BMP/Smad Pathway Is Involved in Lithium Carbonate-Induced Neural-Tube Defects in Mice and Neural Stem Cells.

Neural-tube defects (NTDs) are one type of the most serious birth defects. Studies have shown that inositol deficiency is closely related to the occurrence of NTDs. Bone morphogenetic protein (BMP)-mediated Smad signaling pathways have been implicated in neurogenesis and neural-tube closure. However, the role of the BMP/Smad pathway in inositol-deficiency-induced NTDs remains unclear. Inositol-deficiency models in C57 mice and mouse neural stem cells (mNSCs) were induced with Li2CO3 treatment or inositol withdrawal. The role of the BMP/Smad pathway in the regulation of cell proliferation and the development of NTDs was determined utilizing qRT-PCR, HE staining, Western blot, immunostaining, MTT assay, EdU staining, and flow cytometry. The intraperitoneal injection of Li2CO3 at Embryonic Day 7.5 induced the occurrence of NTDs. The mRNA levels of Bmp2, Bmp4, Smad1, Smad5, Smad8 and Runx2, the phosphorylation of Smad1/5/8, and the nuclear translocation of Runx2 were significantly increased in NTD embryonic brain tissues and mNSCs exposed to Li2CO3 or an inositol-free medium, which were suppressed by BMP receptor selective inhibitor LDN-193189. The Li2CO3-induced phosphorylation of Smad1/5/8 was inhibited by inositol supplementation. Cell proliferation was significantly promoted by Li2CO3 exposure or the absence of inositol in mNSCs, which was reversed by LDN-193189. These results suggest that the activation of the BMP/Smad signaling pathway might play an important role in the development of NTDs induced by maternal Li2CO3 exposure via inositol deficiency.

In vitro and in silico anti-osteoporosis activities and underlying mechanisms of a fructan, ABW90-1, from Achyranthes bidentate.

Achyranthes bidentata is a traditional Chinese medicine used to treat osteoporosis. AB90, a crude saccharide from A. bidentata, showed excellent osteoprotective effects in ovariectomized rats, and ABW90-1, an oligosaccharide purified from AB90, stimulated significant differentiation of osteoblasts. However, the osteogenic effects and underlying mechanisms of ABW90-1 have remained unknown. In the present study, we found that ABW90-1 significantly promoted ALP activity, mineralization, and the expression of osteogenic markers in MC3T3-E1 cells. ABW90-1 showed strong binding with the WNT signaling complex and BMP2 based on number of interactions, hydrogen bond length, and binding energy in silico. ABW90-1 significantly increased the expression of active-ß-catenin, p-GSK-3ß, LEF-1, BMP2, and p-SMAD1. Importantly, the osteogenic effects of ABW90-1 were partially suppressed by DKK-1 and Noggin, which are specific inhibitors of the WNT and BMP signaling pathways, respectively. Collectively, these findings suggest that ABW90-1 has osteogenic effects through crosstalk between WNT/ß-catenin and BMP2/SMAD1 signaling pathways.

Ginkgo Biloba Extract EGB761 Alleviates Warfarin-induced Aortic Valve Calcification Through the BMP2/Smad1/5/Runx2 Signaling Pathway.

ABSTRACT: Calcific aortic valve disease is a common heart disease that contributes to increased cardiovascular morbidity and mortality. There is a lack of effective pharmaceutical therapy because its mechanisms are not yet fully known. Ginkgo biloba extract (EGB761) is reported to alleviate vascular calcification. However, whether EGB761 protects against aortic valve calcification, a disease whose pathogenesis shares many similarities with vascular calcification, and potential molecular mechanisms remain unknown. In this study, porcine aortic valve interstitial cell (pAVIC) calcification was induced by warfarin with or without the presence of EGB761. Immunostaining was performed to establish and characterize the pAVIC phenotype. Calcium deposition and calcium content were examined by Alizarin Red S staining and an intracellular calcium content assay. Alkaline phosphatase activity was detected by the p-nitrophenyl phosphate method. The expression levels of bone morphogenetic protein-2 (BMP2), Runt-related transcription factor 2 (Runx2), homeobox protein MSX-2, and phosphorylated (p)-Smad1/5 were detected by reverse transcription-quantitative polymerase chain reaction (PCR) and Western blot analysis. Consistent with these in vitro data, we also confirmed the suppression of in vivo calcification by EGB761 in the warfarin-induced C57/Bl6 mice. The results indicated that both pAVICs and aortic valves tissue of mice stimulated with warfarin showed increased calcium deposition and expression of osteogenic markers (alkaline phosphatase, BMP2, homeobox protein MSX-2, and Runx2) and promoted p-Smad1/5 translocation from the cytoplasm to the nucleus. The addition of EGB761 significantly inhibited p-Smad1/5 translocation from the cytoplasm to the nucleus, thus suppressing calcification. In conclusion, EGB761 could ameliorate warfarin-induced aortic valve calcification through the inhibition of the BMP2-medicated Smad1/5/Runx2 signaling pathway.

Coicis semen protects against focal cerebral ischemia-reperfusion injury by inhibiting oxidative stress and promoting angiogenesis via the TGFß/ALK1/Smad1/5 signaling pathway.

BACKGROUND: Ischemic stroke is a devastating disease that causes long-term disability. However, its pathogenesis is unclear, and treatments for ischemic stroke are limited. Recent studies indicate that oxidative stress is involved in the pathological progression of ischemic stroke and that angiogenesis participates in recovery from ischemic stroke. Furthermore, previous studies have shown that Coicis Semen has antioxidative and anti-inflammatory effects in a variety of diseases. In the present study, we investigated whether Coicis Semen has a protective effect against ischemic stroke and the mechanism of this protective effect. RESULTS: Coicis Semen administration significantly decreased the infarct volume and mortality and alleviated neurological deficits at 3, 7 and 14 days after MCAO. In addition, cerebral edema at 3 days poststroke was ameliorated by Coicis Semen treatment. DHE staining showed that ROS levels in the vehicle group were increased at 3 days after reperfusion and then gradually declined, but Coicis Semen treatment reduced ROS levels. The levels of GSH and SOD in the brain were increased by Coicis Semen treatment, while MDA levels were reduced. Furthermore, Coicis Semen treatment decreased the extravasation of EB dye in MCAO mouse brains and elevated expression of the tight junction proteins ZO-1 and Occludin. Double immunofluorescence staining and western blot analysis showed that the expression of angiogenesis markers and TGFß pathway-related proteins was increased by Coicis Semen administration. Consistent with the in vivo results, cytotoxicity assays showed that Coicis Semen substantially promoted HUVEC survival following OGD/RX in vitro. Additionally, though LY2109761 inhibited the activation of TGFß signaling in OGD/RX model animals, Coicis Semen cotreatment markedly reversed the downregulation of TGFß pathway-related proteins and increased VEGF levels. METHODS: Adult male wild-type C57BL/6J mice were used to develop a middle cerebral artery occlusion (MCAO) stroke model. Infarct size, neurological deficits and behavior were evaluated on days 3, 7 and 14 after staining. In addition, changes in superoxide dismutase (SOD), GSH and malondialdehyde (MDA) levels were detected with a commercial kit. Blood-brain barrier (BBB) permeability was assessed with Evans blue (EB) dye. Western blotting was also performed to measure the levels of tight junction proteins of the BBB. Additionally, ELISA was performed to measure the level of VEGF in the brain. The colocalization of CD31, angiogenesis markers, and Smad1/5 was assessed by double immunofluorescent staining. TGFß pathway-related proteins were measured by western blotting. Furthermore, the cell viability of human umbilical vein endothelial cells (HUVECs) following oxygen-glucose deprivation/reoxygenation (OGD/RX) was measured by Cell Counting Kit (CCK)-8 assay. CONCLUSIONS: Coicis Semen treatment alleviates brain damage induced by ischemic stroke through inhibiting oxidative stress and promoting angiogenesis by activating the TGFß/ALK1 signaling pathway.

The effects of inhibiting the activation of hepatic stellate cells by lignan components from the fruits of Schisandra chinensis and the mechanism of schisanhenol.

Liver fibrosis is a pathological manifestation induced by chronic liver injury and may cause cirrhosis and liver cancer with the chronic progression of fibrosis. During the onset and progression of liver fibrosis, the activation of hepatic stellate cells (HSCs) is the core mechanism for the secretion of many extracellular matrices to induce fibrosis. Lignans are reportedly the main effective components of Schisandra chinensis with good anti-fibrosis effects. In this study, we compared the inhibiting effects of the seven lignan components from S. chinensis on HSC activation. We found that the seven lignans inhibited the activation of human HSCs (LX-2) in various degrees. Among all lignans, schisanhenol showed the best effect in inhibiting the activation of LX-2 with a dose-effect relationship. Sal also inhibited the phosphorylations of Smad1, Smad2, Smad3, extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), p38, and nuclear transcription factor-κB (NF-κB), as well as downregulated Smad4. All these findings suggested that schisanhenol may ameliorate liver fibrosis by inhibiting the transforming growth factor ß (TGF-ß)/Smad and mitogen-activated protein kinase (MAPK) signaling pathways. Remarkably, schisanhenol may be a potential anti-liver fibrosis drug and warrants further research.

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.

Epigallocatechin-3-gallate inhibits tumor angiogenesis: involvement of endoglin/Smad1 signaling in human umbilical vein endothelium cells.

Strategies targeting endoglin are currently being investigated in clinical trials as an anti-angiogenic therapy. The redundancy between endoglin and vascular endothelial growth factor (VEGF) signaling in angiogenesis was verified. Increased endoglin signaling after an anti-VEGF treatment was observed in patients. Treatment with an endoglin-neutralizing antibody increased VEGF signaling in endothelial cells. Therefore, strategies targeting both the endoglin and VEGF pathways were applied to determine whether the anti-angiogenic effects were increased in vitro. Five possible hits for endoglin were identified from 2000 compounds in the Traditional Chinese Medicine Database using Discovery Studio 4.5 Epigallocatechin-3-gallate (EGCG) attenuates angiogenesis by downregulating VEGF; however, researchers have not determined whether its anti-angiogenic effects are mediated by endoglin/Smad1 signaling. A major contribution of this study is that EGCG significantly inhibited the upregulation of endoglin in semaxanib-treated human umbilical vein endothelial cell. Thus, a combination treatment with EGCG and a VEGF tyrosine kinase inhibitor would be appropriate to reverse drug resistance. EGCG alone significantly decreased endoglin/pSmad1 levels in HUVECs. In the angiogenesis assay, the migration, invasion, and tube formation of HUVECs were markedly suppressed by higher concentrations of EGCG. A combination treatment with EGCG and semaxanib further produced increased anti-angiogenic effects. The main contribution of the study indicated that EGCG significantly decreased the semaxanib-induced overexpression of endoglin. Therefore, a combination treatment including EGCG will probably solve the drug resistance to anti-VEGF treatments.

Protective effects of trans-2,3,5,4'-tetrahydroxystilbene 2-O-ß-d-glucopyranoside on liver fibrosis and renal injury induced by CCl4via down-regulating p-ERK1/2 and p-Smad1/2.

Both as a food and an herbal plant, Polygonum multiflorum (PM) has long been used in food and prescriptions for several centuries in Southeast Asia. trans-2,3,5,4'-tetrahydroxystilbene 2-O-ß-d-glucopyranoside (trans-THSG) is one of the major compounds derived from PM and has been reported to exhibit multiple biological activities such as antioxidation and anti-obesity activities among others. The current study was aimed at investigating the effects of trans-THSG on liver fibrosis and renal injury in a carbon tetrachloride (CCl4) induced rodent model via oral feeding. Research results have demonstrated that administration of trans-THSG (100 and 300 mg kg-1) significantly ameliorated liver fibrosis, manifested by reduced expression of desmin and α-smooth muscle actin (α-SMA) plus collagen deposition. Specifically, treatment with trans-THSG effectively decreased the levels of transforming growth factor-ß (TGF-ß) and reduced the phosphorylation of Smad1/2 (p-Smad1/2) and extracellular signal-regulated kinases 1/2 (p-ERK1/2). Furthermore, we found that trans-THSG significantly down-regulated CCl4-induced excessive collagen secretion and increased the levels of desmin, MMP2 and MMP9 in rat liver tissues, suggesting that trans-THSG prevents liver fibrosis by attenuating the activation of hepatic stellate cells (HSCs) through the inhibition of Smad and ERK signaling pathways. Hence, the present findings demonstrate that trans-THSG is an effective antifibrotic agent in protecting liver from CCl4-induced toxicity.

Protective and therapeutic effects of Pilose antler against kidney deficiency-induced osteoporosis.

This study was carried out to evaluate the preventive and curative effects of Pilose antler against osteoporosis due to kidney deficiency, and investigate its potential mechanism of action. A model of osteoporosis due to kidney deficiency was established in rats using bilateral ovariectomy. Pilose antler polypeptide (PAP), Pilose antler polysaccharide (PAP'), and their mixture (PAP+PAP') were separately administered to the rats for 12 weeks, with progynova and xianlingubao tablets (XLGB) as the positive control groups. We determined the bone mineral density (BMD) and uterus Index of the rats. Osteoblastic bone metabolism-related indices in serum and bone tissue were measured with ELISA. Western blotting and RT-PCR were used to investigate the protein and mRNA expressions of Bmp-2, Smad1, Smad5, Runx2 in bone tissue. The morphology of bone tissue was determined using immunohistochemical methods. Compared with control group, PAP, PAP', PAP+PAP' increased BMD and regulated bone metabolism indices in serum and bone tissue. Treatment with Pilose antler up-regulated the mNRA and protein expressions of Bmp-2, Smad1, Smad5 and Runx2. Immunohistochemical staining showed that Bmp-2, Smad1, Smad5 and Runx2 were stained brown, indicating that all of them were positive. There were abnormal changes in the protein expressions of Bmp-2, Smad1, Smad5 and Runx2 in bone tissue, which may be an important mechanism underlying the development of kidney deficiency osteoporosis. Moreover, PAP, PAP' and PAP+PAP' had some preventive effects on osteoporosis, probably via the activation of the Bmp-2/Smad1 and Smad5/Runx2 signaling pathways through induction of high expressions of their mRNAs and proteins.

Modulation of osteogenic and myogenic differentiation by a phytoestrogen formononetin via p38MAPK-dependent JAK-STAT and Smad-1/5/8 signaling pathways in mouse myogenic progenitor cells.

Formononetin (FN), a typical phytoestrogen has attracted substantial attention as a novel agent because of its diverse biological activities including, osteogenic differentiation. However, the molecular mechanisms underlying osteogenic and myogenic differentiation by FN in C2C12 progenitor cells remain unknown. Therefore the objective of the current study was to investigate the action of FN on myogenic and osteogenic differentiation and its impact on signaling pathways in C2C12 cells. FN significantly increased myogenic markers such as Myogenin, myosin heavy chains, and myogenic differentiation 1 (MyoD). In addition, the expression of osteogenic specific genes alkaline phosphatase (ALP), Run-related transcription factor 2(RUNX2), and osteocalcin (OCN) were up-regulated by FN treatment. Moreover, FN enhanced the ALP level, calcium deposition and the expression of bone morphogenetic protein isoform (BMPs). Signal transduction pathways mediated by p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-related kinases (ERKs), protein kinase B (Akt), Janus kinases (JAKs), and signal transducer activator of transcription proteins (STATs) in myogenic and osteogenic differentiation after FN treatment were also examined. FN treatment activates myogenic differentiation by increasing p38MAPK and decreasing JAK1-STAT1 phosphorylation levels, while osteogenic induction was enhanced by p38MAPK dependent Smad, 1/5/8 signaling pathways in C2C12 progenitor cells.

Pterostilbene Ameliorates Nephropathy Injury in Streptozotocin-Induced Diabetic Rats.

BACKGROUND: Our study investigated the therapeutic role and potential mechanisms of pterostilbene (PS) in diabetic nephropathy (DN) rats. METHODS: DN models were established by high-fat diet after streptozotocin injection. A total of 50 Sprague-Dawley rats were randomly divided into control, DN, PS-treated groups (PS-H, PS-M, PS-L). PS was administered to rats by gavage for 8 weeks at 3 different doses (25, 10, and 5 mg/kg/day). The levels of oxidative stress activity (superoxide dismutase [SOD], malondialdehyde [MDA], glutathione peroxidase [GSH-PX]) and inflammatory factors (tumor necrosis factor [TNF]-α, interleukin (IL)-6, IL-1ß, monocyte chemoattractant factor [MCP]-1) were detected by -ELISA. TGF-ß, Smad1, and fibronectin (FN) were measured through immunohistochemistry. The relative expressions of phospho-IκBα/IκBα, phospho-IκB kinases (IKK)ß/IKKß, phospho-nuclear factor-κB (NF-κB) p65/NF-κB p65 were detected by western blot. RESULTS: Compared with DN group, the levels of TNF-α, IL-6, IL-1ß, and MCP-1 were decreased in the PS-H group (p < 0.05). Meanwhile, the levels of SOD, MDA, GSH-PX improved in kidney and serum in PS-H groups (p< 0.05). PS also significantly decreased the level of phospho-NF-κB p65 and increased the levels of phospho- IKKß and phospho-Iκ-Bα (p < 0.05). The results showed that PS treatment decreased TGF-ß, Smad1, and FN expressions. CONCLUSION: PS had potential therapeutic effects on DN, which may be related to the regulation of NF-κB pathway.

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.

Green Tea Catechins Effectively Altered Hepatic Fibrogenesis in Rats by Inhibiting ERK and Smad1/2 Phosphorylation.

Polyphenols derived from green tea have been reported to have a wide range of profound functions. Tea catechins, including epicatechin, epigallocatechin (EGC), epicatechin-3- O-gallate (ECG), and epigallocatechin-3- O-gallate (EGCG), are considered as the major bioactive polyphenols in tea. The present study was designed to elucidate the potential antifibrogenic role of three abundant tea catechins (ECG, EGC, and EGCG) in a CCl4-induced fibrotic rat and their underlying molecular mechanisms. Tea catechins, especially groups of ECG, EGC, and EGCG, effectively induced several beneficial alterations of liver injury markers, oxidative status, and liver histology. Furthermore, catechins ameliorated liver fibrosis, as evidenced by the reduced expression of desmin, α-smooth muscle actin, transforming growth factor ß (TGF-ß), and downstream ERK1/2 and Smad1/2 phosphorylation. The most significant inhibitory effect on those proteins was observed in ECG (300 mg/kg) and EGCG (300 mg/kg) groups. In addition, catechins conferred their protective role by downregulating the proinflammation cytokines TGF-ß, tumor necrosis factor α, and interleukin 17. It is postulated that tea catechins, particularly ECG and EGCG, are potential therapeutic candidates in antifibrotic therapy.

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.

Salvianolic Acid A, a Component of Salvia miltiorrhiza, Attenuates Endothelial-Mesenchymal Transition of HPAECs Induced by Hypoxia.

Salvianolic acid A (SAA), a polyphenols acid, is a bioactive ingredient from a traditional Chinese medicine called Dan shen (Salvia Miltiorrhiza Bunge). According to previous studies, it was shown to have various effects such as anti-oxidative stress, antidiabetic complications and antipulmonary hypertension. This study aimed to investigate the effect of SAA on pulmonary arterial endothelial-mesenchymal transition (EndoMT) induced by hypoxia and the underlying mechanisms. Primary cultured human pulmonary arterial endothelial cells (HPAECs) were exposed to 1% O2 for 48[Formula: see text]h with or without SAA treatment. SAA treatment improved the morphology of HPAECs and inhibited the cytoskeleton remodeling. A total of 3[Formula: see text][Formula: see text]M SAA reduced migration distances from 262.2[Formula: see text][Formula: see text]m to 198.4[Formula: see text][Formula: see text]m at 24[Formula: see text]h and 344.8[Formula: see text][Formula: see text]m to 109.3[Formula: see text][Formula: see text]m at 48[Formula: see text]h. It was observed that the production of ROS in cells was significantly reduced by the treatment of 3[Formula: see text][Formula: see text]M SAA. Meanwhile, SAA alleviated the loss of CD31 and slightly inhibited the expression of [Formula: see text]-SMA. The mechanisms study shows that SAA treatment increased the phosphorylation levels of Smad1/5, but inhibited that of Smad2/3. Furthermore, SAA attenuated the phosphorylation levels of ERK and Cofilin, which were enhanced by hypoxia. Based on these results, our study indicated that SAA treatment can protect HPAECs from endoMT induced by hypoxia, which may perform via the inhibition on ROS production and further through the downstream effectors of BMPRs or TGF[Formula: see text]R including Smads, ERK and ROCK/cofilin pathways.

Biological Silicon Stimulates Collagen Type 1 and Osteocalcin Synthesis in Human Osteoblast-Like Cells Through the BMP-2/Smad/RUNX2 Signaling Pathway.

Silicon is essential for bone formation. A low-silicon diet leads to bone defects, and numerous animal models have demonstrated that silicon supplementation increases bone mineral density (BMD) and reduces bone fragility. However, the exact mechanism of this action has not been characterized. In this study, we aimed to determine the role of biological silicon in the induction of osteoblast differentiation and the possible underlying mechanism. We examined whether orthosilicic acid promotes collagen type 1 (COL-1) and osteocalcin synthesis through the bone morphogenetic protein-2 (BMP-2)/Smad1/5/runt-related transcription factor 2 (RUNX2) signaling pathway by investigating its effect in vitro at several concentrations on COL-1 and osteocalcin synthesis in human osteosarcoma cell lines (MG-63 and U2-OS). The expression of relevant proteins was detected by Western blotting following exposure to noggin, an inhibitor of BMP-2. In MG-63 cells, immunofluorescence methods were applied to detect changes in the expression of BMP-2, phosphorylated Smad1/5 (P-Smad1/5), and RUNX2. Furthermore, rat bone mesenchymal stem cells (BMSCs) were used to determine the effect of orthosilicic acid on osteogenic differentiation. Exposure to 10 µM orthosilicic acid markedly increased the expression of BMP-2, P-Smad1/5, RUNX2, COL-1, and osteocalcin in osteosarcoma cell lines. Enhanced ALP activity and the formation of mineralized nodules were also observed under these conditions. Furthermore, preconditioning with noggin inhibited the silicon-induced upregulation of P-Smad1/5, RUNX2, and COL-1 expression. In conclusion, the BMP-2/Smad1/5/RUNX2 signaling pathway participates in the silicon-mediated induction of COL-1 and osteocalcin synthesis, and orthosilicic acid promotes the osteogenic differentiation of rat BMSCs.

[Effect of bushen tiaojing recipe on the expressions of Smad1, Smad5, Smad8, and Smad4 in human mural granulosa cells].

OBJECTIVE: To explore the effect of Bushen Tiaojing Recipe (BTR) on the expressions of drosophila mothers against decapentaplegic protein (Smadl), Smad5, Smad8, and Smad4 on human mural granulosa cells. METHODS: Sixty-six patients undergoing in vitro fertilization-embryo transfer (IVF-ET) were randomly assigned to two groups in the ratio of 1:2, the treatment group and the control group. Twenty-three patients in the treatment group were treated with BTR and GnRHa/FSH/hCG, while forty-three patients in the control group were treated with GnRHa/FSH/hCG. The mRNA expressions of Smad1, Smad5, Smad8, and Smad4 on mural granulosa cells of the mature follicle were detected by real-time PCR on the ovum retrieval day. The expressions of Smad1, Smad5, Smad8, and Smad4 at the protein level were observed using cell immunofluorescence method. RESULTS: The mRNA and protein expressions of Smadl in the granulosa cells were significantly higher in the treatment group than in the control group (P <0.05). There was no statistical difference in the mRNA and protein expressions of Smad5, Smad8, and Smad4 between the two groups. CONCLUSION: The mechanisms of BTR for improving the pregnancy rate and the ovarian functions might be correlated with up-regulating mRNA and protein expressions of Smadl of human mural granulosa cells.

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.

Multilayered chromatin analysis reveals E2f, Smad and Zfx as transcriptional regulators of histones.

Histones, the building blocks of eukaryotic chromatin, are essential for genome packaging, function and regulation. However, little is known about their transcriptional regulation. Here we conducted a comprehensive computational analysis, based on chromatin immunoprecipitation-sequencing and -microarray analysis (ChIP-seq and ChIP-chip) data of over 50 transcription factors and histone modifications in mouse embryonic stem cells. Enrichment scores supported by gene expression data from gene knockout studies identified E2f1 and E2f4 as master regulators of histone genes, CTCF and Zfx as repressors of core and linker histones, respectively, and Smad1, Smad2, YY1 and Ep300 as restricted or cell type-specific regulators. We propose that histone gene regulation is substantially more complex than previously thought, and that a combination of factors orchestrate histone gene regulation, from strict synchronization with S phase to targeted regulation of specific histone subtypes.

Down-regulation of transforming growth factor beta 1/activin receptor-like kinase 1 pathway gene expression by herbal compound 861 is related to deactivation of LX-2 cells.

AIM: To investigate the effect of herbal compound 861 (Cpd861) on the transforming growth factor-beta1 (TGF beta 1)/activin receptor-like kinase 1 (ALK1, type I receptor) signaling-pathway-related gene expression in the LX-2 cell line, and the inhibitory mechanism of Cpd861 on the activation of LX-2 cells. METHODS: LX-2 cells were treated with TGF beta 1 (5 ng/mL) Cpd861 (0.1 mg/mL), TGF beta 1 (5 ng/mL) plus Cpd861 (5 ng/mL) for 24 h to investigate the effect of Cpd861 on the TGF beta 1/ALK1 pathway. Real-time PCR was performed to examine the expression of alpha-SMA (alpha-smooth muscle actin), ALK1, Id1 (inhibitor of differentiation 1). Western blotting was carried out to measure the levels of alpha-SMA and phosphorylated Smad1, and immunocytochemical analysis for the expression of alpha-SMA. RESULTS: In LX-2 cells, TGF beta 1/ALK1-pathway-related gene expression could be stimulated by TGF beta 1, which led to excessive activation of the cells. Cpd861 decreased the activation of LX-2 cells by reducing the expression of alpha-SMA mRNA and protein expression. This effect was related to inhibition of the above TGF beta 1/ALK1-pathway-related expression of genes such as Id1 and ALK1, and phosphorylation of Smad1 in LX-2 cells, even with TGF beta 1 co-treatment for 24 h. CONCLUSION: Cpd861 can restrain the activation of LX-2 cells by inhibiting the TGF beta 1/ALK1/Smad1 pathway.