ABSTRACT
Although Radix Achyranthis Bidentatae (RAB) and Radix Cyathulae (RC) are from two different medicinal plants, they are both used as 'Niu-Xi', a widely used traditional Chinese medicine that is believed to stimulate menstruation and affect bone injury. Angiogenesis is actively involved in treating these illnesses. The aim of the present study was to investigate whether the whole extracts of RAB and RC possess pro-angiogenic effects. In order to examine this idea whole extracts of RAB and RC were extracted with boiling water followed by ethanol, respectively. Results from the MTT, wound healing and tube formation assays in human umbilical vein endothelial cells (HUVECs) in vitro revealed that the whole extracts of RAB and RC did not increase cell proliferation or tube formation, but enhanced cell migration. Their angiogenic effects were also confirmed in zebrafish in vivo via increasing the sprout numbers in the sub-intestinal vessel. As determined by quantitative polymerase chain reaction, the whole extracts of RAB and RC both regulated the expression of cell migration-related genes in zebrafish. It is concluded that the whole extracts of RAB and RC induced angiogenesis in HUVECs in vitro and in zebrafish in vivo via increasing cell migration.
ABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Patients suffering from musculoskeletal pain and swellings occupy many hospital beds and demand many rehabilitation facilities. Chinese Medicine is offering many alternatives to ameliorate pain and swelling. However, evidence-based scientific publications supporting their efficacy on pain relief are inadequate. The in vitro and in vivo efficacy of a topical use Chinese herbal bath formula (HB) on anti-inflammation and swelling control was studied. MATERIALS AND METHODS: The therapeutic mechanisms of HB were studied in vitro via anti-inflammatory and pro-angiogenic assays on RAW264.7 and HUVEC cells, respectively. Fibroblast proliferation was also studied with Hs27 cells. The in vivo angiogenic effect of HB was also studied using zebrafish model, while its efficacy of in vivo anti-Inflammation and swelling control were investigated using rat paw edema model. The affected paw was treated by immersing it in the HB or distilled water as control. The sensation of pain, change in paw thickness and inflammation marker in serum were analyzed. RESULTS: In the anti-inflammation assay, HB significantly inhibited nitrite release from RAW264.7 by 47.6% at 800 µg/ml. In the pro-angiogenic assays, it reduced wound area in HUVEC by 8.2% and increased tube formation of HUVEC by 11.5% at 300 µg/ml. HB also stimulated Hs27 proliferation up to 23.5% at 1200 µg/ml. It showed in vivo pro-angiogenic effect by increasing the mean sprout number in the embryos of zebrafish by 2.4 folds. The in vivo therapeutic effects of HB on edema was illustrated by the significant longer thermal withdrawal latency and thinner paw thickness compared with control. After 14 days of treatment, HB also reduced the IL-6 concentration in the serum of rat by 20.9% significantly. CONCLUSIONS: This study showed that HB is effective for swelling control and pain relief from edema due to its anti-inflammatory and pro-angiogenic properties.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Inflammation/drug therapy , Pain/drug therapy , Administration, Topical , Animals , Cell Line , Cell Movement/drug effects , Cell Proliferation , Drugs, Chinese Herbal/administration & dosage , Embryo, Nonmammalian/blood supply , Embryo, Nonmammalian/drug effects , Fibroblasts/drug effects , Fibroblasts/physiology , Macrophages/drug effects , Male , Mice , Neovascularization, Physiologic/drug effects , Rats , Rats, Sprague-Dawley , ZebrafishABSTRACT
Fasciaderived stem cells (FDSCs) were previously isolated from the fascia of the gluteus maximus of the rat. However, the use of FDSCs as a cell source for musculoskeletal tissue engineering has not been compared with that of adiposederived stem cells (ADSCs) and bone marrowderived mesenchymal stem cells (BMSCs). Therefore, the present study aimed to compare the mesenchymal stem cell (MSC) and selfrenewal stem cell markers, proliferative capacity and multilineage differentiation potential of these stem cells in vitro. The MSC and embryonic stem cell (ESC) marker profiles were compared using flow cytometry and quantitative polymerase chain reaction (qPCR). Their proliferative capacities were compared using 5bromo2'deoxyuridine and MTT assays. Their osteogenic, adipogenic and chondrogenic differentiation potentials were compared using standard staining assays and qPCR. The FDSCs possessed similar cell morphology and immunophenotypic profiles with BMSCs and ADSCs. FDSCs demonstrated a similar expression pattern of ESC markers with ADSCs, which has higher expression of sex determining region Ybox (Sox)2 and octamerbinding transcription factor 4, and lower expression of Krüppellike factor 4, when compared with BMSCs. FDSCs exhibited higher proliferation under serumdeprived conditions (0.5% FBS growth medium), and attained higher expression levels of collagen type I, α 2 and type II, α 1 as well as Sox9 mRNA than ADSCs and BMSCs upon chondrogenic induction. An increased amount of proteoglycan deposition was also observed in the FDSC group. However, lower levels of adipogenic and osteogenic marker expression in FDSCs were detected compared with ADSCs and BMSCs upon adipogenic and osteogenic induction, respectively. FDSCs possessed high chondrogenic potential, low osteogenic and adipogenic differentiation potential and were responsive to the induction signals for collagenrich fascial structure regeneration. Therefore, FDSCs may represent an improved alternative cell source to conventional ADSCs and BMSCs for musculoskeletal tissue repair and tissue engineering, particularly for collagenrich structures with poor vasculature.
Subject(s)
Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Animals , Antigens, Surface/metabolism , Biomarkers/metabolism , Cell Differentiation/genetics , Cell Proliferation , Colony-Forming Units Assay , Gene Expression Profiling , Immunophenotyping , Male , RatsABSTRACT
AIM: Carthami Flos (CF) is a Chinese herb traditionally used for cardiovascular disease and bone injury in China with pharmacological effects on improving blood circulation. The aim of this study was to investigate the angiogenic potential of CF whole extract (extracted by boiling with water, followed by ethanol) and the underlying mechanisms in human microvascular endothelial cells (HMEC-1) in vitro and in transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish with transgenic endothelial cells expressing EGFP (Enhanced Green Fluorescent Protein) in vivo. METHODS: Effects of CF whole extract on cell proliferation, migration and tube formation in HMEC-1 cells in vitro were detected by MTT assay, wound healing assay and tube formation assay. Its angiogenic effect in zebrafish was investigated by monitoring the sprout number in the sub-intestinal vessel (SIV), and the underlying mechanisms were tested by quantitative real-time PCR. RESULTS: CF whole extract increased cell proliferation, migration and tube formation in vitro in HMEC-1 cells. Its angiogenic effect was also confirmed in vivo in zebrafish by increasing the sprout number in the SIV. As determined by quantitative real-time PCR, CF whole extract up-regulated the expression of angiogenesis-related genes in zebrafish, including angiogenic and its associated growth factors and receptors (e.g. IGF1, CTGF, NRP2, and VEGFR3), transcription factor (e.g. HIF1A), matrix degradation and endothelial cell migration-related factors (e.g. MMP2, MMP9, TIMP2, PLG and PLAU), cell adhesion molecules (e.g. ITGAV, ITGB3, beta-catenin and PECAM1), tubule formation factors (e.g. ANGPT1, TIE-2, PDGFR-B, CDH5, S1PR1, FGF2, Shh, and TGFRB1), and blood vessel maturation/formation factor (e.g. Ephrin B2). CONCLUSIONS: CF whole extract increased angiogenesis in HMEC-1 cells in vitro and in zebrafish in vivo with multiple mechanisms.
Subject(s)
Angiogenesis Inducing Agents/pharmacology , Carthamus/chemistry , Endothelial Cells/drug effects , Plant Extracts/pharmacology , Animals , Animals, Genetically Modified/embryology , Cell Movement/drug effects , Cell Proliferation/drug effects , Cells, Cultured , Drugs, Chinese Herbal/pharmacology , Embryo, Nonmammalian/drug effects , Humans , Zebrafish/embryologyABSTRACT
ETHNOPHARMACOLOGICAL RELEVANCE: Er-Miao-San (EMS) is a traditional Chinese herbal formulation that contains combinations of Rhizoma Atractylodis (RA) and Cortex Phellodendri (CP). It exhibits analgesic and anti-inflammatory activities and have been used for the treatment of various "Bi Zheng" for thousand years in China. The aims of the present study were to investigate the anti-inflammatory activities of EMS and elucidate the underlying mechanisms with regard to its molecular basis of action for the best combination. MATERIALS AND METHODS: The anti-inflammatory effects of EMS were studied by using lipopolysaccharide (LPS)-stimulated activation of nitric oxide (NO) and pro-inflammatory cytokine production in mouse RAW264.7 macrophages. Expression of inducible NO synthase (iNOS), mitogen-activated protein kinases (MAPKs) phosphorylation, p65 phosphorylation, inhibitor-κBα (IκBα) degradation, and NF-κB DNA-binding activity were further investigated. RESULTS: The present study demonstrated that EMS could suppress the production of NO in LPS-stimulated RAW264.7 macrophages. However, CP and RA did not have significant inhibitory effect on them. EMS also inhibited the production of tumor necrosis factor-alpha, interleukin-1 beta and macrophage chemotactic protein-1. Further investigations showed EMS could suppress iNOs expression and p38 phosphorylation. EMS significantly decreased the content of IκBα, reduced the level of phosphorylated p65 and suppressed the NF-κB DNA-binding activity. All these results suggested the inhibitory effects of EMS on the production of inflammatory mediators through the inhibition of the NF-κB pathway. CONCLUSIONS: Our results indicated that EMS inhibited inflammatory events and iNOS expression in LPS-stimulated RAW264.7 cells through the inactivation of the MAPK and NF-κB pathway. This study gives scientific evidence validating the use of EMS in treatment of patients with "Bi Zheng" in clinical practice in traditional Chinese medicine.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Drugs, Chinese Herbal/chemistry , Inflammation Mediators/antagonists & inhibitors , Macrophages/drug effects , Mitogen-Activated Protein Kinases/antagonists & inhibitors , NF-kappa B/antagonists & inhibitors , Phellodendron/chemistry , Rhizome/chemistry , Animals , Anti-Inflammatory Agents, Non-Steroidal/chemistry , Anti-Inflammatory Agents, Non-Steroidal/isolation & purification , Cells, Cultured , Cytokines/antagonists & inhibitors , Cytokines/biosynthesis , Inflammation Mediators/metabolism , Lipopolysaccharides/pharmacology , MAP Kinase Signaling System/drug effects , Macrophages/metabolism , Medicine, Chinese Traditional , Mice , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesisABSTRACT
Low intensity pulsed ultrasound (LIPUS) was reported to accelerate the rate of fracture healing. When LIPUS is applied to fractures transcutaneously, bone tissues at different depths are exposed to different ultrasound fields. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). Moreover, we have reported that the therapeutic effect of LIPUS is dependent on the axial distance of ultrasound beam in rat fracture model. However, the mechanisms of how different axial distances of LIPUS influence the mechanotransduction of bone cells are not understood. To understand the cellular mechanisms underlying far field LIPUS on enhanced fracture healing in rat model, the present study investigated the effect of ultrasound axial distances on (1) osteocyte, the mechanosensor, and (2) mechanotransduction between osteocyte and pre-osteoblast (bone-forming cell) through paracrine signaling. We hypothesized that far field LIPUS could enhance the osteogenic activities of osteoblasts via paracrine factors secreted from osteocytes. The objective of this study was to investigate the effect of axial distances of LIPUS on osteocytes and osteocyte-osteoblast mechanotransduction. In this study, LIPUS (plane; 2.2 cm in diameter, 1.5MHz sine wave, ISATA=30 mW/cm(2)) was applied to osteocytes (mechanosensor) at three axial distances: 0mm (near field), 60mm (mid-near field) and 130 mm (far field). The conditioned medium of osteocytes (OCM) collected from these three groups were used to culture pre-osteoblasts (effector cell). In this study, (1) the direct effect of ultrasound fields on the mechanosensitivity of osteocytes; and (2) the osteogenic effect of different OCM treatments on pre-osteoblasts were assessed. The immunostaining results indicated the ultrasound beam at far field resulted in more ß-catenin nuclear translocation in osteocytes than all other groups. This indicated that osteocytes could detect the acoustic differences of LIPUS at various axial distances. Furthermore, we found that the soluble factors secreted by far field LIPUS exposed osteocytes could further promote pre-osteoblasts cell migration, maturation (transition of cell proliferation into osteogenic differentiation), and matrix calcification. In summary, our results of this present study indicated that axial distance beyond near field could transmit ultrasound energy to osteocyte more efficiently. The LIPUS exposed osteocytes conveyed mechanical signals to pre-osteoblasts and regulated their osteogenic cellular activities via paracrine factors secretion. The soluble factors secreted by far field exposed osteocytes led to promotion in migration and maturation in pre-osteoblasts. This finding demonstrated the positive effects of far field LIPUS on stimulating osteocytes and promoting mechanotransduction between osteocytes and osteoblasts.
Subject(s)
Osteoblasts/diagnostic imaging , Osteoblasts/physiology , Ultrasonic Therapy , Alkaline Phosphatase/metabolism , Animals , Cell Differentiation , Cell Line , Cell Movement , Cell Proliferation , Enzyme-Linked Immunosorbent Assay , Fracture Healing/physiology , Mechanotransduction, Cellular , Mice , Nitric Oxide/metabolism , Osteocytes/diagnostic imaging , Osteocytes/physiology , Staining and Labeling , Ultrasonography , beta Catenin/metabolismABSTRACT
This study investigated the effect of LIPUS on fracture healing when fractures were exposed to ultrasound at three axial distances: z=0 mm, 60 mm, and 130 mm. We applied LIPUS to rat fracture at these three axial distances mimicking the exposure condition of human fractures at different depths under the soft tissue. Measurement of LIPUS shows pressure variations in near field (nearby transducer); uniform profile was found beyond it (far field). We asked whether different positions of the fracture within the ultrasound field cause inconsistent biological effect during the healing process. Closed femoral fractured Sprague-Dawley rats were randomized into control, near-field (0mm), mid-near field (60 mm) or far-field (130 mm) groups. Daily LIPUS treatment (plane, but apodized source, see details in the text; 2.2 cm in diameter; 1.5 MHz sine waves repeating at 1 kHz PRF; spatial average temporal average intensity, ISATA=30 mW/cm(2)) was given to fracture site at the three axial distances. Weekly radiographs and endpoint microCT, histomorphometry, and mechanical tests were performed. The results showed that the 130 mm group had the highest tissue mineral density; and significantly higher mechanical properties than control at week 4. The 60 mm and 0 mm groups had significantly higher (i.e. p<0.05) woven bone percentage than control group in radiological, microCT and histomorphometry measurements. In general, LIPUS at far field augmented callus mineralization and mechanical properties; while near field and mid-near field enhanced woven bone formation. Our results indicated the therapeutic effect of LIPUS is dependent on the axial distance of the ultrasound beam. Therefore, the depth of fracture under the soft tissue affects the biological effect of LIPUS. Clinicians have to be aware of the fracture depth when LIPUS is applied transcutaneously.
Subject(s)
Femoral Fractures/physiopathology , Femoral Fractures/therapy , Femur/physiopathology , Femur/radiation effects , Fracture Healing/radiation effects , Ultrasonic Therapy/methods , Animals , Female , Femoral Fractures/diagnostic imaging , Femur/diagnostic imaging , High-Energy Shock Waves , Rats , Rats, Sprague-Dawley , Treatment Outcome , UltrasonographyABSTRACT
Low-intensity pulsed ultrasound (LIPUS) with I(SATA)= 30 mW/cm(2) has been proven in facilitating fracture healing, which the spatial average intensity over the on period (I(SATP)) equals 150 mW/cm(2). As active ultrasound wave is only delivered during the on period, we postulate 150 mW/cm(2) is responsible for the beneficial effect of LIPUS. In this study, we compare the biologic effects of 30 mW/cm(2) and 150 mW/cm(2). We propose I(SATA) = 150 mW/cm(2) could further enhance fracture healing process. Closed femoral fractured Sprague-Dawley rats were randomized into control, LIPUS-30 (30 mW/cm(2)) and LIPUS-150 (150 mW/cm(2)) groups. Weekly radiographs and endpoint microCT, histomorphometry, and biomechanical tests were performed. The results show that LIPUS-30 had significantly higher low-density bone volume fraction and woven bone percentage than that of control and LIPUS-150 in microCT and histologic measurements, respectively. Mechanically, failure torque of LIPUS-30 was significantly higher than control and LIPUS-150 at week 6. In conclusion, LIPUS at I(SATA)= 150 mW/cm(2) did not further enhance fracture healing.