Paeonol regulates NLRP3 inflammasomes and pyroptosis to alleviate spinal cord injury of rat.

Spinal cord injury (SCI) is a life-threatening traumatic disorder. Paeonol has been confirmed to be involved in a variety of diseases. The purpose of this study is to investigate the role of paeonol on SCI progression. Sprague Dawley (SD) rat was used for the establishment of SCI model to explore the anti-inflammation, anti-oxidation, and neuroprotective effects of paeonol (60 mg/kg) on SCI in vivo. For in vitro study, mouse primary microglial cells (BV-2) were induced by lipopolysaccharide (LPS)/adenosine triphosphate (ATP) treatment. The effect of paeonol on the polarization of LPS/ATP-induced BV-2 cells was determined by detection the expression inducible nitric oxide synthase (iNOS), tumour necrosis factor alpha (TNF-α), arginase-1 (Arg-1), and interleukin (IL)-10 using qRT-PCR. ELISA was used to assess the levels of IL-1β, IL-18, TNF-α, malondialdehyde (MDA), and glutathione (GSH). Western blotting was conducted to determine the levels of NLRP3 inflammasomes and TLR4/MyD88/NF-κB (p65) pathway proteins. Paeonol promoted the recovery of locomotion function and spinal cord structure, and decreased spinal cord water content in rats following SCI. Meanwhile, paeonol reduced the levels of apoptosis-associated speck-like protein (ASC), NLRP3, active caspase 1 and N-gasdermin D (N-GSDMD), repressed the contents of IL-1β, IL-18, TNF-α and MDA, and elevated GSH level. In vitro, paeonol exerted similarly inhibiting effects on pyroptosis and inflammation. Meanwhile, paeonol promoted BV-2 cells M2 polarization. In addition, paeonol also inactivated the expression of TLR4/MyD88/NF-κB (p65) pathway. Paeonol may regulate NLRP3 inflammasomes and pyroptosis to alleviate SCI, pointing out the potential for treating SCI in clinic.

Zhao H ，Wang X ，Liu S ，Zhang Q ... -  《BMC NEUROSCIENCE》

Nicorandil inhibits TLR4/MyD88/NF-κB/NLRP3 signaling pathway to reduce pyroptosis in rats with myocardial infarction.

Chen F ，Chen ZQ ，Zhong GL ，Zhu JJ ... -  《-》

Paeonol reduces IL-β production by inhibiting the activation of nucleotide oligomerization domain-like receptor protein-3 inflammasome and nuclear factor-κB in macrophages.

Chen G ，Guo T ，Yang L 《-》

TLR4 aggravates microglial pyroptosis by promoting DDX3X-mediated NLRP3 inflammasome activation via JAK2/STAT1 pathway after spinal cord injury.

Toll-like receptor 4 (TLR4) participates in the initiation of neuroinflammation in various neurological diseases, including central nervous system injuries. NLR family pyrin domain containing 3 (NLRP3) inflammasome-mediated microglial pyroptosis is crucial for the inflammatory response during secondary spinal cord injury (SCI). However, the underlying mechanism by which TLR4 regulates NLRP3 inflammasome activation and microglial pyroptosis after SCI remains uncertain. We established an in vivo mouse model of SCI using TLR4-knockout (TLR4-KO) and wild-type (WT) mice. The levels of pyroptosis, tissue damage and neurological function recovery were evaluated in the three groups (Sham, SCI, SCI-TLR4-KO). To identify differentially expressed proteins, tandem mass tag (TMT)-based proteomics was conducted using spinal cord tissue between TLR4-KO and WT mice after SCI. For our in vitro model, mouse microglial BV2 cells were exposed to lipopolysaccharides (1 µg/ml, 8 h) and adenosine triphosphate (ATP) (5 mM, 2 h) to induce pyroptosis. A series of molecular biological experiments, including Western blot (WB), real-time quantitative polymerase chain reaction (RT-qPCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence (IF), immunohistochemical (IHC), chromatin immunoprecipitation (ChIP), Dual-Luciferase Reporter assay (DLA) and co-immunoprecipitation (Co-IP), were performed to explore the specific mechanism of microglial pyroptosis in vivo and in vitro. Our results indicated that TLR4 promoted the expression of dead-box helicase 3 X-linked (DDX3X), which mediated NLRP3 inflammasome activation and microglial pyroptosis after SCI. Further analysis revealed that TLR4 upregulated the DDX3X/NLRP3 axis by activating the JAK2/STAT1 signalling pathway, and importantly, STAT1 was identified as a transcription factor promoting DDX3X expression. In addition, we found that biglycan was increased after SCI and interacted with TLR4 to jointly regulate microglial pyroptosis through the JAK2/STAT1/DDX3X/NLRP3 axis after SCI. Our study preliminarily identified a novel mechanism by which TLR4 regulates NLRP3 inflammasome-mediated microglial pyroptosis in response to SCI-providing a novel and promising therapeutic target for SCI.

Wang J ，Zhang F ，Xu H ，Yang H ，Shao M ，Xu S ，Lyu F ... -  《Clinical and Translational Medicine》

Protective effect of the total flavonoids from Clinopodium chinense against LPS-induced mice endometritis by inhibiting NLRP3 inflammasome-mediated pyroptosis.

Clinopodium chinense (Benth.) O. Kuntze (C. chinense) is a Chinese herbal medicine used in treating gynecological hemorrhagic diseases for hundreds of years. Flavonoids are one kind of the major components in C. chinense. The flavonoids of C. chinense (TFC) have a vital role in treating endometritis but the underlying therapeutic mechanisms of TFC against endometritis have been rarely reported. To elucidate the therapeutic effect and possible mechanisms of TFC against lipopolysaccharide (LPS)-induced endometritis in vivo and LPS-induced primary mouse endometrial epithelial cells (MEECs) injury in vitro. The holistic phytochemicals of the TFC and TFC-contained serum were screened and identified using UPLC-Q-TOF-MS. The model of endometritis was established by intrauterine injection of LPS (5 mg/mL) into female BALB/c mice, and the model mice were treated with TFC for 7 days. The value of MPO was measured by Myeloperoxidase assay kit, the pathological changes in the endometrium were evaluated using H&E staining and transmission electron microscope (TEM), the secretions of IL-18, IL-1β and TNF-α were determined by ELISA kits, the mRNA expressions of IL-18, IL-1β and TNF-α were determined by RT-PCR assay, and the protein levels of TLR4, IKBα, p-IKBα, p65, p-p65, caspase-1, ASC, NLRP3 and GSDMD were measured by Western blot. Subsequently, MEECs were isolated from the uterus of pregnant female mice, injured by LPS for 24 h and incubated with the TFC-contained serum. Finally, cell viability, LDH release, hoechst 33342/PI staining, immunofluorescence staining, scanning electron microscope observation, ELISA assay, RT-PCR detection and Western blot analysis were carried out to further validate the therapeutic effect and the underlying mechanisms of TFC. A total of 6 compounds in the plasma of mice after being intragastric administrated of TFC were identified. The results in vivo showed that TFC significantly reduced MPO value and alleviated pathological injury of the endometrium. Furthermore, TFC significantly decreased the serum IL-18, IL-1β and TNF-α levels, and the mRNA levels of IL-18, IL-1β and TNF-α. TFC also inhibited the expressions of TLR4, p-IKBα, p-p65, caspase-1, ASC, NLRP3 and GSDMD. Besides, compared with the model group in MEECs cells, TFC-contained serum prevented pyroptosis, decreased the levels of IL-18 and IL-1β, and inhibited the mRNA expressions of IL-18, IL-1β and GSDMD. TFC-contained serum also reversed the activation of NLRP3 inflammasome caused by nigericin, and restrainted the translocation of NF-κB into nuclear. TFC protects mice endometritis from the injury of LPS via suppressing the activation of NLRP3 inflammasome and pyroptosis, the underlying mechanisms of which were related to restraining the TLR4/NF-κB/NLRP3 pathway activation.

Li L ，Qi J ，Tao H ，Wang L ，Wang L ，Wang N ，Huang Q ... -  《-》