Trishizukaol A alleviates chronic kidney disease by restraining P2X7/PSME3-driven 20S proteasome hyperactivation

Ethnopharmacological relevance Sarcandra glabra ( S. glabra ) has been traditionally used to promote diuresis and reduce swelling, which is employed to treat facial and lower limb edema, one of the earliest and most visible external signs of chronic kidney disease (CKD). The alleviating effect of S . glabra on CKD and its mechanism remains unclear. Aim of the study This study aims to determine the therapeutic effect of trishizukaol A (TSA), a lindenane sesquiterpenoid isolated from S. glabra , on CKD and investigate its underlying mechanism. Methods First, the biolayer interferometry (BLI) method was used to screen compounds from S . glabra targeting P2X7. Next, the effects of TSA on different stages of CKD were examined. The protein differences between P2X7 KO and WT mice were assessed using proteomic methods. Then, a series of biochemical methods was employed to explore the mechanism by which TSA alleviates CKD through P2X7. Results In mouse models of CKD, TSA significantly reduced urinary albumin excretion and decreased KIM-1 and NGAL, supporting that TSA has a notable ameliorative effect on CKD. Target screening results showed that TSA bound to and inhibited the P2X7 protein. Furthermore, we found that P2X7 was highly expressed in the tubular epithelial cells (TECs) of CKD mice, and blocking this receptor attenuated renal injury. In vivo , proteomic analysis comparing shP2X7 and shCTRL mice identified significant differential protein expression. P2X7 knockdown was predominantly associated with proteasome pathways. In vitro , siP2X7 and siPSME3 inhibited 20s proteasome activation, activated autophagy, and reduced TEC injury. At the same time, activation of autophagy can inhibit the proteasome. These results supported that P2X7 can regulate the crosstalk between proteasome and autophagy and promote TEC injury. In conclusion, TSA alleviates CKD in mice and regulates the P2X7/PSME3 axis, leading to the inactivation of the 20S proteasome hyper-activation and promoting autophagy in TECs. Conclusions This study reveals that TSA alleviates CKD by inhibiting P2X7/PSME3-driven hyperactivation of the 20S proteasome. TSA serves as a mechanistic probe and lead scaffold for dissecting the P2X7-proteasome axis in CKD.