Study On The Mechanism Of Forsythia Fructus In Protecting The Injury Of Small Intestinal Epithelial Cekks Induced By Chemotherapy Based On NLRP3 Inflammatory Pathway And Pyroptosis

Objective:The previous overall animal experiments in our laboratory confirmed that Forsythiae Fructus has a good preventive effect on cisplatin-induced chemotherapy-induced nausea and vomiting(CINV),and its mechanism of action is related to anti-oxidative stress and improvement of gastrointestinal inflammatory damage.In this study,based on previous studies,cisplatininduced rat small intestinal epithelial cell(IEC-6)injury model and mouse macrophage(J774A.1)injury model were used to further investigate the NLRP3 inflammasome and pyroptosis.to explore the mechanism of Forsythiae Fructus in preventing and treating CINV.Methods:1.Detection of chemical composition of Lyophilized powder of Forsythiae Fructus: Prepare lyophilized powder of Forsythiae Fructus,and use ultra-high performance liquid phase-quadrupole-electrostatic field orbitrap high-resolution mass spectrometry(UPLC-Q-Orbitrap-MS)to analyze Lyophilized powder of Forsythiae Fructus chemical composition.2.IEC-6 cell injury model: The IEC-6 cell injury model was constructed with cisplatin and tert-butyl hydroperoxide(t BHP),respectively,to explore the protective effects of Forsythiae Fructus and its main active components,Phillyrin and forsythiaside A.mechanism.Cell viability was detected by CCK8method;Hoechst33342/PI double staining method was used to observe cell morphological changes;DCFH-DA fluorescent probe method was used to determine intracellular reactive oxygen species(ROS)content;q RT-PCR detected the expression levels of Nlrp3,Asc,caspase-1,Il18,Gsdmd,Hmgb1;Western Blot detected the expression levels of NLRP3 inflammatory pathway and pyroptosis-related proteins.3.J774 A.1 cell injury model: The J774 A.1 cell injury model was constructed with cisplatin and LPS/ATP respectively,and the protective mechanism of Forsythiae Fructus and its main active components,Phillyrin n and forsythiaside A,was investigated.Cell viability was detected by CCK8method;Hoechst33342/PI double staining method was used to observe cell morphological changes;DCFH-DA fluorescent probe method was used to determine intracellular ROS content;q RT-PCR detected the expression levels of Nlrp3,Asc,caspase-1,Il1 b,Il18,Gsdmd,Hmgb1;Western Blot detected the expression levels of NLRP3 inflammatory pathway and pyroptosis-related proteins.Results:1.Lyophilized powder of Forsythiae Fructus chemical composition detection and quality control: UPLC-Q-Orbitrap-MS analysis obtained 850 chemical components in Lyophilized powder of Forsythiae Fructus.2.Protective effect on IEC-6 cell injury: Lyophilized powder of Forsythiae Fructus,Phillyrin,and Forsythiaside A with N-acetyl-L-cysteine(NAC)could significantly improve the decrease of IEC-6 cell viability,intracellular ROS level and cell necrosis induced by cisplatin and t BHP.Inhibit overexpression of Nlrp3,Asc,caspase-1,Il18,gsdmd and Hmgb1 genes;Inhibit the phosphorylation of NF-κB protein,decrease the protein levels of NLRP3,ASC,HMGB1 and the ratio of IL-1β/pro-IL-1β,caspase-1/procaspase-1,GSDMD-NT/GSDMD(P<0.05).3.Protective effect on J774 A.1 cell injury: Lyophilized powder of Forsythiae Fructus,Phillyrin,and Forsythiaside A were used in combination with NAC,could significantly inhibit the reduction of J774 A.1 cell viability and decrease caused by cisplatin and LPS/ATP.Intracellular ROS level and cell necrosis induced by cisplatin and LPS/ATP.Inhibit overexpression of Nlrp3,Asc,caspase-1,Il1 b,Il18,gsdmd and Hmgb1 genes;Inhibit the phosphorylation of NF-κB protein,decrease the protein levels of NLRP3,ASC,HMGB1 and the ratio of IL-1β/pro-IL-1β,caspase-1/pro-caspase-1,GSDMDNT/GSDMD(P<0.05).Conclusion:The results of this study show that Forsythiae Fructus and its main active components,Phillyrin and Forsythoside A,can inhibit NLRP3 inflammasome activation and cell pyroptosis,and protect small intestinal epithelial cell damage and macrophage damage caused by cisplatin.Combined with the results of previous animal experiments in our laboratory,it is further demonstrated that the mechanism of Forsythiae Fructus ’s prevention and treatment of CINV is related to its improvement of chemotherapy-induced gastrointestinal inflammatory injury,and inhibition of NLRP3 inflammasome and cell pyroptosis may be an important mechanism for its effect.