The Mechanism Of Remimazolam Inhibiting Cardiomyocyte Pyroptosis In Myocardial Ischemia Reperfusion Injury Via TLR4/MyD88/NF-κB Signaling Pathway

Objective:To investigate the effect of remimazolam on myocardial ischemia reperfusion injury(MIRI)and pyroptosis in rats.Methods:30 Sprague Dawley(SD)rats were randomly divided into Sham group,myocardial ischemia/reperfusion(I/R)model group(I/R group)and remimazolam treatment group(I/R+Rem group),with 10 rats in each group.In Sham group,suture was placed under the left anterior descending(LAD)without ligation;In I/R group,the I/R model was induced with 30 min of LAD occlusion followed by 2 h of reperfusion;In I/R+Rem group,remimazolam(6.2mg/kg/h)was continuous infusion through the tail vein 1 h before operation.After the experiment,left ventricular ejection function(LVEF)and left ventricular fractional shortening(LVFS)were measured by ultrasound for evaluation of cardiac systolic function of rats,and the heart tissue and blood samples of rats were collected for morphological,biochemical and molecular studies.2,3,5-triphenyltetrazolium chloride(TTC)was used to detect the myocardial infarction size of rats;Hematoxylin eosin(HE)staining and transmission electron microscope(TEM)were used to detect myocardial pathological and mitochondrial damage in each group;The serum specific marker enzymes(c Tn T,CK-MB and LDH)in each group were measured using commercial kits;The level of serum inflammatory cytokines(TNF-α,IL-18 and IL-1β)were measured by ELISA kits;The expression of NLRP3,ASC,Cleaved-Caspase-1,GSDMD proteins of rats in each group were detected by Western blotting analysis.Results:1.Cardiac ultrasound results revealed that there was a significant decrease in the activity of the anterior ventricular wall as well as the LVEF and LVFS of the rats in the I/R group compared with the Sham group(p<0.0001),whereas the activity of the anterior ventricular wall of the rats in the I/R+Rem group were enhanced,and the LVEF and LVFS of the rats in the I/R+Rem group were also notably increased when compared with I/R group(p<0.01).2.TTC staining showed that there was a significant decrease in the percent of myocardial infarct size of the rats in the I/R group compared with the Sham group(p<0.001),while the percent of myocardial infarct size in I/R+Rem group were notably reduced compared with the I/R group(p<0.001);3.HE staining results revealed that compared with Sham group,myocardial fiber rupture,myocardial cell swelling,and inflammatory cell infiltration were notably observed in I/R group.whereas compared with I/R group,myocardial fiber rupture,swelling,and inflammatory cell infiltration were not obvious observed in I/R+Rem group;4.Transmission electron microscopy results revealed that there were no obvious membrane rupture and mitochondrial damage in the Sham group,while the majority of mitochondria in the I/R group were notably swollen,and the mitochondria cristae as well as the cell membrane were broken obviously.On the contrary,only a small portion of the mitochondria and mitochondria cristae in the I/R+Rem group were slightly swollen and broken,and the cell membrane were continuous and intact;5.The results of myocardial enzyme showed that the concentration of c Tn T,CK-MB and LDH in I/R group were dramatically increased after I/R when compared with those in Sham group,whereas treated with remimazolam effectively reversed these changes on c Tn T,CK-MB and LDH compared with those in I/R group;6.ELISA results demonstrated that the level of TNF-α,IL-18 and IL-1β in I/R group were dramatically increased compared with the Sham group(p<0.001).On the contrary,treated with remimazolam remarkably attenuated the level of the above inflammatory cytokines compared with the I/R group(p<0.001);7.Western blotting analysis showed that the expression of NLRP3,ASC,Cleaved-Caspase-1 and GSDMD protein in I/R group were notably up-regulated compared with Sham group.By contrast,treated with remimazolam markedly down-regulated the protein expression of ASC,NLRP3,Cleaved-Caspase-1 and GSDMD.Conclusions:Remimazolam exhibited strong favorable cardioprotective effect on myocardial I/R injury which might be related to attenuation of cardiomyocyte pyroptosis.Objective:To screen the potential target and explore the mechanism of remimazolam in alleviating myocardial ischemia-reperfusion injury in rats.Methods:10 SD rats were randomly divided into Sham group or I/R group,with 5 rats in each group.After the completion of modeling,the myocardial tissue RNA were extracted for transcriptome sequencing(RNA-seq).The differentially expressed genes(DEGs)between I/R group and Sham group were visualized by volcano map and heat map;Differential expression pathway and functional enrichment analysis were performed by GO,KEGG,GSEA and PPI analysis;Based on the results of RNA-seq analysis,the potential target genes and regulatory pathway for remimazolam to prevent myocardial ischemia reperfusion injury were screened.In addition,The expression of the key pathway proteins based on the RNA-seq analysis between the three groups were compared by Western blot and q RT-PCR analysis.Results:1.RNA-Seq analysis results demonstrated that there were 21688 DEGs between the two groups.Among them,314 DEGs were up-regulated and 129 DEGs were down-regulated in I/R group when compared with Sham group;2.The results of GO function enrichment analysis indicated that these DEGs were mainly enriched in biological processes such as signal transduction,positive regulation of cell proliferation,and inflammatory response processes;In cell composition,these DEGs were mainly concentrated in membrane,cytoplasmic and membrane integrity components;Among the molecular functions,DEGs were highly focus on protein binding,metal ion binding,and homoprotein binding function.3.The results of KEGG pathway and GSEA analysis demonstrated that the DEGs were highly concentrated in NOD-like signaling pathway,Toll-like receptor signaling pathway,TNF-α signaling pathway and NF-κB signaling pathway.PPI analysis indicated that genes with higher degrees in PPI networks were Ccna2,Cd14,Fos,Fn1,Histlh2 b,Cdk1,etc.The top 10 hub genes screened by Cyto Hubba were Fn1,Myc,Mki67,Ptgs2,Cd68,Tlr4,Fos,Hist1h3 c,Il1b,and Ccl2 in sequence;Based on the RNA-seq analysis,TLR4/My D88/NF-κB signaling pathway has been speculated as a potential regulatory pathway for remimazolam to alleviate myocardial ischemia reperfusion injury in rats;4.Western blot and q RT-PCR analysis were used for validation of TLR4/My D88/NF-κB pathway in each group.Western blot analysis revealed that the expression of TLR4,My D88 and p NF-κBp65 in I/R group were significantly up-regulated when compared with Sham group,whereas treated with remimazolam down-regulated the expression of TLR4,My D88 and p NF-κBp65 proteins when compared with I/R group.Similarly,q RT-PCR analysis showed a significant increase in m RNA levels of TLR4,My D88 and NF-κBp65 in I/R group when compared with Sham group,whereas treated with remimazolam decreased the m RNA levels of TLR4,My D88 and NF-κBp65 when compared with I/R group.Conclusions:Toll-like signaling pathway,NOD-like signaling pathway,TNF-α signaling pathway,NF-κB signaling pathway and inflammatory response were significantly enriched during MIRI.Remimazolam may alleviate myocardial ischemia reperfusion injury and inhibited pyroptosis through TLR4/My D88/NF-κB signaling pathway.Objective:To clarify the effect and mechanism of remimazolam on the hypoxia/reoxygenation injury of H9C2 cells,and clarify the role of TLR4/My D88/NF-κB signaling pathway in pyroptosis of H9C2 cells after hypoxia/reoxygenation injury.Methods:(1)The H9C2 cells hypoxia/reoxygenation(H/R)model was established by exposing to hypoxia conditions(5% CO2,95% N2 without FBS and glucose-deprived DMEM)for 6 hour and reoxygenation conditions(21% O2,5% CO2,and 74% N2)for 6 hours.The cells in remimazolam intervention group(H/R+Rem groups)were pretreated with 6.25 μM,12.5 μM,25 μM,50 μM,100 μM,200 μM,400 μM and 800μM of remimazolam for 20 min,and then treated with H/R;The cells in control groups(Con group)were maintained under normal conditions,and cells in Con+Rem group were pretreated with different doses of remimazolam,the remaining treatment is the same as the Con group.1.CCK-8 assay was used to detect the effect of different concentrations of remimazolam on cell viability of H9C2 cells and H9C2 cells exposed to H/R;LDH kit was used to detect the effects of different concentrations of remimazolam on LDH release of H9C2 cells and H9C2 cells exposed to H/R;2.Hochest33242/PI double staining was used to detect the proportion of pyroptosis cells in each group;The concentration of TNF-α,IL-1β and IL-18 in cell supernatants in each group were measured by ELISA kit;Western blot and immunofluorescence analysis were used to detect the expression of GSDMD,NLRP3,Caspase-1,ASC,TLR4,My D88 and p NF-κBp65 proteins in each group;(2)The TLR4 overexpression model was constructed by lentivirus,and the TLR4 overexpression lentivirus and empty virus were transfected into H9C2 cells.After pretreatment with remimazolam and H/R,H9C2 cells were divided into four groups: H/R group,H/R+Rem group,H/R+Rem+TLR4-OE group,and H/R+Rem+TLR4-NC group.1.The overexpression effect of TLR4 was verified by q RT-PCR.CCK-8 assay was used to detect the effect of overexpression of TLR4 on the cell viability of H9C2 cells after H/R;The effect of overexpression of TLR4 on the LDH release of H9C2 cells after H/R was detected by LDH kit;Hochest33342/PI double staining was used to detect the proportion of pyroptosis cells in each group after overexpression of TLR4;2.ELISA kits was used to examined the levels of TNF-α,IL-18 and IL-1β in each group;The m RNA expression of GSDMD,NLRP3,Caspase-1 and ASC in each group were detected by q RT-PCR analysis;Western blot and q RT-PCR analysis were used to detect the expression of TLR4,My D88 and p NF-κBp65 in each group;3.Molecular docking experiments was used to fit the binding of remimazolam with TLR4 protein,and surface plasmon resonance(SPR)experiments was used to verify the binding and affinity of remimazolam with TLR4 protein.Results:1.CCK-8 assay revealed that 6.25 μM,12.5 μM,25 μM,50 μM,100 μM and200 μM of remimazolam had no negative effect on cell viability of H9C2 cells,while400 μM and 800 μM of remimazolam had a slight inhibitory effect on H9C2 cells;After hypoxia/reoxygenation,the cell survival rate of H9C2 cells in H/R group had a significant decrease compared with Con group;By contrast,after treated with 25 μM,50 μM,100 μM and 200 μM remimazolam,the cell survival rate of H9C2 cells were significantly higher than that of H/R group,in which the cell viability of H9C2 cells treated the 100 μM remimazolam was the highest(p<0.001);There were no significant changes in cell survival rate between H/R group and groups treated with6.25 μM,12.5 μM,400 μM and 800 μM remimazolam;Compared with Con group,the concentration of LDH in the cell cμlture medium of H/R group were significantly increased(p<0.001),whereas the concentration of LDH in H9C2 cells treated with12.5 μM,25 μM,50 μM,100 μM and 200 μM remimazolam significantly decreased when compared with the H/R group,in which the concentration of LDH in H9C2 cells treated with 100 μM remimazolam was the lowest(p<0.001);2.The results of Hochest33342/PI staining showed that the incidence of pyroptosis in H/R group was significantly increased when compared with Con group,whereas the the incidence of pyroptosis in H/R+Rem group was lower than that in H/R group(p<0.01);ELISA results revealed that there was a significant increase in the level of TNF-α,IL-18 and IL-1β in H/R group compared with Con group(p<0.001);By contrast,the level of these cytokines(TNF-α,IL-18 and IL-1β)in H/R+Rem group were notably decreased than that in H/R group(p<0.001);Immunofluorescence analysis demonstrated that there was a significant increase in the intensity of GSDMD,NLRP3,Caspase-1 in H/R group compared with Con group,whereas treated with remimazlam,the intensity of above proteins(GSDMD,NLRP3,Caspase-1)in H/R+Rem group were markedly decreased compared to H/R group;Western blot analysis showed that the expression of TLR4,My D88 and p NF-κBp65in H/R group were significantly up-regulated when compared with Con group,while the expression of TLR4,My D88 and p NF-κBp65 in H/R+Rem group were significantly down-regulated when compared with H/R group(p<0.01);3.CCK-8 assay showed that the cell viability of H/R+Rem group and H/R+Rem+TLR4-NC group were significantly increased when compared with H/R group(p<0.001);After overexpression of TLR4,the cell viability of H/R+Rem+TLR4-OE group was significantly lower than that of H/R+Rem and H/R+Rem+TLR4-NC group(p<0.01);Compared with H/R group,the concentration of LDH of the cell culture medium in H/R+Rem group and H/R+Rem+TLR4-NC group were decreased significantly(p<0.001),while the concentration of LDH in H/R+Rem+TLR4-OE group was markedly increased when compared with H/R+Rem group and H/R+Rem+TLR4-NC group(p<0.001);ELISA analysis revealed that the level of TNF-α,IL-18 and IL-1β in H/R+Rem group and H/R+Rem+TLR4-NC group were markedly decreased when compared with H/R group,while the level of above inflammatory cytokines(TNF-α,IL-18 and IL-1β)in H/R+Rem+TLR4-OE were increased significantly than that of H/R+Rem group and H/R+Rem+TLR4-NC group(p<0.001);4.Western blot and qRT-PCR analysis demonstrated that the expression of TLR4,My D88 and p NF-κBp65 proteins in H/R+Rem group and H/R+Rem+TLR4-NC group were significantly down-regulated compared with H/R group,while the expression of TLR4,My D88 ad p NF-κBp65 in H/R+Rem+TLR4-OE group were up-regulated when compared with H/R+Rem group and H/R+Rem+TLR4-NC group(p<0.001);Similarly,q RT-PCR analysis revealed that there were markedly decreased in the m RNA expression of GSDMD,NLRP3,Caspase-1 and ASC in H/R+Rem group and H/R+Rem+TLR4-NC group compared with H/R group(p<0.001),whereas the m RNA expression of above genes(GSDMD,NLRP3,ASC and Caspase-1)in H/R+Rem+TLR4-OE group were significantly increased compared with H/R+Rem group and H/R+Rem+TLR4-NC group(p<0.001);5.Molecular docking experiments showed that remimazolam had a good binding force with TLR4 protein,and their potential binding sites might be Glutamate38(Gln38)and Aspartic acid 59(Asp59);Similarly,the results of SPR showed that the binding constant(Ka)of remimazolam and TLR4 protein was 1.051×103;The dissociation constant(Kd)of remimazolam and TLR4 protein was 3.698×-3,and the KD value was 3.518×10-6.Molecular docking experiment and SPR experiment showed that remimazolam directly bind to TLR4 protein with a high affinity.Conclusions:Remimazolam can alleviate the hypoxia/reoxygenation injury and pyroptosis of H9C2 cells through TLR4/My D88/NF-κB signaling pathway.