Pyroglutamic acid stimulates DNA synthesis in rat primary hepatocytes through the mitogen-activated protein kinase pathway.

We purified pyroglutamic acid from human placental extract and identified it as a potent stimulator of rat primary hepatocyte DNA synthesis. Pyroglutamic acid dose-dependently stimulated DNA synthesis, and this effect was inhibited by PD98059, a dual specificity mitogen-activated protein kinase kinase 1 (MAP2K1) inhibitor. Therefore, pyroglutamic acid stimulated DNA synthesis in rat primary hepatocytes via MAPK signaling.

Alternative splicing and tissue-specific elastin misassembly act as biological modifiers of human elastin gene frameshift mutations associated with dominant cutis laxa.

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

Successful steroid tapering and partial treatment of suspected immune-mediated haemolytic anaemia in a dog with equine placenta extract supplementation: A case report.

Background: We report on the clinical management and outcome of an 11-year-old dog diagnosed with suspected refractory immune-mediated anemia (IMHA) and treated with equine placental extract supplementation. Case Description: The patient had received standard treatment with subcutaneous infusion of prednisone (2 mg/kg) and oral administration (1.3 mg/kg semel in die [sid]), with limited success as hematocrit (HCT) values continued to fall rapidly, and the patient continued to have severe symptoms of fatigue. The patient was then put on equine placental extract supplements, after which the patient's physical exhaustion was improved, and although the HCT level initially continued to fall, it eventually began to rise and remained near normal for approximately 2 years. A significant reduction in prednisone use was achieved with placental supplementation. Conclusion: Equine placental supplementation may be useful as a new complementary therapy for suspected refractory IMHA.

Evaluating the Impact of a Cream Containing Horse Placental Extract on Eye Corner Wrinkles in Healthy Women: Single-Blind Comparative Study.

BACKGROUND: Placental extract has been mostly used in skin care for cosmetic purposes. However, the use of various placental extracts has been limited due to the lack of established and effective application methods. OBJECTIVE: In this study, we investigated the antiwrinkle effect of a cream formulation-LNC wrinkle eye cream (LNC-EC)-containing horse placental extract as the main ingredient. METHODS: A total of 24 healthy women, aged 37-54 years, with wrinkle grades 1-3, were treated with LNC-EC for 2 weeks. The cream was applied on one-half of the participants' faces, and the results were compared with the untreated half of the face. RESULTS: Visual inspection, using the wrinkle grade standard, showed that the area treated with LNC-EC had a significantly lower wrinkle grade than the untreated area when comparing before and after the application of LNC-EC. In addition, replica analysis showed a significant reduction in both the maximum wrinkle width and the number of wrinkles in the LNC-EC-treated area in comparison to the untreated area before and after the application. These results suggest that LNC-EC has an antiwrinkle effect on the corners of the eyes based on parameters like the maximum wrinkle width and the number of wrinkles. CONCLUSIONS: LNC-EC, with horse placental extract as its main ingredient, was shown to be effective in improving wrinkles at the eye corners, presumably due to a reduction in the maximum wrinkle width and the number of wrinkles. Interpretation of the results is limited because this study was conducted only in the intervention group. A randomized controlled trial with a placebo control group is necessary to verify the antiwrinkle effects of horse placental extract.

Combined treatment with basalt stone and placenta extract to improve the chronic pain and scar after breast cancer surgery: a case report.

BACKGROUND: The changes in body image caused by breast deformities and postoperative pain have a detrimental influence on the physical and mental health of patients with breast cancer. The postoperative quality of life (QOL) of these patients reduces significantly owing to the changes in the breast, an organ unique to women, that occur following breast cancer surgery. CASE PRESENTATION: This case report presents the case of a Asian woman in her early 40 s with postoperative hypertrophic scarring and contraction of the scar following mastectomy; the patient presented with decreased range of motion of the upper arm, hyperpigmentation from radiation burns, changes in breast shape, and chronic pain. The patient received a combination therapy comprising Basalt Stone Treatment and the application of horse placenta extract. As a result of a total of eight sessions conducted once every two weeks, the patient's pain and scar improved. No adverse events were observed after the therapy. CONCLUSION: Combination therapy with Basalt Stone Treatment and horse placenta extract improved the chronic pain and scar after breast cancer surgery.

Successful treatment of ascites accumulation and diarrhea associated with protein-losing enteropathy with oral equine placenta extract supplementation in a dog: A case report.

Background: Protein-losing enteropathy (PLE) is characterized by leakage of serum proteins into the intestinal lumen, indicating hypoproteinemia. Immunosuppressive agents are the mainstay of treatment, but in many cases, patients are forced to taper off early owing to the induction of liver damage. Case Description: An 8-year-old, non-spayed female Chihuahua presented with diarrhea and ascites effusion lasting 2 weeks. Based on the results of radiography and blood tests, a diagnosis of PLE was made. Prednisolone (3 mg/kg semel in die [SID]) and MitoMax (200 mg/day) were administered, but ascites accumulation and diarrhea did not improve. Thus, azathioprine (2 mg/kg/day) was added, but there was no improvement, and liver damage developed. The liver injury did not improve immediately, but diarrhea and ascites effusion improved after serum total protein and serum albumin levels increased after they had decreased. Subsequent tapering of prednisolone from 3 mg/kg SID to 1 mg/kg SID, combined with MitoMax (200 mg/day) and equine placenta extract (eqPE) (2 ml/day), resulted in no recurrence of ascites or diarrhea. Conclusion: In canine PLE with prolonged diarrhea and ascites effusion, supplementation with eqPE may be considered a reasonable additional therapeutic strategy.

Equine placental extract supplement as a night barking remedy in dogs with cognitive dysfunction syndrome.

With the aging of pet dogs, there has been an increasing trend in senility-related diseases; additionally, cognitive disorders accompanied by abnormal behaviours are a major burden for owners. Recently, there have been a series of consultations regarding the fact that night barking, which is an abnormal behaviour, remarkably interferes with the owner's sleep and adversely affects the owner's quality of life. However, there has been no effective solution to this problem. In this study, three aged pet dogs diagnosed with dementia were administered an equine placental extract (eqPE) as pet supplement, which has been shown in laboratory models to improve cognitive function. Consequently, night barking ceased 1 week after the administration of eqPE in case 2 and it was observed to decrease in the other two dogs. Furthermore, night barking disappeared 2 and 3 weeks after the administration of eqPE in cases 1 and 3, respectively. No recurrence or exacerbation of night barking was observed in the three cases treated with the eqPE, and no adverse events were observed. These results suggest that eqPE may be useful for improving night barking in pet dogs with dementia, and it is expected to be a new treatment method.

A farewell to phlebotomy-use of placenta-derived drugs Laennec and Porcine for improving hereditary hemochromatosis without phlebotomy: a case report.

BACKGROUND: Human hepcidin, produced by hepatocytes, regulates intestinal iron absorption, iron recycling by macrophages, and iron release from hepatic storage. Recent studies indicate that hepcidin deficiency is the underlying cause of the most known form of hereditary hemochromatosis. CASE PRESENTATION: A 44-year-old Asian man who developed type 2 diabetes mellitus had elevated serum ferritin levels (10,191 ng/mL). Liver biopsy revealed remarkable iron deposition in the hepatocytes and relatively advanced fibrosis (F3). Chromosomal analysis confirmed the presence of transferrin receptor type 2 mutations (c.1100T>G, c.2008_9delAC, hereditary hemochromatosis type 3 analyzed by Kawabata). The patient received intravenous infusions of Laennec (672 mg/day, three times/week) or oral administration with Porcine (3.87 g/day) for 84 months as an alternative to repeated phlebotomy. At the end of the treatment period, serum ferritin level decreased to 428.4 ng/mL (below the baseline level of 536.8 ng/mL). Hemoglobin A1c levels also improved after treatment with the same or lower dose of insulin (8.8% before versus 6.8% after). Plural liver biopsies revealed remarkable improvements in the grade of iron deposition and fibrosis (F3 before versus F1 after) of the liver tissue. CONCLUSION: The discovery of hepcidin and its role in iron metabolism could lead to novel therapies for hereditary hemochromatosis. Laennec (parenteral) and Porcine (oral), which act as hepcidin inducers, actually improved iron overload in this hereditary hemochromatosis patient, without utilizing sequential phlebotomy. This suggests the possibility of not only improving the prognosis of hereditary hemochromatosis (types 1, 2, and 3) but also ameliorating complications, such as type 2 diabetes, liver fibrosis, and hypogonadism. Laennec and Porcine can completely replace continuous venesection in patients with venesection and may improve other iron-overloading disorders caused by hepcidin deficiency.

Placental extract suppresses the formation of fibrotic deposits by tumor necrosis factor alpha and transforming growth factor beta-induced epithelial-mesenchymal transition in ARPE-19 cells.

OBJECTIVE: Epithelial-mesenchymal transition (EMT) is involved in the development of proliferative vitreoretinopathy (PVR) and subsequent fibrosis. Previously, we demonstrated that placental extract ameliorates fibrosis in a mouse model of non-alcoholic steatohepatitis. In this study, we evaluated whether placental extract influences EMT and fibrosis through cytokine-induced EMT in the retinal pigment epithelial cells, in vitro. RESULTS: Placental extract did not inhibit EMT, but it suppressed excessive mesenchymal reactions and the subsequent fibrosis. These results suggest that placental extract effectively ameliorates EMT-associated fibrosis in PVR. This beneficial effect could be partially attributed to the suppression of excessive mesenchymal reactions.

Horse Placental Extract Enhances Neurogenesis in the Presence of Amyloid ß.

Human placental extract and animal-derived placental extracts from pigs and horses host a wide range of biological activities. Several placental products are used as medicines, cosmetics, and healthcare substances worldwide. However, the use of placental extracts for neuronal functioning is currently not established because the number of relevant studies is limited. A few previous reports suggested the neuroprotective effect and dendrite genesis effect of placental extract. However, no studies have reported on neurogenesis in placental extracts. Therefore, we aimed to investigate the effects of horse placental extract on neurogenesis, and we examined the protective effect of the extract on the onset of memory disorder. A horse placental extract, JBP-F-02, was used in this study. JBP-F-02 treatment dose-dependently increased the number of neural stem cells and dendrite length under Aß treatment in primary cultured cortical cells. The oral administration of JBP-F-02 to a 5XFAD mouse model of Alzheimer's disease at a young age significantly prevented the onset of memory dysfunction. This study suggests that the extract has the potential to prevent dementia.

A Novel Heptapeptide, GPPGPAG Transfers to the Brain, and Ameliorates Memory Dysfunction and Dendritic Atrophy in Alzheimer's Disease Model Mice.

We investigated the effects of a heptapeptide, GPPGPAG, on memory improvement and neuritic regeneration in Alzheimer's disease models to evaluate its potency as a new anti-Alzheimer's disease (AD) therapy. The anti-AD effects of GPPGPAG were evaluated in Aß-treated cortical neurons and 5XFAD, a mouse model of AD. Exposure of cortical neurons to Aß25-35 for 3 days resulted in atrophy of axons and dendrites. Treatment with GPPGPAG improved the dendritic atrophy of Aß-treated cortical neurons, but not axonal atrophy. Postsynaptic and presynaptic densities under Aß1-42 exposure were increased by GPPGPAG post treatment. Oral administration of GPPGPAG to 5XFAD mice for 15 days improved significantly object recognition memory and dendritic density. Direct infusion of GPPGPAG into the lateral ventricle of 5XFAD mice for 28 days improved object recognition memory. Following oral administration of GPPGPAG in mice, the undigested heptapeptide was detected in the plasma and cerebral cortex. Analysis of target protein of GPPGPAG in neurons by DARTS method identified 14-3-3ε as a bound protein. The protective effect of GPPGPAG on Aß1-42-induced dendritic atrophy was canceled by knockdown of 14-3-3ε. Taken together, these results suggest that GPPGPAG is orally available, transfers to the brain, and ameliorates memory dysfunction in AD brain, which is possibly mediated by 14-3-3ε-related dendritic restoration.

Placental extract ameliorates liver fibrosis in a methionine- and choline-deficient diet-induced mouse model of non-alcoholic steatohepatitis.

Non-alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease that is defined by the presence of inflammation and fibrosis, which ultimately leads to cirrhosis and hepatocellular carcinoma. We previously showed that human placental extract (hPE) was intramuscularly injected to ameliorates liver injury in a methionine- and choline-deficient (MCD) diet-induced NASH model. In the present study, we investigated the effects of hPE using dB/dB mice which exhibit obesity and insulin resistance and are thought to reproduce the pathological background of NASH. The MCD-diet induced liver atrophy accompanied by fibrosis around the liver sinusoids. hPE dose-dependently reduced the perivascular fibrosis. Moreover, αSMA-positive activated hepatic stellate cells increased in number in mice on the MCD diet, with this effect reversed by hPE treatment. hPE significantly decreased expression of Acta2, Col1a1, and Tgfb1 genes in hepatic stellate cells, and inhibited Smad phosphorylation. Moreover, hPE treatment increased the expression of the anti-oxidative genes Hmox1, Nqo1, Cat, and Sod1, and significantly enhanced nuclear factor erythroid 2-related factor 2 activity. Furthermore, hPE decreased the expression of Nox4 and attenuated the levels of intracellular reactive oxygen species. These results, along with our previous study, suggest that hPE effectively ameliorates liver fibrosis in NASH. This beneficial effect may, in part, be due to suppression of hepatic stellate cell activation.

Functional rescue of elastin insufficiency in mice by the human elastin gene: implications for mouse models of human disease.

Diseases linked to the elastin gene arise from loss-of-function mutations leading to protein insufficiency (supravalvular aortic stenosis) or from missense mutations that alter the properties of the elastin protein (dominant cutis laxa). Modeling these diseases in mice is problematic because of structural differences between the human and mouse genes. To address this problem, we developed a humanized elastin mouse with elastin production being controlled by the human elastin gene in a bacterial artificial chromosome. The temporal and spatial expression pattern of the human transgene mirrors the endogenous murine gene, and the human gene accurately recapitulates the alternative-splicing pattern found in humans. Human elastin protein interacts with mouse elastin to form functional elastic fibers and when expressed in the elastin haploinsufficient background reverses the hypertension and cardiovascular changes associated with that phenotype. Elastin from the human transgene also rescues the perinatal lethality associated with the null phenotype. The results of this study confirm that reestablishing normal elastin levels is a logical objective for treating diseases of elastin insufficiency such as supravalvular aortic stenosis. This study also illustrates how differences in gene structure and alternative splicing present unique problems for modeling human diseases in mice.

Placental extract suppresses cardiac hypertrophy and fibrosis in an angiotensin II-induced cachexia model in mice.

Cachexia is an intractable metabolic disorder that causes extreme weight loss. It is a symptom of many chronic diseases, including cancer, liver failure, congestive heart failure and chronic kidney disease, and there is as yet no effective treatment. While the mechanisms underlying cachexia are complex, it is often accompanied by elevated angiotensin II (Ang II). Human placental extract (HPE) is a source of numerous biologically active molecules and has been used clinically to treat chronic hepatitis, liver cirrhosis and other chronic diseases. Here, we investigated the effects of HPE in an Ang II-induced cachexia model in mice. HPE treatment preserved both fat mass and lean body mass and suppressed weight loss in the cachexia model, though food intake was unaffected. Ang II infusion also caused cardiac hypertrophy and fibrosis. HPE suppressed these effects as well as Ang II-induced cardiac expression of genes related to heart failure and cardiac remodeling. HPE also reversed Ang II-induced downregulation of mitochondria-related molecules and suppressed cardiac inflammation and oxidative stress. HPE administration may thus be an effective approach to the treatment of cachexia, cardiac hypertrophy and fibrosis.

Development of a mouse iron overload-induced liver injury model and evaluation of the beneficial effects of placenta extract on iron metabolism.

Hepatic iron deposition is seen in cases of chronic hepatitis and cirrhosis, and is a hallmark of a poorer prognosis. Iron deposition is also found in non-alcoholic steatohepatitis (NASH) patients. We have now developed a mouse model of NASH with hepatic iron deposition by combining a methione- and choline-deficient (MCD) diet with an iron-overload diet. Using this model, we evaluated the effects of human placenta extract (HPE), which has been shown to ameliorate the pathology of NASH. Four-week-old male C57BL/6 mice were fed the MCD diet with 2% iron for 12 weeks. In liver sections, iron deposition was first detected around the portal vein after 1 week. From there it spread throughout the parenchyma. Biliary iron concentrations were continuously elevated throughout the entire 12-week diet. As a compensatory response, the diet caused elevation of serum hepcidin, which accelerates excretion of iron from the body. Accumulation of F4/80-positive macrophages was detected within the sinusoids from the first week onward, and real-time PCR analysis revealed elevated hepatic expression of genes related inflammation and oxidative stress. In the model mice, HPE treatment led to a marked reduction of hepatic iron deposition with a corresponding increase in biliary iron excretion. Macrophage accumulation was much reduced by HPE treatment, as was the serum oxidation-reduction potential, an index of oxidative stress. These data indicate that by suppressing inflammation, oxidative stress and iron deposition, and enhancing iron excretion, HPE effectively ameliorates iron overload-induced liver injury. HPE administration may thus be an effective strategy for treating NASH.

Porcine placental extract facilitates memory and learning in aged mice.

Aging induces a decline in both memory and learning ability without predisposing an individual to diseases of the central nervous system, such as dementia. This decline can have a variety of adverse effects on daily life, and it can also gradually affect the individual and the people they are surrounded by. Since recent evidence indicated that placental extract has effects on brain function such as memory, we hypothesized that placental extract could ameliorate the age-associated reduction in cognitive function in aging. Here, we investigated the effect of new modified porcine placental extract (SD-F) on memory ability in aged mice at both the behavioral and molecular levels. Our results revealed that SD-F significantly enhanced memory ability in the object recognition and object location tasks in a dose-dependent manner in aged mice relative to controls. The numbers of Nissl-positive cells in the hippocampal cornu ammonis 3 (CA3) and dentate gyrus (DG) regions were increased in SD-F-treated aged mice relative to controls. RNA-seq analysis of the hippocampus of aged mice identified 542 differentially expressed genes, of which 216 were up-regulated and 326 were down-regulated in SD-F-treated mice relative to controls. Of the 216 up-regulated genes, we identified four characteristic genes directly related to memory, including early growth response protein 1 (Egr1), growth arrest and DNA-damage-inducible, beta (Gadd45b), NGFI-A binding protein 2 (Nab2), and vascular endothelial growth factor a (Vegfa). These results suggest that the efficacy of SD-F involves upregulation of these genes.

Placental extract ameliorates non-alcoholic steatohepatitis (NASH) by exerting protective effects on endothelial cells.

Non-alcoholic steatohepatitis (NASH) is a severe form of fatty liver disease that is defined by the presence of inflammation and fibrosis, ultimately leading to cirrhosis and hepatocellular carcinoma. Treatment with human placental extract (HPE) reportedly ameliorates the hepatic injury. We evaluated the effect of HPE treatment in a mouse model of NASH. In the methione- and choline-deficient (MCD) diet-induced liver injury model, fibrosis started from regions adjacent to the sinusoids. We administered the MCD diet with high-salt loading (8% NaCl in the drinking water) to mice deficient in the vasoprotective molecule RAMP2 for 5 weeks, with or without HPE. In both the HPE and control groups, fibrosis was seen in regions adjacent to the sinusoids, but the fibrosis was less pronounced in the HPE-treated mice. Levels of TNF-α and MMP9 expression were also significantly reduced in HPE-treated mice, and oxidative stress was suppressed in the perivascular region. In addition, HPE dose-dependently increased survival of cultured endothelial cells exposed to 100 µM H2O2, and it upregulated expression of eNOS and the anti-apoptotic factors bcl-2 and bcl-xL. From these observations, we conclude that HPE ameliorates NASH-associated pathologies by suppressing inflammation, oxidative stress and fibrosis. These beneficially effects of HPE are in part attributable to its protective effects on liver sinusoidal endothelial cells. HPE could thus be an attractive therapeutic candidate with which to suppress progression from simple fatty liver to NASH.

Combination of aspartic acid and glutamic acid inhibits tumor cell proliferation.

Placental extract contains several biologically active compounds, and pharmacological induction of placental extract has therapeutic effects, such as improving liver function in patients with hepatitis or cirrhosis. Here, we searched for novel molecules with an anti-tumor activity in placental extracts. Active molecules were separated by chromatographic analysis, and their antiproliferative activities were determined by a colorimetric assay. We identified aspartic acid and glutamic acid to possess the antiproliferative activity against human hepatoma cells. Furthermore, we showed that the combination of aspartic acid and glutamic acid exhibited enhanced antiproliferative activity, and inhibited Akt phosphorylation. We also examined in vivo tumor inhibition activity using the rabbit VX2 liver tumor model. The treatment mixture (emulsion of the amino acids with Lipiodol) administered by hepatic artery injection inhibited tumor cell growth of the rabbit VX2 liver. These results suggest that the combination of aspartic acid and glutamic acid may be useful for induction of tumor cell death, and has the potential for clinical use as a cancer therapeutic agent.

Expression of 36-kDa microfibril-associated glycoprotein (MAGP-36) in human keratinocytes and its localization in skin.

Microfibril-associated glycoprotein-36 (MAGP-36) is a recently isolated elastin-binding protein and considered to be a member of microfibril-associated glycoproteins (MAGPs). We studied the expression of MAGP-36 in cultured normal human keratinocytes and its localization in the skin. MAGP-36 was found to be expressed in cultured human keratinocytes by Western blot and RT-PCR assays. The levels of MAGP-36 (polypeptide and mRNA) and the number of MAGP-36-producing keratinocytes were greatly increased during Ca(2+)-induced differentiation of keratinocytes. Immunohistochemical studies demonstrated that MAGP-36 colocalized with elastic fibers and formed candelabra like-fibers in the superficial dermis of normal skin. In the elderly skin of sun-exposed region, immunoreactivity of MAGP-36 in the superficial dermis disappeared. In the lesional skin of pseudoxanthoma elasticum which is an elastin-related disorder, immunoreactivity of MAGP-36 was found in the accumulation of disintegrated elastic fibers. The results show that MAGP-36 is a component of elastic fibers in the dermis and co-operates with elastin in normal and diseased conditions.

Silicate fiber-based 3D cell culture system for anticancer drug screening.

BACKGROUND: Three-dimensional (3D) in vitro cultures can recapitulate the physiological in vivo microenvironment. 3D Modeling techniques have been investigated and applied in anticancer drug screening. MATERIALS AND METHODS: A silicate fiber scaffold was used for 3D cell cultures, and used to model the efficacy of anticancer drugs, such as mytomicin C and doxorubicin. RESULTS: A unique 3D structure was observed in 13 human tumor cell lines on scaffold, and these cells exhibited higher drug resistance than cells in two-dimensional (2D) cultures. Furthermore, the production of lactate and expression of the nuclear factor-kappa B (NF-κB)-regulated genes B cell lymphoma-2 (BCL2), cyclooxygenase-2 (COX2), and vascular endothelial growth factor (VEGF) were higher in 3D cultures than in 2D cultures. CONCLUSION: These findings suggest that a 3D model using a silicate fiber scaffold can mimic features of cancer, and is also a suitable model for the evaluation of anticancer drugs in vitro.