Reniformin A suppresses non-small cell lung cancer progression by inducing TLR4/NLRP3/caspase-1/GSDMD-dependent pyroptosis.

Pyroptosis is an inflammatory form of programmed cell death that plays an important role in regulating tumor progression. Reniformin A (RA) is a natural compound isolated from the medicinal herb Isodon excisoides that has been applied as folk medicine in the treatment of esophageal cancer. However, whether RA has an individual function in cancer and the molecular mechanisms remain unclear. Here, we show that in non-small-cell lung cancer (NSCLC), RA inhibits tumor growth by functioning as a pyroptosis inducer to promote TLR4/NLRP3/caspase-1/GSDMD axis. Specially, RA treatment increased Toll-like receptor 4 (TLR4) protein expression level by enhancing the TLR4 stability. Based on the molecular docking, we identified that RA directly bound to TLR4 to activate the NLRP3 inflammasome and promote pyroptosis in A549 cells. Moreover, TLR4 is essential for RA-induced pyroptosis, and loss of TLR4 abolished RA-induced pyroptosis and further reduced the inhibitory effect of RA on NSCLC. In vivo experiments confirmed that RA inhibited the growth of lung tumors in mice by affecting pyroptosis in a dose-dependent manner. Furthermore, TLR4 knockdown abolished RA-induced pyroptosis and inhibited the effect of RA chemotherapy in vivo. In conclusion, we propose that RA has a significant anticancer effect in NSCLC by inducing TLR4/NLRP3/caspase-1/GSDMD-mediated pyroptosis, which may provide a potential strategy for the treatment of NSCLC.

The Scutellaria-Coptis herb couple and its active small-molecule ingredient wogonoside alleviate cytokine storm by regulating the CD39/NLRP3/GSDMD signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: A drug pair is a fundamental aspect of traditional Chinese medicine prescriptions. Scutellaria baicalensis Georgi and Coptis chinensis Franch, commonly used as an herb couple (SBCC), are representative heat-clearing and dampness-drying drugs. They possess functions such as clearing heat, drying dampness, purging fire, and detoxifying. These herbs are used in both traditional and modern medicine for treating inflammation. AIM OF THE STUDY: This study investigated the effects of SBCC on cytokine storm syndrome (CSS) and explored its potential regulatory mechanism. MATERIALS AND METHODS: We assessed the impact of SBCC in a sepsis-induced acute lung injury mouse model by administering an intraperitoneal injection of LPS (15 mg/kg). The cytokine levels in the serum and lungs, the wet-to-dry ratio of the lungs, and lung histopathological changes were evaluated. The macrophages in the lung tissue were examined through transmission electron microscopy. Western blot was used to measure the levels of the CD39/NLRP3/GSDMD pathway-related proteins. Immunofluorescence imaging was used to assess the activation of pro-caspase-1 and ASC and their interaction. AMP-Glo™ assay was used to screen for active ingredients in SBCC targeting CD39. One of the ingredients was selected, and its effect on cell viability was assessed. We induced inflammation in macrophages using LPS + ATP and detected the levels of proinflammatory factors. The images of cell membrane large pores were captured using scanning electron microscopy, the interaction between NLRP3 and ASC was detected using immunofluorescence imaging, and the levels of CD39/NLRP3/GSDMD pathway-related proteins were assessed using Western blot. RESULTS: SBCC administration effectively mitigated LPS-induced cytokine storm, pulmonary edema and lung injury. Furthermore, it repressed the programmed death of lung tissue macrophages by inhibiting the NLRP3/GSDMD pyroptosis pathway and regulating the CD39 purinergic pathway. Based on the results of the AMP-Glo™ assay, we selected wogonoside for further valuation. Wogonoside alleviated LPS + ATP-induced inflammatory damage by regulating the inhibiting the NLRP3/GSDMD pyroptosis pathway and regulating the CD39 purinergic pathway. However, its effect on NLRP3 is not mediated though CD39. CONCLUSION: SBCC and its active small-molecule ingredient, wogonoside, improved CSS by regulating the NLRP3/GSDMD pyroptosis pathway and its upstream CD39 purinergic pathway. It is essential to note that the regulatory effect of wogonoside on NLRP3 is not mediated by CD39.

Mahuang Fuzi Xixin decoction alleviates allergic rhinitis by inhibiting NLRP3/Caspase-1/GSDMD-N-mediated pyroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Allergic rhinitis (AR) is a prevalent nasal inflammatory disorder, and pyroptosis plays a crucial role in aggravating AR. Current medications for AR treatment still have deficiencies, and finding new agents is of great interest. Mahuang Fuzi Xixin decoction (MFXD), an ancient Chinese medicine, is now commonly used to treat AR, which has anti-inflammatory and immunomodulatory effects, but its underlying mechanism is unknown. AIM OF THIS STUDY: This study aims to evaluate the effects of MFXD on AR and explore its potential mechanisms in view of the regulatory effect on pyroptosis. METHODS: MFXD, Mahuang, Fuzi, and Xixin water extracts were analyzed using ultra high performance liquid chromatography-Orbitrap-high-resolution accurate mass spectrometry. In in vivo study, the effects of MFXD on AR treatment were evaluated in an ovalbumin-induced mouse model. Mice were administered saline (control and model groups), MFXD (1.375, 2.75 g/kg), and dexamethasone (2.5 mg/kg) for 13 days. AR symptoms were evaluated by blinded observers. Immunoglobulin E (IgE) and histamine levels were measured using enzyme-linked immunosorbent assays. Expression of pyroptosis-related proteins (NLRP3, ASC, Caspase-1 p10/p20, GSDMD-N and IL-1ß) in AR mouse nasal mucosa were estimated by immunohistochemistry. In in vivtro study, the effects of MFXD on pyroptosis were assessed in human nasal epithelial cells (HNEpCs) stimulated with lipopolysaccharide (LPS) and adenosine triphosphate (ATP), and incubated with MFXD (12.5, 25, and 50 µg/mL). Pyroptosis-related protein expression was measured by western blotting. RESULTS: Thirty-three compounds in MFXD were identified, including ephedrine, pseudoephedrine, higenamine, aconine, aconitine, benzoylmesaconitine, benzoylhypaconine and hypaconitine. In the in vivo study, oral taken of MFXD/dexamethasone significantly ameliorated AR symptoms, reduced swelling of the nasal mucosa, and decreased the levels of IgE and histamine in AR mice serum. MFXD/dexamethasone attenuated histopathological changes and reduced the expression of pyroptosis-related proteins in nasal mucosa, indicating the inhibitory effect on nasal epithelial pyroptosis. In the in vitro study, MFXD (50 µg/mL) significantly alleviated cytotoxicity, protected cells from swelling and rupture, and downregulated the expression of pyroptosis-related proteins in LPS/ATP-induced HNEpCs. CONCLUSION: MFXD suppressed nasal epithelial pyroptosis by inhibiting the NLRP3/Caspase-1/GSDMD-N signaling pathway, which alleviates AR. Our results offer valuable insights into potential AR therapies and provide evidence for the clinical utilization of MFXD to treat AR.

Neobractatin induces pyroptosis of esophageal cancer cells by TOM20/BAX signaling pathway.

BACKGROUND: Emerging evidence suggests that pyroptosis, a form of programmed cell death, has been implicated in cancer progression. The involvement of specific proteins in pyroptosis is an area of growing interest. TOM20, an outer mitochondrial membrane protein, has recently garnered attention for its potential role in pyroptosis. Our previous study found that NBT could induce pyroptosis by ROS/JNK pathway in esophageal cancer cells. PURPOSE: This study aims to investigate whether NBT induces pyroptosis and verify whether such effects are involved in up-regulation of TOM20 in esophageal cancer cells. METHODS: The University of ALabama at Birmingham CANcer data analysis Portal (UALCAN) was used to analyze the clinical significance of GSDME in esophageal cancer. MTT assay, morphological observation and Western blot were performed to verify the roles of TOM20 and BAX in NBT-induced pyroptosis after CRISPR-Cas9-mediated knockout. Immunofluorescence was used to determine the subcellular locations of BAX and cytochrome c. MitoSOX Red was employed to assess the mitochondrial reactive oxygen species (ROS) level. KYSE450 and TOM20 knockout KYSE450-/- xenograft models were established to elucidate the mechanisms involved in NBT-induced cell death. RESULTS: In this study, NBT effectively upregulated the expression of TOM20 and facilitated the translocation of BAX to mitochondria, which promoted the release of cytochrome c from mitochondria to the cytoplasm, leading to the activation of caspase-9 and caspase-3, and finally induced pyroptosis. Knocking out TOM20 by CRISPR-Cas9 significantly inhibited the expression of BAX and the downstream BAX/caspase-3/GSDME pathway, which attenuated NBT-induced pyroptosis. The elevated mitochondrial ROS level was observed after NBT treatment. Remarkably, the inhibition of ROS by N-acetylcysteine (NAC) effectively suppressed the activation of TOM20/BAX pathway. Moreover, in vivo experiments demonstrated that NBT exhibited potent antitumor effects in both KYSE450 and TOM20 knockout KYSE450-/- xenograft models. Notably, the attenuated antitumor effects and reduced cleavage of GSDME were observed in the TOM20 knockout model. CONCLUSION: These findings reveal that NBT induces pyroptosis through ROS/TOM20/BAX/GSDME pathway, which highlight the therapeutic potential of targeting TOM20 and GSDME, providing promising prospects for the development of innovative and effective treatment approaches for esophageal cancer.

Substrate specificity and ecological significance of PstS homologs in phosphorus uptake in marine Synechococcus sp. WH8102.

Phosphorus, a vital macronutrient, often limits primary productivity in marine environments. Marine Synechococcus strains, including WH8102, rely on high-affinity phosphate-binding proteins (PstS) to scavenge inorganic phosphate in oligotrophic oceans. However, WH8102 possesses three distinct PstS homologs whose substrate specificity and ecological roles are unclear. The three PstS homologs were heterologously expressed and purified to investigate their substrate specificity and binding kinetics. Our study revealed that all three PstS homologs exhibited a high degree of specificity for phosphate but differed in phosphate binding affinities. Notably, PstS1b displayed nearly 10-fold higher binding affinity (KD = 0.44 µM) compared to PstS1a (KD = 3.3 µM) and PstS2 (KD = 4.3 µM). Structural modeling suggested a single amino acid variation in the binding pocket of PstS1b (threonine instead of serine in PstS1a and PstS2) likely contributed to its higher Pi affinity. Genome context data, together with the protein biophysical data, suggest distinct ecological roles for the three PstS homologs. We propose that PstS1b may be involved in scavenging inorganic phosphorus in oligotrophic conditions and that PstS1a may be involved in transporting recycled phosphate derived from organic phosphate cleavage. The role of PstS2 is less clear, but it may be involved in phosphate uptake when environmental phosphate concentrations are transiently higher. The conservation of three distinct PstS homologs in Synechococcus clade III strains likely reflects distinct adaptations for P acquisition under varying oligotrophic conditions.IMPORTANCEPhosphorus is an essential macronutrient that plays a key role in marine primary productivity and biogeochemistry. However, intense competition for bioavailable phosphorus in the marine environment limits growth and productivity of ecologically important cyanobacteria. In oligotrophic oceans, marine Synechococcus strains, like WH8102, utilize high-affinity phosphate-binding proteins (PstS) to scavenge inorganic phosphate. However, WH8102 possesses three distinct PstS homologs, with unclear substrate specificity and ecological roles, creating a knowledge gap in understanding phosphorus acquisition mechanisms in picocyanobacteria. Through genomic, functional, biophysical, and structural analysis, our study unravels the ecological functions of these homologs. Our findings enhance our understanding of cyanobacterial nutritional uptake strategies and shed light on the crucial role of these conserved nutrient uptake systems in adaptation to specific niches, which ultimately underpins the success of marine Synechococcus across a diverse array of marine ecosystems.

Ursolic acid alleviates chronic prostatitis via regulating NLRP3 inflammasome-mediated Caspase-1/GSDMD pyroptosis pathway.

Ursolic acid (UA) is a naturally occurring pentacyclic triterpenoid widely found in fruits and vegetables. It has been reported that UA has anti-inflammatory effects. However, its efficacy and mechanism of action in the treatment of chronic prostatitis (CP) remain unclear. This study aimed to investigate the efficacy of UA treatment in CP and further explore the underlying mechanism. CP rat and pyroptosis cell models were established in vivo and in vitro, respectively. The efficacy of UA in inhibiting CP was evaluated via haematoxylin-eosin (HE) staining and measurement of inflammatory cytokines. RNA sequencing and molecular docking were used to predict the therapeutic targets of UA in CP. The expression of pyroptosis-related proteins was examined using various techniques, including immunohistochemistry, immunofluorescence, and flow cytometry. UA significantly ameliorated pathological damage and reduced the levels of proinflammatory cytokines in the CP model rats. RNA sequencing analysis and molecular docking suggested that NLRP3, Caspase-1, and GSDMD may be key targets. We also found that UA decreased ROS levels, alleviated oxidative stress, and inhibited p-NF-κB protein expression both in vivo and in vitro. UA improved pyroptosis morphology as indicated by electron microscope and inhibited the expression of the pyroptosis-related proteins NLRP3, Caspase-1, ASC, and GSDMD, reversed the levels of IL-1ß, IL-18, and lactate dehydrogenase in vivo and in vitro. UA can mitigate CP by regulating the NLRP3 inflammasome-mediated Caspase-1/GSDMD pathway. Therefore, UA may be a potential for the treatment of CP.

Jinyinqingre Oral Liquid alleviates LPS-induced acute lung injury by inhibiting the NF-κB/NLRP3/GSDMD pathway.

Acute lung injury (ALI) is a prevalent and severe clinical condition characterized by inflammatory damage to the lung endothelial and epithelial barriers, resulting in high incidence and mortality rates. Currently, there is a lack of safe and effective drugs for the treatment of ALI. In a previous clinical study, we observed that Jinyinqingre oral liquid (JYQR), a Traditional Chinese Medicine formulation prepared by the Taihe Hospital, Affiliated Hospital of Hubei University of Medicine, exhibited notable efficacy in treating inflammation-related hepatitis and cholecystitis in clinical settings. However, the potential role of JYQR in ALI/acute respiratory distress syndrome (ARDS) and its anti-inflammatory mechanism remains unexplored. Thus, the present study aimed to investigate the therapeutic effects and underlying molecular mechanisms of JYQR in ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI and an in vitro RAW264.7 cell model. JYQR yielded substantial improvements in LPS-induced histological alterations in lung tissues. Additionally, JYQR administration led to a noteworthy reduction in total protein levels within the BALF, a decrease in MPAP, and attenuation of pleural thickness. These findings collectively highlight the remarkable efficacy of JYQR in mitigating the deleterious effects of LPS-induced ALI. Mechanistic investigations revealed that JYQR pretreatment significantly inhibited NF-κB activation and downregulated the expressions of the downstream proteins, namely NLRP3 and GSDMD, as well as proinflammatory cytokine levels in mice and RAW2647 cells. Consequently, JYQR alleviated LPS-induced ALI by inhibiting the NF-κB/NLRP3/GSDMD pathway. JYQR exerts a protective effect against LPS-induced ALI in mice, and its mechanism of action involves the downregulation of the NF-κB/NLRP3/GSDMD inflammatory pathway.

Tou Nong powder obstructs ulcerative colitis through the regulation of NF-κB/NLRP3/Caspase-1/GSDMD inflammasome pyroptotic pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Tou Nong Powder (TNP), a classical Chinese medicinal formula originated from the Chinese Ming Dynasty, has been applied to treat skin ulcers in patients with deficient constitutions. According to theory of traditional Chinese medicine, colonic ulcers share similar pathological conditions with skin ulcers, and consequently, TNP has been applied to ulcerative colitis (UC) safely and effectively. AIM OF STUDY: To investigate whether TNP obstructs 2,4,6-trinitrobenzene sulfonic acid (TNBS) induced enteric inflammation through regulation of NLRP3 inflammasome and attenuating enteric pyroptosis. MATERIALS AND METHODS: Network pharmacology and UPLC-Q-TOF/MS were operated to identify compounds and pharmacological potential targets. The therapeutic effects of TNP were assessed on TNBS induced colitis via general symptoms (disease activity index, colonic weight and length) and histopathological observation. The NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway regulation was investigated by Western blot and real time reverse transcription polymerase chain reaction (RT-qPCR). RESULTS: TNP ameliorates the disease activity index, reverses the increase of colonic weight increase, alleviates colonic shortening and colonic histopathological injury. A decrease in tumor necrosis factor α (TNF-α), diamine oxidase (DAO), intercellular adhesion molecule-1 (ICAM-1), and endo-toxin (ET) were investigated in peripheral circulation. Moreover, TNP significantly obstructed the NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway. CONCLUSION: TNP displays a promising therapeutic effect on UC via suppressing NF-κB/NLRP3/Caspase-1/GSDMD signaling pathway and reducing the expression of IL-1ß and IL-18.

[Effects of electroacupuncture on NLRP3 inflammasome and pyroptosis protein GSDMD in uterine tissue in rats with primary dysmenorrhea].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on NLRP3 inflammasome and its downstream protein gastermin D (GSDMD) in rats with primary dysmenorrhea (PDM), and to explore the potential mechanism of EA on the treatment of PDM. METHODS: Forty healthy female SD rats without pregnancy were randomly divided into a control group, a model group, an EA group and an ibuprofen group, 10 rats in each group. PDM model was prepared by injection of estradiol benzoate and oxytocin. Except the control group, the rats in each group were subcutaneously injected with estradiol benzoate for 10 days, and oxytocin was injected on the 11th day. The rats in the EA group were intervened with EA (dense wave, frequency of 50 Hz) at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) at the same time of modeling, once a day, 20 min each time, for 10 consecutive days. The rats in the ibuprofen group were treated with 0.8 mL of ibuprofen by gavage (concentration of ibuprofen solution was 1.25 mg/mL) for 10 consecutive days. After modeling, the writhing reaction was observed. After intervention, the HE staining method was used to observe the histological morphology of uterus and evaluate the pathological damage score of uterus; ELISA method was used to detect the serum levels of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α); Western blot method was used to detect the protein expression of NLRP3, apoptosis related spot like protein (ASC), caspase-1, GSDMD, GSDMD-N and inflammatory factors (interleukin [IL]-1ß, IL-18) in uterine tissue. RESULTS: In the model group, a large number of vacuolar degeneration and death of endometrial epithelial cells, spiral arterioles congestion in lamina propria and neutrophil infiltration were observed. In the EA group, there was a small amount of vacuolar degeneration and death of endometrial epithelial cells, a small amount of spiral arterioles congestion in the lamina propria, and a small amount of neutrophils infiltration. In the ibuprofen group, there was very small number of degeneration and death of endometrial epithelial cells, and no obvious arterial congestion was found in lamina propria, and neutrophil infiltration was occasionally seen. Compared with the control group, in the model group the number of writhing was increased (P<0.01), the writhing reaction score and serum level of PGF2α and PGF2α/PGE2 value were increased (P<0.01), the level of PGE2 was decreased (P<0.01). Compared with the model group, in the EA group and the ibuprofen group the number of writhing were decreased (P<0.05), the latency of writhing was prolonged (P<0.01), the writhing reaction scores and serum levels of PGF2α and PGF2α/PGE2 values were decreased (P<0.05, P<0.01), the levels of PGE2 were increased (P<0.01). Compared with the control group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1ß and IL-18 in the uterine tissues of rats was increased in the model group (P<0.01). Compared with the model group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1ß and IL-18 in the uterine tissues of rats was decreased in the EA group and the ibuprofen group (P<0.01, P<0.05). There was no significant difference between the EA group and the ibuprofen group in the above indexes (P>0.05). CONCLUSION: EA could alleviate pain and uterine tissue injury in rats with PDM. The mechanism may be related to the inhibition of the activation of NLRP3 inflammasome in rat uterine tissues, thereby inhibiting pyroptosis and its inflammatory factors release.

Eugenol Alleviates TGEV-Induced Intestinal Injury via Suppressing ROS/NLRP3/GSDMD-Dependent Pyroptosis.

Transmissible gastroenteritis virus (TGEV), a coronavirus, is one of the main causative agents of diarrhea in piglets and significantly impacts the global swine industry. Pyroptosis is involved in the pathogenesis of coronavirus, but its role in TGEV-induced intestinal injury has yet to be fully elucidated. Eugenol, an essential plant oil, plays a vital role in antiviral innate immune responses. We demonstrate the preventive effect of eugenol on TGEV infection. Eugenol alleviates TGEV-induced intestinal epithelial cell pyroptosis and reduces intestinal injury in TGEV-infected piglets. Mechanistically, eugenol reduces the activation of NLRP3 inflammasome, thereby inhibiting TGEV-induced intestinal epithelial cell pyroptosis. In addition, eugenol scavenges TGEV-induced reactive oxygen species (ROS) increase, which in turn prevents TGEV-induced NLRP3 inflammasome activation and pyroptosis. Overall, eugenol protects the intestine by reducing TGEV-induced pyroptosis through inhibition of NLRP3 inflammasome activation, which may be mediated through intracellular ROS levels. These findings propose that eugenol may be an effective strategy to prevent TGEV infection.

Effects of electroacupuncture on NLRP3 inflammasome and pyroptosis protein GSDMD in uterine tissue in rats with primary dysmenorrhea / 中国针灸

OBJECTIVE@#To observe the effects of electroacupuncture (EA) on NLRP3 inflammasome and its downstream protein gastermin D (GSDMD) in rats with primary dysmenorrhea (PDM), and to explore the potential mechanism of EA on the treatment of PDM.@*METHODS@#Forty healthy female SD rats without pregnancy were randomly divided into a control group, a model group, an EA group and an ibuprofen group, 10 rats in each group. PDM model was prepared by injection of estradiol benzoate and oxytocin. Except the control group, the rats in each group were subcutaneously injected with estradiol benzoate for 10 days, and oxytocin was injected on the 11th day. The rats in the EA group were intervened with EA (dense wave, frequency of 50 Hz) at "Guanyuan" (CV 4) and "Sanyinjiao" (SP 6) at the same time of modeling, once a day, 20 min each time, for 10 consecutive days. The rats in the ibuprofen group were treated with 0.8 mL of ibuprofen by gavage (concentration of ibuprofen solution was 1.25 mg/mL) for 10 consecutive days. After modeling, the writhing reaction was observed. After intervention, the HE staining method was used to observe the histological morphology of uterus and evaluate the pathological damage score of uterus; ELISA method was used to detect the serum levels of prostaglandin E2 (PGE2) and prostaglandin F2α (PGF2α); Western blot method was used to detect the protein expression of NLRP3, apoptosis related spot like protein (ASC), caspase-1, GSDMD, GSDMD-N and inflammatory factors (interleukin [IL]-1β, IL-18) in uterine tissue.@*RESULTS@#In the model group, a large number of vacuolar degeneration and death of endometrial epithelial cells, spiral arterioles congestion in lamina propria and neutrophil infiltration were observed. In the EA group, there was a small amount of vacuolar degeneration and death of endometrial epithelial cells, a small amount of spiral arterioles congestion in the lamina propria, and a small amount of neutrophils infiltration. In the ibuprofen group, there was very small number of degeneration and death of endometrial epithelial cells, and no obvious arterial congestion was found in lamina propria, and neutrophil infiltration was occasionally seen. Compared with the control group, in the model group the number of writhing was increased (P<0.01), the writhing reaction score and serum level of PGF2α and PGF2α/PGE2 value were increased (P<0.01), the level of PGE2 was decreased (P<0.01). Compared with the model group, in the EA group and the ibuprofen group the number of writhing were decreased (P<0.05), the latency of writhing was prolonged (P<0.01), the writhing reaction scores and serum levels of PGF2α and PGF2α/PGE2 values were decreased (P<0.05, P<0.01), the levels of PGE2 were increased (P<0.01). Compared with the control group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the uterine tissues of rats was increased in the model group (P<0.01). Compared with the model group, the protein expression of NLRP3, ASC, caspase-1, GSDMD, GSDMD-N, IL-1β and IL-18 in the uterine tissues of rats was decreased in the EA group and the ibuprofen group (P<0.01, P<0.05). There was no significant difference between the EA group and the ibuprofen group in the above indexes (P>0.05).@*CONCLUSION@#EA could alleviate pain and uterine tissue injury in rats with PDM. The mechanism may be related to the inhibition of the activation of NLRP3 inflammasome in rat uterine tissues, thereby inhibiting pyroptosis and its inflammatory factors release.

Jinyinqingre Oral Liquid alleviates LPS-induced acute lung injury by inhibiting the NF-κB/NLRP3/GSDMD pathway / 中国天然药物

Acute lung injury (ALI) is a prevalent and severe clinical condition characterized by inflammatory damage to the lung endothelial and epithelial barriers, resulting in high incidence and mortality rates. Currently, there is a lack of safe and effective drugs for the treatment of ALI. In a previous clinical study, we observed that Jinyinqingre oral liquid (JYQR), a Traditional Chinese Medicine formulation prepared by the Taihe Hospital, Affiliated Hospital of Hubei University of Medicine, exhibited notable efficacy in treating inflammation-related hepatitis and cholecystitis in clinical settings. However, the potential role of JYQR in ALI/acute respiratory distress syndrome (ARDS) and its anti-inflammatory mechanism remains unexplored. Thus, the present study aimed to investigate the therapeutic effects and underlying molecular mechanisms of JYQR in ALI using a mouse model of lipopolysaccharide (LPS)-induced ALI and an in vitro RAW264.7 cell model. JYQR yielded substantial improvements in LPS-induced histological alterations in lung tissues. Additionally, JYQR administration led to a noteworthy reduction in total protein levels within the BALF, a decrease in MPAP, and attenuation of pleural thickness. These findings collectively highlight the remarkable efficacy of JYQR in mitigating the deleterious effects of LPS-induced ALI. Mechanistic investigations revealed that JYQR pretreatment significantly inhibited NF-κB activation and downregulated the expressions of the downstream proteins, namely NLRP3 and GSDMD, as well as proinflammatory cytokine levels in mice and RAW2647 cells. Consequently, JYQR alleviated LPS-induced ALI by inhibiting the NF-κB/NLRP3/GSDMD pathway. JYQR exerts a protective effect against LPS-induced ALI in mice, and its mechanism of action involves the downregulation of the NF-κB/NLRP3/GSDMD inflammatory pathway.

Structure-Guided Approach to Discover Tuberosin as a Potent Activator of Pyruvate Kinase M2, Targeting Cancer Therapy.

Metabolic reprogramming is a key attribute of cancer progression. An altered expression of pyruvate kinase M2 (PKM2), a phosphotyrosine-binding protein is observed in many human cancers. PKM2 plays a vital role in metabolic reprogramming, transcription and cell cycle progression and thus is deliberated as an attractive target in anticancer drug development. The expression of PKM2 is essential for aerobic glycolysis and cell proliferation, especially in cancer cells, facilitating selective targeting of PKM2 in cell metabolism for cancer therapeutics. We have screened a virtual library of phytochemicals from the IMPPAT (Indian Medicinal Plants, Phytochemistry and Therapeutics) database of Indian medicinal plants to identify potential activators of PKM2. The initial screening was carried out for the physicochemical properties of the compounds, and then structure-based molecular docking was performed to select compounds based on their binding affinity towards PKM2. Subsequently, the ADMET (absorption, distribution, metabolism, excretion and toxicity) properties, PAINS (Pan-assay interference compounds) patterns, and PASS evaluation were carried out to find more potent hits against PKM2. Here, Tuberosin was identified from the screening process bearing appreciable binding affinity toward the PKM2-binding pocket and showed a worthy set of drug-like properties. Finally, molecular dynamics simulation for 100 ns was performed, which showed decent stability of the protein-ligand complex and relatival conformational dynamics throughout the trajectory. The study suggests that modulating PKM2 with natural compounds is an attractive approach in treating human malignancy after required validation.

Adsorption of recalcitrant phosphorus compounds using the phosphate-selective binding-protein PstS.

Currently available wastewater phosphorus (P) treatment technologies target removal of reactive forms of P. Selective adsorption of more recalcitrant soluble non-reactive phosphorus (sNRP) can improve P removal and recovery. A phosphate-selective phosphate-binding protein (PBP), PstS, was immobilized onto NHS-activated beads to assess the ability of this novel bioadsorbent to remove (adsorb) and subsequently recover (desorb) a range of sNRP compounds. Four sNRP compounds representative of wastewater sNRP were selected for use in this study: phytic acid (PA), sodium triphosphate (TrP), beta-glycerol phosphate (BGP), and sodium hexametaphosphate (HMP). Using PBP, adsorption of all sNRP compounds was thermodynamically favorable. The PBP had nearly equivalent binding affinity for PA compared to PBP's typical target, orthophosphate, although it had less affinity for the other sNRP compounds. Adsorption followed pseudo-second order reaction kinetics, with 95% of maximum adsorption occurring within 4 min. This was substantially faster sNRP adsorption compared to other adsorbents in the literature. Adsorption was modeled using the Langmuir isotherm, reflecting that one phosphate molecule attached to one PBP binding site. Notably, this selective 1:1 attachment resulted in higher total P removal for sNRP molecules with high P content. The binding site lost activity with increasing pH, and as such, highest desorption was achieved at pH 12, making the system amenable to sNRP removal as well as controlled recovery.

Biochemical and structural characterization of the cyanophage-encoded phosphate-binding protein: implications for enhanced phosphate uptake of infected cyanobacteria.

To acquire phosphorus, cyanobacteria use the typical bacterial ABC-type phosphate transporter, which is composed of a periplasmic high-affinity phosphate-binding protein PstS and a channel formed by two transmembrane proteins PstC and PstA. A putative pstS gene was identified in the genomes of cyanophages that infect the unicellular marine cyanobacteria Prochlorococcus and Synechococcus. However, it has not been determined whether the cyanophage PstS protein is functional during infection to enhance the phosphate uptake rate of host cells. Here we showed that the cyanophage P-SSM2 PstS protein was abundant in the infected Prochlorococcus NATL2A cells and the host phosphate uptake rate was enhanced after infection. This is consistent with our biochemical and structural analyses showing that the phage PstS protein is indeed a high-affinity phosphate-binding protein. We further modelled the complex structure of phage PstS with host PstCA and revealed three putative interfaces that may facilitate the formation of a chimeric ABC transporter. Our results provide insights into the molecular mechanism by which cyanophages enhance the phosphate uptake rate of cyanobacteria. Phosphate acquisition by infected bacteria can increase the phosphorus contents of released cellular debris and virus particles, which together constitute a significant proportion of the marine dissolved organic phosphorus pool.

[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.

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.

Jiangzhi Ligan Decoction Inhibits GSDMD-Mediated Canonical/Noncanonical Pyroptosis Pathways and Alleviates High-Fat Diet-Induced Nonalcoholic Fatty Liver Disease.

Increasing evidence suggests that gasdermin D (GSDMD) mediated pyroptosis signaling pathways play a vital role in the pathogenesis of nonalcoholic fatty liver disease (NAFLD). Jiangzhi Ligan Decoction (JZLGD) has been verified to prevent NAFLD, but its specific mechanism has not been determined. In this study, an NAFLD model was established in Sprague-Dawley rats by a high-fat diet (HFD). After 12 weeks, JZLGD was orally administered once a day for 6 additional weeks. We investigated the effects of JZLGD on NAFLD rats and determined the GSDMD pathway-associated proteins to explore whether such effects were associated with pyroptosis. Our data show that JZLGD significantly reduced the liver index; improved serum lipid levels, liver function parameters, and lipid droplet content; and relieved NAFLD. We further found that the serum levels of the proinflammatory factors interleukin-1ß (IL-1ß), IL-18, tumor necrosis factor-α, and IL-6 were obviously decreased in the JZLGD group. HFD rats treated with GSDMD exhibited NLRP3, caspase-1, lipopolysaccharide (LPS), and caspase-11 activation; however, these effects were blunted by JZLGD treatment. Taken together, JZLGD may exert hepatoprotective effects against NAFLD in a rat HFD model by regulating GSDMD-mediated canonical/noncanonical pyroptosis pathways.

Salvianolic Acids for Injection alleviates cerebral ischemia/reperfusion injury by switching M1/M2 phenotypes and inhibiting NLRP3 inflammasome/pyroptosis axis in microglia in vivo and in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: After cerebral ischemia/reperfusion injury, pro-inflammatory M1 and anti-inflammatory M2 phenotypes of microglia are involved in neuroinflammation, in which activation of NLRP3 inflammasome and subsequent pyroptosis play essential roles. Salvianolic Acids for Injection (SAFI) is Chinese medicine injection which composed of multiple phenolic acids extracted from Radix Salviae Miltiorrhizae, and has been reported to generate neuroprotective effects after cerebral ischemic insult in clinical and animal studies. AIM OF THE STUDY: The present study was designed to investigate whether SAFI exerts neuroprotective effects by switching microglial phenotype and inhibiting NLRP3 inflammasome/pyroptosis axis in microglia. MATERIALS AND METHODS: The middle cerebral artery occlusion/reperfusion (MCAO/R) model in rats and oxygen-glucose deprivation/reoxygenation (OGD/R) model in co-cultured primary neurons and primary microglia were utilized. The neuroprotective effect of SAFI was evaluated through measuring neurological deficit scores, neuropathological changes, inflammatory factors, cell phenotype markers, and related proteins of NLRP3 inflammasome/pyroptosis axis. RESULTS: The results showed that SAFI treatment was able to: (1) produce a significant increase in neurological deficit scores and decrease in infarct volumes, and alleviate histological injury and neuronal apoptosis in cerebral cortex in MCAO/R model; (2) increase neuronal viability and reduce neuronal apoptosis in the OGD model; (3) reshape microglial polarization patterns from M1-like phenotype to M2-like phenotype; (4) inhibit the activation of the NLRP3 inflammasome and the expression of proteins related to NLRP3 inflammasome/pyroptosis axis in vivo and in vitro. CONCLUSION: These findings indicate that SAFI exert neuroprotective effect, probably via reducing neuronal apoptosis, switching microglial phenotype from M1 towards M2, and inhibiting NLRP3 inflammasome/pyroptosis axis in microglia.

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.