Discovery of Potent Angiotensin-Converting Enzyme Inhibitors in Pomegranate as a Treatment for Hypertension.

Pomegranate (Punica granatum L.) is associated with numerous health benefits due to its high levels of antioxidant polyphenolic substances. Since pomegranate extract has been shown to inhibit angiotensin-converting enzyme (ACE), the potential inhibitory effect of most of its main constituents against ACE is unknown. Therefore, we tested the activities of 24 major compounds, the majority of which significantly inhibited ACE. Notably, pedunculagin, punicalin, and gallagic acid were the most effective ACE inhibitors with IC50 values of 0.91, 1.12, and 1.77 µM, respectively. As demonstrated in molecular docking studies, compounds block ACE by forming multiple hydrogen bonds and hydrophobic interactions with catalytic residues and zinc ions in ACE's C- and N-domains, consequently inhibiting ACE's catalytic activity. Also, the most active pedunculagin stimulated nitric oxide (NO) production, activated the endothelial nitric oxide synthase enzyme (eNOS), and significantly increased eNOS protein expression levels up to 5.3-fold in EA.hy926 cells. Furthermore, pedunculagin increased in cellular calcium (Ca2+) concentration promoted eNOS enzyme activation and reduced the production of reactive oxygen species (ROS). In addition, the active compounds improved glucose uptake in insulin-resistant C2C12 skeletal muscle cells in a dose-dependent manner. The results of these computational, in vitro, and cellular experiments provide further evidence to the traditional medicine that involves using pomegranates to treat cardiovascular diseases like hypertension.

Inhibition of Aldose Reductase by Ginsenoside Derivatives via a Specific Structure Activity Relationship with Kinetics Mechanism and Molecular Docking Study.

This present work is designed to evaluate the anti-diabetic potential of 22 ginsenosides via the inhibition against rat lens aldose reductase (RLAR), and human recombinant aldose reductase (HRAR), using DL-glyceraldehyde as a substrate. Among the ginsenosides tested, ginsenoside Rh2, (20S) ginsenoside Rg3, (20R) ginsenoside Rg3, and ginsenoside Rh1 inhibited RLAR significantly, with IC50 values of 0.67, 1.25, 4.28, and 7.28 µM, respectively. Moreover, protopanaxadiol, protopanaxatriol, compound K, and ginsenoside Rh1 were potent inhibitors of HRAR, with IC50 values of 0.36, 1.43, 2.23, and 4.66 µM, respectively. The relationship of structure-activity exposed that the existence of hydroxyl groups, linkages, and their stereo-structure, as well as the sugar moieties of the ginsenoside skeleton, represented a significant role in the inhibition of HRAR and RLAR. Additional, various modes of ginsenoside inhibition and molecular docking simulation indicated negative binding energies. It was also indicated that it has a strong capacity and high affinity to bind the active sites of enzymes. Further, active ginsenosides suppressed sorbitol accumulation in rat lenses under high-glucose conditions, demonstrating their potential to prevent sorbitol accumulation ex vivo. The findings of the present study suggest the potential of ginsenoside derivatives for use in the development of therapeutic or preventive agents for diabetic complications.

Flavanone glycosides inhibit ß-site amyloid precursor protein cleaving enzyme 1 and cholinesterase and reduce Aß aggregation in the amyloidogenic pathway.

Alzheimer's disease (AD) is a slow but progressive neurodegenerative disease. One of the pathological hallmarks of AD is the progressive accumulation of ß-amyloid (Aß) in the form of senile plaques, and Aß insult to neuronal cells has been identified as one of the major causes of AD onset. In the present study, we investigated the anti-AD potential of four flavonoids, naringenin, didymin, prunin, and poncirin, by evaluating their ability to inhibit acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). All four flavonoids displayed promising inhibitory activity against AChE, BChE, and BACE1. Structure-activity relationships suggested that glycosylation of naringenin at sugar moieties, and at different positions of the glycosidic linkage, might be closely associated with anti-AD potential. Kinetic and docking studies showed the lowest binding energy and highest affinity for the mixed, competitive, and non-competitive type inhibitors didymin, prunin, and poncirin. Hydrophobic interactions and the number of hydrogen bonds determined the strength of the protein-inhibitor interaction. We also examined the neuroprotective mechanisms by which flavonoids act against Aß25-35-induced toxicity in PC12 cells. Exposure of PC12 cells to 10 µM Aß25-35 for 24 h resulted in a significant decrease in cell viability. In addition, pretreatment of PC12 cells with different concentrations of flavonoids for 1 h significantly reversed the effects of Aß. Furthermore, treatment with the most active flavonoid, didymin, significantly reduced BACE1, APPsß, and C99 expression levels in a dose-dependent manner, without affecting amyloid precursor protein (APP) levels in the amyloidogenic pathway. Together, our results indicate that flavonoids, and in particular didymin, exhibit inhibitory activity in vitro, and may be useful in the development of therapeutic modalities for the treatment of AD.

Didymin, a dietary citrus flavonoid exhibits anti-diabetic complications and promotes glucose uptake through the activation of PI3K/Akt signaling pathway in insulin-resistant HepG2 cells.

Didymin is a naturally occurring orally active flavonoid glycoside (isosakuranetin 7-O-rutinoside) found in various citrus fruits, which has been previously reported to possess a wide variety of pharmacological activities including anticancer, antioxidant, antinociceptive, neuroprotective, hepatoprotective, inflammatory, and cardiovascular. However, there have not been any reports concerning its anti-diabetic potential until now. Therefore, we evaluated the anti-diabetic potential of didymin via inhibition of α-glucosidase, protein tyrosine phosphatase 1B (PTP1B), rat lens aldose reductase (RLAR), human recombinant AR (HRAR), and advanced glycation end-product (AGE) formation inhibitory assays. Didymin strongly inhibited PTP1B, α-glucosidase, HRAR, RLAR, and AGE in the corresponding assays. Kinetic study revealed that didymin exhibited a mixed type inhibition against α-glucosidase and HRAR, while it competitively inhibited PTP1B and RLAR. Docking simulations of didymin demonstrated negative binding energies and close proximity to residues in the binding pocket of HRAR, RLAR, PTP1B and α-glucosidase, indicating that didymin have high affinity and tight binding capacity towards the active site of these enzymes. Furthermore, we also examined the molecular mechanisms underlying the anti-diabetic effects of didymin in insulin-resistant HepG2 cells which significantly increased glucose uptake and decreased the expression of PTP1B in insulin-resistant HepG2 cells. In addition, didymin activated insulin receptor substrate (IRS)-1 by increasing phosphorylation at tyrosine 895 and enhanced the phosphorylations of phosphoinositide 3-kinase (PI3K), Akt, and glycogen synthasekinase-3(GSK-3). Interestingly, didymin reduced the expression of phosphoenolpyruvate carboxykinase and glucose 6-phosphatase, two key enzymes involved in the gluconeogenesis and leading to a diminished glucose production. The results of the present study clearly demonstrated that didymin will be useful for developing multiple target-oriented therapeutic modalities for treatment of diabetes, and diabetes-associated complications.

Effects of Onchung-eum, an Herbal Prescription, on 5-Fluorouracil-Induced Oral Mucositis.

In most cancer patients, chemotherapy-induced oral mucositis (OM) is a frequent side effect, leading to low quality of life and delay in therapy. The aim of this study was to evaluate the effects of Onchung-eum, a well-known herbal prescription in traditional medicine comprising 8 herbs that has long been used for skin diseases, on 5-fluorouracil (5-FU)-induced OM in human pharyngeal cells and golden Syrian hamsters. DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, and reactive oxygen species production were measured in vitro. The effects of Onchung-eum on OM of hamster cheek pouches induced by 5-FU were evaluated histologically and using TUNEL assay. In addition, the expression of nuclear factor-κB, caspase-3, and pro-inflammatory cytokines were measured by immunoblotting and immunohistochemistry. Significantly increased cell viability was observed in the Onchung-eum-treated groups compared with the 5-FU-treated control group. In 500 and 1000 mg/kg Onchung-eum-treated groups, the damaged epithelial layers in the cheek pouches of hamsters were significantly recovered. Moreover, at all concentrations, cell death in the cheek pouches of hamsters in the Onchung-eum-treated groups significantly decreased. The expression of pro-inflammatory cytokines, nuclear factor-κB, and caspase-3 also significantly decreased in Onchung-eum-treated groups at 500 and 1000 mg/kg. In conclusion, this study revealed that Onchung-eum can be used to treat chemotherapy-induced OM. However, further studies are required to understand the underlying mechanisms.

Protective effect of Salvia miltiorrhiza Bunge on 5-fluorouracil-induced oral mucositis.

Oral mucositis is a common side-effect caused by chemotherapy or radiotherapy occurring in the majority of cancer patients and is characterized by inflammation and ulcers in the oral mucosa. In the present study, we examined the protective effects of Salvia miltiorrhiza Bunge (SM) on oral mucositis induced by 5-fluorouracil (5-FU) in human pharyngeal cells and golden Syrian hamsters. We investigated the proliferation and antioxidant abilities of SM using MTT, 2-diphenyl-1-picrylhydrazyl (DPPH) and reactive oxygen species (ROS) assays in vitro. Additionally, TUNEL assay was performed, and the expression levels of nuclear factor-κB (NF-κB), caspase-3 and proinflammatory cytokines were assessed by immunoblotting. The results showed that SM increased the cell proliferation rate in human pharyngeal cells up to 128.97±9.7% compared with this rate in the untreated cells and exerted protective effects on mucosal injury caused by 5-FU treatment. In addition, all concentrations of SM increased DPPH scavenging ability and blocked ROS generation in the treated cells. Taken together, following SM treatment, expression of NF-κB and cleaved caspase-3 were significantly decreased followed by inhibition of cell death. These data suggest that SM could be used for the prevention and treatment of oral mucositis caused by cancer therapies.

Effects of herbal Epimedium on the improvement of bone metabolic disorder through the induction of osteogenic differentiation from bone marrow-derived mesenchymal stem cells.

Herbal Epimedium (HE) has been commonly used as a tonic, antirheumatic agent and in the treatment of bone­associated diseases including osteoporosis. Treatment for osteoporosis is important to increase bone mass density and maintain to balance of bone remodeling. The present study was performed to investigate the effects of HE on mouse bone marrow mesenchymal stem cell (mBMMSC) proliferation and osteogenic differentiation, using MTT assays, proliferating cell nuclear antigen (PCNA) detection and apoptosis and differentiation assays. HE was demonstrated to inhibit the proliferation of mBMMSCs up to 45.43±3.33% and to decrease the level of PCNA expression compared with untreated cells. HE also induced late apoptosis at 24 and 48 h after treatment up to 71.93 and 67.03%, respectively, while only 14.93% of untreated cells exhibited apoptosis. By contrast, HE induced differentiation of mBMMSCs into an osteogenic lineage at the beginning of three weeks after commencement of treatment. This suggested that HE is a candidate as an inducer of osteogenesis from bone marrow mesenchymal stem cells, and additionally has potential for use in the treatment of bone metabolic disorders such as osteoporosis.

Astragalus membranaceus augment sperm parameters in male mice associated with cAMP-responsive element modulator and activator of CREM in testis.

Astragalus membranaceus BUNGE (AM; huáng qí) has been widely used as a medicinal herb for different kinds of diseases. AM treatment in vitro enhance sperm motility and ameliorates testicular toxicity, it has demonstrated the ability as a potential treatment for male infertility. In order to gain further insights on the molecular understanding of how AM enhances spermatogenesis, this study investigated whether AM has an affect on sperm parameters associated with cAMP response element modulator (CREM) and activator of CREM in testis (ACT) expression. Five-week-old male ICR mice were divided into four groups; control group and three different concentrations of AM treated groups. Each group was treated for 5 days a week for 5 weeks. Testis samples were collected for real time quantitative PCR and western blot analysis. Epididymis was taken out and used for sperm analysis using the computer assisted semen analysis (CASA) system. To facilitate expression of genes required for spermatogenesis, it is controlled by fine-tuning of CREM and its coactivator, ACT. AM treatment promotes CREM and ACT mRNA expression and also protein expression compared to control. AM enhances sperm values such as sperm count and motility compared to control. Overall, the study highlights, the ability of AM to increases CREM and ACT expression to facilitate sperm development and semen quality.

Trigonellae Semen Enhances Sperm Motility and the Expression of the Cation Sperm Channel Proteins in Mouse Testes.

Genetic defects during spermatogenesis can lead to a reduction in sperm motility and cause male infertility. The cation channels of sperm (CatSper) play a role in the regulation of hyperactivated sperm motility in mouse testes. The effect of Trigonellae Semen (TS) on the male reproductive system and CatSper protein in mouse testes during spermatogenesis was examined. C57BL/c mice were divided into the following five groups: normal, cyclophosphamide- (CP-) only treated (control group), and three groups treated with varying concentrations of TS with CP (100, 500, and 1000 mg/kg TS and 100 mg/kg CP). Real-time PCR, western blot analysis, and a testosterone immunoassay were performed to assess CatSper protein levels in the five groups. Additionally, sperm cell counts and motility were examined. Results indicate that sperm motility and sperm counts increased in the TS treated groups in a dose-dependent manner (p < 0.01). CatSper levels were also significantly higher in the TS treated groups compared to that of the control group (p < 0.001). Therefore, TS treatment could enhance sperm function by promoting spermatogenesis and the expression of CatSper proteins in mouse testes.

Effects of Jae-Seng Acupuncture Treatment on the Improvement of Nasolabial Folds and Eye Wrinkles.

The microneedle therapy system (MTS), a mechanical method involving making minute multiple holes in the skin, reportedly improves skin condition, such as by reducing flushing and melanin. A newly attempted bloodletting therapy, Jae-Seng Acupuncture, has several advantages over traditional mechanical punching methods because it allows the practitioner to regulate the depth and direction of needle stimulations and to choose whether to stimulate the muscle layers. This study was conducted to determine the efficacy of Jae-Seng Acupuncture in the treatment of nasolabial folds and eye wrinkles. The nasolabial folds and eye wrinkles of 107 patients ranging in age from their 20s to their 70s were subjected to DermaVision, a digital skin image analyzer, before the treatment and one to six months after treatment. Additionally, stimulation of the meridians, such as Taeyang, Tongjaryo, Chongmyong, Sungup, Sabaek, Yonghyang, Chichang, Taeyong, was performed to improve the function of the stomach, large intestine. Analyses of the images indicate that Jae-Seng Acupuncture improved nasolabial folds and eye wrinkles, suggesting that this technique is a safe and effective method for the improvement of facial skin conditions.

Panax ginseng induces the expression of CatSper genes and sperm hyperactivation.

The cation channel of sperm (CatSper) protein family plays important roles in male reproduction and infertility. The four members of this family are expressed exclusively in the testis and are localized differently in sperm. To investigate the effects of Panax ginseng treatment on the expression of CatSper genes and sperm hyperactivation in male mice, sperm motility and CatSper gene expression were assessed using a computer-assisted semen analysis system, a Fluoroskan Ascent microplate fluorometer to assess Ca²âº influx, real-time polymerase chain reaction, Western blotting and immunofluorescence. The results suggested that the Ca²âº levels of sperm cells treated with P. ginseng were increased significantly compared with the normal group. The P. ginseng-treated groups showed increased sperm motility parameters, such as the curvilinear velocity and amplitude of lateral head displacement. Taken together, the data suggest that CatSper messenger ribonucleic acid levels were increased significantly in mouse testes in the P. ginseng-treated group, as was the protein level, with the exception of CatSper2. In conclusion, P. ginseng plays an important role in improving sperm hyperactivation via CatSper gene expression.

Differential expression of apoptosis-related factors induces the age-related apoptosis of the gracilis muscle in humans.

In the normal aging process, apoptosis has been implicated as a mechanism responsible for the loss of muscle cells and plays an important role in age-related muscle loss. Several signaling pathways involved in skeletal muscle apoptosis are currently under intense investigation, particularly the caspase-independent pathway. This study investigated the age-related apoptotic changes occurring in the gracilis muscle in humans between 10 and 50 years of age. For this purpose, muscle samples were divided into 5 groups (n=8). Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and immunofluorescence detection were performed to determine the number of apoptotic muscle cells in each group. In addition, the expression levels of apoptosis-related factors, such as Bcl-2, Bax, apoptosis-inducing factor (AIF), caspase-3 and calpain-1 were determined by RT-PCR and western blot analysis. TUNEL assay revealed a significant increase in gracilis muscle apoptosis with aging. The activity of caspase-3 in the gracilis muscle tended to change with age, although the changes were not significant, while the increase in DNA nuclei in muscle from 50 years of age (5.419±0.97) was associated with an increase in the expression of AIF, as observed both at protein (10-30%) and mRNA level (10-60%) in gracilis tissues. Taken together, our results demonstrated that the relative Bcl-2 expression decreased with aging, while Bax expression was upregulated compared to 10-year-olds. In addition, a double-labeling experiment with TUNEL staining and immunofluorescence revealed the co-localization of nuclear AIF-positive and TUNEL-labeled cells. This study suggests that apoptosis in gracilis skeletal muscle in the elderly is partly mediated through the expression of Bcl-2/Bax and the degradation of AIF.

Effects of low level laser therapy on ovalbumin-induced mouse model of allergic rhinitis.

Introduction. This study was designed to investigate the effects of low level laser therapy (LLLT) on experimental allergic rhinitis (AR) models induced by ovalbumin. Materials and Methods. AR was induced by 1% ovalbumin in mice. Twenty-four mice were divided into 4 groups: normal, control, low, and high dose irradiation. Low and high dose LLLT were irradiated once a day for 7 days. Total IgE, cytokines concentrations (IL-4 and IFN- γ ), and thymus and activation regulated chemokine (TARC) were measured. Histological changes in the nasal mucosal tissue by laser irradiation were examined. Results. LLLT significantly inhibited total IgE, IL-4, and TARC expression in ovalbumin-induced mice at low dose irradiation. The protein expression level of IL-4 in spleen was inhibited in low dose irradiation significantly. IL-4 expression in EL-4 cells was inhibited in a dose dependent manner. Histological damages of the epithelium in the nasal septum were improved by laser irradiation with marked improvement at low dose irradiation. Conclusion. These results suggest that LLLT might serve as a new therapeutic tool in the treatment of AR with more effectiveness at low dose irradiation. To determine the optimal dose of laser irradiation and action mechanisms of laser therapy, further studies will be needed.

Aconiti Lateralis Preparata Radix Activates the Proliferation of Mouse Bone Marrow Mesenchymal Stem Cells and Induces Osteogenic Lineage Differentiation through the Bone Morphogenetic Protein-2/Smad-Dependent Runx2 Pathway.

Mesenchymal stem cells have the capacity for self-renewal and under appropriate stimulation give rise to osteogenic, adipogenic, and chondrogenic lineages. To advance the clinical use of stem cell therapy, such as stem cell transplantation, it is important to find substances that promote endogenous stem cell proliferation and differentiation. We investigated whether medicinal herbs have the potential to promote stem cell proliferation and differentiation, using a cell cycle analysis and differentiation assay. We found that Aconiti Lateralis Preparata Radix (ALR) promoted the proliferation rate of mouse bone marrow mesenchymal stem cells (mBMMSCs) up to 122.24% compared to untreated cells. Fluorescence-activated cell sorter analysis showed that the percentage of cells in the G2/M phase increased to 17.33% in ALR-treated cells compared to 5.65% in normal cells. Signaling pathway analysis indicated that this was mediated through the extracellular signal-regulated kinase 1/2 pathway. A differentiation assay showed that ALR induced differentiation of mBMMSCs into an osteogenic lineage 2 weeks after treatment, whereas traditional osteogenic induction medium treatment did not promote differentiation for 3 weeks. This osteogenic differentiation was signaled by the bone morphogenetic protein-2/Smad-dependent Runx2 pathway. We found that ALR could promote mBMMSC proliferation and differentiation into the osteogenic lineage.

Inhibitory effect of Astragalus membranaceus root on matrix metalloproteinase-1 collagenase expression and procollagen destruction in ultraviolet B-irradiated human dermal fibroblasts by suppressing nuclear factor kappa-B activity.

OBJECTIVES: The root of Astragalus membranaceus, regarded as a tonic in traditional Korean medicine, has been prescribed for long periods to treat chronic illness by boosting the immune system. Ultraviolet (UV) irradiation causes damage to skin connective tissue by degrading collagen, which is a major structural component of the extracellular matrix. Such damage is considered to be a cause of the wrinkling observed in premature ageing of the skin. This study has investigated the photo-protective effect of A. membranaceus on UVB radiation-induced activation of nuclear factor kappa-B (NF-κB) activity in human dermal fibroblasts. METHODS: HS68 fibroblast cells cultured with various concentrations of A. membranaceus were exposed to UVB (40 mJ/cm²). Activation of NF-κB P65 and expression of matrix metalloproteinase-1 (MMP-1) and type 1 procollagen were measured by Western blotting. Translocation of NF-κB P65 and MMP-1 regulation were also examined by immunocytochemistry. KEY FINDINGS: Western blotting and immunocytochemistry results showed that A. membranaceus inhibited UVB-induced translocation of NF-κB P65 and MMP-1 expression. The data suggested that A. membranaceus restored type 1 procollagen synthesis by inhibiting NF-κB P65 activity and MMP-1 expression in UVB-exposed human dermal fibroblasts. CONCLUSION: A. membranaceus is a candidate for use in skin protection from UVB-induced skin inflammation and photoageing.

Enhancement of mouse sperm motility by trophinin-binding peptide.

BACKGROUND: Trophinin is an intrinsic membrane protein that forms a complex in the cytoplasm with bystin and tastin, linking it microtubule-associated motor dynein (ATPase) in some cell types. Previously, we found that human sperm tails contain trophinin, bystin and tastin proteins, and that trophinin-binding GWRQ (glycine, tryptophan, arginine, glutamine) peptide enhanced motility of human sperm. METHODS: Immunohistochemistry was employed to determine trophinin protein in mouse spermatozoa from wild type mouse, by using spermatozoa from trophinin null mutant mice as a negative control. Multivalent 8-branched GWRQ (glycine, tryptophan, arginine, glutamine) peptide or GWRQ-MAPS, was chemically synthesized, purified by HPLC and its structure was confirmed by MALDI-TOF mass spectrometry. Effect of GWRQ-MAPS on mouse spermatozoa from wild type and trophinin null mutant was assessed by a computer-assisted semen analyzer (CASA). RESULTS: Anti-trophinin antibody stained the principal (central) piece of the tail of wild type mouse sperm, whereas the antibody showed no staining on trophinin null sperm. Phage particles displaying GWRQ bound to the principal piece of sperm tail from wild type but not trophinin null mice. GWRQ-MAPS enhanced motility of spermatozoa from wild type but not trophinin null mice. CASA showed that GWRQ-MAPS enhanced both progressive motility and rapid motility in wild type mouse sperm. CONCLUSIONS: Present study established the expression of trophinin in the mouse sperm tail and trophinin-dependent effect of GWRQ-MAPS on sperm motility. GWRQ causes a significant increase in sperm motility.

Chlorogenic acid ameliorates brain damage and edema by inhibiting matrix metalloproteinase-2 and 9 in a rat model of focal cerebral ischemia.

Chlorogenic acid (CGA) has been reported to have various beneficial effects on the cardiovascular and central nervous systems. The purpose of the current study was to investigate whether CGA has protective effects against cerebral ischemia and whether these effects are due to modification of brain edema-related vascular factors. In a rat model of transient middle cerebral artery occlusion (MCAo, 2h of occlusion followed by 22 h of reperfusion), we measured infarct volume and performed behavioral test to evaluate the effects of CGA on brain damage and sensory-motor functional deficits. Brain water content and Evans blue extravasation were measured to evaluate brain edema and blood brain barrier (BBB) damage. Lipid peroxidation (LPO) and the expressions and activities of matrix metalloproteinase (MMP)-2 and MMP-9 were measured to investigate the mechanisms of action. Intraperitoneal injection of CGA (3, 10, and 30 mg/kg) at 0 h and 2h after MCAo dose-dependently reduced infarct volume and sensory-motor functional deficits. It also reduced brain water content and Evans blue extravasation. Mechanistically, CGA reduced LPO and MMPs expressions and activities. These results suggest that CGA reduces brain damage, BBB damage and brain edema by radical scavenging activity and the inhibitory effects on MMP-2 and MMP-9.

Astragalus membranaceus ameliorates reproductive toxicity induced by cyclophosphamide in male mice.

The root of Astragalus membranaceus B(UNGE) (AM) is a medicinal herb that has been capable of reducing the adverse effects of conventional chemotherapy. To investigate the effects of AM on cyclophosphamide (CP)-induced reproductive toxicity in mouse testes, 5-week-old male imprinting control region mice were divided into five groups; CP was treated on the first day of each week for 5 weeks (100 mg/kg, i.p.), and AM was treated for 5 days a week for 5 weeks. At the end of the treatment period, the testes were taken out, cleared of the adhering tissues, and weighed. Epididymis was taken out and used for sperm analysis. Testis samples were frozen for real-time quantitative PCR and Western blot analysis. AM treatment increased diminished relative testes weight, and sperm count and motility in mice treated with CP. CP treatment has detrimental effects on the expression of cAMP-responsive element modulator (CREM), a transcription factor that is highly expressed in male germ cells and is crucial to post-meiotic germ cell differentiation. AM restored CREM at both the mRNA and protein levels. AM has beneficial influences and appears able to ameliorate relative testes weight, sperm parameters, and CREM expression against CP-induced reproductive toxicity.

Cistanches herba induces testis cytotoxicity in male mice.

We investigated the effects of Cistanches herba (CH) on the male reproductive system in mice, assessing CREM gene expression and spermatogenesis. Our results demonstrate that CH treatment lead to a significant decrease in sperm count dose-dependently, 298.3 ± 48.9 vs. 296.6 ± 102.4 (250 mg/kg), 236.7 ± 75.1 (500 mg/kg), 223.0 ± 48.7 × 10(6) (1000 mg/kg), respectively. Additionally, serum testosterone levels decreased following CH treatment to as low as ~57% compared with the vehicle-treated group. CREM gene expression was also down-regulated following CH treatment and histological examination of the testicular seminiferous tubules showed severe damage on CH treatment. These results suggest that CH induces cytotoxicity in the male reproductive system, through the inhibition of spermatogenesis, testicular damage, and limited hormonal function.

Early macrophage recruitment and alternative activation are critical for the later development of hypoxia-induced pulmonary hypertension.

BACKGROUND: Lung inflammation precedes the development of hypoxia-induced pulmonary hypertension (HPH); however, its role in the pathogenesis of HPH is poorly understood. We sought to characterize the hypoxic inflammatory response and to elucidate its role in the development of HPH. We also aimed to investigate the mechanisms by which heme oxygenase-1, an anti-inflammatory enzyme, is protective in HPH. METHODS AND RESULTS: We generated bitransgenic mice that overexpress human heme oxygenase-1 under doxycycline control in an inducible, lung-specific manner. Hypoxic exposure of mice in the absence of doxycycline resulted in early transient accumulation of monocytes/macrophages in the bronchoalveolar lavage. Alveolar macrophages acquired an alternatively activated phenotype (M2) in response to hypoxia, characterized by the expression of found in inflammatory zone-1, arginase-1, and chitinase-3-like-3. A brief 2-day pulse of doxycycline delayed, but did not prevent, the peak of hypoxic inflammation, and could not protect against HPH. In contrast, a 7-day doxycycline treatment sustained high heme oxygenase-1 levels during the entire period of hypoxic inflammation, inhibited macrophage accumulation and activation, induced macrophage interleukin-10 expression, and prevented the development of HPH. Supernatants from hypoxic M2 macrophages promoted the proliferation of pulmonary artery smooth muscle cells, whereas treatment with carbon monoxide, a heme oxygenase-1 enzymatic product, abrogated this effect. CONCLUSIONS: Early recruitment and alternative activation of macrophages in hypoxic lungs are critical for the later development of HPH. Heme oxygenase-1 may confer protection from HPH by effectively modifying the macrophage activation state in hypoxia.