The Study Of The Effect And Potential Mechanisms Of Hydrogen Sulfide On Oxidized Low Density Lipoprotein Induced Pyroptosis In Macrophages

Background and Objective:Macrophages,AS the key immune cells in innate immunity and tissue repair,play a decisive role in the development of Atherosclerosis（AS）by forming foam cell-mediated chronic inflammatory response.Recent studies have confirmed that nucleotide binding oligomerization domain-like receptor 3/cysteinyl aspartate specific proteinase 1/gasdermin D mediated pyrodeath of foam macrophages in the subintima of blood vessels plays a key role in the whole process of inflammatory vascular disease-atherosclerosis.Therefore,cell pyroapoptotic can provide a new target for the treatment of AS.Hydrogen sulfide（H₂S）is an important endogenous gas signaling molecule,and a large number of studies have confirmed the cardiovascular protection,anti-inflammatory,anti-AS and anti-hypertension effects of H₂S.However,the regulation of H₂S on pyrophosis of foam macrophages during the occurrence and development of AS and its mechanism remain unclear.Therefore,this study intends to stimulate THP-1 derived macrophages with oxidized low density lipoprotein（ox-LDL）to establish in vitor cell pyroptosis models.To investigate whether the H₂S can inhibit the pyrophosis of macrophages induced by oxidized low density lipoprotein（ox-LDL）by inhibiting the classical cellular pyrophosis pathway.Methods:Human monocyte macrophage leukemia cell（THP-1）were stimulated with 100 ng/mL PMA for 24 h to differentiate into macrophage.THP-1 derived macrophages were then treated with ox-LDL,H₂S donor and endogenous H₂S synthase inhibitor,and were divided into the following 6 groups:（1）blank control group;（2）PAG intervention group;（3）ox-LDL intervention group;（4）ox-LDL+PAG intervention group;（5）ox-LDL+Na HS treatment group;（6）ox-LDL+PAG+Na HS treatment group.CCK-8 method was used to evaluated the effect of Na HS or PAG on THP-1 derived macrophages activity.Lipid deposition was detected by oil red O staining.The morphological characteristics of pyroptosis were observed by inverted microscope.The pyroptosis in each group was detected by Hoechst 33342/PI staining and LDH release.Methylene blue colorimetric method was used to detect the content of H₂S in THP-1 cells.The Caspase-1 activity in cells in each group was detected by Caspase-1 activity assay kit.The levels of IL-1βand IL-18 in the supernatant of THP-1 macrophages were determined by ELISA.Western Blot was used to detect the expression of NLRP3,Pro-Caspase-1,Caspase-1（P20）,GSDMD,GSDMD-N and IL-18 of the pyroptosis signaling pathway in each group of cells.Results:（1）CCK-8 assay results showed that compared with the blank control group NaHS（50,100,200,400μmol/L）had no direct toxicity effect on THP-1 macrophage activity（P<0.05 or P<0.01）;PAG（1,2.5,5,10 mmol/L）significantly decreased THP-1 macrophage activity（P<0.05 or P<0.01）.（2）Oil red O staining results showed that 100 mg/L ox-LDL could induce significant lipid deposition in THP-1macrophages compared with the blank control group,and pretreatment with different concentrations of Na HS could significantly reduce ox-LDL-induced lipid deposition in THP-1 macrophages.（3）The results of the scorch morphology showed that the ox-LDL and ox-LDL+PAG combination interventions resulted in the pyroptosis of cells with obvious swelling and bubble-like protrusions compared to the normal control group,while Na HS significantly inhibited the pyroptosis of cells with obvious swelling and bubble-like protrusions;（4）Hoechst 33342/PI staining and LDH release assay results showed that low concentration ox-LDL（25 and50 mg/L）had no significant effect on LDH release in THP-1 macrophages compared with blank control group（P>0.05）.However,high concentration of ox-LDL（100,150 mg/L）and ox-LDL+PAG combined significantly increased the PI of positive staining and LDH release rate in THP-1 derived macrophages（P<0.05 or P<0.01）.The combined intervention of Na HS and ox-LDL/ox-LDL+PAG significantly reduced the proportion of PI positive staining cells and the LDH release rate（P<0.05 or P<0.01）;（5）The results of the hydrogen sulfide determination assay kit showed that the ox-LDL group and ox-LDL+PAG significantly decreased the content of H₂S in THP-1 derived macrophages compared with the normal control group（P<0.05 or P<0.01）;the combined intervention of Na HS and ox-LDL/ox-LDL+PAG can significantly increased the content of H₂S in THP-1 derived macrophages（P<0.01）;（6）The results of Caspase-1 activity kits showed that the ox-LDL group and ox-LDL+PAG significantly increased the Caspase-1 activity in THP-1derived macrophages compared with the blank control group（P<0.05 or P<0.01）;Na HS and ox-LDL/ox-LDL+PAG combined treatment significantly reduced the Caspase-1 activity in THP-1 derived macrophages（P<0.01）;（7）ELISA results showed that compared with the blank control group,the levels of IL-1βand IL-18 in THP-1 macrophage supernatant in ox-LDL group and ox-LDL+PAG combined intervention were significantly increased（P<0.05 or P<0.01）.However,Na HS combined with ox-LDL/ox-LDL+PAG significantly reduced the levels of IL-1βand IL-18 in the supernatant of THP-1 macrophages（P<0.01）.（8）Western Blot results showed that,100 mg/L and 150 mg/L ox-LDL and ox-LDL+PAG combined treatment significantly up-regulated the specific proteins（NLRP3,Pro-Caspase-1,Caspase-1（P20）,GSDMD,GSDMD-N and IL-18）of pyroptosis in THP-1 derived macrophages compared with the normal control group（P<0.05 or P<0.01）;Compared with the ox-LDL group and ox-LDL+PAG group,the combined intervention of Na HS and ox-LDL/ox-LDL+PAG significantly inhibited the expression of specific proteins of pyroptosis were up-regulated（P<0.05 or P<0.01）.Conclusion:H₂S can inhibit ox-LDL induced pyroptosis of macrophages by acting on classical cell pyroptosis signaling pathway.