Characterization on gut microbiome of PCOS rats and its further design by shifts in high-fat diet and dihydrotestosterone induction in PCOS rats.

Polycystic ovary syndrome (PCOS) is associated with gut microbiota disturbance. Emerging evidence has shown that gut microbiota plays a major role in the development of PCOS. To better understand how the gut microbiota contributes to the development of PCOS, we investigated the influences of high-fat diet and hyperandrogenism, independently or synergistically, have on the gut microbiota in rats. Furthermore, we explored the associations between gut microbiota and hyperandrogenism or other hallmarks of PCOS. Twenty female SD rats were randomized at aged 3 weeks into 4 groups (n = 5, each); HA: PCOS rats fed with ordinary diet; HF: rats with high-fat diet (HFD); HA-HF: PCOS rats fed with HFD; and C: control rats with ordinary diet. PCOS rat model was induced by 5α-dihydrotestosterone (DHT) injection for 6 weeks. The fasting blood glucose (FBG), plasma insulin, testosterone, free testosterone, TNF-α, MDA, SOD, LPS, TLR4, TG, TC, HDL-C, and LDL-C levels were measured. The molecular ecology of the fecal gut microbiota was analyzed by 16S rDNA high-throughput sequencing. The results showed that rats in the HA and HA-HF group displayed abnormal estrous cycles with increasing androgen level and exhibited multiple large cysts with diminished granulosa layers in ovarian tissues. Compare with the C group, relative abundance of the Bacteroidetes phylum decreased significantly in the other groups (P < 0.05). The Chao1 was the highest in the group C and significantly higher than the HA-HF group (P < 0.05). T, FT, insulin, MDA, LPS, and TNF-α levels had the negative correlation with the richness of community (Chao1 index) in the gut. The rats in the HF and HA-HF groups tended to have lower Shannon and Simpson indices than the C group (P < 0.01, respectively). However, there were no significant differences between C group and the HA group in the Shannon and Simpson values. Beta diversity analysis was then performed based on a weighted UniFrac analysis. The PCoA plots showed a clear separation of the C group from the other groups. ANOSIM analysis of variance confirmed that there were statistically significant separations between the C group and the HA, HA-HF, and HF groups (P < 0.01, respectively). These results showed that DHT with HFD could lower diversity of the gut microbial community. Both HFD and DHT could shift the overall gut microbial composition and change the composition of the microbial community in gut. Furthermore, our analyses demonstrated that the levels of TG, MDA, TNF-α, LPS, TLR4, T, FT, FINS, and HDL-C were correlated with the changes of in the gut microbiome. HFD and DHT were associated with the development and pathology of PCOS by shaping gut microbial communities.

Integrative proteomics and immunochemistry analysis of the factors in the necrosis and repair in acetaminophen-induced acute liver injury in mice.

Acetaminophen (APAP) overdose-induced acute liver injury (AILI) is a significant clinical problem worldwide, the hepatotoxicity mechanisms are well elucidated, but the factors involved in the necrosis and repair still remain to be investigated. APAP was injected intraperitoneally in male Institute of Cancer Research (ICR) mice. Quantitative proteome analysis of liver tissues was performed by 2-nitrobenzenesulfenyl tagging, two-dimensional-nano high-performance liquid chromatography separation, and matrix-assisted laser desorption/ionization-time of flight mass spectrometry analysis. Diffrenetial proteins were verified by the immunochemistry method. 36 and 44 differentially expressed proteins were identified, respectively, at 24 hr after APAP (200 or 300 mg·kg -1 ) administration. The decrease in the mitochondrial protective proteins Prdx6, Prdx3, and Aldh2 accounted for the accumulation of excessive reactive oxygen species (ROS) and aldehydes, impairing mitochondria structure and function. The Gzmf combined with Bax and Apaf-1 jointly contributed to the necrosis. The blockage of Stat3 activation led to the overexpression of unphosphorylated Stat3 and the overproduction of Bax. The overexpression of unphosphorylated Stat3 represented necrosis; the alternation from Stat3 to p-Stat3 in necrotic regions represented hepatocytes from death to renewal. The high expressions of P4hα1, Ncam, α-SMA, and Cygb were involved in the liver repair, they were not only the markers of activated HSC but also represented an intermediate stage of hepatocytes from damage or necrosis to renewal. Our data provided a comprehensive report on the profile and dynamic changes of the liver proteins in AILI; the involvement of Gzmf and the role of Stat3 in necrosis were revealed; and the role of hepatocyte in liver self-repair was well clarified.

Suppression of MIF-induced neuronal apoptosis may underlie the therapeutic effects of effective components of Fufang Danshen in the treatment of Alzheimer's disease.

Fufang Danshen (FFDS or Compound Danshen) consists of three Chinese herbs Danshen (Salviae miltiorrhizae radix et rhizome), Sanqi (Notoginseng radix et rhizome) and Tianranbingpian (Borneolum, or D-borneol), which has been show to significantly improve the function of the nervous system and brain metabolism. In this study we explored the possible mechanisms underlying the therapeutic effects of the combination of the effective components of FFDS (Tan IIA, NG-R1 and Borneol) in the treatment of Alzheimer's disease (AD) based on network pharmacology. We firstly constructed AD-related FFDS component protein interaction networks, and revealed that macrophage migration inhibitory factor (MIF) might regulate neuronal apoptosis through Bad in the progression of AD. Then we investigated the apoptosis-inducing effects of MIF and the impact of the effective components of FFDS in human neuroblastoma SH-SY5Y cells. We observed the characteristics of a "Pendular state" of MIF, where MIF (8 ng/mL) increased the ratio of p-Bad/Bad by activating Akt and the IKKα/ß signaling pathway to assure cell survival, whereas MIF (50 ng/mL) up-regulated the expression of Bad to trigger apoptosis of SH-SY5Y cells. MIF displayed neurotoxicity similar to Aß1-42, which was associated with the MIF-induced increased expression of Bad. Application of the FFDS composite solution significantly decreased the expression levels of Bad, suppressed MIF-induced apoptosis in SH-SY5Y cells. In a D-galactose- and AlCl3-induced AD mouse model, administration of the FFDS composite solution significantly improved the learning and memory, as well as neuronal morphology, and decreased the serum levels of INF-γ. Therefore, the FFDS composite solution exerts neuroprotective effects through down-regulating the level of Bad stimulated by MIF.

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.

Protective effects of blocking renin-angiotensin system on the progression of renal injury in glomerulosclerosis.

To investigate the protective effects of blocking rennin-angiotensin system (RAS) on the progression of renal injury in glomerulosclerosis, a glomerulosclerosis model was made for SD rats by unilateral nephrectomy and being injected with Adriamycin into caudal vein. The rats with glomerulosclerosis were randomly divided as ten per group into those without further treatment (group D) and those treated with Benazepril (group DB), Losartan (group DL), or sham-operation (group C), respectively. After 6 weeks of administration of Benazepril or Losartan, the mRNA expressions of TGF-beta1, Col IV, Fn, ET-1 and iNOS in renal cortex were measured by RT-PCR. Besides, the expressions of TGF-beta1, ET-1 and iNOS at protein level were detected by Western blotting and the concentrations of Col IV and Fn were analyzed with immunohistochemistry respectively. Results showed that the rats in group D appeared as obvious proteinuria, hypoalbuminemia and hypercholesterolemia, which had a significant difference compared with group C (p < 0.05), and most of their mesangiums were detected with cellular proliferation and significant increasing for extracellular matrix. Renal cortex TGF-beta1, Col IV, Fn, ET-1 and iNOS in rats of group D were increased by 3.59, 2.57, 2.21, 2.58 and 3.28 times at mRNA level, and by 2.60, 1.40, 0.75, 1.83 and 2.15 times at protein level, respectively, compared with group C. When the animals were treated with Benazepril (group DB) or Losartan (group DL), however, the biochemical and pathological damages were significantly recovered, and protein expressions of TGF-beta1, Col IV, Fn, ET-1 and iNOS were also significantly diminished (p < 0.05). This study suggested that blocking RAS using Benazepril or Losartan can have protective effects on the renal injury in glomerulosclerosis by down-regulating the expressions of TGF-beta1, Col IV, Fn, ET-1 and iNOS.

[Effects of rhein on activity of caspase-3 in kidney and cell apoptosis on the progression of renal injury in glomerulosclerosis].

OBJECTIVE: To investigate the effects of rhein on the progression of renal injury and cell apoptosis in glomerulosclerosis, and further explore the protective mechanism of rhein on glomerulosclerosis. METHODS: Glomerulosclerosis models were made for SD rats by unilateral nephrectomy and being injected with Adriamycin into caudal vein, and randomly divided into control group, renal disease group, Rhein treatment group and Benazepril treatment group, and 6 rats in each group were killed at the 6th, 8th, 10th, 12th week respectively. The apoptosis protease-3 (caspase-3) in renal cortex was determined by immunohistochemistry stain method, and the activity of caspase-3 was measured by colorimetry, and the activity of nuclear factor-kappa B (NF-kappaB) was analyzed by gel electrophoretic mobility shift assay (EMSA), and renal tissue cell apoptosis was tested by terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) in order to observe expressions of caspase-3 and NF-kappaB and cell apoptosis of renal tissue. RESULTS: Renal disease group presented with distinct proteinuria, decreasing of blood albumin content and increasing of cholesterol concentration. Glomerulosclerosis index, apoptosis index, activity of NF-kappaB and expression of caspase-3 in renal disease group were more significantly higher than those in control group (P < 0.05 or P < 0.01) as time passed. Compared with the other time points in renal disease group, there were a great number of TUNEL-positive cells observed at the 10th week, slightly higher than that at the 12th week (9.3 +/- 2.3 vs 8.4 +/- 1.2, P > 0.05), the expression of Caspase-3 was also most obvious at the 10th week, significantly higher than that at the 12th week (11.4 +/- 2.5 vs 8.2 +/- 1.7, P < 0.05), which mainly located around capillary vessel in renal cortex, tending to be consistent with apoptosis cells expression. After the 8 weeks treatment of rhein or Benazepril, the number of TUNEL-positive cells significantly decreased and maintained at a certain level, and the activity of NF-kappaB and expression of caspase-3 decreased (P < 0.05), and renal pathological changes and biochemical changes improved magnificently, moreover, the expression of caspase-3 showed positive correlation with apoptosis index (r = 0.836, P < 0.01). CONCLUSION: Rhein could have significant protective effects on the progression of renal injury, and might regulate pathological changes by influencing the activities of NF-kappaB and caspase-3 in the early phase of glomerulosclerosis. Therefore, down-regulating caspase-3 expression in kidney might be one of the molecular mechanisms in the way that rhein could alleviate renal tissue cell apoptosis in glomerulosclerosis.