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ObjectiveTo investigate the mechanism of Xumingtang in Gu Jin Lu Yan （《古今录验》） in regulating cell pyroptosis through the hypoxia-inducible factor-1α （HIF-1α）/NOD-like receptor pyrin domain-containing protein 3 （NLRP3） pathway in ischemic stroke （IS）. MethodSD rats were randomly divided into a sham operation group， a model group， low- and high-dose Xumingtang groups， and a metformin group， with 20 rats in each group. Oral administration was performed for 3 days， and tissue samples were collected. Differential messenger RNA （mRNA） was screened using high-throughput sequencing， and Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analyses were performed on key differentially expressed genes. The modified neurological severity score （mNSS） and 2，3，5-triphenyltetrazolium chloride （TTC） staining were used to evaluate the effect of brain infarction. Hematoxylin-eosin （HE） staining was used for pathological morphological observation of brain tissue. Enzyme-linked immunosorbent assay （ELISA） was used to compare the levels of interleukin-1β （IL-1β） and interleukin-18 （IL-18） in the ischemic cortical region. Double staining immunohistochemistry was used to detect the co-localization of HIF-1α and NLRP3. Real-time quantitative polymerase chain reaction （PCR） was performed to detect the mRNA expression of NLRP3， HIF-1α， Caspase-1 （CASP-1）， and gasdermin D （GSDMD）. Western blot was used to detect the protein expression of HIF-1α， NLRP3， CASP-1， and GSDMD. ResultA total of 5 705 differentially expressed genes （2 733 downregulated and 2 972 upregulated） were obtained by mRNA sequencing. After conversion to homologous genes and intersection with the pyroptosis gene set， 95 key differentially expressed pyroptosis genes were obtained. Compared with the sham operation group， the model group showed significantly increased mNSS scores， larger brain infarction areas （P<0.01）， diverse neuronal morphology， disordered arrangement， widened cell gaps， significantly increased levels of IL-1β and IL-18 in the ischemic cortical region （P<0.01）， enhanced co-localization fluorescence intensity， and significantly increased mRNA and protein expression levels of HIF-1α， NLRP3， CASP-1， and GSDMD （P<0.01）. Compared with the model group， the high-dose Xumingtang group showed the most significant improvement in neurological function scores and brain infarction areas （P<0.01）. The neuronal integrity and arrangement were more complete， and the cell gaps were narrower in all groups with drug treatment， with significantly reduced co-localization fluorescence intensity. Xumingtang could reduce the levels of IL-1β， IL-18， and the mRNA and protein expression of HIF-1α， NLRP3， CASP-1， and GSDMD （P<0.05， P<0.01）， with the high-dose Xumingtang group showing the most significant effect （P<0.01）. ConclusionXumingtang in Gu Jin Lu Yan can inhibit cell pyroptosis and promote neurological function recovery after IS， which may be related to the inhibition of the HIF-1α/NLRP3 pathway.
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Ulcerative colitis （UC） is a common inflammatory bowel disease （IBD） in clinical practice， characterized by symptoms such as abdominal pain， diarrhea， and bloody mucus in the stool. It is difficult to cure and has a high recurrence rate. The pathogenesis of UC is related to abnormal immune response， oxidative stress in intestinal tissues， and inflammatory reactions. As reported， the abnormal activation of the NOD-like receptor pyrin domain-containing protein 3 （NLRP3） inflammasome is involved in the pathological process of UC. This activation triggers pathological mechanisms such as oxidative stress， pyroptosis， and inflammation in intestinal epithelial cells. Therefore， blocking the abnormal activation of NLRP3 is beneficial for alleviating UC. Currently， western medicine treatment for UC mainly includes salicylic acid derivatives， corticosteroids， and biologics， but the overall efficacy is unsatisfactory. Traditional Chinese medicine （TCM） treatment of this disease has the advantages of significant efficacy and low recurrence rate. In recent years， great advances have been made in the basic research of using TCM methods to treat UC. Studies have found that TCM intervention targeting the NLRP3 inflammasome can significantly promote intestinal mucosal healing and treat UC， and the mechanism of action involves multiple targets， levels， and pathways. This article summarized the experimental research on the impact of TCM targeting the NLRP3 inflammasome on UC in recent years， and found that NLRP3 interacted with factors such as Caspase-1 and nuclear factor-κB （NF-κB）， thereby promoting the release of pro-inflammatory factors and cell pyroptosis in intestinal epithelial cells. This activation triggered oxidative stress， inflammatory reactions， and other pathological mechanisms. TCM acted on the NLRP3 inflammasome and its upstream and downstream factors to block the pathological process of UC， inhibit the pathological damage to the intestinal mucosa， and thereby alleviate colonic ulcers. The findings of this study provide a theoretical basis for the prevention and treatment of UC and further drug development.
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Background Macrophages are essential components of the natural immune system. They play a significant role in resisting foreign bodies in the respiratory tract and maintaining the homeostasis of the internal environment of lung tissue. Objective To investigate the mechanism of macrophage pyroptosis induced by silica dust with different particle sizes. Methods The modified murine macrophage cell line, RAW-ASC cells, was cultured and divided into a blank control group, a lipopolysaccharide (LPS) group (1 μg·mL−1 LPS), a nano-SiO2 group (1 μg·mL−1 LPS+100 μg·mL−1 nano-SiO2), a micro-SiO2 group (1 μg·mL−1 LPS+750 μg·mL−1 micro-SiO2), and a positive control group [1 μg·mL−1 LPS+3 mmol·L−1 adenosine triphosphate (ATP)]. Apart from the blank control group, cells in other groups were pretreated with LPS for 6 h, and then exposed to SiO2 or ATP for 4 h. According to the molecular target NOD-like receptor pyrin domain-containing protein 3 (NLRP3) and reactive oxygen species (ROS), we applied MCC950 (NLRP3 inhibitor) and N-acetyl cysteine (NAC, ROS scavenger) to macrophages. CCK-8 assay was used to detect cell viability; 5-ethynyl-2'-deoxyuridine (EdU) staining was used to detect cell proliferation; lactate dehydrogenase (LDH) assay kit was used to detect LDH in supernatant; calcein AM/PI fluorescent double-staining was applied to evaluate cell rupture; 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescent probe was used to measure the content of ROS; Western blotting was used to measure the expressions of NLRP3, apoptosis-associated speck-like protein containing CARD (ASC), Caspase-1, gasdermin D (GSDMD), and interleukin-1β (IL-1β). Results Compared with the blank group, 100 μg·mL-1nano-SiO2 and 750 μg·mL-1micro-SiO2 dust exposure reduced the cell viability to 40% and 68% (P<0.05), and the cell proliferation rate to 30% and 33% (P<0.01), respectively; they also induced cell lysis and ROS release, upregulated NLRP3, ASC, Caspase-1, GSDMD, and IL-1β at protein level (P<0.05), and induced macrophage pyroptosis. After intervening with MCC950 (10 μmol·L-1) and NAC (10 mmol·L-1), the expressions of NLRP3, ASC, Caspase-1, and IL-1β decreased (P<0.05), and, specifically, NAC effectively reduced ROS levels (P<0.05). Conclusion Both nano- and micro-SiO2 dust have cytotoxicity, can upregulate ROS level, activate NLRP3 inflammasome, and promote the release of cytokines, leading to pyroptosis. These results are helpful to reveal the molecular mechanism of macrophage pyroptosis induced by SiO2 dust.
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BACKGROUND: Nowadays, the ray types, animal species, irradiation modes and sites used in the establishment of animal models of radioactive skin injury in China are not consistent. Meanwhile, there is no uniform standard for the prevention and treatment of radioactive skin injury in clinical practice. OBJECTIVE: To establish an ideal rat model of acute radioactive skin injury. METHODS: Sixty Wistar rats were randomly divided into 32, 38, 45 Gy X-ray groups (n=18) and non-irradiated group (n=6). Three X-ray irradiated groups (32, 38, 45 Gy) received single irradiation of the right posterior buttock, 300 cGy/min, 100 cm between the skin and irradiated source, for 10.67, 12.67, and 15 minutes respectively. No irradiation was given in the non-irradiated group. The study protocol was approved by the Animal Ethic Committee of Shanxi Cancer Hospital (approval No. GDY2018001). RESULTS AND CONCLUSION: There was no accidental death after irradiation. The body mass of the rats decreased within 3 days after irradiation, and then increased. Irradiated wound was severest at about 15 days after irradiation, and the body mass dropped again, and returned to normal 2 days later. Two weeks after radiation, with the increase of X-ray dose, the structures of rat’s skin appendages were destroyed and a large number of inflammatory cells were infiltrated, indicating that the acute radiation skin injury was dose-dependent within a certain range. On the other hand, with the increase of irradiation time, the skin wound in the 38 and 45 Gy groups gradually deepened. At the same dose, the severity of acute radiation skin injury was also positively correlated with the irradiation time. After 6 hours to 15 days of 38 Gy irradiation on the rat skin, macrophages were activated, and the expression of Nod-like receptor pyrin domain-containing protein 3 was enhanced, indicating obvious inflammatory response, and thereby verifying the reliability of the model. To conclude, it is an ideal animal model of acute X-ray skin injury model made by the X-ray linear accelerator, which is easily observed and obtained, with obvious skin inflammation expression. This model is also of high safety and strong tolerance.
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Objective To explore the expression and significance of miR-21 in patients with primary gout. Methods The patients were divided into 4 groups: 35 acute gout patients (AG), 50 intermittent gout patients (IG), 25 chronic gout patients (CG) and 39 healthy patients. Their peripheral blood were collected and laboratory indexes were recorded. The expression of miR-21 and Nod-like receptor pyrin domain-containing protein 3 (NLRP3) mRNA in the peripheral blood mononuclear cells (PBMCs) was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The blood and clinical data of another 5 healthy volunteers were collected, their peripheral blood was stimulated with 100 μg/ml monosodium urate (MSU) for 1 hour, pho-sphate buffer (PBS) was used as controls, then the expression of microRNA (miR)-21, NLRP3, interleukin (IL)-1β mRNA was detected by RT-qPCR. Rank sum test and spearman correlation analysis were used for data analysis. Results In primary gout patients, the expression of miR-21 in AG [12 ×10-4 (8.0 ×10-4)], IG [9.4 ×10-4 (6.9 ×10-4)], CG [7.3 ×10-4 (5.6 ×10-4)] was significantly higher than that in healthy control group [1.0×10-4(2.0×10-4)] (Z=9.83, P=0.02], while the expression of NLRP3 in AG[0.0444(0.0233)], IG[0.0581(0.0326)], CG[0.0314(0.0198)] was significantly lower than that in healthy control group [0.0886(0.0359)] (Z=13.82, P<0.01). In the primary gout of IG group, the expression of miR-21 was positively correlated with NLRP3 mRNA (r=0.449, P=0.016). After stimulated by 100 μg/ml MSU, the expression of miR-21 of the stimulated group [8.78×10-4(14×10-4)] was higher than that in the control group [6.25×10-4(6×10-4)](Z=-2.203, P<0.05), and the expression of IL-1βin stimulated group [3.06(2.00)] was higher than that in the control group [2.64 (1.22] (Z=-2.203, P<0.05). The level of miR-21 in patients with primary gout was positively correlated with the level of uric acid (UA), glutamic oxaloacetic transaminase (AST) and glutamic pyruvic transaminase (ALT) (r=0.473, 0.639, 0.487, P<0.05). Conclusion The increase of miR-21 in patients with primary gout may be involved in the inflammatory reaction of gout.
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Rheumatoid arthritis is an autoinflammatory disease that primarily affects joints and is characterized by pervasive joint inflammation. A20/Tumor necrosis factor, alpha-induced protein 3 (Tnfaip3) inhibits activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappaB) and has been associated with rheumatoid arthritis. However, the precise role of A20 in rheumatoid arthritis remains unclear. Deletion of A20/Tnfaip3 gene in mice elicits impulsive erosive polyarthritis that is similar to rheumatoid arthritis in patients. Recently, it has been shown that A20 protects against arthritis by regulating the NLRP3 inflammasome.