Study On The Mechanism Of Curcumin Regulating Oxidative Stress,Inhibiting Pyroptosis And Improving Inflammatory Microenvironment In The Treatment Of Endometriosis

Objectives:To investigate whether pyroptosis affects the progression of endometriosis disease and to further investigate whether crucumin can treat endometriosis by regulating pyroptosis using network pharmacology and molecular docking techniques.To construct an animal and cellular pyroptosis model of endometriosis and to investigate whether curcumin can treat endometriosis by affecting pyroptosis and oxidative stress.Methods:1.To investigate the differences in expression of endometrial pyroptosis-related proteins(NLRP3,GSDMD,CAPSASE-1,IL-1β,IL-18)between lesions of endometriosis patients and non-EM women by immunohistochemistry;to investigate the differences in expression of IL-1β,IL-18 in peritoneal fluid between lesions of endometriosis patients and non-EM women by ELISA.A model of endometrial pyroptosis cell was constructed by LPS+ATP,and the cell morphology was observed by scanning electron microscopy.To confirm the occurrence of pyroptosis;to construct a mouse model of endometriosis,WB and PCR were used to detect the differences in the expression of proteins and genes related to endometrial pyroptosis between ectopic lesions of model mice and normal mice;ELISA was used to detect the differences in the expression of IL-18 and IL-1β in the peritoneal fluid of ectopic lesions of model mice and normal mice.2.All potential targets of curcumin were screened by SWISS TARGET PREDICTION,Similarity ensemble approach,TCMSP,STITCH multi-platform,all targets of EM were obtained from GeneCards and OMIM database，and curcumin and EM intersection targets were obtained by using Venn diagram.Cytoscape 3.8.0 was used to construct a curcumin-target-disease network map.A protein interaction network map was constructed using the String database.The Geneontology database and KEGG database were analysed to obtain the enriched GO bioanalytical functions of the target genes and the KEGG target gene enrichment signalling pathway,and molecular docking was completed using AutoDockTools 1.5.6 software and AutoDock software.3.A model of endometrial pyroptosis cell was constructed and divided into normal(CON)group,LPS/ATP group,curcumin(CUR)group,NAC group and COCl2 group.WB and PCR were performed to detect the difference in expression of proteins and genes related to pyroptosis in each group;ELISA was performed to detect the difference in expression of IL-18 and IL-1β in cell supernatants of each group.A mouse model of endometriosis was constructed and divided into normal group(sesame oil gavage),simple model group(sesame oil gavage),curcumin group(CUR group)(curcumin sesame oil solution gavage)and dinogestre1 group(dinogestrel sesame oil solution gavage),all groups were continuously gavaged for 21 days,and then executed after day 23.The concentration of IL-1β and IL-18 in the peritoneal lavage fluid of each group was measured,and the differences in the expression of proteins and genes related to pyroptosis between the lesions in each model group and normal mice were detected by WB and PCR.The levels of SOD and MDA in the endothelial membranes of mice were measured in the model and normal mice.Results:1.Immunohistochemical results indicated that the expression of NLRP3,CASPASE-1,GSDMD and other markers of pyroptosis were significantly higher in the ectopic lesions compared with the normal endothelium,suggesting that pyroptosis occurred in the ectopic lesions;while the expression of IL-1β and IL-18 was increased in the ectopic lesions compared with the normal endothelium,suggesting that pyroptosis in the ectopic lesions brought about subsequent inflammation.The combined use of LPS-ATP induced endometrial stromal cell pyroptosis,and the electron microscopic results indicated that the endometrial stromal cells appeared swollen after LPS-ATP treatment,and a large number of membrane pores appeared on the cell membrane,confirming the occurrence of scorching.A mouse model of endometriosis was constructed by.endometrial injection,and the levels of NLRP3,CASPASE-1,GSDMD-N protein and gene expression in the the model mice were significantly increased compared with those of normal mice(p<0.05).The levels of IL-1β and IL-18 in the peritoneal fluid of the model mice were significantly increased compared with those of normal mice(p<0.05),which confirmed the presence of pyroptosis and changes in inflammatory factors associated with pyroptosis in the constructed endometriosis mouse model.2.A total of 180 potential targets of curcumin were obtained through multi-database screening,and 65 potential EM targets of curcumin were obtained after intersection with EM-related targets using venny.GO enrichment analysis and KEGG analysis were performed on the 65 intersected targets.The results returned 129 significant KEGG pathways(p<0.05)and 224 significantly enriched GO terms(p<0.05).signaling pathway(hsa04915),and VEGF signaling pathway(hsa04370),which are involved in various aspects of inflammatory response,oxidative stress,and angiogenesis,while the NOD-like receptor signaling pathway(hsa04621)directly suggests that curcumin could have a therapeutic effect on EM via the inflammatory vesicle pathway and a possible regulatory capacity for pyroptosis.In the GO analysis,regulation of angiogenesis(GO:0045765),cellular response to interleukin-1(GO:0071347),response to oxidative stress(GO:0006979)and other oxidative stress,inflammation and angiogenesis-related biological processes with P-values similarly less than 0.001,corresponding to the KEGG analysis.The binding energy of NLRP3 was-8.0,that of CASPASE-1 was-8.5 and that of GSDMD was-7.4.All three binding energies were less than-5,suggesting that curcumin can form a good docking with key molecules of pyroptosis.The network pharmacology suggests that curcumin has good potential for the treatment of endometriosis and may be linked to the pyroptosis pathway.3.Compared to the LPS-ATP group,the protein and gene expression levels of NLRP3,CASPASE-1,ASC and GSDMD were significantly decreased in the C UR group(p<0.05).After ELISA on cell supernatants,the secretion of IL-1β and IL-18 was significantly lower in the CUR group compared to the LPS-ATP group(p<0.05).The electron microscopic results show ed that the LPS-ATP group showed a significant scorching reaction with swelling of the cells and a large number of pores on the cell membrane compared to the CON group.In the CUR group,the pyroptosis was significantly inhibited,the swelling of cells was not obvious,and there were fewer pores on the cell membrane,indicating that curcumin could inhibit the pyroptosis and reduce the release of inflammatory factors.At the same time,ROS and MDA levels in the CUR group decreased significantly(p<0.05)and SOD levels increased(p<0.05)compared to LPS-ATP,confirming that curcumin could improve the antioxidant capacity of stromal cells.The antioxidant NAC also reduced the level of endosomal stromal cell scorch and enhanced the antioxidant capacity of endosomal stromal cells,with no significant difference from the CUR group.The addition of the pro-oxidant COCl2 to the CUR group resulted in an increase in ROS and MDA levels and a decrease in SOD levels in the COCl2 group,which was significantly different from the CUR group(p<0.05),suggesting that the antioxidant capacity and oxidative stress levels in this group increased.ELISA results indicated that the levels of IL-1β and IL-18 increased again in the COCl2 group compared to the CUR group(p<0.05).It was confirmed that COCl2 could cause pyroptosis again by inhibiting the antioxidant capacity of curcumin.In animal studies,the number of lesions,volume and degree of adhesions decreased significantly in the CUR group compared to the model group(p<0.05),confirming that curcumin could inhibit the progression of endometriosis.The expression levels of NLRP3,GSDMD,GSDMD-N and CASPASE-1 in the ectopic lesions were significantly decreased in the CUR group compared to the model group(p<0.05),the expression of IL-1β and IL-18 in the peritoneal fluid was decreased(p<0.05),the MDA levels in the lesions were decreased and the SOD levels were increased(p<0.05).The results on the animal model were suppressed with the trend of cellular assay results,confirming that curcumin can indeed improve the antioxidant capacity of ectopic lesions,thus reducing pyroptosis and decreasing the release of inflammatory factors,and thus treating endometriosis.Conclusions:1.The combined application of LPS-ATP can successfully construct a model of endometrial stromal cell pyroptosis and the intraperitoneal injection method can successfully construct a mouse model of endometriosis pyroptosis2.Curcumin can treat endometriosis through multiple signaling pathways and directly regulate the NOD-like receptor signaling pathway,which dovetails well with NLRP3,CASPASE-1 and GSDMD and is closely related to the pyroptosis pathway.3.Curcumin can inhibit endothelial stromal cell pyroptosis,reduce the secretion of inflammatory factors in scorched endothelial stromal cells and decrease the level of oxidative stress in endothelial stromal cells.Curcumin can inhibit the growth of ectopic lesions,reduce the degree of abdominal adhesions,decrease the level of ectopic lesion pyroptosis and reduce the level of inflammatory factors in the abdominal fluid in animal models.