The Effect And Mechanism Of Targeted Inhibition Of GSDMD In Sepsis

Purpose:Sepsis is the leading cause of death in ICU patients and has long been a clinical treatment challenge because of the lack of specific treatments.Recent studies have found that GSDMD-mediated pyroptosis is closely associated with sepsis,and targeted inhibition of GSDMD has the potential to be a new therapeutic target.The GSDMD-targeted inhibitors identified in previous studies have not achieved satisfactory results due to their potential drug toxicity and bioavailability,etc.The purpose of our study was identified novel drugs targeting GSDMD inhibition through drug screening and modified it.We hope achieved a better results in the treatment of sepsis.Methods.1 Screening and modifying of GSDMD-targeted inhibitors.1.1 Screening of GSDMD-targeted inhibitors.The compound library was screened using two different pytoptosis stimulation including LPS+Nigericin and LPS+HD,and the supernatant was collected for LDH assayed to find drugs that could inhibit both stimulation.1.2 Modification of GSDMD-targeted inhibitors.1.2.1 Characterisation of TPNs:detection of dynamic light scattering particle size of TPNs,direct observation of TPNs morphology by scanning electron microscopy and transmission electron microscopy,and detection of molecular weight of TPNs by gel permeation chromatography.1.2.2 Safety study of TPNs:inductively coupled plasma mass spectrometry to detect the residual heavy metal Mn²⁺content,detection of the effect on the cell variabilty of different cell lines of TPNs,and HE staining of liver,lung and kidney 7 days after intraperitoneal injection.2 The effect of GSDMD-targeted inhibitors in sepsis.2.1 The effect of TPNs on different inflammasomes:pretreatment of primary macrophages with TPNs,the effect of TPNs on different cellular pyroptosis was examined using NLRP3 inflammasome stimulants:LPS+Nigericin,LPS+MSU and noncanonical inflammasome stimulants:LPS+HD,LPS+lip3000,to detect the effect of TPNs on different pyroptosis stimulation by LDH,PI staining,IL-1α,IL-1β.2.2 Effects of TPNs in sepsis model mice:Sepsis mice model was established using intraperitoneal injection of LPS and observe the effects of TPNs on body temperature,inflammatory factor（IL-1α,IL-1β,IL-6）release,important organ functions（glutamate transaminase（ALT）,glutathione transaminase（AST）,creatinine（CRE））,liver and lung pathology.The effect of GSDMD-targeted inhibitors on the survival rate of mice.3 Mechanistic studies of the effects of GSDMD-targeted inhibitors on sepsis.3.1 Effect of TPNs on GSDMD-N intercalation:TPNs were pretreated with primary macrophages,given different inflammasome stimulantion,GSDMD shedders and oligomers was detected by Western blot.Immunofluorescence was used to detect changes in GSDMD membrane localization.The effect of TPNs on LDH release,GSDMD shear and oligomerization in the above system was also examined by in vitro reconstitution of non-canonical inflammasome induced pyroptosis in 293T cells by transfection with plasmids.3.2 Effect of TPNs on reactive oxygen radicals elimination:The ability of TPNs to scavenge reactive oxygen species（ROS）at the test tube level was examined using DPPH and ABTS^+-scavenging assays,and electron spin resonance spectroscopy.The effect of TPNs on ROS elimination by different ROS agonists in macrophages and renal tubular epithelial cells was probed using immunofluorescence,mitochondrial membrane potential.Cellular antioxidant capacity was assessed using biochemical kits for GSH,Western blot for GPX4.Results.1 Screening and modifying of GSDMD-targeted inhibitors.1.1 Screening of GSDMD-targeted inhibitors.Compound library screening revealed that EGCG significantly inhibited LDH in the supernatant of both LPS+Nigericin and LPS+HD after pyroptosis stimulation.1.2 Optimisation of GSDMD-targeted inhibitors.1.2.1 Characterisation of TPNs:TPNs are self-polymerising nanoparticles with EGCG as the raw material and Mn²⁺as the catalyst.The particle size is around 180 nm,irregularly spherical or sphere-like,and well dispersed.The average molecular mass of the polymers is～3558.1.2.2 Safety studies of TPNs:The residual content of the heavy metal Mn²⁺in the catalyst was 0.8%.TPNs had no significant effect on the cell variabilty and important organ pathology of different cell lines.2 Role of GSDMD-targeted inhibitors in sepsis.2.1 Role of TPNs on different inflammasome:TPNs can inhibit LDH release from the supernatant macrophages and reduce PI-stained cells after stimulation by different inflammasome,as well as reduce the inflammatory factors IL-1α,IL-1β.TPNs can also inhibit the supernatant LDH and IL-1βof THP-1 cells induced by different inflammasome stimulation.2.2 Role of TPNs in sepsis model mice:In animal experiments on sepsis model mice,TPNs could reverse the decrease in body temperature and reduce the release of inflammatory factors such as IL-1α,IL-1βand IL-6,protect the function of vital organs and reduce pathological damage,and improve the survival rate of lethal sepsis model mice.3.Mechanism studies of the GSDMD-targeted inhibitor on sepsis.3.1 Effect of TPNs on GSDMD-N intercalation:Western blot results showed that TPNs could inhibit the formation of GSDMD oligomers without affecting GSDMD shearing.Immunofluorescence results showed that TPNs could reduce GSDMD membrane localization.Meanwhile,The results of reconstruction of non-canonical inflammasome-induced pyroptosis in 293T cells revealed that TPNs could directly reduce the intercalation of GSDMD-NT,not the shedder of GSDMD,cell membrane and oligomer formation.3.2 Effect of TPNs on reactive oxygen radicals elimination:DPPH,ABTS^+-scavenging assays and electron spin resonance spectroscopy results showed that TPNs have efficient ROS scavenging ability at the test tube level.Immunofluorescence,mitochondrial membrane potential results showed that TPNs reduced ROS production in macrophages and renal tubular epithelial cells induced by different ROS agonists.TPNs reversed the down-regulation of GSH and GPX4 in renal epithelial cells after ROS agonist stimulation and restored the cellular antioxidant capacity.Conclusions.1.TPNs are a novel targeted inhibitor of GSDMD.It is simple to prepare and has a good safety profile.2,TPNs can inhibit pyroptosis induced by different inflammasome stimulation,and have a significant protective effect on multi-organ function and survival in sepsis model mice.3,TPNs can simultaneously inhibit the oligomerization of GSDMD-NT without affecting the shearing of GSDMD-NT by inhibiting the direct binding between GSDMD-NT and scavenging ROS,and ultimately inhibit cell pyroptosis with multi-target efficiency.41 Figures,0 tables,78 references...