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1.
Organ Transplantation ; (6): 88-2022.
Article in Chinese | WPRIM | ID: wpr-907038

ABSTRACT

Objective To evaluate the effect of high mobility group box 1 (HMGB1)/ cysteinyl aspartate specific proteinase (Caspase)-1/Gasdermin D (GSDMD) signaling axis-mediated hepatocyte pyroptosis on liver ischemia-reperfusion injury (IRI). Methods C57BL/6 mice were randomly divided into the sham operation group (Sham group), IRI 2 h group, IRI 6 h group, IRI 12 h group, glycyrrhizic acid (GA)+Sham group and GA+IRI 12 h group (n=8 in each group). AML12 cells were evenly divided into the Sham group, IRI 12 h group, GA+Sham group and GA+IRI 12 h group. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), interleukin (IL)-1β and IL-6 in each group were detected by enzyme-linked immune absorbent assay(ELISA). The messenger ribonucleic acid (mRNA) levels of IL-1β and IL-6 were detected by reverse transcription polymerase chain reaction(RT-PCR). The pathological score of liver ischemia and cell apoptosis were compared among all groups. The expression level of HMGB1 in the liver tissues of each group was determined by immunohistochemistry. The expression levels of HMGB1, Caspase-1 and GSDMD proteins in the mouse liver tissues and AML12 cells were measured by Western blot. Results Compared with the Sham group, the serum levels of ALT, AST, IL-1β and IL-6 and the relative expression levels of IL-1β and IL-6 mRNA in the liver tissues were all significantly up-regulated after IRI in each group (all P < 0.05), and showed significant time-dependent pattern along with the prolongation of reperfusion time. Compared with the Sham group, the pathological score of hepatic ischemia and the apoptosis rate of hepatocytes were significantly increased after IRI in each group (all P < 0.05). Immunohistochemical results showed that the expression level of HMGB1 in the liver tissues was significantly up-regulated after IRI, which showed an increasing trend along with the prolongation within the period of 2-12 h. Western blot showed that compared with the Sham group, the relative expression levels of HMGB1, Caspase-1 and GSDMD proteins in vivo and in vitro were up-regulated in the IRI 12 h group. The relative expression level of HMGB1 protein was significantly up-regulated, whereas those of Caspase-1 and GSDMD proteins were significantly down-regulated in the GA+IRI 12 h group compared with those in the IRI 12 h group (all P < 0.05). Conclusions Hepatocytes probably activate the Caspase-1/GSDMD signaling pathway by releasing HMGB1, thereby triggering hepatocyte pyroptosis and leading to liver IRI. Inhibition of extracellular release of HMGB1 by GA may mitigate liver IRI.

2.
Journal of Clinical Hepatology ; (12): 2016-2019, 2022.
Article in Chinese | WPRIM | ID: wpr-942653

ABSTRACT

Objective To investigate the effect of Huatan Qushi Huoxue prescription on lipopolysaccharide (LPS)-induced pyroptosis of RAW264.7 cells and its mechanism. Methods An in vitro cell model of LPS-induced activated RAW264.7 was established and divided into blank group, model group, high-, middle-, and low-dose Huatan Qushi Huoxue prescription groups, and control group. The corresponding drug-containing serum intervention was performed for 24 hours. A scanning electron microscope was used to observe cell morphology, and immunofluorescence assay was used to perform quantitative localization of GSDMD-N. Results The cells in the blank group were round and regular in shape with smooth surface, and those in the control group were swollen, with folds on the surface and gaps in the capsule, which were consistent with the morphology of cell pyroptosis. The cells in the control group had bubbles on the surface with obvious pseudopodia and pores in cell membrane, and those in the high-dose group were not swollen and had a rough surface with pseudopodia, with no obvious pores in cell membrane. The cells in the low- and middle-dose groups were swollen and had a rough surface of cell membrane with pores and pseudopodia. Immunofluorescence assay showed that compared with the blank group, the model group had a significant increase in the positive staining intensity of GSDMD-N, and compared with the model group, the control group and the Traditional Chinese medicine group had a reduction in the positive staining intensity of GSDMD-N. Conclusion Huatan Qushi Huoxue prescription can improve the pyroptosis of macrophages and reduce the expression of GSDMD-N.

3.
Acta Pharmaceutica Sinica ; (12): 2612-2621, 2022.
Article in Chinese | WPRIM | ID: wpr-941520

ABSTRACT

More and more studies have shown that NOD-like receptor protein 3 (NLRP3) inflammasome has become the regulatory factor of inflammatory response and protective immunity, and the assembly and activation of NLRP3 inflammasomes are closely related to the anti-tumor immunity effect. Depending on the cell type and stimuli, activation of the NLRP3 inflammasome can induce immune cells to become polarized, hyperactive, or pyroptotic, releasing interleukin (IL)-1β and IL-18, which leads to cascade immune or inflammatory responses, and its role in tumor immunity has received extensive attention. Here, we review the mechanisms of the NLRP3 inflammasome enhancing CD8+ T cells-mediated anti-tumor immunity by inducing the pyroptosis of tumor cell, the pyroptosis or hyperactive state of dendritic cells (DCs), and the pyroptosis or polarization of the macrophages. Different anti-tumor immune roles of NLRP3 inflammasome activation in tumor cells and immune cells provide new directions for future research and may influence the development of next-generation immunotherapy.

4.
Organ Transplantation ; (6): 647-2022.
Article in Chinese | WPRIM | ID: wpr-941487

ABSTRACT

Liver transplantation is an effective treatment for the end-stage liver disease. However, hepatic ischemia-reperfusion injury (HIRI) will inevitably occur during liver transplantation, which might lead to early graft dysfunction or aggravate rejection. The underlying protective mechanism remains to be further elucidated. Programmed cell death is an important mechanism of HIRI, and multiple novel types of programmed cell death participate in the pathological process of HIRI. In-depth study of programmed cell death is expected to further improve the therapeutic effect of liver transplantation. In this article, research progresses on apoptosis, autophagy and autophagy-dependent cell death, ferroptosis, necroptosis, pyroptosis, pathanatos and other common programmed cell death patterns in HIRI were reviewed, aiming to provide reference for enhancing the success rate of liver transplantation and improving clinical prognosis of the recipients.

5.
Article in Chinese | WPRIM | ID: wpr-940757

ABSTRACT

ObjectiveTo explore the effect of Banxia Xiexintang (BXT) on the NOD-like receptor protein 3 (NLRP3)/cysteinyl aspartate-specific protease-1 (Caspase-1) pyroptosis pathway and its downstream factors in rats with ulcerative colitis (UC), and to explain the mechanism of BXT in the treatment of UC. MethodSD rats were randomly divided into normal control group, model group, low- and high-dose BXT groups (6.3, 12.6 g·kg-1·d-1), and salazosulfapyridine (SASP) group (0.42 g·kg-1·d-1), with 7 rats in each group. The UC model was induced by intragastric administration of 2.5% dextran sodium sulfate (DSS) solution for 10 days, followed by drug intervention for 7 days. The general state of rats was observed during the experiment, and the disease activity index (DAI) score was calculated during the administration period. At the end of the experiment, colonic tissues were collected for hematoxylin-eosin (HE) staining to observe the pathological changes and the curative effect of BXT. Real-time fluorescence-based quantitative polymerase chain reaction (Real-time PCR) was used to detect the mRNA transcriptional levels of NLRP3, Caspase-1, gasdermin D (GSDMD), and interleukin (IL)-1β in colonic tissues. Western blot was used to detect the protein expression of NLRP3, Caspase-1, GSDMD, and IL-1β in colonic tissues to explore the therapeutic mechanism of BXT. ResultCompared with the normal control group, the model group showed increased DAI score, pathological changes in colonic tissues, and up-regulated mRNA and protein expression of NLRP3, Caspase-1, GSDMD, and IL-1β (P<0.05, P<0.01). Compared with the model group, the groups with drug intervention showed reduced DAI scores and improved pathological changes in colonic tissues. The mRNA and protein expression levels of NLRP3, Caspase-1, GSDMD, and IL-1β in colonic tissues of the BXT groups were significantly down-regulated or tended to be down-regulated, especially in the low-dose BXT group (P<0.05, P<0.01). ConclusionBXT can inhibit pyroptosis and alleviate inflammation in rats with UC by regulating the NLRP3/Caspase-1 pathway.

6.
Article in Chinese | WPRIM | ID: wpr-940722

ABSTRACT

ObjectiveTo investigate the regulatory effect of Shoutaiwan on oxidative stress and pyroptosis in lipopolysaccharide (LPS)-induced human extravillous trophoblast (HTR-8/SVneo) cells and provide a new direction for deciphering the mechanism of action of Shoutaiwan. MethodLPS (100 μg∙L-1) was used to induce the injury of HTR-8/SVneo cells (modeling). Five groups were designed in this study, including a blank group, a model group, a Shoutaiwan (10% Shoutaiwan-containing serum) group, an antioxidant (1 mmol·L-1 NAC) group, and NOD like receptor thermoprotein domain 3 (NLRP3) inhibitor (50 μmol·L-1 MCC950) group. Cell viability was detected by cell counting kit-8 (CCK-8) kit. Hochest 33342/PI double fluorescence staining and flow cytometry were employed to observe cell death. The levels of interleukin-18 (IL-18), interleukin-1β (IL-1β), malondialdehyde (MDA), and superoxide dismutase (SOD) in cell supernatant was determined by enzyme-linked immunosorbent assay (ELISA). DCFH-DA probe was used to measure the level of intracellular reactive oxygen species (ROS). Western blot was employed to determine the protein levels of NLRP3, Caspase-1, gastermin D (GSDMD), and IL-1β in cells, and Real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) to measure the mRNA levels of NLRP3 and Caspase-1 in cells. ResultCompared with the blank group, the modeling decreased the cell viability (P<0.01), elevated the levels of IL-1β, IL-18, ROS, and MDA, and weakened the activity of SOD (P<0.01). Furthermore, it up-regulated the protein levels of NLRP3, Caspase-1, GSDMD, and IL-1β and the mRNA levels of NLRP3 and Caspase-1 (P<0.01). Compared with the model group, Shoutaiwan, NAC, and MCC950 increased the cell viability (P<0.01). Further, Shoutaiwan and NAC lowered the levels of MDA and ROS and increased the activity of SOD (P<0.01). Shoutaiwan and MCC950 reduced the IL-1β and IL-18 in cell supernatant (P<0.01), and down-regulated the protein levels of NLRP3, Caspase-1, GSDMD, and IL-1β and the mRNA levels of NLRP3, Caspase-1, and IL-1β (P<0.05,P<0.01). ConclusionShoutaiwan can regulate oxidative stress and pyroptosis to attenuate the LPS-induced damage of HTR-8/SVneo cells, which may be the mechanism of Shoutaiwan in preventing recurrent spontaneous abortion.

7.
Article in Chinese | WPRIM | ID: wpr-940622

ABSTRACT

ObjectiveTo explore the effect of Gegen Qinliantang (GQL) on vulnerable plaque of atherosclerosis based on the macrophage pyroptosis mediated by nuclear factor (NF)-κB/NOD-like receptor protein 3 (NLRP3)/cysteine-aspartic acid protease (Caspase)-1 pathway. MethodA total of 12 normal C57BL/6CNC mice were used as the control group, and 60 ApoE-/- mice of the same line were randomized into 5 groups: model group, low-dose, medium-dose, and high-dose GQL groups (GQL-D, GQL-Z, GQL-G groups, respectively), and western medicine group. The control group and model group were given (ig) equal volume sterile distilled, and GQL-D, GQL-Z, GQL-G and western medicine groups received (ig) corresponding concentration of drugs for 8 weeks. Aortic plaques were observed based on hematoxylin and eosin (HE) staining. Serum levels of interleukin (IL)-1β and IL-18 were detected by enzyme-linked immunosorbent assay (ELISA), protein levels of macrophage mannose receptor (CD206)/apoptosis-associated speck-like protein containing a CARD (ASC) and CD206/NLRP3 by double-labeling immunofluorescence, and C-terminal gasdermin D (GSDMD), N-terminal GSDMD, NLRP3, pro-cysteinyl aspartate specific proteinase 1 (pro-Caspase-1) and NF-κB p65 by Western blot. ResultCompared with the control group, model group demonstrated serious pathological changes, rise of the levels of serum IL-1β and IL-18 and tissue ASC, NLRP3, C-terminal GSDMD, N-terminal GSDMD, pro-Caspase-1, and NF-κB p65, and decrease of CD206 level (P<0.05). As compared with model group, the administration groups showed alleviation of the lesions in aortic wall, decrease in levels of serum IL-1β and IL-18 and tissue ASC, NLRP3, C-terminal GSDMD, N-terminal GSDMD, pro-Caspase-1, and NF-κB p65, and rise of CD206 level, with significant difference between some groups (P<0.05). ConclusionGegen Qinliantang alleviates vulnerable plaque of atherosclerosis by regulating NF-κB/NLRP3/Caspase-1 pathway and further relieving macrophage pyroptosis.

8.
Article in Chinese | WPRIM | ID: wpr-940615

ABSTRACT

ObjectiveTo explore the mechanism of Didangtang (DDT) against the inflammatory cascade triggered by foam cell pyroptosis in high-glucose environment. MethodOxidized low density lipoprotein (ox-LDL, 100 mg·L-1) was used to induce pyroptosis of foam cells. The control group (5.5 mmol·L-1 glucose), foam cell group (100 mg·L-1 ox-LDL), high-glucose group (33.3 mmol·L-1 glucose), DDT group (10% DDT-containing serum), and NOD-like receptor family pyrin domain-containing 3 (NLRP3) inhibitor group (MCC950, 10 nmmol·L-1) were designed. The cell membrane damage was observed by lactate dehydrogenase (LDH) release assay. The expression of cysteinyl aspartate-specific proteinase-1 (Caspase-1) was detected by immunofluorescence method, and expression of key proteins NLRP3, Caspase-1, gastermin D (GSDMD), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in the pyroptosis pathway was determined by Western blot. The release of IL-18 and IL-1β, monocyte chemoattractant protein-1 (MCP-1), and tumor necrosis factor α (TNF-α) in cell supernatant was measured by enzyme-linked immunosorbent assay (ELISA). ResultThe expression of NLRP3, Caspase-1, and GSDMD was up-regulated (P<0.01) and the release of IL-1β, IL-18, MCP-1, IL-1α, and TNF-α was increased (P<0.01) in foam cell group compared with those in the control group. The expression of NLRP3, Caspase-1, and GSDMD was higher (P<0.01) and the release of inflammatory factors was more (P<0.01) in the high-glucose group than in the foam cell group. DDT and MCC950 can inhibit expression of NLRP3, Caspase-1, GSDMD and reduce the release of IL-1β, IL-18, MCP-1, IL-1α, and TNF-α. ConclusionDDT can suppress the pyroptosis of foam cells induced by NLRP3/Caspase-1 pathway in high-glucose environment and thereby alleviate the inflammatory cascade.

9.
Article in Chinese | WPRIM | ID: wpr-940552

ABSTRACT

The increasing incidence of obesity and diabetes has made diabetic kidney disease (DKD) the main cause of chronic kidney disease and end-stage renal disease. Despite current pharmacological interventions for blood glucose control and renin-angiotensin system inhibition, the risk of kidney disease progression and complications remains high. At present, the pathogenesis of DKD has been clarified to be related to chronic inflammatory response, oxidative stress, glucose and lipid metabolism disorders, and hemodynamic abnormalities. According to recent studies, the programmed cell deaths (PCD) of renal intrinsic cells such as pyroptosis and necroptosis play a key role in the occurrence and development of DKD. Pyroptosis and necroptosis, the two newly discovered routes of PCD, can protect the hosts from being invaded by microbial pathogens, but their dysregulation is associated with multiple autoimmunity and autoinflammatory responses. Pyroptosis and necroptosis are closely interlinked and cross-regulated. Different from apoptosis, these two cellular suicide mechanisms cause membrane rupture and release of cell contents through their respective gasdermin D (GSDMD) and mixed lineage kinase domain-like protein (MLKL), with damage-associated molecular patterns (DAMPs) and inflammatory cytokines like interleukin-1β (IL-1β) involved to trigger inflammation, and chronic inflammatory responses are key factors leading to the progression of DKD. Traditional Chinese medicine (TCM) has long been employed for the prevention and treatment of DKD and the resulting clinical outcomes are remarkable. TCM has been proved to exert a protective effect against DKD by affecting the expression of nucleotide oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, receptor-interacting protein kinase 3 (RIPK3), and MLKL. This paper reviewed the relationship of pyroptosis and necroptosis with DKD and its intervention with TCM.

10.
Article in Chinese | WPRIM | ID: wpr-940551

ABSTRACT

ObjectiveTo observe the effect of Danggui Buxuetang on podocyte pyroptosis in diabetic kidney disease (DKD) rats and to explore the possible mechanism of its prevention and treatment of DKD and podocyte pyroptosis. MethodEight of the 50 male SD rats were randomly classified into a normal group, and the remaining 42 were fed a high-glucose and high-fat diet for six weeks and then intraperitoneally injected with 35 mg·kg-1 streptozotocin (STZ) for inducing type 2 diabetes. After successful modeling, they were randomized into the model group, low- (0.72 g·kg-1) and high-dose (1.44 g·kg-1) Danggui Buxuetang group, and irbesartan (0.017 g·kg-1) group and gavaged with the corresponding drugs, while those in the normal group and model group with an equal volume of normal saline, once per day, for 20 weeks. During the medication, the fasting blood glucose (FBG) and 24 h urine protein (24 h-UTP) were measured regularly. After administration, the pathological changes in renal tissues were observed by periodic acid-silver metheramine (PASM) staining, followed by the observation of ultrastructural changes in podocytes under the transmission electron microscope (TEM). Serum levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) were determined by enzyme-linked immunosorbent assay (ELISA). The DNA damage in renal tissue cells of rats was detected by in situ nick end-labeling (TUNEL) assay. The protein expression levels of thioredoxin interacting protein (TXNIP), cysteine-dependent aspartate-directed protease-1 (Caspase-1), and gasdermin D (GSDMD) in renal tissues of rats were detected by immunohistochemistry (IHC), the expression levels of nucleotide binding domain like receptor protein 3 (NLRP3) and Wilms tumor protein-1 (WT-1) in podocytes by immunofluorescent (IF) staining, and the expression levels of TXNIP/NLRP3/Caspase-1/GSDMD pathway proteins and Synaptopodin in renal podocytes by Western blot. ResultCompared with the normal group, the model group exhibited increased FBG and 24 h UTP, glomerular hypertrophy, mesangial hyperplasia, increased extracellular matrix, thickened basement membrane, K-W nodules, vacuolar degeneration in renal tubular epithelial cells, foot process fusion or loss, elevated serum IL-1β and IL-18 levels and TUNEL-positive cells in renal tissue, enhanced NLRP3 but diminished WT-1 expression in podocytes, down-regulated Synaptopodin protein expression, and up-regulated TXNIP/NLRP3/Caspase-1/GSDMD protein expression (P<0.01). Compared with the model group, Danggui Buxuetang high-dose group remarkably lowered FBG, 24-h UTP, and TUNEL-positive cells in renal tissue, improved renal histopathology and podocyte injury and loss, down-regulated NLRP3 expression in podocytes and TXNIP/NLRP3/Caspase-1/GSDMD protein expression levels, and up-regulated WT-1 expression in podocytes and Synaptopodin protein expression (P<0.05, P<0.01). ConclusionDanggui Buxuetang inhibits podocyte pyroptosis to reduce proteinuria and delays the development of DKD possibly by regulating the TXNIP/NLRP3/GSDMD signaling pathway.

11.
Article in Chinese | WPRIM | ID: wpr-940465

ABSTRACT

ObjectiveTo screen the active antitumor components of Gupi Xiaoji decoction by network pharmacology and molecular docking based on the pyroptosis mediated by cysteinyl aspartate-specific protease 1 (Caspase-1) and explore its molecular mechanism in intervening in the pyroptosis of HepG2.2.15 cells through in vitro experiments. MethodThe compounds and targets of Gupi Xiaoji decoction were screened out by Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) to obtain the corresponding gene symbols. The targets of Caspase-1 were collected from GeneCards,online mendelian inheritance in man(OMIM),PharmGKB,and TTD,and the compound-gene target regulatory network was constructed by Cytoscape. The protein-protein interaction(PPI) network was established and analyzed by STRING. The mechanism of the effective components of Gupi Xiaoji decoction on Caspase-1 was predicted by gene ontology(GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses. The molecular docking was verified with AutoDock Vina. The plasma medicated with Gupi Xiaoji Decoction was prepared and HepG2.2.15 cells were cultured in vitro. HepG2.2.15 cells were divided into a blank plasma group,a VX-765 group,a VX-765+medicated plasma group, and a medicated plasma group. After 48 hours of intervention with 15% medicated plasma, the expression and distribution of gasdermin D-N (GSDMD-N) on the surface of the cell membrane were detected by immunofluorescence staining. The release of lactic dehydrogenase (LDH), interleukin(IL)-1β,and IL-18 in the cell supernatant was measured by enzyme-linked immunosorbent assay(ELISA) kits. The expression of Caspase-1 and GSDMD-N was measured by Western blot. ResultThe mitogen-activated protein kinase 14 (MAPK14),MAPK1,protein kinase B1 (Akt1), MAPK8, V-Jun sarcoma virus oncogene homolog (JUN), and TP53 screened by network pharmacology were the main targets. The compounds 7-hydroxy-5,8-dimethoxy-2-phenyl-chromone,wogonin,rhamnazin,moslosooflavone,isorhamnetin,7-O-methylisomucronulatol,formononetin,calycosin,luteolin,quercetin,kaempferol,β-sitosterol,and baicalein screened by network pharmacology were the main active components of Gupi Xiaoji decoction. Go enrichment analysis showed that multiple biological processes were involved, including responses to oxidative stress and metal ions,ubiquitin-like protein ligase binding,and phosphatase binding. KEGG pathway enrichment analysis showed MAPK pathway,nuclear factor(NF)-κB pathway,p53 pathway, and hypoxia-inducible factor-1(HIF-1) pathway were involved. Molecular docking showed that the targets had good binding with the components. In vitro experiments displayed that compared with the blank plasma group,the VX-765 group showed weakened GSDMD-N fluorescence signal,reduced release of LDH,IL-1β,and IL-18,and declining expression of Caspase-1 and GSDMD-N(P<0.01), and the medicated plasma group showed increased GSDMD-N fluorescence signal, increased release of LDH,IL-1β,and IL-18,and up-regulated expression of Caspase-1 and GSDMD-N(P<0.01). ConclusionGupi Xiaoji Decoction can induce the pyroptosis of HepG2.2.15 cells by regulating Caspase-1 through multiple targets and multiple pathways.

12.
Article in Chinese | WPRIM | ID: wpr-940350

ABSTRACT

ObjectiveTo explore the possible mechanism of total flavonoids of peony flower (TFPF) in protecting rats from gouty nephropathy and provide data support for the pharmaceutical research on the treatment of gouty nephropathy. MethodGouty nephropathy rat model was established by adenine combined with ethambutol. Rats were randomly assigned into blank control group, model group, allopurinol (42 mg·kg-1) group, Tongfengshu tablets (600 mg·kg-1, positive control) group, and TFPF (260, 130, and 65 mg·kg-1) groups. Enzyme-linked immunosorbent assay (ELISA) was employed to measure the levels of tumor necrosis factor-α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-1β (IL-1β), and interleukin-18 (IL-18) in rat serum and those of transforming growth factor-β1 (TGF-β1) and IL-1β in renal homogenate. Hematoxylin-eosin(HE) staining was carried out for observation of the morphological changes of renal cells. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was conducted for observation of the DNA damage in renal cells. The expression of NOD-like receptor protein 3 (NLRP3), cysteine aspartic acid protease(Caspase)-1 and IL-1β were observed by immunohistochemistry. The expression levels of NLRP3, Caspase-1 and nuclear transcription factor -κB (NF-κB) in renal tissues were detected by Western blot. ResultCompared with blank group, the contents of TNF-α, MCP-1, IL-1β, IL-18, and TGF-β1 in serum of model group were significantly increased (P<0.01), and the expressions of NLRP3, Caspase-1, NF-κB and IL-1β in kidney of model group were significantly increased (P<0.01). The renal tissue cells showed cytoplasmic swelling, cell membrane rupture, and the number of nuclear pyknotic fracture increased. The positive rate of TUNEL staining was significantly increased in model group (P<0.01), and the contents of IL-1β and TGF-β1 in renal tissue homogenate were significantly increased (P<0.01). Compared with model group, the contents of inflammatory factors TNF-α, IL-1β and IL-18 in serum of rats in TFPF high- and medium-dose groups could be decreased to different degrees (P<0.05, P<0.01), while the content of MCP-1 in TFPF high-dose group was significantly decreased (P<0.01). The content of TGF-β1 in renal tissue homogenate in TFPF high- and medium-dose groups was significantly decreased (P<0.05, P<0.01), and the content of IL-1β in renal tissue homogenate in TFPF medium-dose group was significantly decreased (P<0.01). HE staining showed that each dose group of TFPF could improve the status of renal tubular epithelial cells, reduce cytoplasmic swelling and the number of nuclear pyknosis to varying degrees. The positive rate of TUNEL staining was decreased (P<0.01) and DNA damage was decreased. The expression of NLRP3, Caspase-1, IL-1β and NF-κB protein in renal tissue cells was inhibited (P<0.05, P<0.01). ConclusionTFPF protects rats from gouty nephropathy by inhibiting the secretion of inflammatory cytokines. Specifically, it may inhibit the activation of NF-κB and NLRP3/Caspase-1 pathways to reduce the expression, maturation, and release of inflammatory cytokines such as IL-1β and IL-18 and further inhibit pyroptosis, thereby reversing the inflammatory injury of kidney in gouty nephropathy.

13.
Article in Chinese | WPRIM | ID: wpr-940201

ABSTRACT

ObjectiveTo explore the effect of the serum containing Huanglian Wendantang on pyroptosis in vitro model of insulin resistance and its mechanism. MethodSD rats were randomly divided into two groups, namely Huanglian Wendantang-containing serum group and blank serum group, and given 7.8 g·kg-1·d-1 Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance (IR), and they were randomly divided into control group, model group, metformin hydrochloride group, blank serum group, and Huanglian Wendantang-containing serum high-, medium-, and low-dose groups. After 24 h of cultivation, the cells of each group were treated with insulin for 15 min at concentration of 1×10-7 mol·L-1, and the cell supernatant was collected. The glucose oxidase (GOD-POD) kit was used to determine the glucose content of each group, and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell proliferation, thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group, model group, and Huanglian Wendantang-containing serum group. The levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) in each group were determined by enzyme-linked immunosorbent assay (ELISA), and the mRNA and protein expression levels of NOD like receptor protein 3 (NLRP3) in each group were determined by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and Western blot. In terms of the mechanism, HepG2 cells were randomly divided into control group, empty vector group, NLRP3 overexpression group, empty vector + IR group, empty vector + IR + Huanglian Wendantang-containing serum group, NLRP3 overexpression + IR group, and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells, and calculate the glucose consumption. ELISA was used to determine the release of IL-1β and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 (Caspase-1), gasdermin D (GSDMD), and NLRP3. Immunofluorescence assay was used to detect NLRP3, GSDMD, and Caspase -1 expressions. ResultAs compared with the control group, the glucose consumption in the model group was significantly decreased (P<0.01). As compared with the model group, the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group (P< 0.01). As compared with the control group, the IL-1β and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased (P<0.05, P<0.01). Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1β, IL-18, and NLRP3 mRNA and protein expressions as compared with the model group (P<0.05, P<0.01). Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group (P<0.01), and significantly up-regulated the IL-1β and IL-18 levels and the mRNA and protein levels of NLRP3, Caspase-1, and GSDMD as compared with the empty vector + IR group (P<0.05, P<0.01). Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group (P<0.05, P<0.01). ConclusionHigh fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway, which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus (T2DM).

14.
Article in Chinese | WPRIM | ID: wpr-940195

ABSTRACT

Pyroptosis is a newly discovered programmed cell death. It is an important natural immune response and has obvious anti-infection function. Studies have shown that pyroptosis plays an important role in the occurrence and development of rheumatoid arthritis. Traditional Chinese medicine(TCM) has unique advantages in the prevention and treatment of rheumatoid arthritis. How to guide TCM to effectively prevent and treat rheumatoid arthritis using pyroptosis theory is a new research hotspot in this field. This paper discussed the overview of pyroptosis theory,its mechanism, signal pathway,and application in the treatment of rheumatoid arthritis as well as the research on the activity of TCM based on pyroptosis theory. It was found that the occurrence of pyroptosis was related to Caspase-1-dependent classical inflammatory body pathway and Caspase-1-independent non classical inflammatory body pathway, and pyroptosis produced distinct regulatory effect on the occurrence,development and treatment of rheumatoid arthritis,which would provide a new strategy for the treatment of rheumatoid arthritis. Additionally,TCM recipes such as Miao ethnomedicine prescription Sidaxue and Duhuo Jishengtang, and a variety of effective components such as punicalagin and paeoniflorin monomer derivatives exerted anti-rheumatic and other biological activities by regulating pyroptosis. This provided a theoretical basis and research ideas for the in-depth study of pyroptosis theory and guiding the prevention and treatment of rheumatoid arthritis with TCM.

15.
Article in Chinese | WPRIM | ID: wpr-940163

ABSTRACT

Pyroptosis is a newly discovered programmed cell death. It is an important natural immune response and has obvious anti-infection function. Studies have shown that pyroptosis plays an important role in the occurrence and development of rheumatoid arthritis. Traditional Chinese medicine(TCM) has unique advantages in the prevention and treatment of rheumatoid arthritis. How to guide TCM to effectively prevent and treat rheumatoid arthritis using pyroptosis theory is a new research hotspot in this field. This paper discussed the overview of pyroptosis theory,its mechanism, signal pathway,and application in the treatment of rheumatoid arthritis as well as the research on the activity of TCM based on pyroptosis theory. It was found that the occurrence of pyroptosis was related to Caspase-1-dependent classical inflammatory body pathway and Caspase-1-independent non classical inflammatory body pathway, and pyroptosis produced distinct regulatory effect on the occurrence,development and treatment of rheumatoid arthritis,which would provide a new strategy for the treatment of rheumatoid arthritis. Additionally,TCM recipes such as Miao ethnomedicine prescription Sidaxue and Duhuo Jishengtang, and a variety of effective components such as punicalagin and paeoniflorin monomer derivatives exerted anti-rheumatic and other biological activities by regulating pyroptosis. This provided a theoretical basis and research ideas for the in-depth study of pyroptosis theory and guiding the prevention and treatment of rheumatoid arthritis with TCM.

16.
Article in Chinese | WPRIM | ID: wpr-940104

ABSTRACT

ObjectiveTo explore the effect of the serum containing Huanglian Wendantang on pyroptosis in vitro model of insulin resistance and its mechanism. MethodSD rats were randomly divided into two groups, namely Huanglian Wendantang-containing serum group and blank serum group, and given 7.8 g·kg-1·d-1 Huanglian Wendantang and equal volume of normal saline by intragastric administration according to body surface area. Blank serum and medicated serum with different concentration were extracted and prepared. HepG2 cells were treated with sodium palmitate to construct the model of insulin resistance (IR), and they were randomly divided into control group, model group, metformin hydrochloride group, blank serum group, and Huanglian Wendantang-containing serum high-, medium-, and low-dose groups. After 24 h of cultivation, the cells of each group were treated with insulin for 15 min at concentration of 1×10-7 mol·L-1, and the cell supernatant was collected. The glucose oxidase (GOD-POD) kit was used to determine the glucose content of each group, and calculate the glucose consumption and inhibition rate. The methyl thiazolyl tetrazolium (MTT) assay was used to detect the cell proliferation, thus screening out the optimal dose of serum containing Huanglian Wendantang. HepG2 cells were randomly divided into control group, model group, and Huanglian Wendantang-containing serum group. The levels of interleukin-1β (IL-1β) and interleukin-18 (IL-18) in each group were determined by enzyme-linked immunosorbent assay (ELISA), and the mRNA and protein expression levels of NOD like receptor protein 3 (NLRP3) in each group were determined by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) and Western blot. In terms of the mechanism, HepG2 cells were randomly divided into control group, empty vector group, NLRP3 overexpression group, empty vector + IR group, empty vector + IR + Huanglian Wendantang-containing serum group, NLRP3 overexpression + IR group, and NLRP3 overexpression + IR + Huanglian Wendantang-contain serum group. GOD-POD method was used to measure the glucose content of each group cells, and calculate the glucose consumption. ELISA was used to determine the release of IL-1β and IL-18 in each group. Real-time PCR and Western blot assay were used to determine the mRNA and protein expressions of cysteinyl aspartate specific proteinase-1 (Caspase-1), gasdermin D (GSDMD), and NLRP3. Immunofluorescence assay was used to detect NLRP3, GSDMD, and Caspase -1 expressions. ResultAs compared with the control group, the glucose consumption in the model group was significantly decreased (P<0.01). As compared with the model group, the increase of the glucose consumption of IR-HepG2 cells was the most significant in the Huanglian Wendantang-containing serum high-dose group (P< 0.01). As compared with the control group, the IL-1β and IL-18 release levels and the mRNA and protein expressions of NLRP3 in IR-HepG2 cells were significantly increased (P<0.05, P<0.01). Huanglian Wendantang effectively reduced IR-HepG2 cell supernatant IL-1β, IL-18, and NLRP3 mRNA and protein expressions as compared with the model group (P<0.05, P<0.01). Overexpression of NLRP3 significantly reduced the cell glucose consumption as compared with the control group and the empty vector group (P<0.01), and significantly up-regulated the IL-1β and IL-18 levels and the mRNA and protein levels of NLRP3, Caspase-1, and GSDMD as compared with the empty vector + IR group (P<0.05, P<0.01). Huanglian Wendantang-containing serum effectively reversed the above indicators as compared with the NLRP3 + IR group (P<0.05, P<0.01). ConclusionHigh fat-induced insulin sensitivity of IR-HepG2 cells is closely related to inflammation and NLRP3 expression. Huanglian Wendantang-containing serum improves IR-HepG2 cell pyroptosis through the targeted inhibition of NLRP3/Caspase-1 signaling pathway, which provides new therapeutic targets for the prevention and treatment of IR and type 2 diabetes mellitus (T2DM).

17.
Acta Pharmaceutica Sinica B ; (6): 3139-3155, 2022.
Article in English | WPRIM | ID: wpr-939955

ABSTRACT

Aberrant activation of oncogenic signaling pathways in tumors can promote resistance to the antitumor immune response. However, single blockade of these pathways is usually ineffective because of the complex crosstalk and feedback among oncogenic signaling pathways. The enhanced toxicity of free small molecule inhibitor combinations is considered an insurmountable barrier to their clinical applications. To circumvent this issue, we rationally designed an effective tumor microenvironment-activatable prodrug nanomicelle (PNM) for cancer therapy. PNM was engineered by integrating the PI3K/mTOR inhibitor PF-04691502 (PF) and the broad spectrum CDK inhibitor flavopiridol (Flav) into a single nanoplatform, which showed tumor-specific accumulation, activation and deep penetration in response to the high glutathione (GSH) tumoral microenvironment. The codelivery of PF and Flav could trigger gasdermin E (GSDME)-based immunogenic pyroptosis of tumor cells to elicit a robust antitumor immune response. Furthermore, the combination of PNM-induced immunogenic pyroptosis with anti-programmed cell death-1 (αPD-1) immunotherapy further boosted the antitumor effect and prolonged the survival time of mice. Collectively, these results indicated that the pyroptosis-induced nanoplatform codelivery of PI3K/mTOR and CDK inhibitors can reprogram the immunosuppressive tumor microenvironment and efficiently improve checkpoint blockade cancer immunotherapy.

18.
Article in Chinese | WPRIM | ID: wpr-935753

ABSTRACT

Objective: To explore the role and significance of pyroptosis in gas explosion-induced acute lung injury (ALI) in rats. Methods: In February 2018, 126 SPF male SD rats were selected and randomly divided into blank control group (18 rats) and experimental group (40 m, 80 m, 120 m, 160 m, 200 m and 240 m, 18 per group) . The experimental group carried out gas explosion in the roadway to build the ALI model, the control group did not carry out gas explosion, and other conditions were consistent with the experimental group. Respiratory function indexes such as respiratory frequency (f) , tidal volume (TV) , minute ventilation (MV) and airway stenosis index (Penh) were measured 24 hours after the explosion. 5 rats in each group were sacrificed after anesthesia, Hematoxylin-Eosin (HE) staining was used to observe the pathological morphology of lung tissue. Immunohistochemistry was used to detect the content of Caspase-1. Western blotting was used to detect the content of cell pyroptosis including nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) , Caspase-1, interleukin-1β (IL-1β) and interleukin-18 (IL-18) in lung tissue related protein expression. Results: The f and MV of rats in the experimental group were higher than those in the control group (P<0.05) . Except for the 40 m and 80 m groups, the TV of rats in the other experimental groups were higher than those in the control group (P<0.05) . Except for the 40 m group, the Penh of rats in the experimental groups were lower than those in the control group (P<0.05) . HE staining showed that the lung tissue of the experimental groups at different distance points showed obvious edema of the pulmonary interstitium and alveoli, a large number of red blood cells and inflammatory cells exuded in the alveolar space, thickening of the pulmonary interstitium, and increased lung injury score (P<0.05) . The results of immunohistochemistry showed that the positive expression of Caspase-1 in each experimental group was higher than that in the control group (P<0.05) . Western blotting results showed that the expression of pyroptosis-related proteins in each experimental group was higher than that in the control group (P<0.05) . Conclusion: Pyroptosis is involved in the pathophysiological process of gas explosion-induced ALI in rats.


Subject(s)
Acute Lung Injury/pathology , Animals , Explosions , Lung/pathology , Male , Pyroptosis , Rats , Rats, Sprague-Dawley
19.
Article in Chinese | WPRIM | ID: wpr-933297

ABSTRACT

Objective:To evaluate the effect of hepatic ischemia-reperfusion (I/R) rats-derived exosomes on microglial pyroptosis.Methods:Twenty clean-grade healthy male Sprague-Dawley rats, aged 2-3 weeks, weighing 20-50 g, were divided into 2 groups ( n=10 each) using a random number table method: sham operation group (group S) and hepatic I/R group (group I/R). The serum of rats in S group and I/R group was collected, and exosomes were isolated from the sera using differential centrifugations.Microglial cells were co-cultured with PKH26-labeled exosomes for 6 h. The intake of exosomes in microglial cells was determined using immunofluorescence staining.Primary microglial cells were seeded onto 6-well culture plates at a density of 5×10 5 cells/ml and were divided into 4 groups ( n=6 each) using a random number table method: control group (group C), 10 7 cells/ml I/R-exosomes treated group (group 10 7), 10 8 cells/ml I/R-exosomes treated group (group 10 8), and 10 9 cells/ml I/R-exosomes treated group (group 10 9). Microglia in each group were co-cultured with the corresponding concentration of I/R-exosomes for 6 h. The expression of NOD-like receptor family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein (ASC), cleaved-caspase-1 and gasdermin-D (GSDMD) was detected using Western blot.Primary microglial cells were divided into 3 groups ( n=24 each) by a random number table method: control group (group C), sham operation-exosomes treated group (group S-exosome) and I/R-exosomes treated group (group I/R-exosome). In S-exosome group and I/R-exosome group, exosomes 10 8 cells/ml in S group and I/R group were given, respectively, to incubate cells for 6 h. The expression of NLRP3, ASC and GSDMD mRNA was determined by real-time polymerase chain reaction, and the levels of interleukin-18 (IL-18), IL-1β and tumor necrosis factor-alpha (TNF-α) in the cell culture supernatant were measured by enzyme-linked immunosorbent assay. Results:The results from immunofluorescence staining showed that I/R-exosomes colocalized with microglia.The 10 8 cells/ml I/R-exosomes and 10 9 cells/ml I/R-exosomes up-regulated the expression of NLRP3, ASC, GSDMD and cleaved-caspase-1 in microglial cells ( P<0.01). Compared with group C and group S-exosome, the expression of NLRP3, ASC and GSDMD mRNA in microglial cells was up-regulated, and the levels of IL-18, IL-1β and TNF-α in the supernatant were elevated in group I/R-exosome ( P<0.01). Conclusions:Hepatic I/R rats-derived exosomes can promote microglial pyroptosis.

20.
Chinese Critical Care Medicine ; (12): 284-288, 2022.
Article in Chinese | WPRIM | ID: wpr-931865

ABSTRACT

Objective:To investigate the effect and mechanism of bone marrow mesenchymal stem cell (BMSC) on pyroptosis of rats with kidney injury.Methods:Bone marrow of 4-5 week-old female Sprague-Dawley (SD) rats was isolated in vitro and BMSC was obtained. The third generations of BMSC were used to further experiments. Fifteen 6 week-old SD rats were cluster-randomized divided into control group, kidney injury group and BMSC group (5 rats in each group). Rats in kidney injury group were injected with lipopolysaccharide (LPS) 1 mg/kg via tail vein; the control group was given the same amount of normal saline. BMSC group was injected with 0.5 mL BMSC (including 2×10 6 BMSC) via tail vein after modeling; the kidney injury group received the same amount of normal saline. On day 3 after these injections, serum creatinine (SCr) was detected by picric acid method, and blood urea nitrogen (BUN) was detected by diacetyl monoxime. The levels of cystatin C (Cys C), interleukins (IL-1β and IL-18) in blood were detected by enzyme-linked immunosorbent assay (ELISA). The rats were then sacrificed and their kidneys were removed for subsequent detection. The mRNA expression levels of NOD-like receptor protein 3 (NLRP3) and cysteinyl aspartate-specific protease-1 (caspase-1) of kidney were detected by quantificational real-time quantitative polymerase chain reaction (qRT-PCR). The protein expression levels of NLRP3 and caspase-1 of kidney were detected by Western blotting. Results:In vitro, after bone marrow cell suspension was cultured for 24 hours, a large number of round adherent cells and suspended cells appeared in each culture flask. After 4-5 days of culture, a large number of long spindle cells adhered to the wall, and there were still obvious impurity cells. After trypsin digestion and passage to the third generation, the long spindle adherent cells grew mainly in the culture flask and were basically purified as BMSC. In vivo, compared with the control group, the levels of SCr, BUN, Cys C, IL-1β and IL-18 in kidney injury group were increased [SCr (μmol/L): 85.22±2.29 vs. 21.80±0.59, BUN (mmol/L): 11.50±0.64 vs. 5.86±0.83, Cys C (mg/L): 0.13±0.01 vs. 0.11±0.02, IL-1β (ng/L): 31.49±1.42 vs. 4.74±0.49, IL-18 (ng/L): 29.01±1.95 vs. 1.52±0.03, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly increased [NLRP3 mRNA (2 -ΔΔCt): 3.635±0.296 vs. 1.000±0.002, caspase-1 mRNA (2 -ΔΔCt): 4.020±0.228 vs. 1.001±0.003; NLRP3 protein (NLRP3/β-actin): 1.560±0.868 vs. 0.902±0.036, caspase-1 protein (caspase-1/β-actin): 1.392±0.097 vs. 0.895±0.046, all P < 0.05]. Compared with kidney injury group, the levels of SCr, BUN, IL-1β and IL-18 in BMSC group were significantly decreased [SCr (μmol/L): 51.64±3.84 vs. 85.22±2.29, BUN (mmol/L): 9.90±0.46 vs. 11.50±0.64, IL-1β (ng/L): 24.20±1.45 vs. 31.49±1.42, IL-18 (ng/L): 12.97±1.25 vs. 29.01±1.95, all P < 0.05]. The mRNA and protein expression levels of NLRP3, caspase-1 were significantly decreased [NLRP3 mRNA (2 -ΔΔCt): 1.488±0.136 vs. 3.635±0.296, caspase-1 mRNA (2 -ΔΔCt): 1.643±0.143 vs. 4.020±0.228; NLRP3 protein (NLRP3/β-actin): 1.227±0.053 vs. 1.560±0.868, caspase-1 protein (caspase-1/β-actin): 1.159±0.107 vs. 1.392±0.097, all P < 0.05]. Conclusion:In vivo, BMSC may attenuate pyroptosis in LPS-induced kidney injury rats.

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