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.

[HPLC fingerprints of pig, cattle and sheep biles].

To establish the fingerprints of biles of pig, cattle and sheep, HPLC was used with Acclaim™ RSLC 120 C18 column (3.0 mm×100 mm, 2.2 µm, 120 Å), the column temperature 35 °C, acetonitrile-1% perchloric acid as mobile phase, gradient elution, 0.5 mL·min⁻¹ flow rate, and detection wavelength at 200 nm. The fingerprint was generated by using Similarity Evaluation Software of Chromatographic Fingerprint of Chinese Medicine (2004A Edition). The fingerprint peaks were identified by reference substances and verified by ELSD and LC-MS/MS. Then, the biles of pig, cattle and sheep were detected to contain 14, 9 and 8 common fingerprint peaks respectively, and the similarity was greater than 0.92. To analyze each technical parameter, GHDCA in pig bile and TCA in cattle and sheep bile were selected as reference peak. The precision, repeatability and stability all meet the requirements of fingerprint establishment. The RSD of the relative retention time of the fingerprint peaks was less than 1.5%, and the RSD of the relative peak area was less than 5%. The fingerprint peaks in pig bile were THDCA, TCDCA, GHDCA and GCDCA, and TCA, TCDCA, GCA, GCDCA and GDCA in cattle and sheep bile. The main components of pig, cattle and sheep bile were conjugated bile acids, but there were significant differences in bile acids between pig bile and cattle, sheep biles. The HPLC method established in this paper is simple, rapid and reproducible, and could be applied to the identification and quality control of biles.

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.

[Pro-inflammatory mechanism of lectin from Pinellia pedatisecta on macrophage].

To investigate the mechanism of lectin from Pinellia pedatisecta(PPL) on macrophage-induced inflammation and its association with inflammatory corpuscles NLRP3. Lectin from P. pedatisecta was isolated and purified by gel chromatography, and its purity was analyzed by using SDS-PAGE gel electrophoresis. ELISA was used to investigate the effect of PPL on inflammatory cytokines released by macrophages, with IL-1ß as indicators;and fluorescence probe DCFH-DA fluorometer was used to determine changes in active oxygen ROS of macrophages after application of lectin from P. pedatisecta.RAW264.7 cells were pre-treated with ROS inhibitor N-acetylcysteine (NAC) to investigate the effect on ROS and the release of inflammatory factor IL-1ß from macrophages to research the relationship between them. The protein levels of NLRP3, Caspase-1 p20, ASC and TXNIP were determined by Western blot.The results showed that isolated and purified PPL could reach electrophoretic purity; PPL stimulated macrophages and induced the excessive release of ROS, leading to strong oxidative stress reaction, and the levels of intracellular inflammatory factorsIL-1ß were significantly increased. NAC could inhibit PPL-induced ROS excessive production and significantly reduce the release of IL-1ß. In addition, PPL could induce the increase in protein expression levels of Caspase-1 p20, NLRP3 and ASC, and significantly reduce TXNIP expression. The results showed that PPL could cause a strong oxidative stress response by stimulating macrophages, activate inflammatory corpuscles NLRP3, and result in large amount of IL-1ß release. That is, PPL could lead to inflammatory cascade reaction by promoting the maturation and secretion of IL-1ß through ROS-TXNIP-NLRP3-IL-1ß signaling pathway.

[Antagonistic effect of gingerols against TNF-α release, ROS overproduction and RIP3 expression increase induced by lectin from Pinellia ternata].

To explore the antagonistic effect of gingerols against the inflammation induced by lectin from Pinellia ternata. In this study, ELISA method was used to determine the effect of different extracts from gingerols on the release of inflammatory factor TNF-α from macrophages induced by lectin from P. ternata. The fluorescence probe was used to determine the effect of gingerols on the changes in ROS of macrophages induced by lectin from P. ternata. The western-blot method was applied to study the effect of gingerols on the increase in expression of cell receptor interacting protein RIP3 in macrophages induced by lectin from P. ternata. The scanning electron microscope (SEM) was used to study the effect of gingerols on morphological changes in macrophages induced by lectin from P. ternata. According to the results, gingerols can significantly inhibit the release of inflammatory factor from macrophages induced by lectin from P. ternata, ROS overproduction and increase in RIP3 expression. SEM results showed that gingerols can inhibit the cytomorphosis and necrocytosis induced by lectin from P. ternata. Fresh ginger's detoxication may be related to gingerols' effects in inhibiing release of inflammatory factor, ROS overproduction and increase in RIP3 expression caused by macrophages induced by lectin from P. ternata, which are mainly inflammatory development.

[Optimization of Preparation of Cutting-processed Arecae Pericarpium with Multi-index Orthogonal Method].

OBJECTIVE: 10 optimize the preparation of cutting-processed Arecae Pericarpium by the orthogonal method. METHODS: With four alkaloids such as arecoline, arecaidine, guvacoline, guvacine and yield of water-soluble decoction as indexes, a L9 (3(4)) orthogonal test was adopted to compare the effect of different factors on cutting-processed Arecae Pericarpium. RESULTS: According to the finalized optimal process, Arecae Pericarpium was washed quickly, moisturized, chopped lengthwise in 1 cm segment and then dried for 2.5 h at 50 °C. CONCLUSION: The optimal process method is reasonable and reliable, it can provide basis for the preparation of cutting-processed Arecae Pericarnium.