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

Highlights

• Pinellia pedatisecta lectin (PPL) exerted a pro-inflammatory effect related to ROS.

• PPL induced the activation of MAPKs/NF-κB pathway and the release of IL-1β.

• NLRP3 inflammasome was activated under the stimulation of Pinellia pedatisecta lectin.

• PPL could lead to cell pyroptosis with nuclei pyknotic and plasma membrane rupture.

Abstract

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-1β. The maturation of IL-1β 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-1β. 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.

Introduction

Pinellia pedatisecta Schott, a widely used Chinese medicine for relieving cough and reducing sputum, belongs to the Araceae family [1]. Our previous research had proved that P. pedatisecta demonstrated toxicity due to the Pinellia pedatisecta lectin (PPL), which has been proven to be the main component causing inflammation [2,3]. We have previously verified that PPL could cause mouse peritoneal macrophages to release large amounts of ROS and IL-1β [4]. ROS are known as second messengers in mediating inflammation responses [5]. In the process of inflammation, the production of ROS increases, thereby activating the MAPK and NF-κB signaling pathways [6,7], which results in an abnormal MMP and the release of calcium ions and cytokines [[8], [9], [10], [11]]. Since the MAPK and NF-κB signaling pathways can be activated by certain irritants including excess ROS [12,13], they can increase the level of pro-IL-1β. This, in turn, is activated to become IL-1β, which can exert biological activity [[14], [15], [16]]. Whether the inflammation caused by PPL was related to the MAPKs or to the NF-κB signaling pathway is still under consideration.

Studies have also revealed that ROS may be the key factor regulating the NLRP3 inflammasome [[17], [18], [19]], a multiprotein complex consisted of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), and Caspase-1, but the specific relationship between ROS and NLRP3 remains unclear. As far as what can be determined, thioredoxin (TRX) and thioredoxin-interacting protein (TXNIP) are currently known to be involved in NLRP3 activation [20,21]. After the NLRP3 inflammasome is formed, the caspase-1 may be activated to caspase-1 p20, which can promote pro-IL-1β to become IL-1β [22]. Therefore, we studied whether the proinflammatory response caused by PPL was associated with the activation of the NLRP3 inflammasome. We also studied how PPL acts on the proteins of the inflammasome and on the protein–protein interactions inside the inflammasome. Since caspase-1 p20 can promote pro-IL-1β to become IL-1β, we further researched if that suggested a close relationship between the ROS, MAPK, and NF-κB pathways and the NLRP3 inflammasome during the inflammation caused by PPL.

In our previous study, inflammation caused by PPL was related to the overproduction of ROS and IL-1β [4]. Nevertheless, the signaling pathways that lead to the activation of IL-1β induced by PPL are still little known. In this study, using cultured RAW264.7 cells, we explored whether PPL initiated a chain reaction, including the activation of the MAPK and NF-κB pathways and the NLRP3 inflammasome, the activation of caspase-1, and the production of IL-1β. In addition, we unraveled the mechanism for the proinflammatory activity of PPL. We hope this work provides new perspectives for the development of the detoxification processing of P. pedatisecta.