Anti-Inflammatory Effect of Allicin Associated with Fibrosis in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by pulmonary vascular remodeling. Recent evidence supports that inflammation plays a key role in triggering and maintaining pulmonary vascular remodeling. Recent studies have shown that garlic extract has protective effects in PAH, but the precise role of allicin, a compound derived from garlic, is unknown. Thus, we used allicin to evaluate its effects on inflammation and fibrosis in PAH. Male Wistar rats were divided into three groups: control (CON), monocrotaline (60 mg/kg) (MCT), and MCT plus allicin (16 mg/kg/oral gavage) (MCT + A). Right ventricle (RV) hypertrophy and pulmonary arterial medial wall thickness were determined. IL-1ß, IL-6, TNF-α, NFκB p65, Iκß, TGF-ß, and α-SMA were determined by Western blot analysis. In addition, TNF-α and TGF-ß were determined by immunohistochemistry, and miR-21-5p and mRNA expressions of Cd68, Bmpr2, and Smad5 were determined by RT-qPCR. Results: Allicin prevented increases in vessel wall thickness due to TNF-α, IL-6, IL-1ß, and Cd68 in the lung. In addition, TGF-ß, α-SMA, and fibrosis were lower in the MCT + A group compared with the MCT group. In the RV, allicin prevented increases in TNF-α, IL-6, and TGF-ß. These observations suggest that, through the modulation of proinflammatory and profibrotic markers in the lung and heart, allicin delays the progression of PAH.

Pilose antler aqueous extract promotes the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by stimulating the BMP-2/Smad1, 5/Runx2 signaling pathway.

Peptides from Pilose antler aqueous extract (PAAE) have been shown to stimulate the proliferation and differentiation of bone marrow mesenchymal stem cells (BMSCs). However, the underlying molecular mechanisms are not well understood. Here, PAAE was isolated and purified to explore the molecular mechanisms underlying PAAE's effects on BMSCs as well as its osteoprotective effects in ovariectomized rats. Our results showed that PAAE promoted proliferation and differentiation of BMSCs to become osteoblasts by enhancing ALP activity and increasing extracellular matrix mineralization. The trabecular microarchitecture of ovariectomized rats was also found to be protected by PAAE. Quantitative reverse transcription-polymerase chain reaction (Quantitative RT-PCR) results suggest that PAAE also increased the expression of osteogenic markers including, alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), osteocalcin (OCN), bone morphogenetic protein-2 (BMP-2), and collagen I (COL-I). Immunoblotting results indicated that PAAE upregulated the levels of BMP-2 and Runx2 and was associated with Smad1/5 phosphorylation. PAAE A at the concentration of 200 µg·mL-1 showed the strongest effect on proliferation and osteogenic differentiation of BMSCs after 48 h. Using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), we identified the molecular weight of PAAE A and found that it is less than 3000 Da and showed several significant peaks. In conclusion, PAAE activates the BMP-2/Smad1, 5/Runx2 pathway to induce osteoblastic differentiation and mineralization in BMSCs and can inhibit OVX-induced bone loss. These mechanisms are likely responsible for its therapeutic effect on postmenopausal osteoporosis.

The evaluation of embryotoxicity of Ligusticum chuanxiong on mice and embryonic stem cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Chuanxiong (Chuanxiong Rhizoma, CR), the dried rhizome of Ligusticum chuanxiong Hort, has been used during pregnancy for more than 2000 years. However, the embryotoxicity of CR was not evaluated so far. The purpose of this study was to examine the safety and rational use of CR during pregnancy on mice and mouse embryonic stem cell (ES), and to explore the mechanism of embryotoxicity. AIM OF THE STUDY: This study was carried out to evaluate embryotoxicity of CR decoction in vivo and in vitro, and to explore the mechanism of embryotoxicity from the perspective of bone metabolism. MATERIALS AND METHODS: In animal experiments, pregnant mice were randomly assigned into 5 groups, i.e. mice were orally treated with CR decoction at dosages of 0 (distilled water, as negative controls), 2, 8, 32 g/kg/d (low, medium and high-dose group), and vitamin A (as positive controls), respectively. Maternal and embryo-fetal parameters were registered after cesarean section. The fetal skeletal development was further assessed with the alizarin red S and Hematoxylin-Eosin staining (H&E staining) and fluorescent imaging. Meanwhile, the mouse embryonic stem cell test model (EST model) was established to objectively evaluate the toxicity of CR on the embryo development. The median inhibitory proliferation values (IC50) for both the mouse embryonic stem cell D3 (ES) and mouse embryonic fibroblast 3T3 (3T3) were detected with MTT assays. After removal of inhibiting factor (LIF), mouse embryonic stem cells spontaneously differentiated into cardiomyocytes, the expression of specific myosin heavy chain gene (ß-MHC) contained in cardiomyocytes were detected by q-PCR quantitative analysis, and median inhibitory differentiation concentration (ID50) of ES was obtained. The development toxicity calculation formula was used to determine the embryotoxicity grade of CR decoction. finally, based on the successful induction of osteoblasts, the molecular mechanism of CR embryotoxicity was preliminarily studied based on BMP-Smads signal pathway. RESULTS: Compared with the negative control group, high, medium, and low doses of CR decoction had no significant effect on the maternal body weight and uterine weight (P > 0.05), as well as on the maternal liver, heart, and kidneys. The observation results showed that high dose of CR decoction significantly increase the number of absorbed fetuses (P < 0.05). The EST model was successfully established, the IC50 3T3, IC50 ES and ID50 ES of CR were 9.39 mg/mL, 18.78 mg/mL, and 10.20 mg/mL, respectively. CR was classified as weak embryonic development toxicity by the EST linear discriminant formula. Meanwhile, osteoblasts were successfully induced in vitro, the relative expression levels of BMP2, BMPR2, Smad1, and Smad5 were down-regulated in varying degrees after 3, 6, and 9 days of treatment with different concentration gradients of CR decoction. CONCLUSIONS: Combining in vivo and in vitro experiments, CR showed a potential embryotoxicity. The mechanism of embryotoxicity may be related to inhibiting the expression of key genes in the BMP-SMADs signaling pathway. In the clinical application, the normal dosage of CR is safe to a certain extent. However, when the dosage is too high (160 g/60 kg/d), there may be a risk of embryotoxicity.

Bone-forming peptide-3 induces osteogenic differentiation of bone marrow stromal cells via regulation of the ERK1/2 and Smad1/5/8 pathways.

A bone-remodeling imbalance induced by increased bone resorption and osteoclast formation causes skeletal diseases such as osteoporosis. Induction of osteogenic differentiation of bone marrow stromal cells (BMSCs) leads to bone regeneration. Many researchers have tried to develop new adjuvants as specific stimulators of bone regeneration for therapeutic use in patients with bone resorption. We tried to develop a new adjuvant that has stronger osteogenic differentiation-promoting activity than bone morphogenetic proteins (BMPs). In this study, we identified a new peptide, which we called bone-forming peptide (BFP)-3, derived from the immature precursor of BMP-7. Upon osteogenic differentiation, BMSCs treated with BFP-3 exhibited higher alkaline phosphatase (ALP) activity and mineralization ability and significantly up-regulated expression of osteogenic genes such as ALP, osteocalcin (OC), Osterix, and Runx2 compared with control BMSCs. Furthermore, fluorescence-activated cell sorting (FACS) and immunofluorescence analyses demonstrated that BFP-3 treatment up-regulated CD44 expression. Interestingly, extracellular signal-regulated kinase 1/2 (ERK1/2) and Smad1/5/8 phosphorylation was increased by BFP-3 treatment during osteogenic differentiation. Furthermore, BFP-3-induced osteogenic differentiation was significantly decreased by treatment with ERK1/2- and Smad-specific inhibitors. These results suggest that BFP-3 plays an important role in regulating osteogenic differentiation of BMSCs through increasing levels of osteogenic-inducing factors and regulating the ERK1/2 and Smad1/5/8 signaling pathways. Our finding indicates that BFP-3 may be a potential new therapeutic target for promoting bone formation.

Calcium Supplement Derived from Gallus gallus domesticus Promotes BMP-2/RUNX2/SMAD5 and Suppresses TRAP/RANK Expression through MAPK Signaling Activation.

The present study evaluated the effects of a calcium (Ca) supplement derived from Gallus gallus domesticus (GD) on breaking force, microarchitecture, osteogenic differentiation and osteoclast differentiation factor expression in vivo in Ca-deficient ovariectomized (OVX) rats. One percent of Ca supplement significantly improved Ca content and bone strength of the tibia. In micro-computed tomography analysis, 1% Ca supplement attenuated OVX- and low Ca-associated changes in bone mineral density, trabecular thickness, spacing and number. Moreover, 1% Ca-supplemented diet increased the expression of osteoblast differentiation marker genes, such as bone morphogenetic protein-2, Wnt3a, small mothers against decapentaplegic 1/5/8, runt-related transcription factor 2, osteocalcin and collagenase-1, while it decreased the expression of osteoclast differentiation genes, such as thrombospondin-related anonymous protein, cathepsin K and receptor activator of nuclear factor kappa B. Furthermore, 1% Ca-supplemented diet increased the levels of phosphorylated extracellular signal-regulated kinase and c-Jun N-terminal kinase. The increased expression of osteoblast differentiation marker genes and activation of mitogen-activated protein kinase signaling were associated with significant increases in trabecular bone volume, which plays an important role in the overall skeletal strength. Our results demonstrated that 1% Ca supplement inhibited osteoclastogenesis, stimulated osteoblastogenesis and restored bone loss in OVX rats.

Zinc affects miR-548n, SMAD4, SMAD5 expression in HepG2 hepatocyte and HEp-2 lung cell lines.

MicroRNAs affect disease progression and nutrient status. miR-548n increased 57 % in Zn supplemented plasma from adolescent females (ages 9 to 13 years). The purpose of this study was to determine the effects of Zn concentration in cell culture on the expression of miR-548n, SMAD4 and SMAD5 in hepatocyte (HepG2) and lung epithelium (HEp-2) cell lines. Cells were incubated for 48 h in media containing 10 % Chelex 100-treated FBS (0 µM Zn), or with 15 or 50 µM Zn, before isolation of total RNA and cDNA. Expression of miR-548n, SMAD4 and SMAD5 was measured by qPCR. The ΔΔCT method was used to calculate the fold-change, and 15 µM expression levels were used as reference values. HepG2 miR-548n expression decreased 5-fold, and SMAD4 expression increased 4-fold in the absence of Zn, while HEp-2 miR-548n expression increased 10.5-fold, and SMAD5 expression increased 20-fold in the absence of Zn. HEp-2 miR-548n expression increased 23-fold, while SMAD4 expression decreased twofold, in 50 µM Zn-treated cells. However, SMAD4 and SMAD5 expression was not correlated. These data indicate that miR-548n expression is in part regulated by Zn in a cell-specific manner. SMAD4 and SMAD5 are genes in the TGF-ß/BMP signaling pathway, and SMAD5 is a putative target for miR-548n; Zn participates in regulating this pathway through controlling SMAD4 and SMAD5 expression. However, SMAD5 expression may be more sensitive to Zn than to miR-548n since SMAD5 expression was not inversely correlated with miR-548n expression.

Cistanches Herba aqueous extract affecting serum BGP and TRAP and bone marrow Smad1 mRNA, Smad5 mRNA, TGF-ß1 mRNA and TIEG1 mRNA expression levels in osteoporosis disease.

We studied molecular mechanism of Cistanches Herba aqueous extract (CHAE) in ovariectomized (OVX) rats, as an experimental model of postmenopausal osteoporosis. Female rats were either sham-operated or bilaterally OVX; and at 60 days postoperatively. The OVX group (n = 8) received an ovariectomy and treatment with normal saline for 90 days commencing from 20th post ovariectomy day. The ovariectomized +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy and were treated with Cistanches Herba aqueous extract of 100 mg/kg body weight daily for 90 days commencing from 22nd post ovariectomy day. The ovariectomy +CHAE (OVX + CHAE) group (n = 8) received an ovariectomy, and were treated with the of 200 mg/kg body weight daily for 90 days commencing from 20th post ovariectomy day. Serum BGP and TRAP, E2, FSH and LH level, bone marrow Smad1, Smad5, TGF-ß1 and TIEG1 mRNA expression levels were examined. Results showed that serum BGP and TRAP, FSH and LH levels were significantly increased, whereas E2, Smad1, Smad5, TGF-ß1 and TIEG1 mRNA and proteins expression levels were significantly decreased in OVX rats compared to sham rats. 90 days of CHAE treatment could significantly decrease serum BGP and TRAP, FSH and LH levels, and increase E2, Smad1, Smad5, TGF-ß1 and TIEG1 mRNA and proteins expression levels in OVX rats. It can be concluded that CHAE play its protective effect against OVX-induced bone degeneration partly by regulating some bone metabolism related genes, e.g. Smad1, Smad5, TGF-ß1 and TIEG1.

Human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein-2 during in vitro osteogenic differentiation.

BACKGROUND: Interest in the potential application of adipose-derived stromal cells in cell-mediated tissue engineering of bone and other mesenchymal-derived tissues is growing. This study aimed to investigate the hypothesis that human adipose-derived stromal cells respond to and elaborate bone morphogenetic protein (BMP) 2, which could represent an important target of molecular manipulation to enhance the osteogenic potential of human adipose-derived stromal cells. METHODS: Human adipose-derived stromal cells were differentiated for 10 days toward the osteogenic lineage in osteogenic differentiation media alone or supplemented with recombinant human BMP2 (rhBMP2). Alizarin red staining was quantified by spectrophotometry. Gene expression analyses were performed using quantitative real-time polymerase chain reaction. BMP2 levels in conditioned media were titered by enzyme-linked immunosorbent assay daily during osteogenic differentiation. Human adipose-derived stromal cells were cultured in complete or partially (50 percent) changed osteogenic differentiation media, or unchanged osteogenic differentiation media, to assay for pro-osteogenic secreted factors. In addition, human adipose-derived stromal cells were cultured in osteogenic differentiation media supplemented with BMP2/BMP4-neutralizing antibody. RESULTS: Exogenous rhBMP2 significantly augmented the in vitro osteogenic potential of human adipose-derived stromal cells in a dose-dependent fashion, and significantly increased transcript levels of RUNX2 and osteocalcin. BMP2, BMP4, BMPR1B, and SMAD1/5 expression was significantly increased during differentiation. Enzyme-linked immunosorbent assay demonstrated significantly increased BMP2 elaboration during differentiation. Culture in conditioned osteogenic differentiation media led to significantly increased matrix mineralization. Mineralization was significantly decreased when osteogenic differentiation media was supplemented with a BMP2/BMP4-neutralizing antibody. CONCLUSIONS: These data strongly support that BMP signaling is dynamic and important during normal in vitro osteogenic differentiation of human adipose-derived stromal cells. Thus, BMP2 may be used to enhance the osteogenic differentiation of human adipose-derived stromal cells for bone tissue engineering. Future studies will examine the effect of rhBMP2 on osteogenic differentiation of human adipose-derived stromal cells in vivo.