Anti-oxidative stress mechanism of velvet antler polypeptide in Alzheimer's disease model mice / 解剖学报

Objective To study the effect of velvet antler polypeptides (VAP) on antioxidant in Alzheimer' s disease model mice. Methods Eight months old male amyloid precursor protein (APP)/presenilin-l (PS1) double transgenic mice were selected as Alzheimer' s disease (AD) model and divided into the model group and the VAP intervention group, 12 in each group. Besides, normal mice of the same brood (with no transgene) were recruited as a control group (n= 12).After 6 months of intragastric administration, behavior, morphology and oxidative stress related indicators were detected.SH-SY5 cells were used to establish AD model of damaged by Ap2535. The expression levels of APP and p-secreatase-l(BACE1) protein in mouse hippocampus were detected by Western blotting. VAP intervention group SH-SY5Y cells was cultured with VAP (500 g/L) and amyloid P(Ap) 2535(25 ixmol/L) for 24 hours. Control group cells were normally cultured by DMEM medium. Cell apoptosis, membrane potential, reactive oxygen species (ROS) levels and oxidative stress related indexes were detected. Results In animal models, compared with the model group, the escape latency of mice in the VAP intervention group was shortened (P<0. 05). The neuronal cells in the CA1 region of the hippocampus of the model group were reduced and arranged disorderly. The arrangement of the VAP intervention group was relatively regular, and the morphology was significantly improved. Compared with the model group, senile plaques were decreased in the VAP intervention group. Compared with the model group, the malondialdehyde (MDA) content ol the VAP intervention group increased, and the superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) content increased, the difference was statistically significant. Compared with the control group, the APP and BACE1 content in the model group increased. Compared with the model group, the contents of APP and BACE1 in the VAP intervention group decreased, and the difference was statistically significant (P<0. 05). In the cell model, the apoptosis rates of the VAP intervention group decreased. Compared with the model group, the mitochondrial membrane potential of the VAP intervention group increased, the content ol ROS decreased, the content of MDA decreased, and the content of SOD and GSH-Px increased. The difference were statistically significant (P<0. 05). Conclusion VAP has a protective effect on oxidative stress damage caused by Alzheimer' s disease model animals and cells, which may be achieved by reducing ROS production and increasing the activity of antioxidant enzymes to reduce Ap deposition.

The anti-aging effect of velvet antler polypeptide is dependent on modulation of the gut microbiota and regulation of the PPARα/APOE4 pathway.

We investigated the anti-aging effects of velvet antler polypeptide on D-galactose (D-gal)-induced aging mice. D-gal-induced aging mice were established and randomly divided into five groups, the control, model, vitamin E (VE), velvet antler polypeptide low-dose and velvet antler polypeptide high-dose groups. The Morris water maze test was used to evaluate the learning and memory abilities of aging mice. Hippocampal neurons were observed via hematoxylin-eosin staining and transmission electron microscopy. Biochemical methods were used to detect the activities of superoxide dismutase, malonaldehyde and other enzymes and evaluate the influence of velvet antler polypeptide on the antioxidant capacity of aging mice. Using 16S rRNA gene sequencing and meristem technology, we assessed the effect of velvet antler polypeptide on aging mice's intestinal flora and fatty acid metabolism. The experimental results showed that velvet antler polypeptide could significantly improve aging mice's learning and cognitive abilities, increase the activities of superoxide dismutase, glutathione peroxidase, and catalase in the serum decrease the malonaldehyde content. Intestinal microecological analysis showed that velvet antler polypeptide could significantly increase the beneficial bacterial genus Lactobacillus abundance. Western blot analysis further demonstrated that velvet antler polypeptide could promote fatty acid metabolism by activating peroxisome proliferator-activated receptor α (PPARα) and upregulating the expression of the downstream enzymes carnitine-palmitoyl transferase-1 A and acyl-CoA oxidase 1 while downregulating that of apolipoprotein E4 (APOE4), thereby reducing fatty acid accumulation and increasing adenosine-triphosphate (ATP) production. Therefore, velvet antler polypeptide improves the intestinal microecology and activates the PPARα/APOE4 pathway to regulate fatty acid metabolism.

Antiapoptotic effects of velvet antler polypeptides on damaged neurons through the hypothalamic-pituitary-adrenal axis.

We investigated the effects of velvet antler polypeptide on cognitive impairment and the underlying mechanisms. Hydrogen peroxide-induced cell injury was used to establish an in vitro model of SH-SY5Y cells. In addition, we established an in vivo mouse model of cognitive impairment using intraperitoneal injections of scopolamine hydrobromide in strain mice. We administered three different doses of velvet antler polypeptide in this mouse model and assessed the influence of velvet antler polypeptide on the morphology of hippocampal neurons, hippocampal neuronal apoptosis, adrenocorticotropic hormone, and corticosterone activities in brain tissue samples, and the molecular and biochemical regulation of B-cell lymphoma-2, B-cell lymphoma-2 Associated X-protein, Cysteine-aspartic acid protease-3, glucocorticoid receptor, mineralocorticoid receptor, and corticotropin-releasing hormone in murine hippocampal neurons. Our data suggest that velvet antler polypeptide decreases glucocorticoid receptor, mineralocorticoid receptor, and corticotropin-releasing hormone levels and regulates the hormones released by the hypothalamic-pituitary-adrenal axis, thus suppressing neuronal apoptosis.

Protective Effect and Mechanism of Velvet Antler Peptide on Rotenone-induced SH-SY5Y Cell Damage / 中国实验方剂学杂志

Objective:To investigate the intervening effect of velvet antler peptide （VAP） on rotenone-induced neuroblastoma （SH-SY5Y） cell damage and explore its related mechanism. Method:0.5 μmol·L-1 rotenone was used to SH-SY5Y cells to establish an in vitro model of Parkinson's disease （PD）. A blank control group， a model group， high， medium and low dose VAP groups （150，100，50 mg·L-1， respectively） and a rapamycin group were established. The number of lewy bodies， changes in mitochondrial membrane potential， content of reactive oxygen species （ROS） and α-synuclein （α-syn）， protein kinase B （Akt）， and mammalian target of rapamycin （mTOR） were observed by hematoxylin-eosin（HE） staining， rhodamine 123 staining， DCFH-DA staining and immunohistochemical staining expression respectively. Result:The results of HE staining showed that as compared with the blank group， the number of cells in model group was reduced， the tentacle structure became dull， the shape became round， and eosinophilic Lewy bodies were visible in cytoplasm. As compared with model group， there was no significant difference in cell morphology from rapamycin group and VAP high， medium and low dose groups， but there were fewer Lewy bodies in cytoplasm in these four groups. Rhodamine 123 staining showed that as compared with blank group， the mitochondrial membrane potential was increased significantly in model group （P<0.05）. As compared with the model group， the mitochondrial membrane potential was decreased in rapamycin group and VAP high， medium and low dose groups （P<0.05）. DCFH-DA staining results showed that as compared with blank group， the content of ROS was increased significantly in cells of model group （P<0.05）. As compared with model group， the content of ROS was decreased in rapamycin group and VAP high， medium and low dose groups （P<0.05）. Immunohistochemical staining showed that as compared with blank group， the protein expression levels of α-syn，Akt，and mTOR were increased significantly in model group （P<0.05）. As compared with model group， the protein expression levels of α-syn and mTOR were significantly reduced in rapamycin group and VAP high and medium dose groups （P<0.05）， and the expression levels of Akt were significantly reduced in rapamycin group and VAP high-dose group （P<0.05）. Conclusion:Velvet antler peptides may play a neuroprotective role by regulating the Akt/mTOR signaling pathway and promoting the degradation of α-syn in SH-SY5Y cells.

Velvet antler polypeptide partially rescue facet joint osteoarthritis-like phenotype in adult ß-catenin conditional activation mice.

BACKGROUND: Wnt/ß-catenin signaling pathway is closely related to osteoarthritis. In our preliminary study, ß-catenin conditional activation (cAct) mice that specifically over-express ß-catenin gene in cartilage chondrocyte exhibits osteoarthritis-like phenotype in the lumbar disc and knee joint. Therefore, we used the mice to model FJ-OA and test the potential curative effect of Velvet Antler Polypeptide (VAP) on this mice model. METHODS: We tested the effect of VAP on ß-catenin conditional activation mice, and used Cre negative littermates as controls. Micro-CT, histology and histomorphometry analysis were performed to evaluate the curative effect of VAP on mice facet joint-like phenotype. Expression of ß-catenin and collagen II was detected by immunohistochemistry (IHC) and western-blot., MMP13, ADAMTS4 and ADAMTS5 was detected by immunofluorescence (IF). RT-PCR analysis was preformed to detect mRNA expression of cartilage degrading enzymes, such as MMP13, ADAMTS4 and ADAMTS5. RESULTS: Results of micro-CT (µCT) analysis showed that VAP could partially reverse lumbar disc osteophyte formation observed in ß-catenin(ex3)Col2ER mice. Histology data revealed VAP partially improved facet joint cartilage tissue invades. Histomorphometry analysis showed an increase in total cartilage area after VAP treatment. IHC show that VAP reduced ß-catenin protein levels and moderately up-regulated collagen II protein levels. RT-PCR and IF data showed that VAP down-regulated the expression of extracellular matrix synthesis (ECM) degradation enzymes MMP13, ADAMTS4 and ADAMTS5. CONCLUSION: Taken together, VAP may modulate ECM by inhibits MMP13, ADAMTS4 and ADAMTS5 via Wnt /ß-catenin signaling pathway. Velvet Antler Polypeptide may be a potential medicine for FJ-OA.

Promotive effect of velvet antler polypeptide-collagen/chitosan composite materials on fracture healing of mandibular defect of rabbits and its mechanism / 吉林大学学报(医学版)

Objective:To prepare the velvet antler polypeptide-collagen/chitosan composite materials,and to investigate its promotive effect on cicatrization of mandibular defect and possible mechanism.Methods:The collagen and chitosan solution were mixed.The composite material was prepared by glutaraldehyde crosslinking method.The microstructure of the composite material was observed by transmission electron microscope (SEM).The unilateral mandibular defect models of 36 rabbits were established.The rabbits were divided into experiment and control groups,and each group was divided into 4-,8-and 12-week subgroups,and there were 6 rabbits in each sub group.The rabbits in experiment group were implanted with velvet antler polypeptide-collagen /chitosan composite materials and the rabbits in control group were treated.4,8 and 12 weeks after operation,the histology of bone defect and peripheral nerve reconstruction of the rabbit models were detected by CT;the expression of vascular endothelial growth factor (VEGF) in bone tissue of the rabbits was detected by immunohistochemistry;the ultrastructure of bone defect was observed by SEM.Results:The structure of composite materials had layered folds and the inner diameter of the stent became larger and mainly dominated by sheet structure,which was the ideal structure of biological materials.4 weeks after operation,the new bone was formatted in experiment group,most of the new bone like-tissue materials were degraded,and the VEGF expression showed an increasing trend;8 weeks after operation,the trabecular bone in the bone defect of the rabbits in experiment group was increased obviously and the expression of VEGF was decreased.12 weeks after operation,the new bone formation and the density in experiment group was consistent with the normal tissue,and the expression level of VEGF returned to normal.At each the point after operation,the degree of bone defect healing and bone formation rate in experiment group were obviously prior to control group.Conclusion:Velvet antler polypeptide-collagen /chitosan composite material has the promotive effect on the fracture healing of mandibular defect of the rabbits and its possible mechanism may be related to promoting the expression of VEGF.

A recombinant polypeptide from velvet antler of Cervus nippon Temminck exhibits similar immunomodulatory effects as its natural counterpart.

CONTEXT: Velvet antler (VA) is recognized as one of the most important Chinese traditional medical herbs. To date, the immunoactivity of the single component of VA is rarely studied though its compound extracts have been well analyzed. OBJECTIVE: The current study was designed to evaluate the immunomodulatory effects of a recombinant polypeptide (rVAP32) based on the VA of the sika deer by comparison with its natural counterpart (nVAP32). MATERIALS AND METHODS: Splenocytes proliferation and NK-cell cytotoxicity assay was evaluated by the WST-8 colorimetric method. CD4+/CD8+ cell subpopulations regulation was screened by the flowcytometry method and the Th1 or Th2-related cytokine production was measured by ELISA. RESULTS: In vitro tests showed that both rVAP32 and nVAP32 could significantly stimulate splenocytes proliferation and enhance the NK-cell cytotoxicity and CD4+/CD8+ cell subpopulations when compared with the irrelevant peptide and blank control groups. Also, they demonstrated a significant capacity in up- and down-regulating the expression of Th1- and Th2-related cytokines, respectively. There is no statistically significant difference found between the rVAP32 tested group and nVAP32 control group. DISCUSSION AND CONCLUSION: The results obtained herein indicate that rVAP32 has the similar immunomodulatory effects on the immune system of mice as its counterpart nVAP32 in vitro. The further test in vivo is qualified and rVAP32 is promised for developing a new biopharmaceutical product as a substitute for nVAP32.

Effects of velvet antler polypeptide on sexual behavior and testosterone synthesis in aging male mice / 亚洲男科学杂志(英文版)

Twenty-four-month-old male C57BL/6 mice with low serum testosterone levels were used as a late-onset hypogonadism (LOH) animal model for examining the effects of velvet antler polypeptide (VAP) on sexual function and testosterone synthesis. These mice received VAP for 5 consecutive weeks by daily gavage at doses of 100, 200, or 300 mg kg-1 body weight per day (n = 10 mice per dose). Control animals (n = 10) received the same weight-based volume of vehicle. Sexual behavior and testosterone levels in serum and interstitial tissue of testis were measured after the last administration of VAP. Furthermore, to investigate the mechanisms of how VAP affects sexual behavior and testosterone synthesis in vivo, the expression of steroidogenic acute regulatory protein (StAR), cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in Leydig cells was also measured by immunofluorescence staining and quantitative real-time PCR. As a result, VAP produced a significant improvement in the sexual function of these aging male mice. Serum testosterone level and intratesticular testosterone (ITT) concentration also increased in the VAP-treated groups. The expression of StAR, P450scc, and 3β-HSD was also found to be enhanced in the VAP-treated groups compared with the control group. Our results suggested that VAP was effective in improving sexual function in aging male mice. The effect of velvet antler on sexual function was due to the increased expression of several rate-limiting enzymes of testosterone synthesis (StAR, P450scc, and 3β-HSD) and the following promotion of testosterone synthesis in vivo.

The anti-osteoporotic effect of velvet antler polypeptides from Cervus elaphus Linnaeus in ovariectomized rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The deer velvet antler is well known for its traditional medicinal value, and is widely used in the clinic. It is recorded in the Compendium of Materia Medica that the deer velvet antler replenishes vital essence and strengthens the bone. AIM OF THE STUDY: The goal of this study was to investigate the anti-osteoporotic effect of total velvet antler polypeptides from Cervus elaphus Linnaeus (TVAPL) on ovariectomized rats (OVX), and their possible mechanism of the action. MATERIALS AND METHODS: Wistar rats were divided into five groups: sham-operated group, OVX group, and OVX rats treated with 20, 40, or 60 mk/kg TVAPL for 12 weeks. Calcium and phosphorus levels, bone weight coefficient (BWC), bone mineral density (BMD), and bone mineral content (BMC) were evaluated. The MTT assay was used to measure the activities of interleukin-1 (IL-1) and interleukin-6 (IL-6). In addition, cartilage cells and osteoblast-like cells were exposed to TVAPL, natural velvet antler polypeptides (nVAP), and synthetic velvet antler polypeptides (sVAP), to determine their effects on cell proliferation using the tritiated thymidine incorporation assay. Finally, the enzyme-linked immunosorbent assay was used to determine the effects of nVAP and sVAP on cytokines related to bone metabolism. RESULTS: The administration of TVAPL for 12 weeks significantly reversed osteoporosis in OVX rats, thereby improving the BWC, BMD, BMC, and bone microarchitecture. IL-1 and IL-6 were significantly activated in the OVX group, and their activation was inhibited by TVAPL. In addition, nVAP and sVAP promoted the proliferation of cartilage and osteoblast-like cells (p<0.01 or p<0.001), and inhibited the secretion of IL-1α from THP-1 monocytic cells in vitro. CONCLUSION: These results suggest that TVAPL are effective in preventing bone loss in OVX rats. The effect of TVAPL on osteoporosis is due to inhibition of IL-1 and IL-6 by nVAP, and promotion of mitosis. sVAP has similar bioactivity as nVAP. Thus, both TVAPL and sVAP may be potential therapeutic agents for the treatment of postmenopausal osteoporosis.

Experimental study of VAP-chitosans-honey suspension on the healing of decubitus ulcer in swines / 中国矫形外科杂志

[Objective]To study the influence of VAP(velvet antler polypeptide)-chitosan-honey suspension on decubitus ulcer.[Method]Swines'pressure ulcers were used as decubitus ulcer model.Honey was used as solvent carrier.VAP and chitosan were put into the honey in different proportion.The suspension was applied to the ulcer once a day for seven days.The dressings were changed once every other day.The healing state of the ulcer was observed and the area of the ulcer was calculated.The changes of the ulcer histopathology were observed.[Result]In the group of the suspension proportion of the VAP to the chitosan was 4:1,the wounds had little effusion and the granulation tissues grew fast with the scars falling off early and Absolutely.Pathology results indicated that in the group of the suspension proportion of the VAP to chitosan was 4:1,none necrosis was found,the epithelization was apparent,and the inflammatory cells were fewer.There was no edema,but more newly born blood vessles.[Conclusion]The VAP-chitosan-honey suspension could apparently promote the healing of decubitus ulcer,but the possible mechanism needs to be further studied.

EFFECT OF VELVET ANTLER POLYPEPTIDE(VAP) ON DIFFERENTIATION OF RAT BRAIN-DERIVED STEM CELLS IN VITRO / 解剖学报

Objective To investigate the probabilities of brain-derived stem cells from fetal rats differentiating into neurons and astrocytes by velvet antler polypeptide(VAP) in vitro. Methods Neural stem cells from E12-14d rats were cultured for 7 days until neural stem cells (NSCs) aggregations were formed into neurospheres. The neurospheres were cultured at different concentrations of VAP, and immunocytochemistry was used to detect the differentiation of neural stem cells. Results The differentiated cells in 50?g/L VAP group are more than that in control group; the number of NSE positive cells in 50?g/L,100?g/L and 200?g/L groups is more than that in control group.Conclusion Neural stem cells can be successfully induced into neurons by VAP in vitro, which could provide a basis for regeneration of nerve system.;