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.

Two novel polysaccharides from rhizomes of Cibotium barometz promote bone formation via activating the BMP2/SMAD1 signaling pathway in MC3T3-E1 cells.

Cibotium barometz, an important traditional Chinese medicine, is used in strengthening bones and tendons. We found that C. barometz crude polysaccharides (CB70) could alleviate bone loss and markedly improve the biomechanical properties of OVX rats. Thus, to clarify biological active ingredient(s) of CB70, two homogeneous polysaccharides (CBP70-1-1 and CBP70-1-2) were purified from CB70. A combination of monosaccharide composition, FT-IR, GC-MS and NMR analysis indicated that CBP70-1-1 was composed of →6)-D-Galp-(1→, D-Glcp-(1→, →3,6)-D-Manp-(1→, →4)-D-Glcp-(1→ and →6)-D-Glcp-(1→ with relative molecular weights of 12,724 Da, and CBP70-1-2 was composed of →4)-D-Glcp-(1→, D-Glcp-(1→, →3,6)-D-Manp-(1→, →6)-D-Galp-(1→, →4,6)-D-Glcp-(1→ and →3)-L-Araf-(1→ with relative molecular weights of 3611 Da. Morphological analyses revealed that CBP70-1-1 and CBP70-1-2 appeared as a sheet that were irregular in size and shape, while the surface of CBP70-1-1 was full of sharp protuberances and CBP70-1-2 was smooth. Furthermore, the effects of CBP70-1-1 and CBP70-1-2 on the proliferation, differentiation and mineralization of mouse pre-osteoblastic MC3T3-E1 cells were assessed via CCK-8 assay, alkaline phosphatase activity assay, and alizarin red-based assay, respectively. These results revealed that CBP70-1-1 and CBP70-1-2 significantly promoted the proliferation, differentiation and mineralization of MC3T3-E1 cells, even better than E2. More importantly, quantitative real-time PCR and Western blot analysis indicated that CBP70-1-2 pronouncedly promoted the expression of osteogenic-related marker genes (Runx2, Osx, Ocn and Opn) and proteins (BMP2, RUNX2, OSX and p-SMAD1), which implies that the osteogenic activity of CBP70-1-2 is accomplished mainly by activating the BMP2/SMAD1 signaling pathway. These findings suggest CBP70-1-2 as a potential natural anti-osteoporotic agent for pharmacotherapy.

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.

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.

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.

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.

Sizn1 is a novel protein that functions as a transcriptional coactivator of bone morphogenic protein signaling.

Bone morphogenic proteins (BMPs) play pleotrophic roles in nervous system development, and their signaling is highly regulated at virtually every step in the pathway. We have cloned a novel gene, Sizn1 (Smad-interacting zinc finger protein), which functions as a transcriptional coactivator of BMP signaling. It positively modulates BMP signaling by interacting with Smad family members and associating with CBP in the transcription complex. Sizn1 is expressed in the ventral embryonic forebrain, where, as we will show, it contributes to BMP-dependent, cholinergic-neuron-specific gene expression. These data indicate that Sizn1 is a positive modulator of BMP signaling and provide further insight into how BMP signaling can be modulated in neuronal progenitor subsets to influence cell-type-specific gene expression and development.

Chinese herbal formula Qilong-Lishui granule improves puromycin aminonucleoside-induced renal injury through regulation of bone morphogenetic proteins.

BACKGROUND: The Chinese herbal formula Qilong-Lishui granule (QLG) is an effective natural product for treatment of renal disorder. It was composed of six Chinese herbs according to our clinical practice in the treatment of patients with kidney disease. However, molecular and cellular mechanisms of QLG are still unclear. Therefore, the objective of the current study is to investigate molecular and cellular mechanisms of QLG in puromycin aminonucleoside (PAN)-induced nephrotic syndrome. METHOD: Wistar rats were divided into six groups of sham operation, PAN model, PAN model with high-dosage QLG (QLG-H), PAN model with median-dosage QLG (QLG-M), PAN model with low-dosage QLG (QLG-L), and PAN model with fosinopril (FP). The PAN model was induced by jugular vein injection of PAN at a dose of 5 mg/100 g body weight. Quantities of 24 h urinary protein excretion were examined on days 5, 10, 15, 20, 25 and 30. All rats were sacrificed on day 31 for blood biochemistry, kidney histology and reverse transcriptase-polymerase chain reaction analysis. RESULTS: PAN-induced nephrotic syndrome was successfully produced in rats. Treatment of QLG significantly reduced protein excretion and blood urea nitrogen and creatinine. QLG and FP treatments also improved protein content in blood, and reduced total cholesterol and triglyceride in blood. Moreover, QLG and FP improved the damage of interstitial induced by PAN. Furthermore, CYP and FP were able to reverse BMPRII and Smad1 mRNAs abundance caused by PAN. CONCLUSION: QLG attenuates PAN-induced kidney injury possibly through the bone morphogenetic protein signal transduction pathway.