〔Abstract〕 Objective To explore the role of mitochondrial autophagy in ovarian inflammation associated with polycystic ovary syndrome(PCOS) based on the NOD-like receptor thermoprotein domain-related protein 3(NLRP3) pathway. Methods Human ovarian granulosa cell line SVOG was treated with 25 nmol/L dihydrotestosterone(DHT) for 24 h to establish PCOS cell model. SVOG cells were transfected with adenovirus carrying NLRP3(Ad-NLRP3) and negative vector(Ad-EV) or NLRP3 shRNA(sh-NLRP3) and negative control(sh-NC) to overexpress or knockdown NLRP3. Mito-Tracker staining and GFP-LC3 staining were used to evaluate mitochondrial autophagy in cells. TUNEL staining, JC-1 staining and Mito-SOX staining were used to analyze the apoptosis, mitochondrial membrane potential and mitochondrial-derived superoxide production. 32 female BALB/c mice were randomly divided into three groups: control(Con) group, DHEA group, DHEA+sh-NC group and DHEA+sh-NLRP3 group, with 8 mice in each group. Except the control group, all other groups treated mice with dehydroepiandrosterone(DHEA) to establish PCOS mouse model. DHEA+sh-NLRP3 group and DHEA+sh-NC group were administrated with sh-NLRP3 or sh-NC encapsulated in lentivirus at a concentration of 1×109TU/mlviatail vein injection. The ultrastructure of mitochondria in ovarian tissue of mice in each group was observed by transmission electron microscope. Results Compared with DHT+sh-NC group, the level of NLRP3 of SVOG cells in DHT+sh-NLRP3 group decreased(P<0.05). The co-location of GFP-LC3 and mitochondria in SVOG cells in DHT+sh-NLRP3 group was higher than that in DHT+sh-NC group(P<0.05). Compared with DHT+sh-NC group, the number of TUNEL positive cells and Mito-SOX fluorescence density of SVOG cells in DHT+sh-NLRP3 group decreased, and the ratio of polymer JC-1 to monomer JC-1 increased(P<0.05). Compared with Con+Ad-EV group, the level of NLRP3, the number of TUNEL-positive cells and the fluorescence density of mito-SVOG in Con+Ad-NLRP3 group increased(P<0.05), and the co-location level of GFP-LC3 and mitochondria decreased; the ratio of polymer JC-1 to monomer JC-1 decreased(P<0.05). Compared with the control group, TUNEL positive cells, relative ROS intensity and percentage of damaged mitochondria in the ovarian tissue of mice in DHEA group increased(P<0.05). Compared with DHEA+sh-NC group, TUNEL positive cells, relative ROS intensity and percentage of damaged mitochondria in DHEA+SH-NLRP group decreased(P<0.05). Conclusion Inhibition of mitochondrial autophagy induced by activation of NLRP3 leads to mitochondrial dysfunction and promotes mitochondrial-related apoptosis in GCs. Knockdown of NLRP3 is beneficial to mitochondrial homeostasis and improves the resistance of GCs to oxidative stress injury, thus promoting the recovery of PCOS.