Salvianolic Acid F Regulates Bax/Caspase-3/GSDME Signaling Pathway to Inhibit Pyroptosis of HK-2 Cells / 中国实验方剂学杂志

ObjectiveTo investigate the mechanism of salvianolic acid F （Sal F） in repairing the high glucose-induced injury in human kidney-2 （HK-2） cells via the B-cell lymphoma-2 （Bcl-2）-associated X protein （Bax）/cysteinyl aspartate-specific proteinase 3 （Caspase-3）/gasdermin-E （GSDME） pathway. MethodThe cell counting kit-8 （CCK-8） was used to measure the relative viability of HK-2 cells exposed to high glucose and different concentrations （2.5， 5， 10， 20 μmol·L-1） of Sal F and the relative viability of HK-2 cells treated with Sal F for different time periods. The levels of lactate dehydrogenase （LDH） and interleukin-1β （IL-1β） in the supernatant of the cell culture were measured by the LDH assay kit and enzyme-linked immunosorbent assay （ELISA） kit， respectively. Flow cytometry combined with Annexin V-FITC/propidium iodide （PI） and Hoechst 33342/PI staining was employed to reveal the proportion of PI-positive HK-2 cells exposed to high glucose. Western blotting was employed to determine the protein levels of Bax， Bcl-2， cytochrome C， cysteinyl aspartate-specific proteinase （Caspase）-9， Caspase-3， and GSDME in the HK-2 cells exposed to high glucose and treated with Sal F. The 2，7-dichlorodihydrofluorescein diacetate fluorescence probe （DCFH-DA） and mitochondrial membrane potential assay kit （JC-1） were used to determine the production of reactive oxygen species （ROS） and the mitochondrial membrane potential in the HK-2 cells exposed to high glucose and treated with Sal F. ResultCompared with the blank group， the model group showed decreased cell viability （P<0.01）， elevated levels LDH and IL-1β， increased proportion of PI-positive cells （P<0.01）， up-regulated protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME （P<0.01）， down-regulated protein level of Bcl-2 （P<0.01）， decreased mitochondrial membrane potential， and excessive ROS accumulation. Compared with the model group， Sal F repaired the high glucose-induced injury in HK-2 cells （P<0.05）， lowered the levels of LDH and IL-1β （P<0.05， P<0.01）， and decreased the proportion of PI-positive cells （P<0.01）. In addition， Sal F down-regulated the protein levels of Bax， cytochrome C， Caspase-9， Caspase-3， and GSDME and up-regulated the protein level of Bcl-2 （P<0.05， P<0.01）， increased the mitochondrial membrane potential， and decreased the accumulation of ROS in HK-2 cells. ConclusionSal F can reduce the production of ROS， restore the balance of mitochondrial membrane potential， and inhibit pyroptosis via the Bax/Caspase-3/GSDME signaling pathway to repair the high glucose-induced injury in HK-2 cells.

Anti-glioma mechanism of pterostilbene by regulating apoptosis and GSDME-mediated pyroptosis pathways: a study based on network pharmacology and experimental research / 中国中药杂志

This study aimed to explore the anti-glioma effect of natural compound pterostilbene(PTE) through regulating pyroptosis and apoptosis pathways, and to analyze the possible anti-glioma pathways and targets of PTE by network pharmacology and molecular docking. In this study, the action targets of PTE and the glioma targets were obtained by network pharmacology to construct a target network and a protein-protein interaction(PPI) network to predict the possible action targets of PTE against glioma. Molecular docking was performed on the core targets by AutoDock and the action pathways of PTE against glioma were predicted by enrichment analysis. In addition, the effect of PTE on the viability of U87MG and GL261 glioma cells was detected by CCK-8 assay. Clone formation assay and cell scratching assay were used to explore the effect of different concentrations of PTE on the proliferation and migration, respectively of glioma cells. Hoechst staining was used to observe PTE-induced apoptosis in glioma cells. The changes in mitochondrial membrane potential were detected by JC-1 staining. The pyroptosis-inducing effect of PTE on glioma cells was observed by inverted microscopy and lactate dehydrogenase(LDH) assay. Hoechst 33342/PI dual staining assay was performed to detect the integrity of glioma cell membranes. The expressions of pyroptosis and apoptosis-related proteins in glioma cells after PTE induction were determined by Western blot. In this study, 37 anti-glioma targets of PTE were obtained, and enrichment analysis suggested that PTE exerted anti-glioma effects through various signaling pathways including cancer pathway, proteoglycan in cancer, PI3K/AKT pathway, and apoptosis regulatory pathway. Molecular docking revealed that PTE had good binding activity with the main targets. Compared with the control group, PTE significantly reduced the viability as well as the proliferation, migration and adhesion abilities of U87MG and GL261 cells; it induced the apoptosis of the two glioma cells and the decrease of mitochondrial membrane potential in U87MG cells, and the effects increased with the increase of drug concentration. Compared with the conditions in the control group, glioma cells in the PTE group had increased pyroptosis-specific appearance and gradually increased LDH release; the number of PI positive cells was significantly elevated with the increase of PTE concentration as revealed by Hoechst 33342/PI staining; the expression levels of apoptosis-related factors cleaved PARP1 and B-cell lymphoma-2(Bcl-2) associated X(BAX) in the PTE group were markedly up-regulated, while the expression level of Bcl-2 was markedly down-regulated; the activation levels of pyroptosis-related proteins cleaved caspase-3 and gasdermin E-N(GSDME-N) had a remarkable rise in the PTE group, while no significant changes were found in the activation levels of gasdermin D-N(GSDMD-N) and cleaved caspase-1. In summary, PTE plays an anti-glioma role by inhibiting cell viability, proliferation, and migration and activating the caspase-3/GSDME-mediated pyroptosis pathway and mitochondrial apoptosis pathway.

Regulation of Pyroptosis in Gastric Cancer by Traditional Chinese Medicine： A Review / 中国实验方剂学杂志

Pyroptosis， a new type of inflammatory programmed cell death， is different from apoptosis， necrosis， cytosis， ferroptosis， and autophagy. Pyroptosis is dependent on the activation of cysteine aspartate-specific protease （Caspase）， which cleaves key mediator proteins to form pores in the cell membrane and induces the maturation and release of the proinflammatory cytokines interleukin-1β and interleukin-18 into the extracellular environment， resulting in a cascade of inflammatory reactions. Gastric cancer as a malignant tumor of the digestive tract is refractory and has poor prognosis， and the chemoradiotherapy of this disease may lead to a variety of complications. At present， the pathogenesis of gastric cancer remains unclear. Studies have proved that pyroptosis is associated with the occurrence and development of gastric cancer， which has attracted wide attention. Pyroptosis is a double-edged sword for gastric cancer. On the one hand， it can release the contents of proinflammatory cells to amplify or maintain inflammation and induce the "inflammation-cancer" transformation of cells. On the other hand， pyroptosis can enhance the sensitivity of drugs for chemotherapy to improve the therapeutic effect and survival. In recent years， the anti-tumor mechanism of traditional Chinese medicine （TCM） has become a research hotspot as TCM has demonstrated significant effects in clinical application. Therefore， the regulation of pyroptosis by TCM may be a new direction for the treatment of gastric cancer in the future. Based on the available studies， this paper introduces the roles of pyroptosis-associated key proteins in the occurrence and development of gastric cancer. Furthermore， this paper summarizes the effects of TCM prescriptions and active ingredients on alleviating gastric mucosal damage， reducing the incidence of gastric cancer， and preventing tumor metastasis and recurrence by mediating pyroptosis pathways， aiming to provide new ideas for deciphering the mechanism of pyroptosis and exploring the TCM treatment of gastric cancer in the future.

Natural Medicinal Components Mediating Pyroptosis by GSDMs in Anti-tumor Therapy： A Review / 中国实验方剂学杂志

Pyroptosis， an atypical new cell death mode other than apoptosis and necrosis， has been discovered in recent years. Pyroptosis depends on the cleavage of gasdermins （GSDMs） by Caspases. The activated GSDMs act on the plasma membrane to form a perforation， which results in cell lysis and triggers inflammation and immune response. Pyroptosis can be induced by four distinct signaling pathways， including canonical and non-canonical inflammasome pathways， apoptosis-associated Caspases-mediated pathway， and granzyme pathway. In these signaling pathways， GSDMs are the executors of pyroptosis. Pyroptosis is associated with the death of tumor cells and the inflammatory damage of normal tissues. Recent studies have demonstrated that moderate pyroptosis can lead to tumor cell death to exert an anti-tumor effect， and meanwhile stimulate the tumor immune microenvironment， while it can promote tumor development. Despite the good performance， drug-based anti-tumor therapies such as tumor immunotherapy， chemotherapy， and targeted therapy have some shortcomings such as drug resistance， recurrence， and damage to normal tissues. The latest research shows that a variety of natural compounds have anti-tumor effects in the auxiliary treatment of tumors by mediating the pyroptosis pathways in a multi-target and multi-pathway manner， which provide new ideas for the study of anti-tumor therapy. We reviewed the molecular mechanism of pyroptosis and the regulatory role of pyroptosis in tumors and tumor immune microenvironment， and summarized the recent research progress in the natural medicinal components regulating pyroptosis in anti-tumor therapy， with a view to providing ideas for the research on the anti-tumor therapy based on pyroptosis.