Cangfudaotan decoction inhibits mitochondria-dependent apoptosis of granulosa cells in rats with polycystic ovarian syndrome

Abstract

Polycystic ovary syndrome (PCOS) is a universal endocrine and metabolic disorder prevalent in reproductive aged women. PCOS is often accompanied with insulin resistance (IR) which is an essential pathological factor. Although there is no known cure for PCOS, cangfudaotan (CFDT) decoction is widely used for the treatment of PCOS; nevertheless, the underlying mechanism is not clear. In this study, 40 Sprague-Dawley (SD) rats (female) were randomized to 4 groups, namely the control group, PCOS group, PCOS+CFDT group, and PCOS+metformin group. The rats in the control group were fed a normal-fat diet, intraperitoneally injected with 0.5% carboxymethyl cellulose (CMC, 1 mL/kg/d) for 21 days and orally given saline (1 mL/kg/d) for the next 4 weeks. The rats in the PCOS group, PCOS+CFDT group, and PCOS+Metformin group were fed a high-fat diet (HFD) and intraperitoneally injected with letrozole (1.0 mg/kg) for 21 days. During this period, we recorded the body weight, estrous cycles, and rate of pregnancy in all rats. We also observed the ovarian ultrastructure. Blood glucose indices, serum hormones, and inflammatory factors were also recorded. Then, we detected apoptotic and mitochondrial function, and observed mitochondria in ovarian granular cells by transmission electron microscopy. We also detected genes of ASK1/JNK pathway at mRNA and protein levels. The results showed that CFDT alleviated pathohistological damnification and apoptosis in PCOS rat model. In addition, CFDT improved ovarian function, reduced inflammatory response, inhibited apoptosis of granular cells, and inhibited the operation of ASK1/JNK pathway. These findings demonstrate the occurrence of ovary mitochondrial dysfunction and granular cell apoptosis in PCOS. CFDT can relieve mitochondria-dependent apoptosis by inhibiting the ASK1/JNK pathway in PCOS rats.

Keywords: apoptosis; cangfu daotan decoction; granulosa cell; insulin resistance; mitochondrial dysfunction; polycystic ovary syndrome.

Figure 1

CFDT decoction improved the weight, rate of pregnancy, and the rate of abnormal estrous cycle in PCOS rats model. Rats were treated with CFDT decoction (15.0 g/kg.d) and metformin (50 mg/kg) followed by feeding with HFD and intraperitoneal injection with of letrozole (1 ml/kg/d) for 21 days. (A) Weight; (B) The rate of pregnancy (Female rats of each groups were sent to mate with male rats with a ratio of 1:1 for 12 h. The mixture of sperm and vaginal smears were seen on the next morning indicated the success of pregnancy, and this was considered as the 0.5th day of gestation. The pregnant rats were euthanized on the 13.5th day of the gestation) (n=4); (C) The rate of abnormal estrous cycle, estrous cycle of the rats (Estrous interval period: Vagina smear with white blood cells mainly; Preestrus period: With nuclear epithelial cells mainly; Estrous period: With Keratinized epithelial cells mainly; Late estrous period: See Keratinized epithelial cells and white blood cells) (n=6). Data are shown as mean ± SD. *p < 0.05 versus control group, ^# p < 0.05 versus PCOS group, ^△ p < 0.05 versus PCOS+ CFDT group. δ The groups between the PCOS+ CFDT and PCOS+metformin do not have statistical difference.

Figure 2

CFDT decoction improved the ovarian index, ovarian diameter, the number of all phases of follicles, serum hormones, blood glucose indices, and inflammatory factors in PCOS rats model. Rats were treated with CFDT decoction (15.0 g/kg.d) and metformin (50 mg/kg) followed by feeding with HFD and intraperitoneal injection with of letrozole (1 ml/kg/d) for 21 days. (A) Ovarian index (Ovarian index= wet weight of bilateral ovaries (mg)/body weight (g)×100%, the size of the ovary in PCOS group was significantly reduced compared with the control group) and ovarian diameter, the scale bars represent a length of 1 cm on histology; (B) Histological assessment of the ovarian tissue using hematoxylin-eosin (HE) staining [All phases of follicles (primary follicles, secondary follicles, and atretic follicles) and corpora lutea were counted], the scale bars represents a length of 20 μm on histology; (C) Serum hormones (LH, FSH, T, and E₂); (D) Blood glucose indices (FBG, FINS, and HOMA-IR); (E) Serum inflammatory factors (TNF- α, IL-1β, IL-6, and CRP). Data are shown as mean ± SD. *p < 0.05 versus control group, ^# p < 0.05 versus PCOS group, ^△ p < 0.05 versus PCOS+ CFDT group. (n=6).

Figure 3

CFDT decoction improved mitochondrial function in PCOS rats model. Rats were treated with CFDT decoction (15.0 g/kg.d) and metformin (50 mg/kg) followed by feeding with HFD and intraperitoneal injection with of letrozole (1 ml/kg/d) for 21 days. (A) Electron microscope pictures (10,000×; 20,000×; 40 000×) of the ovary in PCOS rats, the scale bars represents a length of 2 μm, 1 μm, and 500 nm on histology respectively. Abnormal mitochondrial (paired yellow arrow) morphology showed mitochondrial membrane rupture or swellings, and normal mitochondrial (single yellow arrow) morphology type showed smooth mitochondrial membrane and distinct inner carinulae and percentage of damaged mitochondria; (B) The MMP (ratio of red/green), the opening of mPTP (%), the mitochondrial ROS, the mtDNA damage (ratio of long/short fragments), the mitochondrial RCR, mitochondrial oxygen consumption rate; (C) The mitochondrial respiratory chain complex enzymes (I, II, III, IV, and V) and ATP were recorded above. Data are shown as mean ± SD. *p < 0.05 versus control group, ^# p < 0.05 versus PCOS group, ^△ p < 0.05 versus PCOS+ CFDT group. (n=6).

Figure 4

CFDT decoction improved mitochondrial biogenesis and dynamics in PCOS rats model. Rats were treated with CFDT decoction (15.0 g/kg.d) and metformin (50 mg/kg) followed by feeding with HFD and intraperitoneal injection with of letrozole (1 ml/kg/d) for 21 days. We used real-time qPCR and western blot to detect mitochondrial biogenesis and dynamics. We chose OPA1, Mfn1, and Mfn2 to represent mitochondrial biogenesis function, PGC-1 α to represent the dynamic mitochondrial fusion, and Drp1 and Fis1 to represent mitochondrial fission. The expression of OPA1, Mfn1, Mfn2, PGC-1 α, Drp1, and Fis1 at mRNA (A) and protein (B) levels. Data are shown as mean ± SD. *p < 0.05 versus control group, ^# p < 0.05 versus PCOS group, ^△ p < 0.05 versus PCOS+ CFDT group. (n=6).

Figure 5

CFDT decoction improved mitochondrial function and inhibited apoptosis via inhibiting the ASK1/JNK pathway. Rats were treated with CFDT decoction (15.0 g/kg.d) and metformin (50 mg/kg) followed by feeding with HFD and intraperitoneal injection with of letrozole (1 ml/kg/d) for 21 days. (A) Graphical abstract: Mitochondrial dysfunction of the ovarian granular cells occurs following PCOS and can lead to granular cells apoptosis. CFDT decoction can relieve granular cell apoptosis by improving mitochondrial function via inhibiting the ASK1/JNK pathway in vivo. We suggested that CFDT decoction be used as a potential therapeutic agent for PCOS and mitochondria are seen as a potential therapeutic target. We used real-time qPCR and western blot to detect the target genes of the ASK1/JNK pathway at mRNA (B) and protein (C) levels. (D) TUNEL positive cells, the scale bars represent a length of 50 μm on histology; (E) The activity of caspase-9/3. Data are shown as mean ± SD. *p < 0.05 versus control group, ^# p < 0.05 versus PCOS group, ^△ p < 0.05 versus PCOS+ CFDT group. (n=6).