Objective To investigate the effect of picroside Ⅱ (PIC Ⅱ) on the pyroptosis and thioredoxin-interacting protein (TXNIP)/nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) signaling pathway in alveolar epithelial cells of severe pneumonia rats. Methods A severe pneumonia rat model was constructed and all experimental rats were divided into a control group, a severe pneumonia group, low, medium, and high dose PIC Ⅱ groups (PIC Ⅱ-L, PIC Ⅱ-M, PIC Ⅱ-H groups), and a high-dose PIC Ⅱ+TXNIP/NLRP3 pathway activator trimethylamine oxide group (PIC Ⅱ-H+TMAO group). The levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) were detected by ELISA; Wright’s staining was applied to detect eosinophil count (EOS), lymphocyte count (LYM), and neutrophil count (NEU) in the sediment of alveolar lavage fluid. Hematoxylin-eosin staining was used to observe the pathological changes of lung tissue. The expressions of cysteine aspartate protease 1 (Caspase-1) and dermatin D (GSDMD) were detected by immunohistochemistry. The expressions of TXNIP, NLRP3 and apoptosis-associated microprotein (ASC) were detected by Western blot. Results Compared with the control group, the severe pneumonia group had severe lung tissue injury, obvious inflammatory cell infiltration, and increased expressions of TNF-α, IL-1β, IL-6, EOS, LYM, NEU, Caspase-1, GSDMD, TXNIP, NLRP3 and ASC (all P<0.05). Compared with the severe pneumonia group, lung tissue injury in PIC Ⅱ-L, PIC Ⅱ-M and PIC Ⅱ-H groups was reduced successively, and inflammatory cell infiltration was gradually reduced. The expressions of TNF-α, IL-1β, IL-6, EOS, LYM, NEU, Caspase-1, GSDMD, TXNIP, NLRP3 and ASC were decreased successively (all P<0.05). Compared with the PIC Ⅱ-H group, the PIC Ⅱ-H+TMAO group showed increased lung tissue damage and obviously increased inflammatory cell infiltration, the expression of TNF-α, IL-1β, IL-6, EOS, LYM, NEU, Caspase-1, GSDMD, TXNIP, NLRP3, and ASC were obviously increased (all P<0.05). Conclusion PIC Ⅱ inhibits pyroptosis of alveolar epithelial cells in severe pneumonia rats by inhibiting the TXNIP/NLRP3 pathway.
Immunoglobulin A nephropathy (IgAN) is an immune-mediated chronic inflammatory disease with a complex pathogenesis and diverse clinical manifestations. Currently, there is no specific treatment plan. Programmed cell death is an active and orderly way of cell death controlled by genes in the body, which maintains the homeostasis of the body and the development of organs and tissues by participating in various molecular signaling pathways. In recent years, programmed cell death has played an important regulatory role in the occurrence and development of IgAN, involving complex signaling pathways. Under pathological conditions, it may relieve kidney damage through various pathways such as reducing oxidative stress, inhibiting inflammation, and improving energy metabolism. This article provides a review of the research progress of IgAN in apoptosis, autophagy, pyroptosis, ferroptosis,and cuproptosis in order to provide new therapeutic targets for IgAN.
Objective To investigate the role of cysteinyl aspartate specific proteinase-3 (Caspase-3)/ gasdermin-E (GSDME)-mediated pyroptosis in skeletal muscle atrophy induced by cigarette smoke in mice.Methods To construct a mouse model of COPD, C57BL/6 mice were exposed to cigarette smoke (CS) for 24 weeks. HE staining was used to observe the changes in the morphology of the gastrocnemius muscle in mice. Immunohistochemistry was used to detect the expression of pyroptosis-related proteins in gastrocnemius muscle. To construct a model of skeletal muscle cell atrophy in vitro, C2C12 myoblasts were induced to differentiate into skeletal muscle cells with 2% horse serum, and then skeletal muscle cells were treated with cigarette smoke extract (CSE). Skeletal muscle cells were further treated with the caspase-3 inhibitor Z-DEVD-FMK and the GSDME inhibitor Dimethyl fumarate (DMF) to explore the effects of inhibition of caspase-3/GSDME on CSE-induced skeletal muscle cell atrophy. To observe the effects of TNF-α on the expression of caspase-3 and GSDME proteins as well as the impact on myotubes, skeletal muscle cells were stimulated with tumor necrosis factor-alpha (TNF-α). Western blotting was applied to detect protein expression levels of caspase-3 and GSDME in skeletal muscle cells. Hoechst 33342/ Hoechst33342/ Propidium Iodide (PI) staining was applied to detect the PI-positive rate of skeletal muscle cells. The lactate dehydrogenase (LDH) release of C2C12 myotubes was measured by LDH release test. Immunofluorescence was used to detect changes in myotube diameter. Results CS-induced skeletal muscle atrophy was observed in mice, accompanied by increased pyroptosis- associated proteins (c-caspase-3 and GSDME-N) (P<0.05). CSE also induced elevated c-caspase-3 and GSDME-N expression in C2C12 cells , resulting in increased LDH release, positive ratio of PI, along with reduced myotube diameter (P<0.05). In addition, TNF-α promotes myotube atrophy and the expression of cleaved-caspase-3 and GSDME-N proteins in skeletal muscle cells. ConclusionCS can induce skeletal muscle atrophy through activated TNF-α/Caspase-3/GSDME-mediated pyroptosis.
ObjectiveTo investigate the regulatory role of MSC-derived exosomes in obliterative bronchiolitis after lung transplantation. MethodsThe murine lung transplantation model was established with male C57BL/6 mice, and the mice were divided into a sham group (sham, n=6), a surgery group (OB, n=6), and a treatment group (OB+MSC-exo, n=6). The in vitro model was created by stimulating RAW264.7 with lipopolysaccharide+nigericin (LPS+Nigericin), and comprised a PBS group, a LPS+Nigericin group, and a LPS+Nigericin+MSC-exo group. Immunofluorescence and hematoxylin-eosin (HE) staining were used to analyze gasdermin D (GSDMD) expression, as well as lumen stenosis in lung grafts. Bioinformatics methods were employed to predict and screen target gene collagen type V alpha 1 (COL5A1). Q-PCR was used to measure mRNA expression levels of interleukin (IL)-1β, IL-18, IL-6, tumor necrosis factor-α (TNF-α), and COL5A1 in lung grafts and macrophages. Western blot was performed to detect Cleaved-Caspase 1 protein expression in lung grafts and GSDMD protein expression in macrophages. ResultsImmunofluorescence and HE staining revealed that in vivo infusion of MSC-exo reduced GSDMD expression in grafts, ameliorated tracheal epithelial cilia loss and lumen stenosis, and decreased Cleaved-Caspase 1 protein as well as IL-1β and IL-18 mRNA expression. MSC-exo treatment or COL5A1 knockdown reduced IL-1β, IL-18, IL-6, and TNF-α mRNA expression in macrophages, with comparable efficacy. MSC-exo infusion also decreased the number of COL5A1+ cells and mRNA expression levels in lung grafts. ConclusionMSC-derived exosomes alleviate obliterative bronchiolitis after lung transplantation by inhibiting COL5A1.
Objective To investigate the role and mechanisms of trimetazidine (TMZ) in intensive care unit-acquired weakness (ICU-AW). Methods Seventy wild-type male C57BL/6 mice were selected and the ICU-AW mouse model was constructed by intraperitoneal injection of different concentrations of lipopolysaccharide (LPS). The body weights, grip strengths, and 96-hour survival rates of each group were observed, and the optimal concentration of LPS and time of sampling were screened out, the mRNA and protein expression of the gastrocnemius muscle atrophic proteins Atrogin-1 and muscle-specific RING finger protein 1 (MuRF1) were further detected to verify the success of modelling, and LPS (12 mg/kg) was used as the subsequent modelling concentration according to the preliminary results. After successful modelling, another 70 mice were randomly divided into normal control group (Normal group), LPS solvent (Vehicle) group, LPS group, LPS+TMZ solvent group, LPS+TMZ group, LPS+TMZ+AC-YVAD-CMK (AC) solvent group, and LPS+TMZ+AC group, with 10 mice in each group. The Normal group did not have any intervention; the Vehicle group was injected intraperitoneally with an equal volume of saline with LPS; the remaining groups were injected intraperitoneally with LPS (12 mg/kg); after the completion of the LPS injection, the LPS+TMZ group, the LPS+TMZ+AC solvent group, and the LPS+TMZ+AC group were given TMZ (5 mg/kg) by gastric gavage once a day for 4 days. The LPS+TMZ solvent group was given TMZ equivalent saline gavage once a day for 4 days. The LPS+TMZ+AC group was injected intraperitoneally with the cysteinyl aspartate specific proteinase 1 (Caspase-1) inhibitor AC-YVAD-CMK (AC, 6.5 mg/kg) 1 h before LPS injection, and the LPS+TMZ+AC solvent group was injected with an equal amount of AC solvent phosphate buffer. At the end of TMZ treatment, body weight, grip strength, 96-hour survival rate, mRNA and protein expression of MuRF1, Atrogin-1, Caspase-1, and gasdermin D (GSDMD) in gastrocnemius muscle, as well as serum IL-1β and IL-18 concentrations in mice were detected in each group, and the gastrocnemius muscle was stained with HE to observe histopathological changes. Results Compared with the Normal group, mice in the LPS (12 mg/kg) and LPS (14 mg/kg) groups showed significant decreases in body weight and grasping strength and the weakening was most obvious at 3 - 5 d (P<0.05), but the survival rate of the LPS (12 mg/kg) group was higher than that of the LPS (14 mg/kg) group (P<0.05), the HE staining of gastrocnemius muscle showed that the mice in the LPS (12 mg/kg) group was significantly atrophied compared with that of the Normal group, and the gene and protein expression of MuRF1 and Atrogin-1 were significantly elevated (P<0.05), and the mice injected with LPS (12 mg/kg) for 4 days (96 h) were finally selected as the conditions for subsequent experimental modelling and sampling.The mRNA and protein expression of Caspase-1 and GSDMD in skeletal muscle was significantly higher in the LPS group compared with the Normal and Vehicle groups (P<0.01), and the concentrations of serum IL-1β and IL-18 were significantly higher(P<0.01). Mice in the TMZ group showed significant improvement in body weight, grip strength, survival rate, and degree of muscle atrophy compared with the LPS and TMZ solvent groups (P<0.05); gene and protein levels of MuRF1, Atrogin-1, Caspase-1, and GSDMD in the gastrocnemius muscle were significantly reduced (P<0.05); and levels of serum IL-1β and IL-18 were significantly reduced (P<0.05) ); the mice in the LPS+TMZ+AC group had significantly improved body weight, grip strength, survival rate, and muscle atrophy compared with the LPS+TMZ group and the LPS+TMZ+AC solvent group (P<0.05), and the gene and protein contents of MuRF1, Atrogin-1, Caspase-1, and GSDMD in the gastrocnemius muscle were reduced (P<0.05), and the serum IL-1β and IL -18 concentrations were reduced (P<0.05). Conclusion TMZ is able to exert a skeletal muscle protective effect by inhibiting Caspase-1/GSDMD-mediated pyroptosis, which is an important reference for the prevention and treatment of ICU-AW.
Objective To investigate the effects of wedelolactone (WEL) on lipopolysaccharide (LPS)-induced pyroptosis of alveolar epithelial cells and AMP-activated protein kinase/nucleotide binding oligomeric domain like receptor 3 (NLRP3)/cysteinyl aspartate specific proteinase-1 (Caspase-1) signaling pathway. Methods Human lung epithelial cells BEAS-2B were treated with 5 - 200 μmol/L wedelolactone, and cell activity was detected using MTT assay. The alveolar epithelial cells were divided into control group, lipopolysaccharide group (LPS group), 10 μmol/L wedelolactone group (WEL-L group), 20 μmol/L wedelolactone group (WEL-M group), 40 μmol/L wedelolactone group (WEL-H group), 40 μmol/L wedelolactone+10 μmol/L AMPK inhibitor Compound C group (WEL-H+Compound C group), and 20 μmol/L Caspase-1 inhibitor Z-YVAD-FMK group (Z-YVAD-FMK group). Transmission electron microscopy was applied to observe the microstructure of cells. ELISA was applied to detect levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-8 (IL-8). Immunofluorescence was applied to detect Caspase-1 and gasdermin family proteins (DGSDMD). Western blot was applied to detect protein expression levels of AMPK, NLRP3, and Caspase-1. Results Wedelolactone concentrations of 10, 20 and 40 μmol/L were selected for follow-up experiments. Compared with Control group, LPS group showed decreased cell activity, severe damage, cell contraction, mitochondrial ridge breakage and decreased number, increased levels of TNF-α, IL-1β, IL-8 and GSDMD, NLRP3, Caspase-1 expression, and decreased p-AMPK/AMPK expression (P<0.05). Wedelolactone treatment could significantly improve LPS-induced pyrosis of alveolar epithelial cells (P<0.05). Compound C could partially reverse the effect of wedelactone on LPS-induced pyrodeath of alveolar epithelial cells (P<0.05). Z-YVAD-FMK treatment also significantly improved LPS-induced pyroptosis of alveolar epithelial cells (P<0.05). Conclusion Wedelolactone can inhibit LPS-induced pyroptosis of pulmonary alveolar epithelial cells by inhibiting AMPK/NLRP3/Caspase-1 signaling pathway.
Objectives To observe the expression of key proteins in the NLRP3/Caspase-1 pathway of pyroptosis in the mouse model of hepatic Echinococcus multilocularis (Em) infection and explore its correlation. Methods Twenty-five BALB/c mice were randomly divided into the control group and the infected group. The infected group was injected with 0.2 mL suspension of protoscolex (including 3 000 protoscoleces) injected under the liver capsule to establish a model of secondary infection with hepatic Em. The control group was treated without any treatments and conventional feeding was conducted. The mice were sacrificed at 1, 2, 3, and 5 months after infection. The liver was harvested and observed for gross morphology. HE staining and transmission electron microscopy were performed to observe the histopathological changes. The expressions of key proteins in the NLRP3/Caspase-1 pathway of pyroptosis and the IL-1β, a downstream factor of pyroptosis in the liver were detected by immunohistochemistry, Western blot and ELISA. Results Compared with the control group, the cystic lesions on the surface of liver tissues in the infected group mice gradually increased and protruded from the liver surface with the extension of infection time. HE staining showed various pathological changes such as inflammatory cell infiltration and fibrous hyperplasia in the liver lesions to varying degrees. After 2 months of Em infection, transmission electron microscope observation showed that the cell membrane of hepatocytes were broken and discontinuous, conforming to the "punching" phenomenon of pyroptosis. The results of ELISA showed that the concentration of IL-1β in liver homogenate of mice after 1, 2, 3 and 5 months of Em infection were significantly higher than that of the control group, and the difference was statistically significant (F=127.2, P<0.05). Immunohistochemical examination showed that the positive cell ratios of Caspase-1 and NLRP3 in liver of mice infected with Em at 1, 2, 3 and 5 months, were higher than that of the control group, and the difference were statistically significant (F=114.6, P<0.05; F=85.89, P<0.05). The Western blot results showed that the relative expression levels of Caspase-1, Xiaopi D, and NLRP3 proteins in the liver of infected mice showed a trend of first increasing (the expression of Caspase-1 and GSDMD reached their peak at 1 month of infection, while the expression of NLRP3 reached its peak at 2 months of infection) and then decreasing. There were statistically significant differences between the infection groups at different time points and the control group, as well as comparison between the infection groups at different time points there were also statistically significant differences (all P<0.05). Conclusion It is feasible to establish mouse Em infection model by “skin incision and liver puncture through abdominal muscle layer”. There is a new type of programmed cell death, pyroptosis, after Em infection in mouse liver. It may play a role in inflammation amplification through pyroptosis NLRP3/Caspase-1 pathway.
ObjectiveTo investigate the effect of Gasdermin D(GSDMD) gene knockout on lung injury and reactive oxygen species (ROS)/nod-like receptor protein 3 (NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1) pyroptosis pathway of pneumonia mice. Methods20 wild-type (WT) male C57BL/6J mice were randomly divided into WT control group and WT model group. 20 GSDMD gene knockout (KO) mice were randomly divided into KO control group and KO model group, each group consisted of 10 animals. The pneumonia model was induced by puncturing the nasal mucosa with a sterile needle and slowly instilling Streptococcus pneumoniae suspension in WT model group and KO model group. Serum and bronchoalveolar lavage fluid (BALF) were collected 7 days after modeling, and the levels of interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were detected; the lung tissues were collected and the dry wet ratio (W/D), pathological changes, the levels of malondialdehyde (MDA), total antioxidant capacity (T-AOC), superoxide dismutase (SOD), ROS activity, and the protein expression levels of GSDMD, GSDMD-N, NLRP3, Caspase-1 were detected. ResultsThe lung tissue of WT model group showed cell infiltration with alveolar septal thickening, the ratio of W/d in lung tissue, the levels of IL-1β, IL-6 and TNF-α in serum and BALF, the levels of MDA, ROS, NLRP3 and Caspase-1 in lung tissue were all higher than those in WT control group, the levels of T-AOC and SOD in lung tissue were lower than those in WT control group (P<0.05), and the levels of serum, BALF and lung tissue in KO control group were not significantly different from those in WT control group (P>0.05) There was no expression of GSDMD-N in the lung tissue of KO model group, lung tissue w/d ratio, serum and BALF levels of IL-1β, IL-6, TNF-α, MDA, ROS activity, GSDMD-N, NLRP3 and Caspase-1 protein expression were lower than those in WT model group, the levels of T-AOC and SOD in lung tissue were higher than those in WT Model Group (P<0.05). ConclusionGSDMD gene knockout attenuates lung injury and inhibits ROS/NLRP3/Caspase-1 pathway in pneumonia mice.
ObjectiveTo summarize the research advances of pyroptosis in hepatic ischamia-reperfusion injury (IRI).MethodThe literatures about the studies of mechanism of pyroptosis in hepatic IRI were retrieved and analyzed.ResultsPyroptosis, also known as inflammatory necrocytosis, was proven to play an important role in the hepatic IRI. When hepatic ischemia-reperfusion occurred, the classical pathway of pyroptosis dependenting on caspase-1 and the non-classical pathway of pyroptosis dependenting on caspase-11 were initiated by specific stimulants, and leaded to the activation of gasdermin D, releases of proinflammatory factors such as interleukin-1β, interleukin-18, etc., and the recruitment and activation of neutrophils. Consequently, pyroptosis caused more severe hepatic inflammation and aggravated existing cell injury and dysfunction of liver during hepatic IRI.ConclusionsPyroptosis plays an important role in liver IRI. Further researches about mechanism of pyroptosis will be beneficial to the prevention and treatment of the pyroptosis of related diseases.
Objective To investigate the inhibitory effects and related mechanisms of NOD like receptor protein 3 (NLRP-3) inflammasome inhibitor MCC950 on oxidative stress, inflammation, and pyroptosis in human esophageal epithelial cells (HEECs). MethodsHEECs cells were passaged and divided into blank control group, acid stimulation group (stimulated 3 times a day with pH 4 acidic medium for 15 minutes each time, cultured for 48 hours), bile salt stimulation group (stimulated 3 times a day with 400 μmol/L bile salt mixture for 15 minutes each time, cultured for 48 hours), lipopolysaccharide (LPS) group (stimulated with 10 μL of 100 ng/mL LPS for 48 hours), MCC950 group (stimulated with 10 μL of 7.5 ng/mL MCC950 for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS for 48 hours), and N-acetyl-L-cysteine (NAC) group (stimulated with 1 mmol/L NAC for 4 hours, then stimulated with acid, bile hydrochloric acid, and LPS 48 hours). Three culture dishes were used in each group to detect the mRNA and protein expression levels of oxidative protein/antioxidant protein [Nox-4 (NADPH oxidase 4), nuclearfactor erythroidderived 2-like 2 (Nrf-2), heme oxygenase-1 (HO-1)], NLRP-3 signaling pathway [NLRP-3/caspase-1/intereukin (IL)-1β/IL-18], and cell apoptosis pathway [caspase-4/caspase-5/GSDMD] using real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting experiments. Cell apoptosis were observed through Hoechst33342 staining. ResultsMCC950 intervention (0.023) and NAC intervention (0.031) effectively inhibited HEECs apoptosis induced by acid (0.042), bile salt (0.047), and LPS (0.054). The results of RT-PCR experiments showed that MCC950 intervention and NAC intervention significantly inhibited the high expression of Nox-4 mRNA (MCC950: 1.68; NAC: 1.62) in HEECs cells induced by acid (2.40), bile salt (3.07), and LPS (3.52), and significantly upregulated the mRNA expression levels of antioxidant proteins Nrf-2 (MCC950: 0.72; NAC: 0.57) and HO-1 (MCC950: 0.74; NAC: 0.57). MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA expression levels of NLRP-3 (MCC950: 1.58; NAC: 1.47), ASC (MCC950: 1.56; NAC: 1.93), caspase-1 (MCC950: 1.64; NAC: 1.96), IL-1β (MCC950: 1.66; NAC: 1.82), IL-18 (MCC950: 1.58; NAC: 1.84) in HEECs cells induced by acid stimulated, bile salt stimulated, and LPS. MCC950 intervention and antioxidant NAC intervention effectively inhibited the mRNA expression levels of apoptosis pathway markers such as caspase-4 (MCC950: 1.51; NAC: 1.61), caspase-5 (MCC950: 1.38; NAC: 1.64), and GSDMD (MCC950: 1.41; NAC: 1.54) induced by acid stimulation, bile salt stimulation, and LPS in HEECs cells. The electrophoresis results were similar with RT-PCR. ConclusionAcid, bile salt, and LPS can all induce the overexpression of oxidative stress markers in HEECs, reduce the expression of antioxidant proteins, and activate the NLRP-3 inflammasome signaling pathway and cell pyroptosis pathway, promoting cellular inflammatory damage, but MCC950 has a protective effect.