown that 9 circRNAs promote TRPM3 expression by inhibiting hsa-miR-130a-3p in CAD patients.

Typhonium giganteum Lectin Exerts A Pro-Inflammatory Effect on RAW 264.7 via ROS and The NF-κB Signaling Pathway.

Typhonii rhizoma, a widely used herb in traditional Chinese medicine, has acute irritating toxicity related to Typhonium giganteum lectin (TGL). TGL exhibits acute inflammatory effects, but the underlying molecular mechanisms are largely unknown. This paper is designed to assess the pro-inflammatory response of TGL on RAW 264.7 cells. RAW 264.7 treated with 6.25, 12.5, 25, and 50 µg/mL TGL showed elevated levels of inflammatory factors (TNF-α, IL-1ß) and of p-IκB and p-p65, all dose-dependent, indicating that TGL had a substantial inflammatory effect and mobilized the nuclear factor-κB (NF-κB) pathway. All four TGL treatments also induced the up-regulation of reactive oxygen species (ROS) and cytosolic free Ca2+ and down-regulation of mitochondrial membrane potential (MMP). The production of cytokines and p-IκB, p-p65 were reduced by N-acetylcysteine (NAC), an ROS scavenger, which somewhat abrogated ROS production. The results showed the TGL-activated inflammatory signaling pathway NF-κB to be associated with the overproduction of ROS. Moreover, 50 µg/mL treatment with TGL led to cell apoptosis after 1 h and increased necrosis over time. These results provided potential molecular mechanisms for the observed inflammatory response to TGL including up-regulation of ROS and cytosolic free Ca2+, down-regulation of MMP, the mobilization of the NF-κB pathway, and the subsequent overproduction of pro-inflammatory factors resulting in apoptosis. Long-term stimulation with TGL resulted in strong toxic effects related to inflammation that induced necrosis in macrophages.