Grape Seed Procyanidin B2 Protects Porcine Ovarian Granulosa Cells against Oxidative Stress-Induced Apoptosis by Upregulating let-7a Expression.

Oxidative stress is a causal factor and key promoter of all kinds of reproductive disorders related to granulosa cell (GC) apoptosis that acts by dysregulating the expression of related genes. Various studies have suggested that grape seed procyanidin B2 (GSPB2) may protect GCs from oxidative injury, though the underlying mechanisms are not fully understood. Therefore, whether the beneficial effects of GSPB2 are associated with microRNAs, which have been suggested to play a critical role in GC apoptosis by regulating the expression of protein-coding genes, was investigated in this study. The results showed that GSPB2 treatment protected GCs from a H2O2-induced apoptosis, as detected by an MTT assay and TUNEL staining, and increased let-7a expression in GCs. Furthermore, let-7a overexpression markedly increased cell viability and inhibited H2O2-induced GC apoptosis. Furthermore, the overexpression of let-7a reduced the upregulation of Fas expression in H2O2-treated GCs at the mRNA and protein levels. Dual-luciferase reporter assay results indicated that let-7a directly targets the Fas 3'-UTR. Furthermore, the overexpression of let-7a enhanced the protective effects of GSPB2 against GC apoptosis induced by H2O2. These results indicate that GSPB2 inhibits H2O2-induced apoptosis of GCs, possibly through the upregulation of let-7a.

Effect of hyperbaric oxygen on the permeability of the blood-brain barrier in rats with global cerebral ischemia/reperfusion injury.

OBJECTIVE: The aim of this study was to investigate the effects of hyperbaric oxygen on the permeability of the blood-brain barrier in rats with global cerebral ischemia/reperfusion injury and explore possible mechanisms. METHODS: A rat model of global cerebral ischemia/reperfusion injury established via Pulsinelli four-vessel occlusion method and a total of 162 Wistar rats were randomly divided into three groups, including sham group, global cerebral ischemia/reperfusion group (IR group) and hyperbaric oxygen treated group (HBO group). Permeability of the blood-brain barrier of these rats were evaluated by Evans Blue staining. The expression of caveolin-1 and tight junction protein ZO-1 was examined by Immunohistochemistry staining and western-blotting. RESULTS: Successfully establishment of the rat model was verified by W:D ratio, and significantly increased Evans Blue level was found in IR group compared to control group, whereas hyperbaric treatment could result in decreased Evans Blue level in HBO group. Increased expression of caveolin-1 and tight junction protein ZO-1 were found in rats with hyperbaric oxygen exposure compared to those in IS group. CONCLUSIONS: Hyperbaric oxygen exposure improved the permeability of the blood-brain barrier in rats with global cerebral ischemia/reperfusion injury, and increased expression of caveolin-1 and tight junction protein ZO-1 were involved in the mechanisms.

HPPR encodes the hydroxyphenylpyruvate reductase required for the biosynthesis of hydrophilic phenolic acids in Salvia miltiorrhiza.

Salvia miltiorrhiza is a medicinal plant widely used in the treatment of cardiovascular and cerebrovascular diseases. Hydrophilic phenolic acids, including rosmarinic acid (RA) and lithospermic acid B (LAB), are its primary medicinal ingredients. However, the biosynthetic pathway of RA and LAB in S. miltiorrhiza is still poorly understood. In the present study, we accomplished the isolation and characterization of a novel S. miltiorrhiza Hydroxyphenylpyruvate reductase (HPPR) gene, SmHPPR, which plays an important role in the biosynthesis of RA. SmHPPR contained a putative catalytic domain and a NAD(P)H-binding motif. The recombinant SmHPPR enzyme exhibited high HPPR activity, converting 4-hydroxyphenylpyruvic acid (pHPP) to 4-hydroxyphenyllactic acid (pHPL), and exhibited the highest affinity for substrate 4-hydroxyphenylpyruvate. SmHPPR expression could be induced by various treatments, including SA, GA3, MeJA and Ag+, and the changes in SmHPPR activity were correlated well with hydrophilic phenolic acid accumulation. SmHPPR was localized in cytoplasm, most likely close to the cytosolic NADPH-dependent hydroxypyruvate reductase active in photorespiration. In addition, the transgenic S. miltiorrhiza hairy roots overexpressing SmHPPR exhibited up to 10-fold increases in the products of hydrophilic phenolic acid pathway. In conclusion, our findings provide a new insight into the synthesis of active pharmaceutical compounds at molecular level.

Critical Role of FoxO1 in Granulosa Cell Apoptosis Caused by Oxidative Stress and Protective Effects of Grape Seed Procyanidin B2.

Reactive oxygen species (ROS) are closely related to the follicular granulosa cell apoptosis. Grape seed procyanidin B2 (GSPB2) has been reported to possess potent antioxidant activity. However, the GSPB2-mediated protective effects and the underlying molecular mechanisms in granulosa cell apoptosis process remain unknown. In this study, we showed for the first time that GSPB2 treatment decreased FoxO1 protein level, improved granulosa cell viability, upregulated LC3-II protein level, and reduced granulosa cell apoptosis rate. Under a condition of oxidative stress, GSPB2 reversed FoxO1 nuclear localization and increased its level in cytoplasm. In addition, FoxO1 knockdown inhibited the protective effects of GSPB2 induced. Our findings suggest that FoxO1 plays a pivotal role in regulating autophagy in granulosa cells, GSPB2 exerts a potent and beneficial role in reducing granulosa cell apoptosis and inducing autophagy process, and targeting FoxO1 could be significant in fighting against oxidative stress-reduced female reproductive system diseases.

Computational identification and systematic classification of novel GRAS genes in Isatis indigotica.

Isatis indigotica Fort., belonging to Cruciferae, is one of the most commonly used plants in traditional Chinese medicine. The accumulation of the effective components of I. indigotica is related with its growth conditions. The GRAS genes are members of a multigene family of transcriptional regulators that play a crucial role in plant growth. Although the activities of many GRAS genes have long been recognized, only in recent years were some of them identified and functionally characterized in detail. In the present study, 41 GRAS genes were identified from I. indigotica through bioinformatics methods for the first time. They were classified into ten groups according to the classification of Arabidopsis and rice. The characterization, gene structure, conserved motifs, disordered N-terminal domains, and phylogenetic reconstruction of these GRASs were analyzed. Forty-three orthologous gene pairs were shared by I. indigotica and Arabidopsis, and interaction networks of these orthologous genes were constructed. Furthermore, gene expression patterns were investigated by analysis in methyl jasmonate (MeJA)-treated I. indigotica hairy roots based on RNA-seq data. In conclusion, this comprehensive analysis would provide rich resources for further studies of GRAS protein functions in this plant.

Deep Sequencing Reveals the Effect of MeJA on Scutellarin Biosynthesis in Erigeron breviscapus.

BACKGROUND: Erigeron breviscapus, a well-known traditional Chinese medicinal herb, is broadly used in the treatment of cerebrovascular disease. Scutellarin, a kind of flavonoids, is considered as the material base of the pharmaceutical activities in E. breviscapus. The stable and high content of scutellarin is critical for the quality and efficiency of E. breviscapus in the clinical use. Therefore, understanding the molecular mechanism of scutellarin biosynthesis is crucial for metabolic engineering to increase the content of the active compound. However, there is virtually no study available yet concerning the genetic research of scutellarin biosynthesis in E. breviscapus. RESULTS: Using Illumina sequencing technology, we obtained over three billion bases of high-quality sequence data and conducted de novo assembly and annotation without prior genome information. A total of 182,527 unigenes (mean length = 738 bp) were found. 63,059 unigenes were functionally annotated with a cut-off E-value of 10(-5). Next, a total of 238 (200 up-regulated and 38 down-regulated genes) and 513 (375 up-regulated and 138 down-regulated genes) differentially expressed genes were identified at different time points after methyl jasmonate (MeJA) treatment, which fell into categories of 'metabolic process' and 'cellular process' using GO database, suggesting that MeJA-induced activities of signal pathway in plant mainly led to re-programming of metabolism and cell activity. In addition, 13 predicted genes that might participate in the metabolism of flavonoids were found by two co-expression analyses in E. breviscapus. CONCLUSIONS: Our study is the first to provide a transcriptome sequence resource for E. breviscapus plants after MeJA treatment and it reveals transcriptome re-programming upon elicitation. As the result, several putative unknown genes involved in the metabolism of flavonoids were predicted. These data provide a valuable resource for the genetic and genomic studies of special flavonoids metabolism and further metabolic engineering in E. breviscapus.

[Effect of Cordyceps sinensis powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats].

OBJECTIVE: To observe the effect of Cordyceps sinensis (CS) powder on renal oxidative stress and mitochondria functions in 5/6 nephrectomized rats, and to primarily explore its possible mechanisms. METHODS: Totally 30 male Sprague-Dawley rats were divided into the sham-operation group, the model group, and the treatment group by random digit table, 10 in each group. A chronic kidney disease (CKD) rat model was prepared by one step 5/6 nephrectomy. Rats in the treatment group were intragastrically administered with CS powder solution at the daily dose of 2 g/kg, once per day. Equal volume of double distilled water was intragastrically administered to rats in the sham-operation group and the model group. All medication lasted for 12 weeks. The general condition of rats, their body weight, blood pressure, 24 h proteinuria, urinary N-acetyl-ß-D-glucosaminidase (NAG), serum creatinine (SCr) , and blood urea nitrogen (BUN) were assessed before surgery, at week 2, 4, 6, 8, 10, and 10 after surgery. Pathological changes of renal tissues were observed under light microscope. Morphological changes of mitochondria in renal tubular epithelial cells were observed under transmission electron microscope. Activities of antioxidant enzymes including reduced glutathione (GSH), manganese superoxide dismutase (MnSOD), and malondialdehyde (MDA) in fresh renal tissue homogenate were detected. Mitochondria of renal tissues were extracted to detect levels of mitochondrial membrane potential and changes of reactive oxygen species (ROS). And expressions of cytochrome-C (Cyto-C) and prohibitin in both mitochondria and cytoplasm of the renal cortex were also measured by Western blot. RESULTS: (1) Compared with the sham-operation group, body weight was significantly decreased at week 2 (P <0. 01), but blood pressure increased at week 4 (P <0. 05) in the model group. Compared with the model group, body weight was significantly increased at week 12 (P <0. 01), but blood pressure decreased at week 8 (P < 0. 01) in the treatment group. (2) Compared with the sham-operation group, 24 h proteinuria, urinary NAG, blood SCr and BUN significantly increased in the model group (all P <0. 01). Compared with the model group, blood and urinary biochemical indices all significantly decreased in the treatment group (all P <0. 01). (3) Results of pathological renal scoring: Glomerular sclerosis index, scoring for tubulointerstitial fibrosis, degree of tubulointerstitial inflammatory infiltration were all obviously higher in the model group than in the sham-operation group (all P <0. 01). All the aforesaid indices were more obviously improved in the treatment group than in the model group (all P <0. 01). (4) Compared with the sham-operation group, activities of MnSOD and GSH-Px were significantly reduced, but MDA contents obviously increased in the renal cortex of the model group (all P <0. 01). Compared with the model group, activities of MnSOD and GSH-Px obviously increased (P <0. 05, P <0. 01), but MDA contents obviously decreased in the renal cortex of the treatment group (P <0. 01). (5) Compared with the sham-operation group, the mitochondrial membrane potential significantly decreased, but ROS levels significantly increased in the model group (all P <0.01). Compared with the model group, mitochondrial transmembrane potential increased in the treatment group, thereby inhibiting the tendency of increased production of ROS (both P < 0. 01). (6) Results of Western blot showed that, compared with the sham-operation group, expression levels of mitochondrial Cyto-C and Prohibitin were significantly reduced in the renal cortex (P <0. 01), but significantly elevated in the cytoplasm of the model group (P <0. 01). Compared with the model group, each index was obviously improved in the treatment group with statistical difference (P <0. 05, P <0. 01). CONCLUSION: CS powder had renal protection, and its mechanism might partially depend on in- hibition of oxidative stress and protection for mitochondria.

Inhibition of TGF-ß1/Smad signal pathway is involved in the effect of Cordyceps sinensis against renal fibrosis in 5/6 nephrectomy rats.

UNLABELLED: The present study aimed to investigate the effects of Cordyceps sinensis on renal fibrosis and its possible mechanisms. Sprague-Dawley rats were randomly divided into three groups: sham operation (SHAM) group, 5/6 subtotal nephrectomy (SNx) untreated group, and 5/6 subtotal nephrectomy treated with C. sinensis (2.0 g/kg d) (CS) group. Rats were studied 12 weeks after the surgery, and the CS group presented with significantly lower proteinuria, and better renal function compared with the SNx group (p<0.05). Pathological study showed that the glomerulosclerosis tubulointerstitial injury score was significantly reduced in the CS group compared with the SNx group. Furthermore, the mRNA expression of TGF-ß1, Smad2 and Smad3 and the protein expression of TGF-ß1, TßRI, TßRII and p-Smad2/3 were attenuated by the C. sinensis treatment. In constrast, the mRNA and protein expression of Smad7 was upregulated. Furthermore, the expression of α-SMA and FSP1 was also significantly attenuated, accompanied by the increasing expression of E-cadherin, suggesting the inhibition of the epithelial-mesenchymal transition (EMT). IN CONCLUSION: C. sinensis exerted its antifibrotic effect on the SNx rats through the inhibition of the TGF-ß1/Smad pathway.

Pirfenidone inhibits macrophage infiltration in 5/6 nephrectomized rats.

Tubulointerstitial macrophage infiltration is a hallmark of chronic kidney disease involved in the progression of renal fibrosis. Pirfenidone is a newly identified antifibrotic drug, the potential mechanism of which remains unclear. The aim of this study was to investigate the effects of pirfenidone on M1/M2 macrophage infiltration in nephrectomized rats. Nephrectomized rats were treated with pirfenidone by gavage for 12 wk. Twenty-four hour urinary protein, N-acetyl-ß-D-glycosaminidase (NAG) activity, systolic blood pressure, and C-reactive protein were determined. Paraffin-embedded sections were stained for CD68, CCR7, and CD163 macrophages. Monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α (MIP-1α), as well as M1 and M2 macrophages secretory markers, were evaluated by real-time RT-PCR and Western blotting analysis. Pirfenidone significantly improved the elevated proteinuria and NAG activity from week 2 onward after surgery. Pirfenidone attenuated interstitial fibrosis and decreased expression of fibrotic markers including transforming growth factor-ß(1), connective tissue growth factor, α-smooth muscle actin, fibronectin, and fibroblast-specific protein-1. Pirfenidone significantly decreased the infiltrating macrophages. The number of M1 and M2 macrophages was significantly lower after pirfenidone treatment. MCP-1 and MIP-1α were increased in nephrectomized rats at mRNA and protein levels. Pirfenidone treatment significantly inhibited their expression. The TNF-α, IL-6, and nitric oxide synthases-2 expressed by M1 macrophages were increased in nephrectomized rats, and pirfenidone significantly attenuated their expression. Pirfenidone treatment also significantly decreased arginase-1, dectin-1, CD206, and CD86 expressed by M2 macrophages. Thus pirfenidone inhibits M1 and M2 macrophage infiltration in 5/6 nephrectomized rats, which suggests its efficacy in the early and late periods of renal fibrosis.

Molecular characterization and association analysis of porcine adipose triglyceride lipase (PNPLA2) gene.

The adipose triglyceride lipase (PNPLA2, also known as ATGL) is a novel triacylglycerol (TG) lipase which specifically removes the first fatty acid from the triglyceride molecule generating free fatty acid and diglyceride (DG) in mammalian cells. Here we describe the molecular characterization of the porcine ATGL gene. The full-length cDNA sequence contains a 1,461 bp open reading frame encoding a protein of 486 amino acids with a calculated molecular mass of 53.2 kDa and an isoelectric point of 7.90. The porcine ATGL protein shares high identity with other mammalian ATGL. The ATGL gene contains 9 coding exons, spans approximately 6 kb. The porcine ATGL mRNA was expressed predominantly in backfat, mildly in muscle, small intestine and heart, and almost absent in liver, spleen, lung, stomach, kidney and ovary. Statistical analysis showed the ATGL gene polymorphism (G/A(392)) was different between Chinese indigenous and introduced commercial western pig breeds, and was highly associated with almost all the fat deposition and carcass traits, including subcutaneous fat thickness, viscera adipose tissue, lean percentage, loin eye traits and even rib numbers.