Effects of hyaluronic acid on differentiation of human amniotic epithelial cells and cell-replacement therapy in type 1 diabetic mice.

Our hypothesis is that hyaluronic acid may regulate the differentiation of human amniotic epithelial cells (hAECs) into insulin-producing cells and help the treatment of type 1 diabetes. Herein, a protocol for the stepwise in vitro differentiation of hAECs into functional insulin-producing cells was developed by mimicking the process of pancreas development. Treatment of hAECs with hyaluronic acid enhanced their differentiation of definitive endoderm and pancreatic progenitors. Endodermal markers Sox17 and Foxa2 and pancreatic progenitor markers Pax6, Nkx6.1, and Ngn3 were upregulated an enhanced gene expression in hAECs, but hAECs did not express the ß cell-specific transcription factor Pdx1. Interestingly, hyaluronic acid promoted the expression of major pancreatic development-related genes and proteins after combining with commonly used inducers of stem cells differentiation into insulin-producing cells. This indicated the potent synergistic effects of the combination on hAECs differentiation in vitro. By establishing a multiple injection transplantation strategy via tail vein injections, hAECs transplantation significantly reduced hyperglycemia symptoms, increased the plasma insulin content, and partially repaired the islet structure in type 1 diabetic mice. In particular, the combination of hAECs with hyaluronic acid exhibited a remarkable therapeutic effect compared to both the insulin group and the hAECs alone group. The hAECs' paracrine action and hyaluronic acid co-regulated the local immune response, improved the inflammatory microenvironment in the damaged pancreas of type 1 diabetic mice, and promoted the trans-differentiation of pancreatic α cells into ß cells. These findings suggest that hyaluronic acid is an efficient co-inducer of the differentiation of hAECs into functional insulin-producing cells, and hAECs treatment with hyaluronic acid may be a promising cell-replacement therapeutic approach for the treatment of type 1 diabetes.

Glucose-lowering and hypolipidemic activities of polysaccharides from Cordyceps taii in streptozotocin-induced diabetic mice.

BACKGROUND: Hyperglycemia and dyslipidemia are classic features of patients with diabetes mellitus (DM). Cordyceps taii, a folk medicinal fungus native to southern China, possesses various pharmacological activities. This study aimed to assess the glucose-lowering and hypolipidemic effects of polysaccharides from C. taii (CTP) in streptozotocin (STZ)-induced diabetic mice. METHODS: Kunming mice were intraperitoneally injected with STZ at a dose of 100 mg/kg body weight. After induction of diabetes, diabetic mice were randomly divided into five groups: diabetic mellitus group (DM), metformin-treated group, low, medium, and high-dose CTP-treated group (CTP-L, CTP-M, and CTP-H). Normal mice served as the control group. After treatment for 28 days, body weight, fasting serum insulin (FSI), fasting blood glucose (FBG), homeostasis model assessment-insulin resistance (HOMA-IR), triglyceride (TG), total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), high-density lipoprotein-cholesterol (HDL-C), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and C-reactive protein (CRP) levels were measured. Histological analysis of pancreatic tissue and immune organ indices was also performed to evaluate the anti-diabetes effect of CTP. SPSS (version 21.0) software was used for statistical analysis, and statistical differences were considered significant at p < 0.05. RESULTS: Compared with the DM group, the body weight and FSI level of CTP-H group increased by 36.13 and 32.47%, whereas the FBG and HOMA-IR decreased by 56.79 and 42.78%, respectively (p < 0.05). Histopathological examination of the pancreas revealed that CTP improved and repaired the impaired islet ß-cells in pancreatic tissue. Compared with the DM group, the levels of TC, TG, and LDL-C decreased by 13.84, 31.87, and 36.61%, whereas that of HDL-C increased by 28.60% in CTP-H (p < 0.05). Further study showed that the thymus index in CTP-H was elevated by approximately 54.96%, and the secretion of pro-inflammatory cytokines TNF-α, IL-6, and CRP was inhibited by approximately 19.97, 34.46, and 35.41%, respectively (p < 0.05). CONCLUSION: The anti-diabetes effect of CTP is closely associated with immunoregulation and anti-inflammation, and CTP may be considered as a therapeutic drug or functional food for DM intervention.

[Pharmacokinetics of ganoderic acids].

Ganoderic acid(GA) is one of main bioactive components produced by Ganoderma lucidum,which a traditional Chinese herbal medicine and a kind of tracyclic triterpene lanosterol derivatives with highly oxidized structure. It has a variety of important pharmacological activities,such as anticancer,immunoregulation,anti-oxidation,anti-diabetes and anti-HIV. At present,the studies of GA mainly focus on biosynthesis,fermentation control,isolation and purification,structure identification and pharmacological effects.However,there are a fewer pharmacokinetic studies of GA,although it is closely related to the clinical application. Recent studies have shown that GA can be absorbed rapidly by gastrointestinal tract and distributed in various tissues and organs after oral intake. GA is metabolized by liver at phase Ⅰ and phase Ⅱ,and then mainly excreted by bile. In this paper,the pharmacokinetic characteristics of GA and its absorption,distribution,metabolism and excretion(ADME) will be systematically summarized,in order to provide scientific basis for the application and development studies of Ganoderma triterpenoid drugs and their rational clinical use.

Dynamic changes in chromatin and microtubules at the first cell cycle in SCNT or IVF goat embryos.

We investigated the dynamic changes in chromatin and microtubules at the first cell cycle in goat somatic cell nuclear transfer (SCNT)-derived and in vitro fertilization (IVF)-derived embryos. Stage-dependent and characteristic changes to chromatin and microtubules occurred in SCNT-derived embryos at different times after activation. About half donor nuclei underwent premature chromosome condensation (PCC) at 1 h post activation, and furtherly reached telophase at 2 h after activation. However, we discovered that the separated chromosomes reaggregated, not keeping two independent nuclei; and formed one pronucleus at 2.5 h after activation. One pronucleus was found in all reconstructed oocytes except other no nucleus oocytes from 3 to 22 h after activation. Reconstructed oocytes reached the first mitotic metaphase at 23 h post activation, which was later than that of IVF-derived embryos at 16 h after insemination. SCNT-derived embryos showed significantly higher abnormalities in the first mitotic metaphase spindle, compared with IVF-derived embryos. Abnormal spindles included multi polar and half spindles. SCNT-derived embryos began to cleave at 24 h after activation, which was later than that of IVF-derived embryos at 21 h after insemination. SCNT-derived embryos showed delayed conversion from telophase to interphase than IVF-derived embryos during cleavage. These might lead to poor development in SCNT-derived embryos.

Hyaluronic acid enhances proliferation of human amniotic mesenchymal stem cells through activation of Wnt/ß-catenin signaling pathway.

This study investigated the pro-proliferative effect of hyaluronic acid (HA) on human amniotic mesenchymal stem cells (hAMSCs) and the underlying mechanisms. Treatment with HA increased cell population growth in a dose- and time-dependent manner. Analyses by flow cytometry and immunocytochemistry revealed that HA did not change the cytophenotypes of hAMSCs. Additionally, the osteogenic, chondrogenic, and adipogenic differentiation capabilities of these hAMSCs were retained after HA treatment. Moreover, HA increased the mRNA expressions of wnt1, wnt3a, wnt8a, cyclin D1, Ki-67, and ß-catenin as well as the protein level of ß-catenin and cyclin D1 in hAMSCs; and the nuclear localization of ß-catenin was also enhanced. Furthermore, the pro-proliferative effect of HA and up-regulated expression of Wnt/ß-catenin pathway-associated proteins - wnt3a, ß-catenin and cyclin D1 in hAMSCs were significantly inhibited upon pre-treatment with Wnt-C59, an inhibitor of the Wnt/ß-catenin pathway. These results suggest that HA may positively regulate hAMSCs proliferation through regulation of the Wnt/ß-catenin signaling pathway.

New cytochalasins from medicinal macrofungus Crodyceps taii and their inhibitory activities against human cancer cells.

Three new cytochalasins (1-3) together with two known cytochalasin analogues (4 and 5) were isolated from the chloroform fraction of ethanolic extract of a medicinal macrofungus Cordyceps taii. The structures of the new compounds were elucidated on the basis of spectroscopic analysis, including HRESIMS, 1D and 2D NMR experiments. The cytotoxicities of Compounds 1-5 were investigated by the sulforhodamine B (SRB) method in vitro against human highly metastatic lung cancer cell 95-D, human lung cancer cell line A-549 and normal hepatocyte HL-7702. The results revealed that Compounds 4 and 5 showed potent antitumor activities against human lung cancer cell 95-D with IC50 value of 3.67 and 4.04 µM, respectively. In comparison with cisplatin, the first-line chemotherapy drug, they had little or no cytotoxicity on normal cells, but showed stronger cytotoxic effects on cancer cells 95-D.

Antitumor and antimetastatic activities of chloroform extract of medicinal mushroom Cordyceps taii in mouse models.

BACKGROUND: Cordyceps taii, an entomogenous fungus native to south China, is a folk medicine with varieties of pharmacological activities including anticancer effect. To validate the ethnopharmacological claim against cancer, the antitumor and antimetastatic activities of chloroform extract of C. taii (CFCT) were investigated in vivo. METHODS: The in vitro cytotoxic activities of CFCT against human lung cancer (A549) and gastric cancer (SGC-7901) cells were evaluated using the Sulforhodamine B (SRB) assay. In vivo anti tumor and antimetastatic activities, Kunming mice bearing sarcoma 180 and C57BL/6 mice bearing melanoma B16F10 were employed, respectively. The antitumor effects of CFCT were completely evaluated on the basis of the tumor weight, survival time, histologic analysis, and immune organ indices. The histopathological change, metastatic foci and malignant melanoma specific marker HMB45 in the lung tissue were detected for the evaluation of the antimetastatic activity of CFCT. RESULTS: CFCT exhibited dose- and time-dependent cytotoxicities against A549 and SGC-7901 cells with the IC50 values of 30.2 and 65.7 µg/mL, respectively. Furthermore, CFCT at a dose of 50 or 100 mg/kg could significantly inhibit the tumor growth in vivo and prolonged the survival time in two different models as compared with the model group, especially when combined with the CTX at a low dose rate. And it also increased spleen index of Kunming mice and thymus index of C57BL/6 mice. Meanwhile, histologic analysis illustrated that CFCT alone or in combination with CTX could induce tumor tissue necrosis of both models. In addition, CFCT at a dose of 50 or 100 mg/kg inhibited the lung metastasis of melanoma B16F10 in tumor-bearing C57BL/6 mice. The antimetastatic effect was also observed when CFCT was used in combination with CTX. In comparison to any other groups, CFCT at a dose of 100 mg/kg could effectively enhance the GSH-Px activities of various tissues in tumor-bearing C57BL/6 mice. CONCLUSIONS: These findings demonstrate that CFCT has potent in vivo antitumor and antimetastatic activities, and may be helpful to the development of anticancer chemopreventive agents from C. taii.

[Research progress on hypoglycemic effect and its mechanism of action of medicinal fungal polysaccharides].

Due to substantial morbidity and high complications, diabetes mellitus is considered as the third "killer" in the world. Medicinal fungal polysaccharides, as water-soluble macromolecular substances with low toxicity, exhibit diversified pharmacological actions such as immune regulation, anti-tumor, antivirus, antioxidant, anti-aging, hypoglycemic effect and improving liver and kidney function. In recent year, a number of investigators reported medicinal fungal polysaccharides showed good anti-diabetes and hypoglycemic activity, and their acting mechanisms involved in glycometabolism and ß cell function, e. g. promoting glycogen synthesis, promoting glycolysis, inhibiting the activity of α-glucosidase, promoting insulin secretion, increasing insulin sensitivity, enhancing antioxidation. Therefore, the hypoglycemic activity and its mechanisms of action of medicinal fungal polysaccharides showed characteristics of multiple effects, multi-target, and multi-pathway regulation. These finding suggest that medicinal fungal polysaccharides are a promising source for the development of discovery of anti-diabetic agent.

[The mechanism of inhibition of the increase in intracellular calcium concentration by the islet amyloid polypeptide in high glucose-stimulated INS-1 cells].

OBJECTIVE: To explore the potential mechanism of the inhibition of increased intracellular free calcium concentration ([Ca²âº]i) by short-term exposure to the islet amyloid polypeptide (IAPP) in high glucose-stimulated pancreatic ß cells. METHODS: The pancreatic ß cells were loaded with calcium sensitive fluorescent indicator Fluo-4/AM. The fluorescence intensity, which represented [Ca²âº]i, was measured in time by laser scanning confocal microscope before and after stimulated by glucose, KCl, caffeine and carbachol. RESULT: The fluorescence intensity F/F0 in INS-1 cells, increased to about 2 folds after glucose stimulation. After the exposure to the IAPP with different concentration, the fluorescence intensity F/F0 was decreased slightly in the pretreated cells by 16.7 mmol/L glucose with 0.5 µmol/L IAPP. However, after the pretreatment of IAPP with the concentration of 1.0, 5.0, 10.0 µmol/L, the fluorescence intensity F/F0 showed a dose-dependent decrease with statistical difference. The fluorescence intensity F/F0 in the cells increased rapidly in a peak pattern after the stimulation of 30 mmol/L KCl. But with the pretreatment of 10.0 µmol/L IAPP, the fluorescence intensity F/F0 decreased with statistical difference. With 20 mmol/L caffeine and 100 µmol/L carbachol which stimulated Ca²âº release respectively from internal ryanodine receptor (RYR) and inositol triphosphate (IP3) Ca²âº storage, the fluorescence intensity F/F0 curve presented a peak pattern. After 10 µmol/L IAPP pretreatment, the fluorescence intensity F/F0 showed no statistical difference from the control group. CONCLUSIONS: The short-term effect of IAPP on pancreatic ß cells has no influence on the caffeine and carbachol stimulated Ca²âº release from endoplasmic reticulum RYR and IP3 Ca²âº storage. The inhibition of calcium increase in INS-1 cells by short-term exposure to IAPP may mainly via inhibiting the voltage-gated L-calcium channels with intact release capacity of Ca²âº storage.

Galangin induces the osteogenic differentiation of human amniotic mesenchymal stem cells via the JAK2/STAT3 signaling pathway.

The regulation of stem cell directional differentiation is a core research topic in regenerative medicine, and modulating the fate of stem cells is a promising strategy for precise intervention through the utilization of naturally small molecule compounds. The present study aimed to explore the potential pro-osteogenic differentiation effect of galangin, a flavonoid derived from Alpinia officinarum, on human amniotic mesenchymal stem cells (hAMSCs) and the underlying molecular mechanism. The results showed that galangin had no cytotoxicity towards hAMSCs when the concentration was less than 50 µM. Treatment with 10 µM galangin significantly increased alkaline phosphatase (ALP) secretion and calcium deposition in hAMSCs. Meanwhile, galangin upregulated the mRNA and protein expression of early osteoblast-specific markers, namely ALP, RUNX2, and OSX, and late osteoblast-specific markers, CoL1α1, OPN, and OCN, in hAMSCs. Furthermore, signaling pathway screening studies showed that galangin enhanced the phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). In addition, molecular docking results suggest there is a promising interaction between galangin and JAK2. Finally, treatment with the JAK2 specific inhibitor AG490 effectively reversed the induction of osteogenic differentiation, upregulation of osteoblast-specific marker expression, and activation of JAK2/STAT3 signaling induced by galangin. These results show that galangin induces the osteogenic differentiation of hAMSCs through the JAK2/STAT3 signaling pathway and could serve as a promising small molecular osteoinducer for application to hAMSCs in regenerative medicine.

Protective effects of Ligularia fischeri root extracts against ulcerative colitis in mice through activation of Bcl-2/Bax signalings.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease characterized by high levels of proinflammatory cytokines and epithelial barrier dysfunction. The root of Ligularia fischeri (Ledeb.) Turcz. is a traditional Chinese medicinal herb with diverse therapeutic properties, which has been successfully used to treat inflammation-related diseases. However, little is known about its effect and mechanism against UC. PURPOSE: To investigate the efficacy and mechanism of L. fischeri root extracts against UC. METHODS: L. fischeri root samples were prepared using the alcohol extraction method and liquid-liquid extraction method. A dextran sodium sulfate-induced UC mouse model and a lipopolysaccharide (LPS)-induced inflammatory cell model were employed in the present study. Cell apoptosis was detected by TUNEL staining, and an enzyme-linked immunosorbent assay was used to quantify the abundance of inflammatory factors in tissues. Hematoxylin and eosin staining and Masson staining were employed to analyze drug toxicity to the liver and kidney. A myeloperoxidase (MPO) assay kit was used to detect neutrophil infiltration in colon tissues. RT-qPCR was then employed to quantify the transcriptional levels of proinflammatory and apoptotic-related genes, while tight junction and apoptosis-related proteins were quantified via western blotting. Gas Chromatography/Mass Spectrometry analysis was then performed to identify the natural compounds in L. fischeri root extracts. RESULTS: The water decoction extract, methanol extract, and especially the chloroform extract (CE) exerted potent therapeutic effects in UC mice. Similar to the positive control group (5-aminosalicylic acid), oral administration of CE (30, 60, and 90 mg/kg/d) elicited distinct therapeutic effects on UC mice in the medium- and high-dose groups. CE decreased disease activity index, histopathological score, and MPO level significantly, and effectively retained the colon length. Furthermore, CE significantly reduced the levels of proinflammatory cytokines, including interleukin (IL)-1ß, IL-6, and tumor necrosis factor-α and enhanced the expression of tight junction proteins, such as zonula occludens (ZO)-1, ZO-2, claudin-1, and occludin, as well as the transcriptional levels of mucins, such as MUC-1 and MUC-2, in UC mice. Notably, CE prevented apoptosis of colonic epithelial cells by up-regulating Bcl-2 and down-regulating Bax. Also, CE inhibited the secretion of pro-inflammatory cytokines and apoptosis in LPS-induced RAW264.7 macrophages via the activation of Bcl-2/Bax signals. CONCLUSIONS: Collectively, L. fischeri root extracts, especially CE, have obvious therapeutic effects against UC. CE reduces inflammation and protects the intestinal epithelial cells and intestinal epithelial barrier via activation of the Bcl-2/Bax signaling pathway, and may be a promising therapeutic agent for UC treatment.

Evaluation of fertilization capability of frozen-thawed completely immotile spermatozoa collected from a white bengal tiger after interspecific ICSI with bovine oocytes.

The objective of this study was to evaluate the fertilization capability of White Bengal Tiger frozen-thawed completely immotile spermatozoa after interspecific intracytoplasmic sperm injection (ICSI) with bovine oocytes. The fertilization status of presumptive zygotes was assessed 18 h after ICSI by immunofluorescence staining and confocal microscopy. The fertilization rate was 34.8% (8/23), as confirmed by the extrusion of two polar bodies, or male and female pronuclei formation. For unfertilized oocytes (65.2%, 15/23), one activated oocyte had an activated spermatozoon but most were unactivated oocytes with unactivated spermatozoa (1/15, 6.7% vs 10/15, 66.7%, respectively, p < 0.05). These results showed that White Bengal Tiger frozen-thawed completely immotile spermatozoa retained the capacity to fertilize bovine oocytes after interspecific ICSI. This is the first report of in vitro produced zygotes using tiger immotile sperm with bovine oocytes by interspecific ICSI technique, which provides an efficient and feasible method for preservation and utilization of endangered feline animals.

A new ganoderic acid from Ganoderma lucidum mycelia.

A new ganoderic acid (GA), 7-O-ethyl ganoderic acid O (GA-O) (1), together with two known compounds, GA-T (2) and GA-Me (3), was isolated and purified from fermented mycelia of Ganoderma lucidum. The structure of the new triterpenoid was elucidated on the basis of the interpretation of extensive spectroscopic data (HR-MS, IR, UV, 1D and 2D NMR) as 3 alpha,15 alpha,22-triacetoxy-7 alpha-ethoxy-5 alpha-lanost-8,24E-dien-26-oic acid. The new compound was found to contain a rare ethoxyl group at C-7. In addition, its cytotoxicity against 95D and HeLa human cancer cell lines was also evaluated.

RASL11B gene enhances hyaluronic acid-mediated chondrogenic differentiation in human amniotic mesenchymal stem cells via the activation of Sox9/ERK/smad signals.

This study aimed to elucidate the molecular mechanisms, whereby hyaluronic acid, a main extracellular matrix component of articular cartilage, promotes the chondrogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs). Our previous findings indicated that hyaluronic acid combined with hAMSCs showed a marked therapeutic effect against rat osteoarthritis. In the present study, hyaluronic acid markedly enhanced the expression of chondrocyte-specific markers including Col2α1, Acan, and Sox9 in hAMSCs, with strong synergistic effects on chondrogenic differentiation, in combination with the commonly used inducer, transforming growth factor ß3 (TGF-ß3). Microarray analysis showed that Ras-like protein family member 11B (RASL11B) played a pivotal role in the process of hyaluronic acid-mediated chondrogenesis of hAMSCs. This directional differentiation was significantly inhibited by RASL11B knockdown, but RASL11B overexpression dramatically promoted the expression of Sox9, a master chondrogenesis transcriptional factor, at the levels of transcription and translation. Increased Sox9 expression subsequently resulted in high expression levels of Col2α1 and Acan and the accumulation of cartilage-specific matrix components, such as type 2 collagen and glycosaminoglycans. Moreover, we observed that RASL11B activated the signal molecules such as ERK1/2, and Smad2/3 in the presence of hyaluronic acid during TGF-ß3-induced chondrogenesis of hAMSCs. Taken together, these findings suggest that hyaluronic acid activates the RASL11B gene to potentiate the chondrogenic differentiation of hAMSCs via the activation of Sox9 and ERK/Smad signaling, thus providing a new strategy for cartilage defect repairing by hyaluronic acid-based stem cell therapy.

Hyaluronic acid ameliorates the proliferative ability of human amniotic epithelial cells through activation of TGF-ß/BMP signaling.

Human amniotic epithelial cells (hAECs) are a useful and noncontroversial source of stem cells for cell therapy and regenerative medicine, but their limited proliferative ability hinders the acquisition of adequate quantities of cells for clinical use due to not expressing telomerase in hAECs. Our previous study showed that hyaluronic acid (HA), an important component of the extracellular matrix, promoted the proliferation of human amniotic mesenchymal stem cells. Herein, we hypothesize that HA might improve the proliferative capability of hAECs. In the present study, the role of HA on the proliferation of human amniotic epithelial cells (hAECs) in vitro was investigated for the first time. HA at molecular weight of 300 kDa showed an obvious pro-proliferation effect on hAECs. Furthermore, HA not only kept phenotypic characteristics and differentiation capabilities of hAECs, but significantly promoted the secretion of the anti-inflammatory factors such as IL-10 and TGF-ß1, and the expression of stem cell pluripotent factors such as Oct4 and Nanog. Analysis of PCR microarray data and RT-qPCR validation showed that TGF-ß/BMP signaling was activated in the presence of HA. Further study showed that SB431542, an inhibitor of the TGF-ß/BMP signaling, significantly suppressed the mRNA expression of TGFBR3, BMP4, BMP7, BMPR1B, SMAD3, SMAD4, and the pro-proliferative effect of HA on hAECs. These data suggest that HA is a safe and effective enhancer for in vitro expansion of hAECs, whose regulatory mechanism involves the TGF-ß/BMP signaling.

Cocktail of Hyaluronic Acid and Human Amniotic Mesenchymal Cells Effectively Repairs Cartilage Injuries in Sodium Iodoacetate-Induced Osteoarthritis Rats.

Osteoarthritis (OA) is one of the most common refractory degenerative articular cartilage diseases. Human amniotic mesenchymal cells (hAMSCs) have emerged as a promising stem cell source for cartilage repair, and hyaluronic acid (HA) has proven to be a versatile regulator for stem cell transplantation. Herein, an effective and straightforward intra-articular injection therapy using a cocktail of hAMSCs and HA was developed to treat knee OA in a rat model. The injured cartilage was remarkably regenerated, yielding results comparable to normal cartilage levels after 56 days of treatment. Both hAMSCs and HA were indispensable organic components in this therapy, in which HA could synergistically enhance the effects of hAMSCs on cartilage repair. The regenerative mechanism was attributed to the fact that the addition of HA comprehensively enhances the activities of hAMSCs, including chondrogenic differentiation, proliferation, colonization, and regenerative modulation. This cocktail paves a new avenue for injection therapy to treat OA, holding the potential to realize rapid clinical translation.

Effect of CD44 on differentiation of human amniotic mesenchymal stem cells into chondrocytes via Smad and ERK signaling pathways.

CD44 antigen (CD44) is a transmembrane protein found in cell adhesion molecules and is involved in the regulation of various physiological processes in cells. It was hypothesized that CD44 directly affected the chondrogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs). In the present study, the expression of chondrocyte­associated factors was detected in the absence and presence of the antibody blocker anti­CD44 antibody during the chondrogenic differentiation of hAMSCs. Following inhibition of CD44 expression, the transcriptional levels of chondrocyte­associated genes SRY­box transcription factor 9, aggrecan and collagen type II α 1 chain, as well as the production of chondrocyte markers type II collagen and aggrecan were significantly decreased in hAMSCs. Further investigation indicated that there was no significant change in total ERK1/2 expression following inhibition of CD44 expression; however, phosphorylated (p)­ERK1/2 expression was decreased. The expression of p­Smad2/3 was also upregulated following CD44 inhibition. These data indicated that CD44 may affect the differentiation of hAMSCs into chondrocytes by regulating the Smad2/3 and ERK1/2 signaling pathway.

Hyaluronic acid promotes osteogenic differentiation of human amniotic mesenchymal stem cells via the TGF-ß/Smad signalling pathway.

AIMS: This study investigated the effects of hyaluronic acid (HA), a commonly used osteogenic medium referred to as DAG, and the combined administration of HA and DAG (CG) on the osteogenic differentiation of human amniotic mesenchymal stem cells (hAMSCs), and the underlying mechanism. MAIN METHODS: The phenotype of hAMSCs was detected by flow cytometry and immunocytochemical staining. Alkaline phosphatase (ALP) and calcium deposition assays were employed for evaluating the osteogenic differentiation of hAMSCs. The expression of osteogenesis-related genes and proteins was determined by quantitative reverse transcription PCR (qRT-PCR) and Western blotting, respectively. Meanwhile, the molecular mechanism of osteogenic differentiation of hAMSCs was detected by PCR array and qRT-PCR. KEY FINDINGS: The results showed that treatment with CG could significantly stimulate hAMSC ALP activity and calcium deposition compared to treatment with DAG, while HA had little effect. The expression of osteogenesis-related molecules and stemness-related molecules was up-regulated at the mRNA and protein levels in all three groups, and this up-regulation was most significant in the CG group. In addition, treatment with CG significantly increased the gene expressions involved in regulation of the TGF-ß/Smad signalling pathway compared to treatment with DAG. Furthermore, the pro-osteogenic differentiation effects as well as the up-regulated expression of genes observed in the CG treatment group were significantly inhibited when the cells were pre-treated with SB431542, an inhibitor of the TGF-ß/Smad pathway. SIGNIFICANCE: These results suggest that HA in combination with DAG could significantly enhance the osteogenic differentiation of hAMSCs, potentially via the TGF-ß/Smad signalling pathway.

Synergistic antitumor efficacy of antibacterial helvolic acid from Cordyceps taii and cyclophosphamide in a tumor mouse model.

The antibacterial agent helvolic acid, which was isolated from the active antitumor fraction of Cordyceps taii, showed potent cytotoxicity against different human cancer cells. In the present study, the in vivo antitumor effect of helvolic acid was investigated in murine sarcoma S180 tumor-bearing mice. Doses of 10 and 20 mg/kg/day helvolic acid did not exert significant antitumor activity. Interestingly, co-administration of 10 mg/kg/day helvolic acid and 20 mg/kg/day cyclophosphamide (CTX) - a well-known chemotherapy drug - showed promising antitumor activity with a growth inhibitory rate of 70.90%, which was much higher than that of CTX alone (19.5%). Furthermore, the combination markedly prolonged the survival of tumor-bearing mice. In addition, helvolic acid enhanced the immune organ index. The protein expression levels of ß-catenin, cyclin D1, and proliferating cell nuclear antigen were significantly suppressed in mice treated with 20 mg/kg/day helvolic acid and in those receiving combination therapy. Taken together, these results indicated that helvolic acid in combination with CTX showed potent in vivo synergistic antitumor efficacy, and its mechanism of action may involve the Wnt/ ß-catenin signaling pathway.