Forsythiae Fructus attenuates cisplatin-induced cytotoxicity in IEC-6 ​cells and J774A.1 macrophages by inhibiting NLRP3/caspase-1/GSDMD mediated pyroptosis

Abstract

Objective: Forsythiae Fructus (lian qiao in Chinese), the dried fruit of Forsythia suspensa (Thunb.) Vahl, is a commonly used traditional Chinese medicine known for its diverse biological activities, including antiemetic, anti-inflammatory, antioxidant, antiviral, and neuroprotective properties. This study investigated the protective effects of Forsythiae Fructus and its primary components, phillyrin and forsythoside A, against cisplatin-induced cytotoxicity in vitro, specifically focusing on the intestinal epithelial cells (IEC-6) and the J774A.1 macrophage cell line. Methods: Cisplatin and tert-butyl hydroperoxide (tBHP) were used to induce stress in IEC-6 ​cells, while cisplatin and lipopolysaccharides (LPS)/adenosine triphosphate (ATP) were employed for J774A.1 macrophages. The protective effects of Forsythiae Fructus aqueous extract (FAE), phillyrin, and forsythoside A against cytotoxicity in these cultured cells were evaluated. Cell viability was assessed using the Cell Counting Kit-8 assay, while cell membrane permeability was determined through Hoechst 33342 and propidium iodide staining. Intracellular reactive oxygen species (ROS) levels were investigated using DCFH-DA, and the expression of mRNA and protein related to the NLRP3 inflammasome and GSDMD-induced pyroptosis was quantified through qRT-PCR and western blotting. Results: In IEC-6 ​cells, combining FAE, phillyrin, or forsythrin A with a subthreshold dose of the antioxidant N-acetyl-L-cysteine (NAC) significantly mitigated cisplatin- or tBHP-induced cell necrosis and restored impaired cell viability. Additionally, the upregulation of NF-κB, ASC, NLRP3, caspase-1, GSDMD, and HMGB1 at both mRNA and protein levels induced by cisplatin or tBHP was markedly reversed with the joint intervention of FAE, phillyrin, or forsythrin A with NAC. Similarly, in cisplatin- or LPS/ATP-treated J774A.1 macrophages, the effects on cell necrosis, cell viability, and the NLRP3/caspase-1/GSDMD pathway mirrored our previous findings in IEC-6 ​cells. Conclusion: The study suggests that the alleviating effect of Forsythiae Fructus and its primary components, phillyrin and forsythoside A, against cisplatin-induced cytotoxicity may be attributed to inhibiting oxidative stress, downregulating the NLRP3/caspase-1/GSDMD pathway, and inhibiting pyroptosis.

Keywords

• Forsythiae fructus
• Cisplatin
• Cytotoxicity
• NLRP3 inflammasome
• Pyroptosis