BACKGROUNDCholestasis (CT) is a group of disorders caused by impaired production, secretion or excretion of bile. This may result in the deposition of bile components in the blood and liver, which in turn causes damage to liver cells and other tissues. If untreated, CT can progress to severe complications, including cirrhosis, liver failure, and potentially life-threatening conditions.OBJECTIVEThis research was intended to elucidate the function and mechanism of Paeoniflorin (PF) in ameliorating ANIT-induced pyroptosis in CT.METHODSCT models were established in SD rats and HepG2 cells through ANIT treatment. Histological examination was conducted using haematoxylin and eosin (HE) staining to assess the histopathological alterations in the liver. Network pharmacology was employed to identify potential PF targets in CT treatment. To evaluate pyroptosis levels, various methods were used, including serum biochemical analysis, Enzyme-Linked Immunosorbent Assay (ELISA), immunofluorescence (IF), immunohistochemistry (IHC), Western blotting, transmission electron microscopy (TEM), and scanning electron microscopy (SEM). The HuProt™ 20K Chip was utilized to pinpoint potential PF-binding targets. PF's direct mechanisms in CT treatment were explored using molecular docking (MD), molecular dynamics simulations (MDS), Cellular Thermal Shift Assay (CETSA), and Surface Plasmon Resonance (SPR).RESULTSPF administration was found to alleviate ANIT-induced liver pathology, enhance liver function markers, and improve cell viability. Network pharmacology and pyroptosis inhibitor studies suggested that PF might mitigate CT via the NLRP3-dependent pyroptosis pathway. This hypothesis was further supported by Western blotting, IF, and IHC analyses, which indicated PF's potential to inhibit NLRP3-dependent pyroptosis in CT. GSDMD was identified as a target through HuProt™ 20K Chip screening. The binding affinity of PF to GSDMD was validated through MD, MDS, CETSA, and SPR techniques. Additionally, the regulatory impact of GSDMD on downstream inflammatory pathways was confirmed by ELISA and IHC.CONCLUSIONPF exhibited a hepatoprotective effect in ANIT-induced CT, primarily by targeting GSDMD, thereby suppressing ANIT-induced pyroptosis and the subsequent release of inflammatory mediators.

01 Oct 2024·Brain and Behavior

The Activation of PKM2 Induces Pyroptosis in Hippocampal Neurons via the NLRP3/Caspase‐1/GSDMD Pathway in Neonatal Rats With Hypoxic‐Ischemic Brain Injury

Article

Author: Zhou, Ruiyu ; Ouyang, Ying ; Ruan, Yanghao ; Jin, Ni ; Sha, Sha

ABSTRACTIntroductionThe presence of hypoxic‐ischemic brain damage (HIBD) in neonates triggers a strong neuroinflammatory reaction. Pyroptosis, a programmed cell death mechanism associated with inflammation, plays a crucial role in HIBD. Pyruvate kinase M2 (PKM2) plays a significant role in connecting metabolic processes and inflammatory responses, but whether it affects hippocampus pyroptosis in HIBD is unclear. The aim of this study is to elucidate the role of PKM2 in HIBD and to propose a novel therapeutic approach for neonatal ischemic‐hypoxic encephalopathy.MethodsIn this study, we employed neonatal 7‐day‐old Sprague Dawley rats to establish a model of HIBD using the Rice–Vannucci surgical technique and a hypoxia device. To inhibit the elevation of PKM2, we utilized the PKM2 inhibitor shikonin. The rats were categorized into four groups: Sham, Shikonin, HIBD, and Shikonin + HIBD. Behavioral tests, hematoxylin eosin staining, immunofluorescence staining, ELISA (IL‐1β, IL‐18), and LDH were conducted in each group to evaluate neurological function, hippocampal damage, the occurrence of neuronal pyroptosis, and the neuroinflammation. Western blot was used to assess the expression levels of PKM2, NLRP3, Caspase‐1, Cleaved Caspase‐1, GSDMD, GSDMDN, and IL‐1β.ResultsThe expression of PKM2 elevated in hippocampal tissues of the HIBD model and the localization of PKM2 in the hippocampus was activated in neurons instead of microglia during the HIBD. Meanwhile, the inhibition of PKM2 improved the behavioral test scores and the body weight of rats, the neuronal damage in the CA1 region of hippocampal tissue was also attenuated. In addition, inhibiting PKM2 alleviated neuronal pyroptosis by decreasing the expression of PKM2, NLRP3, Caspase‐1, Cleaved Caspase‐1, GSDMD, GSDMDN. Furthermore, serum levels of LDH and inflammatory factors IL‐1β and IL‐18 decrease with PKM2 inhibition.ConclusionsBased on these findings, we can conclude that PKM2 plays a crucial role in regulating hippocampal neuronal pyroptosis of HIBD rats via NLRP3/Caspase‐1/GSDMD pathway. Therefore, inhibiting PKM2 could be a promising therapeutic strategy for the treatment of neonatal ischemic‐hypoxic encephalopathy.

01 Oct 2024·Shock

GSDMD KNOCKOUT ALLEVIATES SEPSIS-ASSOCIATED SKELETAL MUSCLE ATROPHY BY INHIBITING IL18/AMPK SIGNALING

Article

Author: Zhao, Hong ; Wang, Dongfang ; Wang, Chuntao ; Zhang, Yongsheng ; Liu, Yukun ; Li, Zhanfei ; Xu, Ligang ; Bai, Xiangjun ; Wang, Yuchang ; Li, Tonghan

ABSTRACTBackground: Sepsis commonly leads to skeletal muscle atrophy, characterized by substantial muscle weakness and degeneration, ultimately contributing to an adverse prognosis. Studies have shown that programmed cell death is an important factor in the progression of muscle loss in sepsis. However, the precise role and mechanism of pyroptosis in skeletal muscle atrophy are not yet fully comprehended. Therefore, we aimed to examine the role and mechanism of action of the pyroptosis effector protein GSDMD in recognized cellular and mouse models of sepsis. Methods: The levels of GSDMD and N-GSDMD in skeletal muscle were evaluated 2, 4, and 8 days after cecal ligation and puncture. Sepsis was produced in mice that lacked the Gsdmd gene (Gsdmd knockout) and in mice with the normal Gsdmd gene (wild-type) using a procedure called cecal ligation and puncture. The degree of muscular atrophy in the gastrocnemius and tibialis anterior muscles was assessed 72 h after surgery in the septic mouse model. In addition, the architecture of skeletal muscles, protein expression, and markers associated with pathways leading to muscle atrophy were examined in mice from various groups 72 h after surgery. The in vitro investigations entailed the use of siRNA to suppress Gsdmd expression in C2C12 cells, followed by stimulation of these cells with lipopolysaccharide to evaluate the impact of Gsdmd downregulation on muscle atrophy and the related signaling cascades. Results: This study has demonstrated that the GSDMD protein, known as the “executive” protein of pyroptosis, plays a crucial role in the advancement of skeletal muscle atrophy in septic mice. The expression of N-GSDMD in the skeletal muscle of septic mice was markedly higher compared with the control group. The Gsdmd knockout mice exhibited notable enhancements in survival, muscle strength, and body weight compared with the septic mice. Deletion of the Gsdmd gene reduced muscular wasting in the gastrocnemius and tibialis anterior muscles caused by sepsis. Studies conducted in living organisms (in vivo) and in laboratory conditions (in vitro) have shown that the absence of the Gsdmd gene decreases indicators of muscle loss associated with sepsis by blocking the IL18/AMPK signaling pathway. Conclusion: The results of this study demonstrate that the lack of Gsdmd has a beneficial effect on septic skeletal muscle atrophy by reducing the activation of IL18/AMPK and inhibiting the ubiquitin-proteasome system and autophagy pathways. Therefore, our research provides vital insights into the role of pyroptosis in sepsis-related skeletal muscle wasting, which could potentially lead to the development of therapeutic and interventional approaches for preventing septic skeletal muscle atrophy.

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Indication	Highest Phase	Country/Location	Organization	Date
Sepsis	Preclinical	CN	Ruijin Hospital, Shanghai Jiaotong University School of Medicine	02 Nov 2023
Sepsis	Preclinical	CN	Shanghai Xiandai Medicine Preparation Engineering Research Centre Co., Ltd.	02 Nov 2023

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