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Matrix metalloproteinase 13 is one of three mammalian collagenases that are capable of initiating the degradation of interstitial collagens during wound healing. Herein, we report for the first time the molecular cloning of the catalytic domain (CD) of sika deer MMP-13, followed by protein expression in Escherichia coli and purification by affinity chromatography. The final yield was approximately 90.4 mg per liter of growth culture with a purity of 91.6%. The mass recovery during the purification and renaturation were 70.2% and 81.5%, respectively. Using gelatin zymography and a degradation assay, we found that the refolded sika deer MMP-13 (CD) could digest gelatin. The optimal pH and temperature for the enzyme bioactivity was 8.0 and 37 °C, respectively. The Km value for the enzyme-catalyzed digestion of gelatin was 136+/-8 μg/mL, and the Vmax was 4.12 × 10(3) U/μg. sdMMP13 (CD) was able to completely degrade collagen II and gelatin, and partially degrade fibronectin. The sdMMP-13 (CD) activity was significantly inhibited by several chemicals including 1, 10-phenanthroline, EDTA, Fe(2+), Cu(2+), and Mn(2+)., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Cervical cancer is a common gynecological malignancy afflicting women all over the world. Ginsenoside Rh2 (GRh2), especially 20(S)-GRh2, is a biologically active component in the natural plant ginseng, which can exhibit anticancer effects. Here, we aimed to investigate the effect of 20(S)-GRh2 on cervical cancer and elucidate the underlying mechanism through RNA-seq. In this study, the CCK-8 assay showed that 20(S)-GRh2 inhibited HeLa cell viability in a time- and dose-dependent manner. Caspase 3 activity and Annexin V staining results showed that 20(S)-GRh2 induced apoptosis of HeLa cells. Gene function enrichment analysis revealed that the biological process gene ontology (GO) terms were associated with the apoptotic signaling pathway. Biological process GO terms' similarity network indicated that apoptosis might be from endoplasmic reticulum stress (ERs). Kyoto Encyclopedia of Genes and Genomes enrichment analysis revealed that 20(S)-GRh2 primarily modulates apoptosis pathway genes. Combined protein–protein interaction network, hub gene screening, and qPCR validation data showed that ERs-related genes (ATF4 and DDIT3) and the downstream apoptotic genes (JUN, FOS, BBC3, and PMAIP1) were potential novel targets of 20(S)-GRh2-inducing cervical cancer cell apoptosis. Differential transcript usage analysis indicated that DDIT3 is also a differential transcript and its usage of the isoform (ENST00000552740.5) was reduced by 20(S)-GRh2. Molecular docking suggested that 20(S)-GRh2 binds to the targets (ATF4, DDIT3, JUN, FOS, BBC3, and PMAIP1) with high affinity. In conclusion, our findings indicated that 20(S)-GRh2 might promote ERs-related apoptosis of cervical cancer cells by regulating the DDIT3-based targets' signal pathway. The role of 20(S)-GRh2 at the transcriptome level provides novel targets and evidence for the treatment of cervical cancer. [ABSTRACT FROM AUTHOR]

Background: Cancer cells through autophagy-mediated recycling to meet the metabolic demands of growth and proliferation. The steroidal saponin 20(S)-ginsenoside Rh2 effectively inhibits the growth and survival of a variety of tumor cell lines and animal models, but the effects of Rh2 on autophagy remain elusive. Methods: Cell viability was measured by CCK-8 (cell counting kit-8) assays. Apoptosis, ROS generation and mitochondrial membrane potential were analyzed by flow cytometry. Western blot analyses were used to determine changes in protein levels. Morphology of apoptotic cells and autophagosome accumulation were analyzed by DAPI staining and transmission electron microscopy. Autophagy induction was monitored by acidic vesicular organelle staining, EGFP-LC3 and mRFP-GFP-LC3 transfection. Atg7 siRNA and autophagy regulator was used to assess the effect of autophagy on apoptosis induced by G-Rh2. Results: In this study, we found that low concentration G-Rh2 attenuated cancer cell growth and induced apoptosis upon serum-free starvation. Caspase 3 inhibitors failed to block apoptosis in G-Rh2-treated cells, indicating a caspase-independent mechanism. G-Rh2-treated cells in serum-deprived conditions showed impaired mitochondrial function, increased release and nuclear translocation of apoptosis-inducing factor, but little changes in the mitochondrial and cytoplasmic distributions of cytochrome C. Annexin A2 overexpression in 293T cells inhibited G-Rh2-induced apoptosis under serum-starved conditions. Meanwhile, G-Rh2 reduced lysosomal activity and inhibited the fusion of autophagosome and lysosome, leading to a block of autophagic flux. Knockdown Atg7 significantly inhibited autophagy and triggered AIF-induced apoptosis in serm free condition. The autophagy inducer significantly decreased the apoptosis levels of G-Rh2-treated cells in serum-free conditions. Conclusions: Under nutrient deficient conditions, G-Rh2 represses autophagy in cervical cancer cells and enhanced apoptosis through an apoptosis-inducing factor mediated pathway. [ABSTRACT FROM AUTHOR]

Historical literature and pharmacological studies demonstrate that ginseng, one of the most popular herbal medicines in China, holds potential benefits for Parkinson's disease (PD). Studies in Drosophila melanogaster (Dm) have highlighted mitochondrial dysfunction upon loss of PTEN-induced putative kinase 1 (PINK1) as a central mechanism of PD pathogenesis. Using PINK1 B9 mutant Dm, we aimed to explore the therapeutic action of ginseng total protein (GTP) on PD and provide in-depth scientific interpretation about the traditional efficacy of ginseng. We first used gel chromatography to purify GTP and confirmed its molecular weight by SDS-PAGE. Effects of GTP on PINK1 B9 mutants, which were supplied with standard diet from larvae to adult stages, were assayed in flies aged 3–6 (I), 10–15 (II), and 20–25 (III) days. Parkinson-like phenotypes were analyzed by evaluating lifespan, dopaminergic neurons, dopamine levels, and locomotor ability. Mitochondrial function was assessed by evaluating ATP production, respirometry, and mitochondrial DNA. In addition, reactive oxygen species were measured using dihydroethidium and 2′,7′-dichlorodihydrofluorescein diacetate staining. PD-related oxidative stress was simulated by paraquat and rotenone, and mitochondrial membrane potential was measured using JC-10 reagent. Protein and mRNA expression was detected by Western blot and real-time quantitative reverse transcription polymerase chain reaction, respectively. This study demonstrates for the first time that GTP treatment delays the onset of a Parkinson-like phenotype in PINK1 B9 Dm, including prolongation of lifespan and rescue of climbing ability, as well as rescue of the progressive loss of a cluster of dopaminergic neurons in the protocerebral posterior lateral 1 region, which was accompanied by a significant increase of dopamine content in the brain. In addition, GTP notably reduced the penetrance of abnormal wing position, indicating a strong inhibitory effect on indirect flight muscle degeneration. We further showed that GTP could promote maintenance of mitochondrial function and protect mitochondria from PD-associated oxidative stress by activating the mitochondrial unfolded protein response (UPRmt). GTP protected against mitochondrial dysfunction and neurodegeneration by inducing UPRmt in the Dm PINK1 B9 model of PD. Our results suggest that GTP is a promising candidate for PD, and reveal a new mechanism by which ginseng is neuroprotective. Image 1 [ABSTRACT FROM AUTHOR]

Panax ginseng C.A. Mey (PG) is famous for "Qi-tonifying" effect, which has a medicinal history of more than 2 millennia. Modern pharmacology has confirmed that the "Qi-tonifying" effect of PG may be closely related to its pharmacological properties such as anti-oxidation, antineoplastic and treatment of cardiovascular disease. As one of the earliest cells affected by oxidative stress, RBCs are widely used in the diagnosis of diseases. Ginseng polysaccharide (GPS), is one of the major active components of PG, which plays an important role in resisting oxidative stress, affecting energy metabolism and other effects. However, the molecular mechanism explaining the "Qi-tonifying" effect of GPS from the perspective of RBCs oxidative damage has not been reported. This study aimed to investigate the protective effect of GPS on oxidatively damaged RBCs using in vitro and in vivo models and explore the molecular mechanisms from the perspective of glycolysis and gluconeogenesis pathways. To provides a theoretical basis for the future research of antioxidant drugs. Established three different in vitro and in vivo research models: an in vitro model of RBCs exposed to hydrogen peroxide (H 2 O 2) (40 mM), an in vivo model of RBCs from rats subjected to exhaustive swimming, and an in vitro model of BRL-3A cells exposed to H 2 O 2 (25 μM). All three models were also tested in the presence of different concentrations of GPS. The findings showed that GPS was the most potent antagonist of H 2 O 2 -induced hemolysis and redox inbalance in RBCs. In exhaustive exercise rats, GPS ameliorated RBVs hemolysis, including reducing whole-blood viscosity (WBV), improving deformability, oxygen-carrying and -releasing capacities, which was related to the enhancing of antioxidant capacity. Moreover, GPS promoted RBCs glycolysis in rats with exhaustive exercise by recovering the activities of glycolysis-related enzymes and increasing band 3 protein expression, thereby regulating the imbalance of energy metabolism caused by oxidative stress. Furthermore, we demonstrated that GPS improved antioxidant defense system, enhanced energy metabolism, and regulated gluconeogenesis via activating PPAR gamma co-activator 1 alpha (PGC-1α) pathway in H 2 O 2 -exposed BRL-3A cells. Mechanistically, GPS promoted glycolysis and protected RBCs from oxidative injury was partly dependent on the regulation of gluconeogenesis, as inhibition of gluconeogenesis by metformin (Met) attenuates the regulation of antioxidant enzymes and key enzymes of glycolytic by GPS in exhaustive exercise rats. This study demonstrates that GPS protects RBCs from oxidative stress damage by promoting RBCs glycolysis and liver gluconeogenesis pathways. These results may contribute to the study of new RBCs treatments to boost antioxidant capacity and protect RBCs against oxidative stress. [Display omitted] Effect of ginseng "qi-tonifying" is related to antioxidant and energy metabolism. Ginseng polysaccharide protects red blood cell and hepatocytes under oxidative stress Ginseng polysaccharide against oxidative stress via glycolysis and gluconeogenesis Ginseng polysaccharide promotes glycolysis is related to band-3 protein Ginseng polysaccharide improves gluconeogenesis via PPAR gamma co-activator 1 alpha [ABSTRACT FROM AUTHOR]

Ethnopharmacological relevanceQuercetin is the active component of the higher content in PCP, which exerts various biological activities such as anti-obesity effect, anti-inflammatory and anti-oxidant activities in alcoholic liver disease (ALD). P2X7 receptor (P2X7R) plays an important role in health and disease, which can be activated by extracellular ATP to induce a variety of downstream events, including lipid metabolism, inflammatory molecule release, oxidative stress. However, whether the mechanism of quercetin on ethanol-induced hepatic steatosis via P2X7R-mediated haven't been elucidated. Zebrafish transgenic (fabp10: EGFP) larvae were treated with 100 μM, 50 μM, 25 μM quercetin for 48 h at 3 days post fertilization (dpf), then soaked in 350 mmol/L ethanol for 32 h, treated with 1 mM ATP (P2X7R activator) for 30min. Serum lipids, liver steatosis, oxidative stress factors were respectively detected. The mRNA levels in the related pathways were measured by quantitative Real-Time PCR (RT-qPCR) to investigate the mechanisms. Quercetin improved the liver function via decreasing ALT, AST and γ-GT level of zebrafish with acute ethanol-induced hepatic steatosis and attenuated hepatic TG, TC accumulation. Additionally, quercetin significantly reduced the MDA content and suppressed the ethanol-induced reduction of hepatic oxidative stress biomarkers GSH, CAT and SOD and significantly down-regulated the expression of P2X7R, and up-regulated the expression of phosphatidylinositol 3-kinase (PI3K), Kelch like ECH associated protein1 (Keap1), Nuclear Factor E2 related factor 2 (Nrf2). Moreover, ATP stimulation activated P2X7R, which further mediated the mRNA expressions of PI3K, Keap1 and Nrf2. Quercetin exhibited hepatoprotective capacity in zebrafish model, via regulating P2X7R-mediated PI3K/Keap1/Nrf2 oxidative stress signaling pathway. Image 1 • Quercetin alleviated lipid accumulation in zebrafish. • Quercetin suppressed alcohol hepatosteatosis via PI3K/Keap1/Nrf2 signaling pathways. • ATP-activated P2X7R were involved in regulating oxidative stress in zebrafish with alcohol hepatosteatosis. [ABSTRACT FROM AUTHOR]

Clinical applications and pharmacological research suggest that Dioscorea opposita Thunb. (Chinese yam), a well-known traditional Chinese medicine which has been used for more than 2000 years to nourish kidney-yang and protect the male reproductive system, might be efficacious for the treatment of erectile dysfunction (ED). This study aimed to investigate the active component extract of Chinese yam, determine its effectiveness in hydrocortisone-induced "kidney-yang deficiency syndrome" (KDS-Yang) rats and in oxidatively damaged TM3 cells and explore the underlying mechanism on restoring erectile function. We clarified the Chinese yam cold-soaking extract (CYCSE) as the main active extract of Chinese yam by a CCK8 assay and further identified its composition. The KDS-Yang rats were induced by intragastric administration of hydrocortisone. After 10 d of CYCSE intervention, cavernous and testis morphology were stained with hematoxylin and eosin. Inducible nitric oxide synthase (iNOS), cyclic guanosine monophosphate (cGMP), testosterone, 8-hydroxy-2-deoxyguanosine (8-OHdG) and superoxide dismutase (SOD) levels were detected by enzyme-linked immunosorbent assay kits. Leydig cells were performed using immunohistochemistry. Reactive oxygen species were measured using a DCFH-DA fluorescent probe, and testicular collagenous fibers were stained with a Masson kit. Detection of testicular apoptosis was performed by a TUNEL assay. Nrf2 and NQO1 mRNA expression levels were measured by qRT-PCR. The protein expression levels of Nrf2, HO-1, TGF-β1 and SMAD2/3 were analyzed by Western blot. We demonstrated in KDS-Yang rats and oxidatively damaged TM3 cells that CYCSE successfully restored erectile function through ameliorating testicular function. Our data suggested that CYCSE can stimulate the NO/cGMP pathway and restore the cavernous morphology to protect against KDS-Yang-induced ED. It also protected testis morphology, increased Leydig cell proliferation and stimulated testosterone secretion. In the damaged testes, excessive increases in 8-OHdG and inhibition of SOD activity were ameliorated, and the Nrf2/HO-1 signaling pathway was enhanced after treatment with CYCSE, indicating that the antioxidant defense system was activated. These findings were also validated in vitro. Additionally, fibrosis of the testes and TM3 cells was reversed by CYCSE through the TGF-β1/SMAD2/3 pathway. CYCSE has a therapeutic effect on KDS-Yang-induced ED, and the mechanism includes stimulation of testosterone secretion, resistance to oxidative stress and prevention of fibrosis. These findings provide a new scientific verification for the application of Chinese yam in the treatment of KDS-Yang-induced ED. Image 1 [ABSTRACT FROM AUTHOR]