Objective To investigate the effects of paeonol on the nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway and pyroptosis levels in ectopic lesion tissues of endometriosis(EMS)mice and mouse endometrial epithelial cells(mEECs).Methods An allograft transplantation method was used to establish an EMS mouse model.Thirty-five Balb/c mice were divided into a sham group,a model group,a 12.5 mg/kg paeonol group,a 25 mg/kg paeonol group,a 50 mg/kg paeonol group,a 0.5 mg/kg gestrinone group(positive control group),and a 50 mg/kg paeonol+Nrf2 inhibitor ML385(30 mg/kg)group,with 5 mice in each group.mEECs were divided into a cell control group,a 10 ng/ml transforming growth factor-β1(TGF-β1)or 100 ng/ml lipopolysaccharide(LPS)model group,a 50 μmol/Lpaeonol group,a 100 μmol/L paeonol group,and a 100 μmol/L paeonol+ML385 group.Enzyme-linked immunosorbent assay(ELISA)kits were used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β,and IL-18 in the serum of EMS mice and the culture supernatant of mEECs.Hematoxylin and eosin(H&E)staining was used to detect histopathological changes in ectopic lesion tissues of mice.Western blot experiments were conducted to detect the protein expression levels of NOD-like receptor thermal protein domain-associated protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),caspase-1,gasdermin D protein N-terminal fragment(GSDMD-N),Nrf2,heme oxygenase-1(HO-1),and quinone oxidoreductase-1(NQO-1)in ectopic lesion tissues and mEECs.The MTT method was used to detect the cell survival rate of mEECs,and the EdU staining method was used to detect the proliferation ability of mEECs.Wound healing assay and Transwell chambers were used to detect the migration and invasion abilities of mEECs,respectively.The FAM-FLICA? Caspase-1 kit was used to detect Caspase-1 levels in mEECs.AutoDock Vina software was used for molecular docking of paeonol with Nrf2 to evaluate their binding ability.Results Compared with the model group,the paeonol groups and the gestrinone group showed reduced ectopic lesion volumes in EMS mice(P<0.05),decreased serum levels of TNF-α,IL-6,IL-1β,and IL-18,decreased expression of pyroptosis marker proteins NLRP3,ASC,Caspase-1,and GSDMD-N in ectopic lesion tissues,and increased expression levels of Nrf2,HO-1,and NQO-1.Compared with the TGF-β1 model group,the paeonol group showed reduced proliferation,migration,and invasion abilities of mEECs.Compared with the LPS model group,the paeonol group showed decreased levels of inflammatory factors TNF-α,IL-6,IL-1β,and IL-18 in mEECs,decreased expression levels of NLRP3,ASC,Caspase-1,and GSDMD-N,and increased expression levels of Nrf2,HO-1,and NQO-1.In the EMS mouse model,the Nrf2 inhibitor ML385 reversed the inhibitory effects of 50 mg/kg paeonol on pyroptosis and inflammatory levels in ectopic lesion tissues.In the in vitro cell model,ML385 reversed the effects of 100 μmol/L paeonol on mEECs.The binding score of paeonol with Nrf2 was-6.1 kcal/mol,and paeonol formed hydrogen bonds with the VAL-604 and LEU-365 amino acid residues of Nrf2.Conclusion Paeonol can alleviate inflammatory responses in EMS mice,reduce NLRP3 inflammasome activation and pyroptosis levels in ectopic lesion tissues,and activate the Nrf2/HO-1 signaling pathway in ectopic lesion tissues of EMS mice.

Objective To investigate the effects of paeonol on the nuclear factor erythroid 2-related factor 2(Nrf2)signaling pathway and pyroptosis levels in ectopic lesion tissues of endometriosis(EMS)mice and mouse endometrial epithelial cells(mEECs).Methods An allograft transplantation method was used to establish an EMS mouse model.Thirty-five Balb/c mice were divided into a sham group,a model group,a 12.5 mg/kg paeonol group,a 25 mg/kg paeonol group,a 50 mg/kg paeonol group,a 0.5 mg/kg gestrinone group(positive control group),and a 50 mg/kg paeonol+Nrf2 inhibitor ML385(30 mg/kg)group,with 5 mice in each group.mEECs were divided into a cell control group,a 10 ng/ml transforming growth factor-β1(TGF-β1)or 100 ng/ml lipopolysaccharide(LPS)model group,a 50 μmol/Lpaeonol group,a 100 μmol/L paeonol group,and a 100 μmol/L paeonol+ML385 group.Enzyme-linked immunosorbent assay(ELISA)kits were used to detect the levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1β,and IL-18 in the serum of EMS mice and the culture supernatant of mEECs.Hematoxylin and eosin(H&E)staining was used to detect histopathological changes in ectopic lesion tissues of mice.Western blot experiments were conducted to detect the protein expression levels of NOD-like receptor thermal protein domain-associated protein 3(NLRP3),apoptosis-associated speck-like protein(ASC),caspase-1,gasdermin D protein N-terminal fragment(GSDMD-N),Nrf2,heme oxygenase-1(HO-1),and quinone oxidoreductase-1(NQO-1)in ectopic lesion tissues and mEECs.The MTT method was used to detect the cell survival rate of mEECs,and the EdU staining method was used to detect the proliferation ability of mEECs.Wound healing assay and Transwell chambers were used to detect the migration and invasion abilities of mEECs,respectively.The FAM-FLICA? Caspase-1 kit was used to detect Caspase-1 levels in mEECs.AutoDock Vina software was used for molecular docking of paeonol with Nrf2 to evaluate their binding ability.Results Compared with the model group,the paeonol groups and the gestrinone group showed reduced ectopic lesion volumes in EMS mice(P<0.05),decreased serum levels of TNF-α,IL-6,IL-1β,and IL-18,decreased expression of pyroptosis marker proteins NLRP3,ASC,Caspase-1,and GSDMD-N in ectopic lesion tissues,and increased expression levels of Nrf2,HO-1,and NQO-1.Compared with the TGF-β1 model group,the paeonol group showed reduced proliferation,migration,and invasion abilities of mEECs.Compared with the LPS model group,the paeonol group showed decreased levels of inflammatory factors TNF-α,IL-6,IL-1β,and IL-18 in mEECs,decreased expression levels of NLRP3,ASC,Caspase-1,and GSDMD-N,and increased expression levels of Nrf2,HO-1,and NQO-1.In the EMS mouse model,the Nrf2 inhibitor ML385 reversed the inhibitory effects of 50 mg/kg paeonol on pyroptosis and inflammatory levels in ectopic lesion tissues.In the in vitro cell model,ML385 reversed the effects of 100 μmol/L paeonol on mEECs.The binding score of paeonol with Nrf2 was-6.1 kcal/mol,and paeonol formed hydrogen bonds with the VAL-604 and LEU-365 amino acid residues of Nrf2.Conclusion Paeonol can alleviate inflammatory responses in EMS mice,reduce NLRP3 inflammasome activation and pyroptosis levels in ectopic lesion tissues,and activate the Nrf2/HO-1 signaling pathway in ectopic lesion tissues of EMS mice.