[Effect of electroacupuncture on autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on the autophagy of ovarian granulosa cells in rats with premature ovarian insufficiency (POI), and explore the mechanism of EA in improving POI. METHODS: Thirty-two female SD rats were randomly divided into a blank group (n=8) and a model making group (n=24). The rats in the model making group were injected intraperitoneally with cyclophosphamide for 15 days to establish the POI model (the dosage on the 1st day was 50 mg/kg, and 8 mg/kg from the 2nd day to 15th day). The successfully modeled rats were then randomly divided into a model group, an EA group, and an estradiol (E2) group, with 8 rats in each group. Rats in the EA group received EA at bilateral "Gongsun" (SP 4) with continuous wave, frequency of 2 Hz, and current intensity of 0.1 to 1 mA, 20 min per treatment, once daily for 14 days. Rats in the E2 group were administered with E2 (0.01 mg/mL) by gavage (10 mL/kg), once daily for 14 days. The changes in estrous cycle were observed by rapid Giemsa staining before and after modeling. After intervention, ovarian tissue morphology was observed by HE staining; serum levels of follicle-stimulating hormone (FSH), E2, anti-Mullerian hormone (AMH), and inhibin B (INHB) were detected by ELISA; immunofluorescence staining was used to observe the expression of p62 in ovarian granulosa cells; the ultrastructure of ovarian granulosa cells was observed by transmission electron microscopy, and the number of autophagosomes and autolysosomes was compared; Western blot and real-time fluorescence quantitative PCR were used to detect the protein and mRNA expression of p62, Beclin-1, and microtubule-associated protein 1A/1B-light chain 3 (LC3) in ovarian tissue. RESULTS: The results of vaginal smears in the blank group showed regular cyclical changes; the rats in the model group showed prolonged estrous cycle or cycle arrest, mostly in proestrus or metestrus, with overall ovarian atrophy, disordered structure, and decreased granulosa cells. Compared with the blank group, rats in the model group showed increased serum FSH level (P<0.01), decreased serum levels of E2, AMH, and INHB (P<0.01), decreased positive expression of p62 in ovarian granulosa cells (P<0.01), with obvious swelling of ovarian granulosa cells, mild to moderate swelling of mitochondria, slight expansion of rough endoplasmic reticulum, and hypertrophy of Golgi apparatus; the number of autophagosomes and autolysosomes in the ovaries was increased (P<0.01), the expression of p62 protein and mRNA was decreased (P<0.01), and the expression of Beclin-1 and LC3 protein and mRNA in ovarian tissue was increased (P<0.01). Compared with the model group, rats in the EA group and the E2 group showed decreased serum FSH levels (P<0.01), increased levels of E2, AMH, and INHB (P<0.01), increased positive expression of p62 in ovarian granulosa cells (P<0.01), alleviated degree of ovarian granulosa cell damage, with relatively intact organelle morphology, and decreased number of autophagosomes and autolysosomes in the ovaries (P<0.01); the rats also showed increased expression of p62 protein and mRNA (P<0.01), and decreased expression of Beclin-1 and LC3 protein and mRNA (P<0.01) in ovarian tissue. CONCLUSION: EA at "Gongsun" (SP 4) could improve ovarian reserve function in POI rats by reducing the number of autophagosomes and autolysosomes, up-regulating p62 expression, and down-regulating Beclin-1 and LC3 expression, thus inhibiting autophagy of ovarian granulosa cells, and regulating the serum levels of FSH, E2, AMH, and INHB.

High-sugar high-fat treatment induces autophagy of retinal microvascular endothelial cells.

OBJECTIVE: To investigate the role of high-sugar high-fat treatment in inducing autophagy of rat retinal microvascular endothelial cells. METHODS: The optimal concentrations and time points of glucose and oxidized low-density lipoprotein (ox-LDL) in inducing rat retinal microvascular endothelial cells were determined by examining the proliferate rate by CCK-8 assay. They were divided into control group (blank control), model group (treatment of 50 mM glucose and 10 µg/ml ox-LDL for 24 h), chloroquine group (treatment of 20 µM chloroquine, 50 mM glucose and 10 µg/ml ox-LDL for 24 h), resveratrol group (treatment of 50 µM resveratrol, 50 mM glucose and 10 µg/ml ox-LDL for 24 h) and MITO-Tempol group (treatment of 20 µM MITO-Tempol, 50 mM glucose and 10 µg/ml ox-LDL for 24 h). Reactive oxygen species (ROS) level in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment was measured by flow cytometry. In addition, protein levels of cathepsin B and cathepsin D in rat retinal microvascular endothelial cells induced with high sugar high-fat treatment were examined by immunofluorescence, and protein levels of LC3 A/B and the autophagy substrate P62 were detected by Western blot. RESULTS: Primary retinal microvascular endothelial cells were isolated from neonatal Sprague-Dawley (SD) rats. ROS level was significantly higher in model group than that of control group (P < 0.05). Compared with that of model group, ROS level was significantly reduced in chloroquine group and MITO-Tempol group, which was significantly elevated in resveratrol group (P < 0.05). Positive expressions of cathepsin B and cathepsin D were significantly reduced in model group than those of control group (P < 0.05). They were significantly elevated in chloroquine group and MITO-Tempol group, and reduced in resveratrol group than those of model group (P < 0.05). LC3 A/B and P62 were significantly upregulated in model group than those of control group (P < 0.05). Compared with those of model group, LC3 A/B and P62 were significantly downregulated in chloroquine group and MITO-Tempol group, and upregulated in resveratrol group (P < 0.05). CONCLUSION: High-sugar high-fat treatment induces autophagy of rat retinal microvascular endothelial cells, which can be intervened to a certain extent by chloroquine and MITO-Tempol.