L-AP Alleviates Liver Injury in Septic Mice by Inhibiting Macrophage Activation via Suppressing NF-κB and NLRP3 Inflammasome/Caspase-1 Signal Pathways.

Liver injury and progressive liver failure are severe life-threatening complications in sepsis, further worsening the disease and leading to death. Macrophages and their mediated inflammatory cytokine storm are critical regulators in the occurrence and progression of liver injury in sepsis, for which effective treatments are still lacking. l-Ascorbic acid 6-palmitate (L-AP), a food additive, can inhibit neuroinflammation by modulating the phenotype of the microglia, but its pharmacological action in septic liver damage has not been fully explored. We aimed to investigate L-AP's antisepticemia action and the possible pharmacological mechanisms in attenuating septic liver damage by modulating macrophage function. We observed that L-AP treatment significantly increased survival in cecal ligation and puncture-induced WT mice and attenuated hepatic inflammatory injury, including the histopathology of the liver tissues, hepatocyte apoptosis, and the liver enzyme levels in plasma, which were comparable to NLRP3-deficiency in septic mice. L-AP supplementation significantly attenuated the excessive inflammatory response in hepatic tissues of septic mice in vivo and in cultured macrophages challenged by both LPS and ATP in vitro, by reducing the levels of NLRP3, pro-IL-1ß, and pro-IL-18 mRNA expression, as well as the levels of proteins for p-I-κB-α, p-NF-κB-p65, NLRP3, cleaved-caspase-1, IL-1ß, and IL-18. Additionally, it impaired the inflammasome ASC spot activation and reduced the inflammatory factor contents, including IL-1ß and IL-18 in plasma/cultured superannuants. It also prevented the infiltration/migration of macrophages and their M1-like inflammatory polarization while improving their M2-like polarization. Overall, our findings revealed that L-AP protected against sepsis by reducing macrophage activation and inflammatory cytokine production by suppressing their activation in NF-κB and NLRP3 inflammasome signal pathways in septic liver.

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.

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.

Astragaloside IV Alleviates Doxorubicin-Induced Cardiotoxicity by Inhibiting Cardiomyocyte Pyroptosis through the SIRT1/NLRP3 Pathway.

Doxorubicin (DOX) is a powerful anthracycline antineoplastic drug used to treat a wide spectrum of tumors. However, its clinical application is limited due to cardiotoxic side effects. Astragaloside IV (AS IV), one of the major compounds present in aqueous extracts of Astragalus membranaceus, possesses potent cardiovascular protective properties, but the underlying molecular mechanisms are unclear. Thus, the aim of this study was to investigate the effect of AS IV on DOX-induced cardiotoxicity (DIC). Our findings revealed that DOX induced pyroptosis through the caspase-1/gasdermin D (GSDMD) and caspase-3/gasdermin E (GSDME) pathways. AS IV treatment significantly improved the cardiac function and alleviated myocardial injury in DOX-exposed mice by regulating intestinal flora and inhibiting pyroptosis; markedly suppressed the levels of cleaved caspase-1, N-GSDMD, cleaved caspase-3, and N-GSDME; and reversed DOX-induced downregulation of silent information regulator 1 (SIRT1) and activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome in mice. The SIRT1 inhibitor EX527 significantly blocked the protective effects of AS IV. Collectively, our results suggest that AS IV protects against DIC by inhibiting pyroptosis through the SIRT1/NLRP3 pathway.

Study on the therapeutic mechanism of HJ granules in a rat model of urinary tract infection caused by Escherichia coli.

ETHNOPHARMACOLOGICAL RELEVANCE: Urinary tract infections (UTIs) are globally prevalent infectious diseases, predominantly caused by uropathogenic Escherichia coli (UPEC). The misuse of antibiotics has led to the emergence of several drug-resistant strains. Traditional Chinese Medicine (TCM) has its own advantages in the treatment of UTIs. HJ granules is a herbal formula used for the treatment of UTIs. However, its mechanism of action is not clear. AIM OF THE STUDY: The aim of this study was to investigate the therapeutic efficacy and mechanism of action of HJ granules in a rat model of UTI caused by Escherichia coli (E coli) CFT073. MATERIALS AND METHODS: SD rats were selected to establish a rat UTI model by injecting UPEC strain CFT073 into the bladder using the transurethral placement method. HJ granules were administered to rats after modelling and the efficacy of HJ granule was investigated by measuring urinary decanalogue, inflammatory factors in bladder tissue and pathological changes in the bladder after 3d of administration. Expression of sonic hedgehog (SHH), NOD-like receptor thermoprotein domain 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and activation of cysteinyl aspartate specific proteinase-1 (caspase-1) were detected by western blotting and immunofluorescence staining in rat bladder tissue. NLRP3, ASC and caspase-1, a cysteine-containing aspartic protein, were expressed and activated. RESULTS: The results showed that infection of rats with UPEC resulted in increased pH and erythrocytes in bladder irrigation fluid; increased expression of IL-1ß, IL-6 and SHH and decreased expression of IL-10 in bladder tissue; and significant upregulation of the expression of both SHH and NLRP3 inflammasom and significant activation of NLRP3 inflammasom. HJ granules significantly increased the concentration of IL-10 in the bladder, inhibited the expression of SHH and NLRP3 inflammasom in bladder tissue, and suppressed the activation of NLRP3 inflammasom, thereby reducing inflammatory lesions in bladder tissue. CONCLUSION: HJ granules may improve bladder injury and treat UTIs by inhibiting the expression and activation of NLRP3 inflammasom.

Protopine ameliorates OVA-induced asthma through modulatingTLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis.

BACKGROUND: Chronic airway inflammation and hyperresponsiveness are characteristics of asthma. The isoquinoline alkaloid protopine (PRO) has been shown to exert anti-inflammatory effects, but its mechanism of action in asthma is not known. PURPOSE: Investigate the protective properties of PRO upon asthma and elucidate its mechanism. STUDY DESIGN AND METHODS: The effects of PRO in asthma treatment were assessed by histology, biochemical analysis, and real-time reverse transcription-quantitative polymerase chain reaction. Then, we integrated molecular docking, western blotting, cellular experiments, immunohistochemistry, immunofluorescence analysis, flow cytometry, and metabolomics analysis to reveal its mechanism. RESULTS: In vivo, PRO therapy reduced the number of inflammatory cells (eosinophils, leukocytes, monocytes) in bronchoalveolar lavage fluid (BALF), ameliorated pathologic alterations in lung tissues, and inhibited secretion of IgG and histamine. Molecular docking showed that PRO could dock with the proteins of TLR4, MyD88, TRAF6, TAK1, IKKα, and TNF-α. Western blotting displayed that PRO inhibited the TLR4/NF-κB signaling pathway. PRO regulated expression of the pyroptosis-related proteins NLR family pyrin domain containing 3 (NLRP3) inflammasome, gasdermin D, caspase-1, and drove caspase-1 inactivation to affect inflammatory responses by inhibiting the NLRP3 inflammasome. In vitro, 24 h after treatment with PRO, cell activity, as well as levels of reactive oxygen species (ROS) and interleukin (IL)-1ß and IL-18, decreased significantly. Immunofluorescence staining showed that PRO decreased expression of TLR4 and MyD88 in vitro. PRO decreased nuclear translocation of NF-κB p65. Twenty-one potential biomarkers in serum were identified using metabolomics analysis, and they predominantly controlled the metabolism of phenylalanine, tryptophan, glucose, and sphingolipids. CONCLUSION: PRO reduced OVA-induced asthma. The underlying mechanism was associated with the TLR4/MyD88/NF-κB pathway and NLRP3 inflammasome-mediated pyroptosis.

Guarea microcarpa C. DC. extract inhibits NLRP3 inflammasome by suppressing its ATPase activity.

ETHNOPHARMACOLOGICAL RELEVANCE: Guarea genus comprises tropical and subtropical terrestrial herbs inhabiting Central and South America. These plants, including Guarea guidonia (L.) Sleumer, have anti-inflammatory, analgesic, antibacterial, antiviral, and immune-enhancing properties. AIM OF THE STUDY: Although various species of the Guarea genus are known for their medicinal properties, comprehensive data on their anti-inflammatory effects remain limited. Therefore, we investigated the NLRP3 inflammasome-inhibiting effects of the Guarea genus in this study. MATERIALS AND METHODS: To evaluate the anti-inflammatory activities of 18 members of the Guarea genus, we treated NLRP3 inflammasome activators with their extracts in LPS-primed J774A.1 and THP-1 cells. Cell viability was determined by water soluble tetrazolium salt (WST) and cytokine production, protein expression, and nuclear fractionation were determined by western blotting. Reactive oxygen species (ROS) production and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) oligomerization were measured using confocal microscopic analysis. Inflammation-induced zebrafish was used in the in vivo experiments. RESULTS: Among the 18 Guarea members tested, Guarea microcarpa C. DC. extract (GM) exhibited no cytotoxicity and specifically suppressed the activation of the NLRP3 inflammasome, but not of the AIM2 or NLRC4 inflammasomes, by inhibiting the ATPase activity of NLRP3. This was achieved without affecting NF-κB signaling, potassium efflux, or intracellular ROS production, all of which are involved in NLRP3 activation. The reduced ATPase activity of NLRP3 led to decreased ASC oligomerization. Furthermore, GM exhibited anti-inflammatory effects in vivo. Additionally, GM treatment alleviated inflammation at the organismal level in an LPS-induced inflammation model using zebrafish embryos. CONCLUSION: Our results demonstrate the anti-inflammatory effects of GM via suppressing the NLRP3 inflammasome. Therefore, GM can be a potential therapeutic candidate for various inflammatory diseases caused by aberrant NLRP3 inflammasome activation.

A novel mechanism for regulating lung immune homeostasis: Zukamu granules alleviated acute lung injury in mice by inhibiting NLRP3 inflammasome activation and regulating Th17/Treg cytokine balance.

ETHNOPHARMACOLOGICAL RELEVANCE: Acute lung injury (ALI) is a severely acute lung inflammation with high morbidity and mortality. Zukamu granules (ZKMG) is one of the Uygur patent drugs commonly used in clinic, which is included in the National Essential Drugs List (2018 edition). Clinical studies have shown that ZKMG has a significant effect on acute upper respiratory tract infection, and has better anti-inflammatory and antipyretic effects. However, the immunomodulatory mechanism of ZKMG on ALI is still not clear. AIM OF THE STUDY: The aim of this study is to investigate the lung protective effect and immunomodulatory mechanism of ZKMG on lipopolysaccharide (LPS) -induced ALI mice, and to provide an important basis for the treatment strategy and theoretical basis of ALI. MATERIALS AND METHODS: First, network pharmacology was used to predict the potential signaling pathways and biological processes of ZKMG related to immunology. Molecular docking technique was used to predict the possibility between the core components of ZKMG acting on NLRP3 protein. In addition, protein levels of F4/80 in lung tissues were assessed by Immunohistochemistry (IHC). The contents of IL-1ß, IL-18, IL-17A and IL-10 in the lung tissue and serum, MPO in the lung tissue were detected by enzyme-linked immunosorbent assay (ELISA). Real-time quantitative PCR analysis (RT-qPCR) was used to detect NLRP3 mRNA in lung tissue. Protein levels of NLRP3, Caspase-1, Cleaved caspase-1 p20, ASC, and GSDMD were detected by Western blot (WB). RESULTS: The results of network pharmacology showed that the immune pathways of ZKMG were mainly Th17 signaling pathway, IL-17 signaling pathway, NOD-like receptor signaling pathway, etc. Molecular docking results showed that the core components of ZKMG had good binding ability to NLRP3 protein. The verification experiments showed that ZKMG can reduce the degree of lung injury, and reduce the level of inflammatory infiltration of neutrophils and macrophages by reducing the content of MPO and F4/80. In addition, ZKMG can reduce NLRP3 mRNA, inhibit the expression of NLRP3/Caspase-1/GSDMD and other related pathway proteins, and reduce inflammatory factors such as IL-1ß and IL-18. It can also reduce the content of pro-inflammatory cytokine IL-17A, increase the content of anti-inflammatory cytokine IL-10 in lung tissue. CONCLUSION: ZKMG can reduce the degree of lung tissue injury in ALI by inhibiting NLRP3/Caspase-1/GSDMD signaling pathway and restoring the IL-17A/IL-10 cytokine balance, and its protective mechanism may be related to the regulation of lung immune homeostasis. It will provide a new strategy for studying the regulation of lung immune homeostasis.

The marine Penicillium sp. GGF16-1-2 metabolite dicitrinone G inhibits pancreatic angiogenesis by regulating the activation of NLRP3 inflammasome.

Citrinin derivatives have been found to have various pharmacological activities, such as anti-inflammatory, anti-tumor, and antioxidant effects. Dicitrinone G (DG) was a new citrinin dimer isolated from marine-derived fungus Penicillium sp. GGF 16-1-2 which has potential activity. Here, we aim to investigate whether DG has anti-pancreatic cancer activity. In xenograft tumor model, 2 × 106 BXPC-3 cells were injected into the hind flank of NU/NU nude mice by subcutaneously for 2 weeks followed by treating with DG (0.25, 0.5, 1 mg/kg) and 5-FU (30 mg/kg) for 4 weeks. Tumor volume and weight were measured, and the expression of CD31, IL-18, NLRP3, and Caspase-1 in tumor tissue were detected. In vitro, HUVECs were treated with conditioned medium (CM) derived from BXPC-3 cells, the effects of DG on angiogenesis were detected by tube formation and western blot analysis. In vivo studies showed that the tumor growth and angiogenesis were greatly suppressed. The tumor weight inhibition rates of DG and 5-FU groups were about 42.36%, 38.94%, 43.80%, and 31.88%. Furthermore, the expression of CD31 and Caspase-1 were decreased. In vitro, CM derived from BXPC-3 cells which treated with DG could inhibit the tube formation and expression of pro-angiogenic NICD in HUVECs. Our study suggests that DG could suppress angiogenesis via the NLRP3/IL-18 pathway and may have the potential to inhibit tumor development.

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.

Mechanism of DaiTongXiao in the treatment of gouty arthritis through the NLRP3 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: DaiTongXiao (DTX) is a traditional Chinese Dai folk formulation utilized for gouty arthritis treatment, with substantial evidence supporting its anti-inflammatory properties. The NLRP3 inflammasome disorder is tightly linked to the development of many inflammatory diseases. AIM OF THE STUDY: To elucidate the therapeutic efficacy of DTX in gouty arthritis and reveal its potential underlying mechanism. MATERIALS AND METHODS: The primary active constituents in DTX were determined through ultraviolet spectrophotometry and gas chromatography. Rats underwent induction with monosodium urate (MSU), followed by treatment of J774A.1 cells with adenosine triphosphate (ATP) activation and lipopolysaccharide (LPS) induction and the subsequent culture in Dulbecco's modified Eagle's medium. The degree of foot joint swelling in rats was assessed, and ankle joints were evaluated through H&E staining. Enzyme-linked immunosorbent assay was performed to measure the levels of interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor (TNF)-α in both serum and cells. Reverse transcription-polymerase chain reaction (RT-PCR) was performed to determine the relative mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 macrophages. The expression of NLRP3, ASC, Caspase-1, and NF-κB was examined by western blotting. RESULTS: DTX could alleviate MSU-induced joint swelling in rats, as evidenced by a reduction in joint inflammation. Moreover, DTX effectively enhanced the survival rate of J774A.1 cells following LPS induction and ATP activation. Furthermore, DTX significantly reduced IL-1ß, IL-6, IL-8, and TNF-α levels in both cell culture medium and rat serum. RT-PCR results revealed that DTX notably downregulated the mRNA expression levels of NLRP3, ASC, Caspase-1, and NF-κB in J774A.1 cells. Additionally, DTX downregulated NLRP3, ASC, NF-κB, and Caspase-1 expression in the joint tissue. CONCLUSIONS: DTX exerts a significant anti-gouty arthritis effect, with its mechanism being tightly linked to the NLRP3 inflammatory signaling pathway. This pathway may be modulated by inhibiting IL-1ß differentiation and maturation by downregulating NLRP3, ASC, Caspase-1, and NF-κB protein expression. This, in turn, leads to a reduction in the release of IL-6, IL-8, and TNF-α, ultimately impeding gouty arthritis progression.

[Acacetin protects rats from cerebral ischemia-reperfusion injury by regulating TLR4/NLRP3 signaling pathway].

This study aims to investigate the mechanism of acacetin in protecting rats from cerebral ischemia-reperfusion injury via the Toll-like receptor 4(TLR4)/NOD-like receptor protein 3(NLRP3) signaling pathway. Wistar rats were randomized into sham, model, low-and high-dose acacetin, and nimodipine groups, with 10 rats in each group. The rat model of middle cerebral artery occlusion(MCAO) was established with the improved suture method in other groups except the sham group. The neurological deficit score and cerebral infarction volume of each group were evaluated 24 h after modeling. Enzyme-linked immunosorbent assay(ELISA) was employed to measure the levels of interleukin-1ß(IL-1ß), IL-6, tumor necrosis factor-α(TNF-α), malondialdehyde(MDA), supe-roxide dismutase(SOD), and glutathione(GSH). Western blot was employed to determine the expression levels of B-cell lymphonoma-2(Bcl-2), Bcl-2-associated X protein(Bax), and TLR4/NLRP3 signaling pathway-related proteins(TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß) in the rat brain tissue. Hematoxylin-eosin(HE) staining was employed to reveal the histopathological changes in the ischemic area. Compared with the sham group, the modeling of MCAO increased the neurological deficit score and cerebral infarction volume, elevated the IL-1ß, IL-6, TNF-α, and MDA levels and lowered the SOD and GSH levels in the brain tissue(P<0.05). Compared with the MCAO model group, low-and high-dose acacetin and nimodipine decreased the neurological deficit score and cerebral infarction volume, lowered the IL-1ß, IL-6, TNF-α, and MDA levels and elevated the SOD and GSH levels in the brain tissue(P<0.05). Compared with the sham group, the model group showed up-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and down-regulated protein level of Bcl-2 in the brain tissue(P<0.05). Compared with the MCAO model group, the acacetin and nimodipine groups showed down-regulated protein levels of Bax, TLR4, p-NF-κB/NF-κB, NLRP3, pro-caspase-1, cleaved caspase-1, pro-IL-1ß, and cleaved IL-1ß and up-regulated protein level of Bcl-2 in the brain tissue(P<0.05). In conclusion, acacetin regulates the TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress, thus exerting the protective effect on cerebral ischemia-reperfusion injury in rats.

Recent advances in pyroptosis, liver disease, and traditional Chinese medicine: A review.

In recent years, the incidence of liver disease has increased, becoming a major cause of death. Various liver diseases are intricately linked to pyroptosis, which is one of the most common forms of programmed cell death. As a powerful weapon in the fight against liver diseases, traditional Chinese medicine (TCM) can affect pyroptosis via a number of routes, including the classical, nucleotide oligomerization domain-like receptors protein 3/caspase-1/gasdermin D (GSDMD) pathway, the nonclassical lipopolysaccharide/caspase-11/GSDMD pathway, the ROS/caspase-3/gasdermin E pathway, the caspase-9/caspase-3/GSDMD pathway, and the Apaf-1/caspase-11/caspase-3 pathway. In this review, we provide an overview of pyroptosis, the interplay between pyroptosis and liver diseases, and the mechanisms through which TCM regulates pyroptosis in liver diseases. The information used in the text was collected and compiled from the databases of PubMed, Web of Science, Scopus, CNKI, and Wanfang Data up to June 2023. The search was not limited with regard to the language and country of the articles. Research and review articles were included, and papers with duplicate results or unrelated content were excluded. We examined the current understanding of the relationship between pyroptosis and liver diseases as well as the advances in TCM interventions to provide a resource for the identification of potential targets for TCM in the treatment of liver diseases.

[Effect of multi-glycosides of Tripterygium wilfordii on renal injury in diabetic kidney disease rats through NLRP3/caspase-1/GSDMD pyroptosis pathway].

This study investigated the effect of multi-glycosides of Tripterygium wilfordii(GTW) on renal injury in diabetic kidney disease(DKD) rats through Nod-like receptor protein 3(NLRP3)/cysteine-aspartic acid protease-1(caspase-1)/gsdermin D(GSDMD) pyroptosis pathway and the mechanism. To be specific, a total of 40 male SD rats were randomized into the normal group(n=8) and modeling group(n=34). In the modeling group, a high-sugar and high-fat diet and one-time intraperitoneal injection of streptozotocin(STZ) were used to induce DKD in rats. After successful modeling, they were randomly classified into model group, valsartan(Diovan) group, and GTW group. Normal group and model group were given normal saline, and the valsartan group and GTW group received(ig) valsartan and GTW, respectively, for 6 weeks. Blood urea nitrogen(BUN), serum creatinine(Scr), alanine ami-notransferase(ALT), albumin(ALB), and 24 hours urinary total protein(24 h-UTP) were determined by biochemical tests. The pathological changes of renal tissue were observed based on hematoxylin and eosin(HE) staining. Serum levels of interleukin-1ß(IL-1ß) and interleukin-18(IL-18) were detected by enzyme-linked immunosorbent assay(ELISA). Western blot was used to detect the expression of pyroptosis pathway-related proteins in renal tissue, and RT-PCR to determine the expression of pyroptosis pathway-related genes in renal tissue. Compared with the normal group, the model group showed high levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), low level of ALB(P<0.01), severe pathological damage to kidney, and high protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01). Compared with the model group, valsartan group and GTW group had low levels of BUN, Scr, ALT, and 24 h-UTP and serum levels of IL-1ß and IL-18(P<0.01), high level of ALB(P<0.01), alleviation of the pathological damage to the kidney, and low protein and mRNA levels of NLRP3, caspase-1, and GSDMD in renal tissue(P<0.01 or P<0.05). GTW may inhibit pyroptosis by decreasing the expression of NLRP3/caspase-1/GSDMD in renal tissue, thereby relieving the inflammatory response of DKD rats and the pathological injury of kidney.

["Trichosanthis Fructus-Allii Macrostemonis Bulbus" combination inhibits NLRP3 inflammasome activation and reduces inflammatory response in RAW264.7 macrophage by inducing autophagy].

This study aims to explore the effect of "Trichosanthis Fructus-Allii Macrostemonis" combination(GX) on the activation of NOD-, LRR-, and pyrin domain-containing protein 3(NLRP3) inflammasome, the release of inflammatory cytokines, and the level of autophagy in RAW264.7 macrophage damaged by lipopolysaccharide(LPS), and the mechanism of GX against inflammatory response in macrophages. To be specific, LPS was used to induce the injury of RAW264.7 cells. Cell Counting Kit-8(CCK-8) assay was employed to measure the survival rate of cells, and Western blot to detect the protein expression of NLRP3, apoptosis-associated speck-like protein(ASC), cysteine-aspartic acid protease(caspase)-1, interleukin(IL)-18, IL-1ß, microtubule-associated protein light chain 3(LC3)-Ⅱ, and selective autophagy junction protein p62/sequestosome 1 in RAW264.7 macrophages. ELISA was used to measure the levels of IL-18 and IL-1ß in RAW264.7 cells. Transmission electron microscopy was applied to observe the number of autophagosomes in RAW264.7 cells. Immunofulourescence staining was used to detect the expression of LC3-Ⅱ and p62 in RAW264.7 cells. The result showed that GX significantly reduced the protein expression of NLRP3, ASC, and caspase-1 in RAW264.7 cells, significantly increased the protein expression of LC3Ⅱ, decreased the expression of p62, significantly inhibited the secretion of IL-18 and IL-1ß, significantly increased the number of autophagosomes, significantly enhanced the immunofluorescence of LC3Ⅱ, and reduced the immunofluorescence of p62. Furthermore, 3-methyladenine(3-MA) could reverse the inhibitory effect of GX on NLRP3, ASC, and caspase-1 and reduce the release of IL-18 and IL-1ß. In summary, GX can increase of the autophagy activity of RAW264.7 and inhibit the activation of NLRP3 inflammasome, thereby reducing the release of inflammatory cytokines and suppressing inflammatory response in macrophages.

Paeonia lactiflora Pall. Polysaccharide alleviates depression in CUMS mice by inhibiting the NLRP3/ASC/Caspase-1 signaling pathway and affecting the composition of their intestinal flora.

ETHNOPHARMACOLOGICAL RELEVANCE: Paeonia lactiflora Pall. (PL) has been commonly used to de-stressing the liver and relieve depression in traditional Chinese medicine for over a thousand years. Recently, it has been widely used in studies on anti-depressant, anti-inflammatory and regulation of intestinal flora. However, the polysaccharide component has received less attention than the saponin component of PL. AIM OF THE STUDY: This study aimed to elucidate the effects of Paeonia lactiflora polysaccharide (PLP) on depressive behavior in mice in a chronic unpredictable mild stress (CUMS) model and its possible action mechanisms. MATERIALS AND METHODS: A model of chronic depression induced by the CUMS approach. Behavioral experiments were used to assess the success of the CUMS model and the therapeutic impact of PLP. Then the extent of damage to the colonic mucosa was assessed by H&E staining; the extent of neuronal damage was assessed by Nissler staining. Inflammatory factor expression was assessed at different sites in the mouse by enzyme-linked immunoassay (Elisa). The alterations of faecal microflora were detected by 16S rRNA gene sequencing. In the colonic tissues, NLRP3, ASC and Caspase-1 mRNA and protein levels detected by quantitative real-time PCR (qRT-PCR) and Western blot(WB). RUSULTS: PLP can improve depressive behavior in CUMS mice, and colonic mucosal and neuronal damage. Elisa assay showed that PLP could reduce interleukin-1ß (IL-1ß), interleukin-6 (IL-6), tumour necrosis factor-α (TNF-α) levels, and increase 5-Hydroxytryptamine(5-HT) levels in CUMS mice. 16S sequencing analysis showed that PLP could regulate the intestinal flora of CUMS mice and increase their species richness. In addition, PLP significantly inhibited NLRP3/ASC/Caspase-1 signalling pathways activation in the colonic tissues of CUMS mice. CONCLUSIONS: PLP modulates depression-related intestinal ecological dysregulation, increases species richness, and inhibits inflammatory factors levels and NLRP3 inflammasome activation to reduce colonic mucosal and neurons damage, thereby improving depression-like behavior and neurotransmitter release in CUMS mice.

Eleutheroside E from pre-treatment of Acanthopanax senticosus (Rupr.etMaxim.) Harms ameliorates high-altitude-induced heart injury by regulating NLRP3 inflammasome-mediated pyroptosis via NLRP3/caspase-1 pathway.

Eleutheroside E, a major natural bioactive compound in Acanthopanax senticosus (Rupr.etMaxim.) Harms, possesses anti-oxidative, anti-fatigue, anti-inflammatory, anti-bacterial and immunoregulatory effects. High-altitude hypobaric hypoxia affects blood flow and oxygen utilisation, resulting in severe heart injury that cannot be reversed, thereby eventually causing or exacerbating high-altitude heart disease and heart failure. The purpose of this study was to determine the cardioprotective effects of eleutheroside E against high-altitude-induced heart injury (HAHI), and to study the mechanisms by which this happens. A hypobaric hypoxia chamber was used in the study to simulate hypobaric hypoxia at the high altitude of 6000 m. 42 male rats were randomly assigned to 6 equal groups and pre-treated with saline, eleutheroside E 100 mg/kg, eleutheroside E 50 mg/kg, or nigericin 4 mg/kg. Eleutheroside E exhibited significant dose-dependent effects on a rat model of HAHI by suppressing inflammation and pyroptosis. Eleutheroside E downregulated the expressions of brain natriuretic peptide (BNP), creatine kinase isoenzymes (CK-MB) and lactic dehydrogenase (LDH). Moreover, The ECG also showed eleutheroside E improved the changes in QT interval, corrected QT interval, QRS interval and heart rate. Eleutheroside E remarkably suppressed the expressions of NLRP3/caspase-1-related proteins and pro-inflammatory factors in heart tissue of the model rats. Nigericin, known as an agonist of NLRP3 inflammasome-mediated pyroptosis, reversed the effects of eleutheroside E. Eleutheroside E prevented HAHI and inhibited inflammation and pyroptosis via the NLRP3/caspase-1 signalling pathway. Taken together, eleutheroside E is a prospective, effective, safe and inexpensive agent that can be used to treat HAHI.

Dapagliflozin Attenuates NLRP3/Caspase-1 Signaling Pathway-Mediated Pyroptosis of Vascular Smooth Muscle Cells by Downregulating CTSB.

Background: Atherosclerosis is a chronic inflammatory disease. Pyroptosis triggers and amplifies the inflammatory response and plays an important role in atherosclerosis. Cathepsin B (CTSB) can promote atherosclerosis and activate NOD-like receptor protein 3 (NLRP3) to mediate pyroptosis. Dapagliflozin (DAPA) can inhibit cell pyroptosis to improve atherosclerosis. This study aimed to explore the effect of DAPA on oxidized low-density lipoprotein (ox-LDL)-induced pyroptosis of vascular smooth muscle cells (VSMCs) and its underlying mechanism. Objective: We aimed to investigate the effect of DAPA on ox-LDL-induced pyroptosis of VSMCs in mice and its underlying mechanism. Methods: VSMCs were transfected with CTSB-overexpressing and -silencing lentiviral vectors. VSMCs were treated with different concentrations of ox-LDL (0, 50, 100 and 150 µg/ml ). Then, Hoechst 33342/PI double staining, interleukin (IL)-1ß and lactate dehydrogenase (LDH) release assay were used to detect cell pyroptosis. Western blotting was used to detect pyroptosis indicators protein, based on which the appropriate concentration of ox-LDL was selected. After VSMCs were treated with different concentrations of DAPA (0.1 µM, 1.0 µM, 5.0 µM, 10 µM, 25 µM and 50 µM), the proliferative activity of VSMCs was detected using Cell Counting Kit-8 (CCK8) assay. After VSMCs were pretreated with different DAPA concentrations (0.1 µM, 1.0 µM, 5.0 µM and 10 µM) for 24 hours and then treated with 150 µg/mL ox-LDL for 24 hours, the effects of different concentrations of DAPA on pyroptosis of VSMCs were detected, based on which the appropriate DAPA concentration was selected. After lentivirus transfected VSMCs were treated with 150 µg/mL ox-LDL for 24 hours, the effects of overexpression and silencing of CTSB in pyroptosis were observed. On the basis of DAPA (0.1 µM)- and ox-LDL(150 µg/mL)-treated VSMCs, overexpression and silencing of CTSB were used to observe the effects of DAPA and CTSB on ox-LDL-mediated VSMCs pyroptosis. Results: (1) VSMCs stably transfected with CTSB-overexpressing and -silencing lentiviruses were obtained; 150 µg/mL was the optimal concentration of ox-LDL for inducing pyroptosis of VSMCs, and 0.1 µM was the optimal concentration of DAPA for ameliorating pyroptosis of VSMCs. (2) Ox-LDL-induced pyroptosis of VSMCs was worsened by CTSB overexpression but suppressed by CTSB silencing. (3) DAPA attenuated ox-LDL-induced pyroptosis of VSMCs through downregulating CTSB and NLRP3. (4) Overexpression of CTSB based on DAPA intervention aggravated ox-LDL-induced pyroptosis of VSMCs. Conclusion: DAPA attenuates NLRP3/caspase-1 pathway-mediated pyroptosis of VSMCs through downregulating CTSB.

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.

Protection of Oxygen Glucose Deprivation-Induced Human Brain Vascular Pericyte Injury: Beneficial Effects of Bellidifolin in Cellular Pyroptosis.

Pericytes play critical roles in the maintenance of brain vascular homeostasis. However, very little is currently known about how pericytes regulate ischemic stroke-induced brain injury. Inflammation is a key event in the pathobiology of stroke, in which the nod-like receptor protein-3 (NLRP3) inflammasome is involved in, triggering sterile inflammatory responses and pyroptosis. In the current study, an immortalized cell line derived from human brain vascular pericytes (HBVPs) was constructed, and it showed that HBVPs challenged with oxygen glucose deprivation (OGD) displays pronounced cellular excretion of LDH, IL-1ß, IL-18 and increased PI positive staining. Mechanistically, upon OGD treatment, NLRP3 forms an inflammasome with its adaptor protein apoptosis-associated speck-like protein, containing a caspase recruitment domain (ASC) and caspase-1, manifested as much more co-stainings of NLRP3, ASC and Caspase-1 in HBVPs, accompanied by the increased protein levels of NLRP3, ASC, caspase-1 as well as the pyroptosis-associated protein gasdermin D (GSDMD). Intriguingly, GSDMD-N shuttled to the mitochondrial membrane triggered by OGD exposure, which promoted massive mitochondria-derived ROS generation. Importantly, the invention value of the specific targets was evaluated by treatment with bellidifolin, a kind of ketone compound derived from Swertia chirayita in traditional Tibetan medicine. It showed that bellidifolin exerts beneficial effects and attenuates the formation of NLRP3/ASC/Caspase-1 complex, thereby impeding GSDMD-N shuttling and resultant ROS generation, protecting against OGD-induced HBVPs pyroptosis. Overall, these findings unravel the potential mechanisms of pericyte injury induced by OGD and indicate that bellidifolin may exert its beneficial effects on pyroptosis, thus providing new therapeutic insights into stroke.