Effect of High-Fat Diet on the Intestinal Flora in Letrozole-Induced Polycystic Ovary Syndrome Rats.

AIM: The aim of this study was to explore whether letrozole and high-fat diets (HFD) can induce obese insulin-resistant polycystic ovary syndrome (PCOS) with intestinal flora dysbiosis in a rat model. We compared the changes in the intestinal flora of letrozole-induced rats fed with HFD or normal chow, to explore the effects of HFD and letrozole independently and synergistically on the intestinal flora. METHODS: Five-week-old female Sprague Dawley (SD) rats were divided into four groups: control (C) group fed with regular diet; L1 group administered with letrozole and fed with regular diet; L2 group received letrozole and fed with HFD; and HFD group fed with HFD. At the end of the experiment, ovarian morphology, hormones, metabolism, oxidative stress, and inflammatory status of all rats were studied. 16S rDNA high-throughput sequencing was used to profile microbial communities, and various multivariate analysis approaches were used to quantitate microbial composition, abundance, and diversity. RESULTS: Compared to the C group, the increased plasma fasting insulin and glucose, HOMA-IR, triglyceride, testosterone, and malondialdehyde were significantly higher in the L2 group, while high-density lipoprotein cholesterol was significantly lower in the L1 group and L2 group. The indices of Chao1 and the Abundance-based Coverage Estimator (ACE) (α-diversity) in the L2 and HFD groups were significantly lower than that in the C group. Bray-Curtis dissimilarity based principal coordinate analysis (PCoA) plots and analysis of similarities (ANOSIM) test showed obvious separations between the L2 group and C group, between the HFD group and C group, and between the L2 and HFD groups. At the phylum level, Firmicutes and ratio of Firmicutes and Bacteroidetes (F/B ratio) were increased in the L2 group; Bacteroidetes was decreased in the L2 and HFD groups. No significant differences in bacterial abundance between the C group and L1 group were observed at the phylum level. Based on linear discriminant analysis (LDA) effect size (LEfSe) analysis, the bacterial genera (the relative abundance > 0.1%, LDA > 3, p < 0.05) were selected as candidate bacterial signatures. They showed that the abundance of Vibrio was significantly increased in the L1 group; Bacteroides and Phascolarctobacterium were enriched in the HFD group, and Bacteroides, Phascolarctobacterium, Blautia, Parabacteroides, Akkermansia [Ruminococcus]_torques_group, and Anaerotruncus were enriched in the L2 group. CONCLUSION: The effect of letrozole on intestinal flora was not significant as HFD. HFD could destroy the balance of intestinal flora and aggravate the intestinal flora dysbiosis in PCOS. Letrozole-induced rats fed with HFD have many characteristics like human PCOS, including some metabolic disorders and intestinal flora dysbiosis. The dysbiosis was characterized by an increased Firmicutes/Bacteroidetes ratio, an expansion of Firmicutes, a contraction of Bacteroidetes, and the decreased microbial richness. Beta-diversity also showed significant differences in intestinal microflora, compared with control rats.

[Effect of Jianpi Shengxue Granule on Fertility and Early Embryo Development Toxicity of Rats].

Objective To assess the effects of Jianpi Shengxue Granule (JSG) on the fertility and early embryo development in rats. Methods Totally 176 SD rats were stratified by sex, and divided into 4 groups, i.e., the control group, low, medium, high dose JSG treatment groups. Rats in the 3 JSG treatment groups were administered with JSG at 0, 1. 16, 3. 48, 5. 80 g/kg per day by gastrogavage, re- spectively. After 2 weeks of administration for females and 4 weeks of administration for males, males and females were caged in the ratio of 1:1. Females were administered to the gestation day 7. Males were administered for 59 -63 days until the day before anatomy. All parental generations were anatomized to observe signs and morphologies. Pathological examination was performed. General toxicity was detected. The testis and epididymis were weighed. Spermatozoa number was counted from epididymis, and repro- duction toxicity of sperm motility was checked. The numbers of corpus luteum, live fetus, dead fetus, and absorbed fetus were counted. The implantation number was calculated to observe early embryo de- velopment toxicity. Results (1) General toxicity: The body weight growth slowed down in male rats of the high dose JSG treatment group. No abnormality was found in low and medium dose JSG treatment groups. (2)Fertility toxicity: There was no obvious toxic effect on testis, epididymis, and spermatozoa number from epididymis sperm in all JSG treatment groups. (3) Early embryo development toxicity: No significant effect on the formation of early embryos was found in all JSG treatment groups. Conclusions Certain gastrointestinal toxicity of JSG might exist to some extent. The dose for non-adverse effect of JSG on fertility and early embryo development was 3. 38 g/kg per day. No obvious fertility or early embryo development toxicity occurred in each JSG treatment group.

[Effective Ingredients of Yangjing Zhongyu Decoction Regulated Androgen Biosyntheses by Mitogen-Activated Protein Kinase Pathway in Porcine Granulose Cells].

OBJECTIVE: To study the molecular mechanism of Yangjing Zhongyu Decoction (YZD) n-butanol extracts (ZDC) and ethyl acetate extracts (YSYZ) in reducing androgen in porcine granulose cells by mitogen-activated protein kinase (MAPK) pathway. METHODS: Porcine granulose cells were isolated and cultured. They were inoculated by MAPK inhibitor PD98059 at different concentrations, and then they were divided into the blank control group (0), 1, 3, 10, and 25 micromol/L groups. After 24-h culture the cytochrome P450c17a (CYP17) mRNA expression level was detected using Real-time fluorescent quantitative PCR. Contents of androgen (testosterone) in the supernate were detected using RIA and optimal PD98059 concentration screened. After intervened by 10 micromol/L PD98059 for 24 h, the culture solution was intervened by effective ingredients of with or without YZD or YSYZ at various concentrations (0, 1 , 5, 25, 50 mg/mL) at various time points (3, 6, 18, 24 h). Expression levels of p-ERK1/2, c-Fos and CYP17 were detected by Western blot. Testosterone content in the supernate was determined by radioimmunoassay (RIA). RESULTS: Ten pLmol/L PD98059 could obviously decrease p-ERK1/2 protein expression and increase CYP17 mRMA expression, and elevate testosterone content in the supernate (P < 0.05). ZDC and YSYZ at 25 ng/mL could increase p-ERK1/2 protein expression and c-Fos levels, and reduce CYP17 protein expression, and lower testosterone content in the supernate after 6-h intervention (P < 0.01). CONCLUSION: Effective ingredients of YZD could reduce androgen production in porcine granulose cells through increasing activities of MAPK.

[Chronic toxicity test of fangyouling by transdermal administration in rabbits].

OBJECTIVE: To observe the toxicity of fangyouling after one month' s transdermal administration in rabbits and evaluate its security. METHODS: Forty rabbits were randomly divided into 4 groups including a control group and low, middle and high dose groups of fangyouling. The rabbits in the control group were administered with sunflower oil, and the other rabbits were administrated dermally with fangyouling of 50,300 and 2,000 mg/kg respectively once a day for 4 weeks. The general condition, the skin irritation reaction, body weight, food consumption, hematology, blood biochemistry, organ coefficients and histopathological changes of all the rabbits were observed. RESULTS: There was no obvious effect on the general condition in all the rabbits. However, the mild skin irritation was observed in 2 rabbits of the middle dose group and 4 rabbits of the high-dose group. The decreases of body weight and food consumption were noted in the high dose group. No changes were detected of hematology, blood biochemistry or viscera pathological at all dose levels. CONCLUSION: The dose of non-toxic response of fangyouling is 50 mg/kg at this study condition.

[Effect of low-frequency electroacupuncture intervention on oxidative stress and glucose metabolism in rats with polycystic ovary syndrome].

OBJECTIVE: To observe the effect of low-frequency electroacupuncture (EA) treatment on oxidative stress and metabolism disorder in polycystic ovary syndrome (PCOS) rats, so as to study its underlying mechanisms in improving PCOS. METHODS: Thirty female SD rats were randomized into control group, model group and EA group (n = 10 in each group). The PCOS model was established by subcutaneous injection of testosterone propionate (1 mg/100 g, 10 mg/mL) for 8 weeks. EA (2 Hz) was applied to "Zhongwan" (CV 12) , "Guanyuan" (CV 4) and bilateral "Sanyinjiao" (SP 6) or "Housanli" (ST 36) for 30 min, once daily (except weekends) for 5 weeks. Serum testosterone (T) , sex hormone-binding globulin (SHBG) and fasting serum insulin (FINS) contents were detected by ELISA, serum malondialdehyde (MDA) and superoxide dismutase (SOD) levels were assayed by thiobarbituric acid (TBA) and xanthine oxidation (XTO) methods, respectively, and fasting blood glucose (FBG) was determined by roche glucose meter. In addition, homeostasis model assessment for insulin resistance (HOMA-IR) and modified B cell function index (MBCI) were calculated. RESULTS: Compared with the control group, serum T, FBG, FINS, HOMA-IR, and serum MDA levels were significantly increased in the model group (P < 0.01), while serum SHBG, MBCI and SOD levels were considerably decreased in the model group (P < 0.01). Following EA treatment, all the increased serum T, FBG, FINS, HOMA-IR, and serum MDA levels, and the decreased serum SHBG, MBCI and SOD levels were reversed obviously (P < 0.05, P < 0.01). CONCLUSION: EA treatment may normalize insulin sensitivity, ameliorate insulin resistance and hyperinsulinemia in PCOS rats, probably by regulating the function of pancreatic islets ß cell and by reducing oxidative stress and free androgen.

[Effect of Yangjing Zhongyu Decoction on mRNA and protein expression of PCNA, StAR, and FSHR in ovarian granulosa cells cultured by excess androgen].

OBJECTIVE: To observe the effect of Yangjing Zhongyu Decoction (YZD) on mRNA and protein expression of PCNA, StAR, and FSHR in ovarian granulose cells (GCs) cultured by excess androgen. METHODS: Ovarian GCs from porcine follicles were isolated and cultured in vitro. Follicular stimulating hormone (FSH) or YZD was added in the GCs treated by excess testosterone propionate. Totally 48 h later mRNA and protein expression of PCNA, StAR, and FSHR were detected by RT-PCR and Western blot. RESULTS: Excess androgen inhibited mRNA and protein expression of PCNA, StAR, and FSHR of GCs. FSH and YZD could antagonize inhibition of excess androgens, and promote mRNA and protein expression of PCNA, StAR, and FSHR in GCs. CONCLUSION: YZD could antagonize the inhibition of excess androgen on mRNA and protein expression of PCNA, StAR and FSHR in GCs. Thus, we inferred that YZD could improve the follicle dysplasia by promoting mRNA and protein expression of PCNA, StAR and FSHR in GCs.

Effectiveness of abdominal acupuncture for patients with obesity-type polycystic ovary syndrome: a randomized controlled trial.

OBJECTIVE: To assess the effectiveness of abdominal acupuncture at the endocrine and metabolic level in patients with obesity-type polycystic ovary syndrome (PCOS). METHODS: Eighty-six women from the First Affiliated Hospital of Guangzhou Medical College with a diagnosis of PCOS (body-mass index [BMI] ≥25 kg/m(2)) were randomly assigned to receive 6 months of abdominal acupuncture (once a day) or oral metformin (250 mg three times daily in the first week, followed by 500 mg three times daily thereafter). BMI, waist-to-hip ratio (WHR), ovarian volume, menstrual frequency, homeostasis model assessment for insulin resistance (HOMA-IR), and Ferriman-Gallwey score were measured at the beginning of the study and after 6 months of treatment. Luteotrophic hormone (LH), testosterone, follicle-stimulating hormone (FSH), fasting blood glucose, 2-hour Postprandial blood glucose, fasting insulin, 2-hour postprandial blood insulin, total cholesterol, triglycerides, low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) were also assessed. RESULTS: According to the results at baseline and 6 months, BMI, WHR, Ferriman-Gallwey score, ovarian volume, luteotrophic hormone, ratio of luteotrophic hormone to follicle-stimulating hormone, testosterone, LDL-C, triglycerides, total cholesterol, fasting blood glucose, 2-hour postprandial blood glucose, fasting insulin, 2-hour postprandial blood insulin, and HOMA-IR were reduced significantly in the two groups (p<0.05). Menstrual frequency and HDL-C (p<0.05) increased significantly in both groups; follicle-stimulating hormone also increased in both groups, but the change was not significant (p>0.05). The acupuncture group showed considerable advantages over the metformin group in terms of reduced BMI and WHR and increases in menstrual frequency (p<0.05). CONCLUSION: Abdominal acupuncture and metformin improved the endocrine and metabolic function of patients with obesity-type PCOS. Abdominal acupuncture may be more effective in improving menstrual frequency, BMI, and WHR, with few adverse effects.

[Observation on therapeutic effect of acupoint sticking at Neiguan (PC 6) and Xinshu (BL 15) for treatment of bradycardia].

OBJECTIVE: To observe the therapeutic effect of acupoint sticking of Wuzhuyu (Evodia Rutaecarpa) for treatment of bradycardia. METHODS: Sixty cases were randomly divided into an observation group and a control group, 30 cases in each group. The observation group was treated with acupoint sticking of Wuzhugu (Evodia Rutaecarpa) at Neiguan (PC 6) and Xinshu (BL 15) once each day. The control group was treated with oral administration of Xinbao pills, 3 pills each time, thrice each day. The therapeutic effects and changes of 24 hours' holter were observed after 4 weeks. RESULTS: After treatment, 24 hours' average heart rate was significantly improved in the two groups, with significant differences as compared with those before treatment (both P<0.01) and with no significant difference between the two groups (P>0.05). The total effective rate was 86.7% (26/30) in the observation group and 83.3% (25/30) in the control group, their therapeutic effect being similar. CONCLUSION: Acupoint sticking of Wuzhugu (Evodia Rutaecarpa) can significantly raise the levels of 24 hours' average heart rate in the patients of bradycardia. This therapy and Xinbao pills have similar therapeutic effect on the improvement of clinical symptom and heart rate levels.

[The apoptosis-inducing activity of human selenoprotein P shorter isoform].

OBJECTIVE: Human selenoprotein P (HSelP) is unique protein that contains 10 selenocysteines encoded by 10 inframe UGA, which typically function as stop codon. The function of HSelP remains unclear, in part due to the inability to express it by gene recombinant technique. This study is to investigate expression and purification of recombinant HSelP in prokaryotic expression system, and its activity to induce apoptosis in vitro. METHODS: The shorter HSelP isoform was cloned. After the selenocysteine (SeCys) at 40th position from N terminus of the HSelP shorter isoform was mutated into cysteine by PCR, it was expressed in E. coli. The expressed product was purified with DEAE column and identified by Western blot. Subsequently, its function on induction of mitochondrial apoptotic activity was studied. RESULTS: The mutant HSelP shorter isoform expressed in prokaryotic system was purified by DEAE column to 90% homogeneity. The purified product, HSelP280m, induced the opening of mitochondrial permeability transition pore (PTP) and decreased the transmembrane potential in a dose-dependent manner. These events could be abolished by PTP specific inhibitors. CONCLUSION: HSelP280m can induce the opening of mitochondrial PTP, which provides a basis for investigating the structure and function of recombinant HSelP.