Effects of electroacupuncture on Sirt3/NLRP3/GSDMD signaling pathway in the substantia nigra of midbrain of rats with Parkinson's disease. / ç”µé’ˆå¯¹å¸•é‡‘æ£®ç— å¤§é¼ ä¸­è„‘é»‘è´¨Sirt3/NLRP3/GSDMD信号通路的影响.

OBJECTIVES: To observe the effects on tyrosine hydroxylase (TH), α-synaptic nucleoprotein (α-syn), sirtuin 3 (Sirt3), NOD-like receptor 3 (NLRP3) and gasdermin-D (GSDMD) in the substantia nigra of midbrain after electroacupuncture (EA) at "Fengfu"(GV16), "Taichong" (LR3) and "Zusanli" (ST36) in rats of Parkinson's disease (PD), so as to explore the mechanism of EA in treatment of PD. METHODS: SD rats were randomly divided into control, model and EA groups, with 10 rats in each group. The PD model was established by injecting rotenone into the neck and back, lasting 28 days. In the EA group, EA was applied to GV16, LR3 and ST36, 30 min each time, once daily, consecutively for 28 days. The open-field test was adopted to detect the total distance of autonomic movement of rats, and the pole climbing test was used to detect the body coordination ability of rats. In the substania nigra of midbrain, the positive expression of TH was determined using immunohistochemistry, the mRNA expression levels of α - syn, Sirt3, NLRP3 and GSDMD were detected by quantitative real-time fluorescence PCR, and the protein expression levels of NLRP3, apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) and cysteinyl aspartate specific proteinase (Caspase)-1 were detected by Western blot. RESULTS: Compared with the control group, the total distance of autonomous movement was decreased (P<0.01) in the model group, and the score of pole climbing experiment was increased (P<0.01)；in the midbrain substantia nigra the positive expression of TH was decreased (P<0.01)；the mRNA expression level of Sirt3 was decreased (P<0.01), and those of α-syn, NLRP3 and GSDMD were increased (P<0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 were increased (P<0.01). When compared with the model group, the total distance of autonomous movement in open field experiment was increased (P<0.01) in the EA group and the score of pole climbing experiment was lower (P<0.05)；in the midbrain substantia nigra the positive expression of TH was increased (P<0.01)；the mRNA expression level of Sirt3 in the midbrain substantia nigra was increased (P<0.01), and those of α-syn, NLRP3 and GSDMD were reduced (P<0.01)；while the protein expression levels of NLRP3, ASC and Caspase-1 decreased (P<0.01, P<0.05). CONCLUSIONS: EA at "GV16" "LR3" and "ST36" can repair the neuronal injury, clear the abnormal accumulation of α-syn in the substania nigra of midbrain, and ameliorate mitochondrial damage in PD rats, which may be obtained by regulating Sirt3/NLRP3/GSDMD signaling pathway, so as to delay the occurrence and development of Parkinson's disease.

NcRNA Regulated Pyroptosis in Liver Diseases and Traditional Chinese Medicine Intervention: A Narrative Review.

Pyroptosis is a novel pro-inflammatory mode of programmed cell death that differs from ferroptosis, necrosis, and apoptosis in terms of its onset and regulatory mechanisms. Pyroptosis is dependent on cysteine aspartate protein hydrolase (caspase)-mediated activation of GSDMD, NLRP3, and the release of pro-inflammatory cytokines, interleukin-1 (IL-1ß), and interleukin-18 (IL-18), ultimately leading to cell death. Non-coding RNA (ncRNA) is a type of RNA that does not encode proteins in gene transcription but plays an important regulatory role in other post-transcriptional links. NcRNA mediates pyroptosis by regulating various related pyroptosis factors, which we termed the pyroptosis signaling pathway. Previous researches have manifested that pyroptosis is closely related to the development of liver diseases, and is essential for liver injury, alcoholic fatty liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), liver fibrosis, and liver cancer. In this review, we attempt to address the role of the ncRNA-mediated pyroptosis pathway in the above liver diseases and their pathogenesis in recent years, and briefly outline that TCM (Traditional Chinese Medicine) intervene in liver diseases by modulating ncRNA-mediated pyroptosis, which will provide a strategy to find new therapeutic targets for the prevention and treatment of liver diseases in the future.

Xiaoqinglong decoction improves allergic rhinitis by inhibiting NLRP3-mediated pyroptosis in BALB/C mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Xiaoqinglong decoction (XQLD), first recorded in Shang Han Lun, is a traditional Chinese medicine prescribed for the treatment of allergic rhinitis (AR). XQLD alleviates the clinical symptoms of AR by inhibiting the occurrence of an inflammatory response, but the specific regulatory mechanism remains unclear. AIM OF THE STUDY: NLRP3-mediated pyroptosis is closely related to AR pathogenesis. Hence, this study aimed to explore the potential role of NLRP3-mediated pyroptosis pathway in the AR-associated pharmacological mechanism of XQLD. MATERIALS AND METHODS: BALB/C mice models of AR was established by using ovalbumin (OVA) and aluminum hydroxide sensitization. After intragastric administration of different dosages of XQLD, nasal allergic symptoms were observed. The expression of OVA-sIgE and Th2 inflammatory factors (IL-4, IL-5, and IL-13) in serum was detected by ELISA. The histopathological morphology and expression of inflammatory factors in nasal mucosa along with pyroptosis were investigated. Molecular docking was performed to analyze the binding of representative compounds of XQLD with NLRP3. Activation of the NLRP3 inflammasome was detected by immunofluorescence and western blotting. RESULTS: XQLD significantly improved the nasal allergic symptoms of mice, reduced the degree of goblet cell proliferation, mast cell infiltration, and collagen fiber hyperplasia in nasal mucosa. Meanwhile, it could downregulate the expression of Th2 inflammatory factors (IL-4, IL-5, and IL-13) in serum and nasal mucosa. XQLD significantly reduced the number of GSDMD and TUNEL double-positive cells and IL-1ß and IL-18 expression. Molecular docking confirmed that seven representative compounds of XQLD had good binding properties with NLRP3 and were able to inhibit the activation of the NLRP3 inflammasome. CONCLUSIONS: The representative compounds of XQLD might inhibit pyroptosis in nasal mucosa mediated by the NLRP3 inflammasome to helping the recovery of AR, which provides a new modern pharmacological proof for XQLD to treat AR.

Emodin attenuates cardiomyocyte pyroptosis in doxorubicin-induced cardiotoxicity by directly binding to GSDMD.

BACKGROUND: Doxorubicin (Dox), which is an anticancer drug, has significant cardiac toxicity and side effects. Pyroptosis occurs during Dox-induced cardiotoxicity (DIC), and drug inhibition of this process is one therapeutic approach for treating DIC. Previous studies have indicated that emodin can reduce pyroptosis. However, the role of emodin in DIC and its molecular targets remain unknown. HYPOTHESIS/PURPOSE: We aimed to clarify the protective role of emodin in mitigating DIC, as well as the mechanisms underlying this effect. METHODS: The model of DIC was established via the intraperitoneal administration of Dox at a dosage of 5 mg/kg per week for a span of 4 weeks. Emodin at two different doses (10 and 20 mg/kg) or a vehicle was intragastrically administered to the mice once per day throughout the Dox treatment period. Cardiac function, myocardial injury markers, pathological morphology of the heart, level of pyroptosis and mitochondrial function were assessed. Protein microarray, biolayer interferometry and pull-down assays were used to confirm the target of emodin. Moreover, GSDMD-overexpressing plasmids were transfected into GSDMD-/- mice and HL-1 cells to further verify whether emodin suppressed GSDMD activation. RESULTS: Emodin therapy markedly enhanced cardiac function and reduced cardiomyocyte pyroptosis in mice induced by Dox. Mechanistically, emodin binds to GSDMD and inhibits the activation of GSDMD by targeting the Trp415 and Leu290 residues. Moreover, emodin was able to mitigate Dox-induced cardiac dysfunction and myocardial injury in GSDMD-/- mice overexpressing GSDMD, as shown by increased EF and FS, decreased serum levels of CK-MB, LDH and IL-1ß and mitigated cell death and cell morphological disorder. Additionally, emodin treatment significantly reduced GSDMD-N expression and plasma membrane disruption in HL-1 cells overexpressing GSDMD induced by Dox. In addition, emodin reduced mitochondrial damage by alleviating Dox-induced GSDMD perforation in the mitochondrial membrane. CONCLUSION: Emodin has the potential to attenuate DIC by directly binding to GSDMD to inhibit pyroptosis. Emodin may become a promising drug for prevention and treatment of DIC.

Xuebijing alleviates LPS-induced acute lung injury by downregulating pro-inflammatory cytokine production and inhibiting gasdermin-E-mediated pyroptosis of alveolar epithelial cells.

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is characterized by diffuse alveolar injury primarily caused by an excessive inflammatory response. Regrettably, the lack of effective pharmacotherapy currently available contributes to the high mortality rate in patients with this condition. Xuebijing (XBJ), a traditional Chinese medicine recognized for its potent anti-inflammatory properties, exhibits promise as a potential therapeutic agent for ALI/ARDS. This study aimed to explore the preventive effects of XBJ on ALI and its underlying mechanism. To this end, we established an LPS-induced ALI model and treated ALI mice with XBJ. Our results demonstrated that pre-treatment with XBJ significantly alleviated lung inflammation and increased the survival rate of ALI mice by 37.5%. Moreover, XBJ substantially suppressed the production of TNF-α, IL-6, and IL-1ß in the lung tissue. Subsequently, we performed a network pharmacology analysis and identified identified 109 potential target genes of XBJ that were mainly involved in multiple signaling pathways related to programmed cell death and anti-inflammatory responses. Furthermore, we found that XBJ exerted its inhibitory effect on gasdermin-E-mediated pyroptosis of lung cells by suppressing TNF-α production. Therefore, this study not only establishes the preventive efficacy of XBJ in ALI but also reveals its role in protecting alveolar epithelial cells against gasdermin-E-mediated pyroptosis by reducing TNF-α release.

[Anti-glioma mechanism of pterostilbene by regulating apoptosis and GSDME-mediated pyroptosis pathways: a study based on network pharmacology and experimental research].

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.

Dietary tryptophan supplementation enhances mitochondrial function and reduces pyroptosis in the spleen and thymus of piglets after lipopolysaccharide challenge.

The thymus and spleen, the main reservoirs for T lymphocytes, modulate the innate immune response. Oxidative stress, excessive inflammation and abnormal pyroptosis can cause dysfunction of these organs. This study aimed to examine whether tryptophan supplementation can improve growth performance and mitochondrial function via the adenosine 5'-monophosphate-activated protein kinase (AMPK)/sirtuin1 (Sirt1)/peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) signalling pathway and decrease pyroptosis via the nucleotidebinding oligomerisation domain-like receptor protein 3 (NLRP3)/caspase-1/gasderminD (GSDMD) signalling pathway in the spleen and thymus of piglets after lipopolysaccharide (LPS) challenge. Eighteen weaned piglets were allotted to three treatment groups: non-challenged control, LPS-challenged control and LPS + 0.2% tryptophan. On day 35, the pigs in the LPS and LPS + 0.2% tryptophan groups were injected with 100 µg/kg BW LPS, whereas those in the control group were administered with sterile saline. At 4 h postchallenge, the weaned piglets were sacrificed, and their thymuses and spleens were collected. Results showed that tryptophan enhanced growth performance and antioxidant status by increasing catalase, glutathione peroxidase and total superoxide dismutase activities and decreasing malondialdehyde and reactive oxygen species contents. Tryptophan also reduced the mRNA levels of proinflammatory cytokine genes and enhanced mitochondrial function by increasing the mRNA levels of mitochondrial transcription factor A, nuclear respiratory factor-1, mitochondria transcription factor B1, AMPKα1, AMPKα2, Sirt1 and PGC1α and the protein expression of phosphorylated AMPK, Sirt1 and PGC1α. It also reduced pyroptosis by decreasing the mRNA levels of NLRP3, apoptosis-associated speck-like protein containing CARD, caspase-1 and GSDMD and the protein expression of NLRP3, caspase-1 and GSDMD. These results indicate that tryptophan supplementation enhances growth performance and mitochondrial function via the AMPK/Sirt1/PGC1α signalling pathway and decreases pyroptosis via the NLRP3/caspase-1/GSDMD signalling pathway in the spleen and thymus of LPS-challenged piglets.

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.

Qingchang Wenzhong Decoction Prevents the Occurrence of Intestinal Tumors by Regulating Intestinal Microbiota and Gasdermin E.

Background: Intestinal tumors are the third most common malignant tumors worldwide, accounting for approximately 10% of all new cancer cases worldwide. Cancer prevention is a promising way to limit the intestinal tumor incidence rate; however, challenges remain. Qingchang Wenzhong decoction (QCWZD) can clinically treat mild to moderate ulcerative colitis symptoms. Moreover, the mechanism by which it prevents intestinal tumors has not been clarified. In this study, we explored the mechanism by which QCWZD prevents the occurrence of intestinal tumors. Methods: To study the preventive mechanism of QCWZD on intestinal tumors, we used two model mice with azoxymethane/dextran sodium sulfate (AOM/DSS)- and Apcmin/+-induced intestinal tumor formation. The two models exhibited colitis-associated cancer and familial adenomatous polyposis, respectively. Colon and small intestine tissues were collected and analyzed based on histopathology and immunohistochemistry analyses. Fecal samples were collected, and 16S rRNA sequencing was used to analyze the correlation between intestinal microbiota and the prevention of intestinal tumors. Results: In the AOM/DSS mice, the QCWZD reduced the number and size of tumors, as well as tumor load. Similarly, in the Apcmin/+ mice, QCWZD can also reduce the number of tumors and the tumor load. The results of 16S rRNA sequencing confirmed that QCWZD altered the composition of intestinal microbiota in mice, a phenomenon that may prevent the occurrence of intestinal tumors by aiding the increase in the abundance of beneficial bacteria, such as Ralstonia and Butyricicoccus, and reducing that of pathogenic bacteria, such as Desulfobacterota and Bacteroides, in the intestine. Further, immunohistochemistry reveald that QCWZD can improve the expression of intestinal barrier-related proteins and inhibit pyroptosis-related proteins. Conclusions: QCWZD has the potential to prevent the occurrence of intestinal tumors. The anti-tumor activity may be achieved by regulating the intestinal microbiota, improving the function of the intestinal barrier, and inhibiting GSDME mediated pyroptosis.

Drug D, a Diosgenin Derive, Inhibits L-Arginine-Induced Acute Pancreatitis through Meditating GSDMD in the Endoplasmic Reticulum via the TXNIP/HIF-1α Pathway.

Acute pancreatitis (AP) is one of the most common causes of hospitalization for gastrointestinal diseases, with high morbidity and mortality. Endoplasmic reticulum stress (ERS) and Gasdermin D (GSDMD) mediate AP, but little is known about their mutual influence on AP. Diosgenin has excellent anti-inflammatory and antioxidant effects. This study investigated whether Diosgenin derivative D (Drug D) inhibits L-arginine-induced acute pancreatitis through meditating GSDMD in the endoplasmic reticulum (ER). Our studies were conducted in a mouse model of L-arginine-induced AP as well as in an in vitro model on mouse pancreatic acinar cells. The GSDMD accumulation in ER was found in this study, which caused ERS of acinar cells. GSDMD inhibitor Disulfiram (DSF) notably decreased the expression of GSDMD in ER and TXNIP/HIF-1α signaling. The molecular docking study indicated that there was a potential interaction between Drug D and GSDMD. Our results showed that Drug D significantly inhibited necrosis of acinar cells dose-dependently, and we also found that Drug D alleviated pancreatic necrosis and systemic inflammation by inhibiting the GSDMD accumulation in the ER of acinar cells via the TXNIP/HIF-1α pathway. Furthermore, the level of p-IRE1α (a marker of ERS) was also down-regulated by Drug D in a dose-dependent manner in AP. We also found that Drug D alleviated TXNIP up-regulation and oxidative stress in AP. Moreover, our results revealed that GSDMD-/- mitigated AP by inhibiting TXNIP/HIF-1α. Therefore, Drug D, which is extracted from Dioscorea zingiberensis, may inhibit L-arginine-induced AP by meditating GSDMD in the ER by the TXNIP /HIF-1α pathway.

Honokiol alleviates ulcerative colitis by targeting PPAR-γ-TLR4-NF-κB signaling and suppressing gasdermin-D-mediated pyroptosis in vivo and in vitro.

Ulcerative colitis (UC) is a chronic, idiopathic relapsing inflammatory bowel disease. Honokiol is a major active component of the traditional Chinese medicinal herb Magnolia officinalis, which has been widely used in traditional prescriptions to treat tumors, inflammation, and gastrointestinal disorders. In this study, we investigated the ability of this polyphenolic compound to suppress UC in mice and the possible regulatory mechanism. A mouse model of UC induced with dextran sulfate sodium (DSS) in 40 male C57BL/6J mice was used for the in vivo study, and in vitro experiments were performed in mouse RAW264.7 macrophages. Lipopolysaccharide was used to induce the inflammatory response. The mouse bodyweights, stool consistency, and bleeding were determined and the disease activity indices calculated. RAW264.7 macrophages were cultured with or without either honokiol or lipopolysaccharide. Gene and protein expression was analyzed with RT-PCR and western blotting, respectively. GW6471 and GW9662 were used to interrupt the transcription of peroxisome proliferator activated receptor alpha (PPAR-α) and peroxisome proliferator activated receptor gamma (PPAR-γ). Both the in vivo and in vitro experimental results showed that the oral administration of honokiol markedly attenuated the severity of UC by reducing the inflammatory signals and restoring the integrity of the colon. Honokiol dramatically reduced the proinflammatory cytokines TNF-α, IL6, IL1ß, and IFN-γ in mice with DSS-induced UC. It also upregulated PPAR-γ expression, and downregulated the TLR4-NF-κB signaling pathway. Moreover, honokiol inhibited gasdermin-D-mediated cell pyroptosis. These findings demonstrate for the first time that honokiol exerts a strong anti-inflammatory effect in a mouse model of UC, and that its underlying mechanism is associated with the activation of the PPAR-γ-TLR4-NF-κB signaling pathway and gasdermin-D-mediated macrophage pyroptosis. Therefore, honokiol may be a promising new drug for the clinical management of UC.

Aloe-Emodin Induces Mitochondrial Dysfunction and Pyroptosis by Activation of the Caspase-9/3/Gasdermin E Axis in HeLa Cells.

Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-anthraquinone), derived from some Chinese edible medicinal herbs, exerts a potential anticancer activity on various cancer cells, making it a drug candidate for cancer therapy. Yet, the role of aloe-emodin in pyroptosis, a new type of cell death, is uncharacterized. In this study, we explored the molecular mechanisms of aloe-emodin-triggered pyroptosis. Aloe-emodin inhibited proliferation and migration and triggered caspase-dependent cell death of HeLa cells in a dose-dependent manner. Aloe-emodin caused mitochondrial dysfunction and induced pyroptosis by activating the caspase-9/3/GSDME axis. Transcriptional analysis showed extensive changes in gene expressions in cellular pathways, including MAPK, p53, and PI3K-Akt pathways when treated with aloe-emodin. This study not only identified a novel role of aloe-emodin in pyroptotic cell death, but also performed a systematical genome-wide analysis of cellular pathways responding to aloe-emodin, providing a theoretical basis for applying anthraquinone derivatives in the treatment of GSDME-expressing cancers.

Germacrone induces caspase-3/GSDME activation and enhances ROS production, causing HepG2 pyroptosis.

Liver cancer is a highly lethal malignancy. Despite considerable efforts made in recent years, the prognosis of patients with liver cancer remains poor. Curcuma zedoaria (known as Ezhu in Chinese) is widely prescribed in traditional Chinese medicine. Germacrone (GM) is a sesquiterpene constituent derived from the essential oil of Ezhu, and exerts anti-carcinogenic effects by inducing apoptosis in various cancer cells. The present study investigated the potential mechanism of GM in HepG2 cells. Cell Counting Kit-8, colony-formation and lactate dehydrogenase-release assays, as well as cell death assays using flow cytometry, were performed to evaluate HepG2 cell proliferation following GM treatment. HepG2 cells were transfected with caspase-3 small interfering RNA and then treated with GM. Caspase-3 expression levels were determined by reverse transcription-quantitative PCR and western blotting. The present study showed that GM inhibited the growth of HepG2 cells and induced the proteolytic cleavage of caspase 3, with concomitant cleavage of gasdermin E (GSDME), by markedly increasing the production of reactive oxygen species (ROS). This led to caspase 3-dependent cleavage of GSDME, thereby promoting pyroptosis in HepG2 cells. However, these changes were rescued by ROS scavengers, such as N-acetylcysteine. Furthermore, GM inhibited tumor growth by promoting the cleavage of caspase 3 and GSDME in HepG2 cell xenograft models. These results indicated that GM induced GSDME-dependent pyroptosis through caspase 3 activation, at least in part, by damaging the mitochondria and enhancing ROS production, thereby supporting the possible development of GM as a candidate for the prevention and treatment of liver cancer.

[Effect of electroacupuncture preconditioning on expression of Gasdermin D, Caspase-1 and IL-1ß in rats with myocardial ischemia reperfusion injury].

OBJECTIVE: To observe the effect of electroacupuncture(EA) preconditioning on expression of Caspase-1, Gasdermin D(GSDMD) and interleukin-1ß(IL-1ß) in myocardial tissue of myocardial ischemia reperfusion injury (MIRI) rats in order to explore its underlying mechanisms in resisting MIRI. METHODS: Forty male rats were randomly divided into 4 groups: normal control (normal), sham operation (sham), MIRI model and EA groups. The MIRI model was established by ligation of the left anterior descending branch of the left coronary artery for 30 min and perfusion. EA (2 Hz/100 Hz, 1 mA) was applied to bilateral "Neiguan" (PC6) for 20 min, once a day for 3 consecutive days. The echocardiography was used to analyze the left ventricular end-diastolic dimension (LVEDD), left ventricular end-systolic dimension (LVESD) and left ventricular ejection fraction (LVEF, by using Teichholz formula) 4 h after modeling. The myocardial TTC staining was used to observe the proportion of the infarct area, and Western blot was used to detect the expression levels of GSDMD, Caspase-1, IL-1ß proteins in the myocardium. RESULTS: Compared with the normal group, the immunoactivity of GSDMD was increased in the sham group (P<0.05). Compared with the sham group, the LVEF was significantly decreased (P<0.000 1), while the myocardial infarction area, immunoactivity of GSDMD, and the expression levels of Caspase-1, GSDMD and IL-1ß proteins were considerably increased in the model group (P<0.000 1, P<0.001). In comparison with the model group, the decreased ejection fraction and the increased myocardial infarction area, and Caspase-1, GSDMD and IL-1ß expression were reversed in the EA group (P<0.001, P<0.000 1, P<0.01). CONCLUSION: EA preconditioning may ameliorate myocardial injury in MIRI rats which may be associated with its function in down-regulating the expression of myocardial Caspase-1 protein to reduce cardiomyocyte pyroptosis.

Evaluation of the healing properties of Garcinia brasiliensis extracts in a cutaneous wound model.

ETHNOPHARMACOLOGICAL RELEVANCE: Wound healing is a complex process that can leave pathological scars, especially in case of infections from opportunistic microorganisms. In this context, herbal medicines open up great possibilities for investigation. One of the species of interest native to Brazil is Garcinia brasiliensis ("bacupari"). Traditionally known for treating wounds and ulcers, G. brasiliensis presents anti-inflammatory, antioxidant and antimicrobials properties. But, its wound healing profile in experimental models, in order to validate its efficacy, is still litle studied. AIM OF THE STUDY: Thus, the objective of this work was to evaluate, in an infected cutanous wound model, the potential of formulations incorporated with G. brasiliensis leaves extracts. MATERIALS AND METHODS: Crude extract (CE), Ethyl Acetate Fraction (EAF) and Hexanic Fraction (HF) were submitted to phytochemical assays, high performance thin layer chromatography (HTPLC) and cytotoxicity studies. CE and EAF were also tested for microbicidal properties and incorporated in cream and gel formulations at 10% concentration. After stability testing, the gel formulations with CE or EAF at 10% were selected and applied to skin wounds infected or not with Staphylococcus aureus in Wistar rats. The healing potenttial of the extracts was verified by the expression of the protein Annexin A1 (AnxA1), related to the processes of inflammation and antifibrotic function, the cells immunostaining for Gasdermin-D (GSDM-D), a marker of pyroptotic cell death, and the dosage of interleukin-10 (IL-10) and monocyte chemotactic protein (MCP)-1 inflammatory mediators. RESULTS: Phytochemical studies indicated the presence of compounds of pharmacological interest, including Catechin, Quercetin and Berberine in addition to low cytotoxicity of CE and EAF at 10%. After the 6-day topical treatments, CE and EAF gel formulations demonstrated to control the pruritus formation process. The treatments decreased AnxA1 expression and the amount of cells immunostained for GSDM-D, and increased the expression of MCP-1 in infected wounds. CONCLUSIONS: Together, the results show important anti-inflammatory profile and skin healing potential of CE and EAF from G. brasiliensis leaves, even in infected lesions, with therapeutic perspectives.

Intrinsic Radical Species Scavenging Activities of Tea Polyphenols Nanoparticles Block Pyroptosis in Endotoxin-Induced Sepsis.

Sepsis, a life-threating illness caused by deregulated host immune responses to infections, is characterized by overproduction of multiple reactive oxygen and nitrogen species (RONS) and excessive pyroptosis, leading to high mortality. However, there is still no approved specific molecular therapy to treat sepsis. Here we reported drug-free tea polyphenols nanoparticles (TPNs) with intrinsic broad-spectrum RONS scavenging and pyroptosis-blocking activities to treat endotoxin (LPS)-induced sepsis in mice. The RONS scavenging activities originated from the polyphenols-derived structure, while the pyroptosis blockage was achieved by inhibiting gasdermin D (GSDMD) mediating the pore formation and membrane rupture, showing multifunctionalities for sepsis therapy. Notably, TPNs suppress GSDMD by inhibiting the oligomerization of GSDMD rather than the cleavage of GSDMD, thus displaying high pyroptosis-inhibition efficiency. As a result, TPNs showed an excellent therapeutic efficacy in sepsis mice model, as evidenced by survival rate improvement, hypothermia amelioration, and the organ damage protection. Collectively, TPNs present biocompatible candidates for the treatment of sepsis.

Gasdermin D in pyroptosis.

Pyroptosis is the process of inflammatory cell death. The primary function of pyroptosis is to induce strong inflammatory responses that defend the host against microbe infection. Excessive pyroptosis, however, leads to several inflammatory diseases, including sepsis and autoimmune disorders. Pyroptosis can be canonical or noncanonical. Upon microbe infection, the canonical pathway responds to pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), while the noncanonical pathway responds to intracellular lipopolysaccharides (LPS) of Gram-negative bacteria. The last step of pyroptosis requires the cleavage of gasdermin D (GsdmD) at D275 (numbering after human GSDMD) into N- and C-termini by caspase 1 in the canonical pathway and caspase 4/5/11 (caspase 4/5 in humans, caspase 11 in mice) in the noncanonical pathway. Upon cleavage, the N-terminus of GsdmD (GsdmD-N) forms a transmembrane pore that releases cytokines such as IL-1ß and IL-18 and disturbs the regulation of ions and water, eventually resulting in strong inflammation and cell death. Since GsdmD is the effector of pyroptosis, promising inhibitors of GsdmD have been developed for inflammatory diseases. This review will focus on the roles of GsdmD during pyroptosis and in diseases.

Vitexin exerts protective effects against calcium oxalate crystal-induced kidney pyroptosis in vivo and in vitro.

BACKGROUND: Nephrolithiasis is a common urinary disease with a high recurrence rate of secondary stone formation. Several mechanisms are involved in the onset and recurrence of nephrolithiasis, e.g., oxidative stress, inflammation, apoptosis, and epithelial-mesenchymal transition (EMT). Vitexin, a flavonoid monomer derived from medicinal plants that exert many biological effects including anti-inflammatory and anticancer effects, has not been investigated in nephrolithiasis studies. Moreover, pyroptosis, a form of programmed cell death resulting from inflammasome-associated caspase activation, has not been studied in mice with nephrolithiasis. PURPOSE: We aimed to investigate the protective effect and underlying mechanisms of vitexin in nephrolithiasis, and the related role of pyroptosis in vivo and in vitro. METHODS: Mouse models of nephrolithiasis were established via intraperitoneal injection of glyoxylate, and cell models of tubular epithelial cells and macrophages were established using calcium oxalate monohydrate (COM). Crystal deposition and kidney tissue injury were evaluated by hematoxylin and eosin, and von Kossa staining. Renal oxidative stress indexes including malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and catalase (CAT), were analyzed. The renal expression of interleukin-1 beta (IL-1ß), gasdermin D (GSDMD), osteopontin (OPN), CD44, and monocyte chemotactic protein 1 (MCP-1), and EMT-related proteins in renal tubular epithelial cells was assessed. Cell viability and the apoptosis ratio were evaluated. RESULTS: In vivo, vitexin alleviated crystal deposition and kidney tissue injury, and decreased the level of MDA, and increased the levels of SOD, GSH, and CAT. Vitexin also reduced the levels of the pyroptosis-related proteins GSDMD, NLRP3, cleaved caspase-1, and mature IL-1ß, which were elevated in mice with nephrolithiasis, and repressed apoptosis and the expression of OPN and CD44. Moreover, vitexin mitigated F4/80-positive macrophage infiltration and MCP-1 expression in the kidneys. Furthermore, an in vitro study showed that vitexin increased the viability of HK-2 cells and THP-1-derived macrophages, which was impaired by treatment with COM crystals, decreased the medium lactate dehydrogenase (LDH) level, and inhibited the expression of pyroptosis-related proteins in HK-2 cells and macrophages. Vitexin repressed EMT of HK-2 cells, with increased expression of pan-cytokeratin (Pan-ck) and decreased expression of Vimentin and alpha-smooth muscle actin (α-SMA), and downregulated the Wnt/ß-catenin pathway. Moreover, vitexin suppressed tumor necrosis factor-α (TNF-α) and IL-1ß mRNA expression, which was upregulated by COM in macrophages. CONCLUSION: Vitexin exerts protective effects against nephrolithiasis by inhibiting pyroptosis activation, apoptosis, EMT, and macrophage infiltration. In addition, GSDMD-related pyroptosis mediates nephrolithiasis.

Proteomics reveals distinct mechanisms regulating the release of cytokines and alarmins during pyroptosis.

A major pathway for proinflammatory protein release by macrophages is inflammasome-mediated pyroptotic cell death. As conventional secretion, unconventional secretion, and cell death are executed simultaneously, however, the cellular mechanisms regulating this complex paracrine program remain incompletely understood. Here, we devise a quantitative proteomics strategy to define the cellular exit route for each protein by pharmacological and genetic dissection of cellular checkpoints regulating protein release. We report the release of hundreds of proteins during pyroptosis, predominantly due to cell lysis. They comprise constitutively expressed and transcriptionally induced proteins derived from the cytoplasm and specific intracellular organelles. Many low-molecular-weight proteins including the cytokine interleukin-1ß, alarmins, and lysosomal-cargo proteins exit cells in the absence of cell lysis. Cytokines and alarmins are released in an endoplasmic reticulum (ER)-Golgi-dependent manner as free proteins rather than by extracellular vesicles. Our work provides an experimental framework for the dissection of cellular exit pathways and a resource for pyroptotic protein release.

PORIMIN: The key to (+)-Usnic acid-induced liver toxicity and oncotic cell death in normal human L02 liver cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Usnic acid (UA) is one of the well-known lichen metabolites that induces liver injury. It is mainly extracted from Usnea longissima and U. diffracta in China or from other lichens in other countries. U. longissima has been used as traditional Chinese medicine for treatment of cough, pain, indigestion, wound healing and infection. More than 20 incidences with hepatitis and liver failure have been reported by the US Food and Drug Administration since 2000. UA is an uncoupler of oxidative phosphorylation causing glutathione and ATP depletion. Previous histological studies observed extensive cell and organelle swellings accompanied with hydrotropic vacuolization of hepatocytes. AIM OF THE STUDY: This study was to investigate the mechanism of UA-induced liver toxicity in normal human L02 liver cells and ICR mice using various techniques, such as immunoblotting and siRNA transfection. MATERIALS AND METHODS: Assays were performed to evaluate the oxidative stress and levels of GSH, MDA and SOD. Double flouresencence staining was used for the detection of apoptotic cell death. The protein expressions, such as glutathione S transferase, glutathione reductase, glutathione peroxidase 4, catalase, c-Jun N-terminal protein kinase, caspases, gastamin-D and porimin were detected by Western blotting. Comparisons between transfected and non-transfected cells were applied for the elucidation of the role of porimin in UA-induced hepatotoxicity. Histopathological examination of mice liver tissue, serum total bilirubin and hepatic enzymes of alanine aminotransferase and aspatate aminotransferase were also studied. RESULTS: The protein expressions of glutathione reductase, glutathione S transferase and glutathione peroxidase-4 were increased significantly in normal human L02 liver cells. Catalase expression was diminished in dose-dependent manner. Moreover, (+)-UA did not induce the activation of caspase-3, caspase-1 or gasdermin-D. No evidence showed the occurrence of pyroptosis. However, the porimin expressions were increased significantly. In addition, (+)-UA caused no cytotoxicity in the porimin silencing L02 cells. CONCLUSIONS: In conclusion, (+)-UA induces oncotic L02 cell death via increasing protein porimin and the formation of irreversible membrane pores. This may be the potential research area for future investigation in different aspects especially bioactivity and toxicology.