Acupotomy ameliorates subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis by regulating bone morphogenetic protein 2-Smad1 pathway.

OBJECTIVE: To investigate the effects of acupotomy on the subchondral bone absorption and mechanical properties in rabbits with knee osteoarthritis (KOA). METHODS: The rabbits were divided into blank control, model, acupotomy and electroacupuncture (EA) groups, with 12 rabbits in each. Modified Videman's method was used to prepare KOA model. The acupotomy and EA group were given indicated intervention for 3 weeks. The behavior of rabbits in each group was recorded. Subsequently, cartilage-subchondral bone units were obtained and morphological changes were observed by optical microscope and micro computed tomography. Compression test was used to detect the mechanical properties of subchondral bone, Western blot and real-time polymerase chain reaction (RT-PCR) were applied to detect the expression of bone morphogenetic protein 2-Smad1 (BMP2-Smad1) pathway in subchondral bone. RESULTS: Compared with the control group, rabbits in the KOA group showed lameness, knee pain, and cartilage degradation; the subchondral bone showed active resorption, the mechanical properties decreased significantly and the BMP2-Smad1 pathway downregulated significantly. Both acupotomy and EA intervention could increase the thickness of trabecular bone (Tb. Th), the bone volume fraction (BV/TV) and the thickness of subchondral bone plate, reduce the separation of trabecular bone (Tb. Sp), improve the maximum load and elastic modulus of subchondral bone, and effectively delay cartilage degeneration in KOA rabbits. This process may be achieved through upregulation the related proteins of BMP2-Smad1 pathway. The maximum load and elastic modulus of subchondral bone in the acupotomy group were slightly better than those in the EA group. CONCLUSIONS: Acupotomy could effectively protect cartilage by inhibiting abnormal bone resorption and improving mechanical properties of subchondral bone thorough the related proteins of BMP2-Smad1 pathway in KOA rabbits.

[Effect of Bushen Chushi() decoction combined with platelet-rich plasma on regulating TGF-ß1 and Smad-1 expression in serum of knee osteoarthritis patient].

OBJECTIVE: To investigate the effect of Bushen Chushi decoction combined with platelet-rich plasma(PRP) to treat knee osteoarthritis(KOA) in early and middle stage and its regulation on TGF-ß1 and Smad-1 expression in serum. METHODS: Total of 45 patients with KOA in early and middle stage from May 2020 to April 2022 were treated and divided into control group and observation group. In control group, there were 30 patients including 12 males and 18 females, aged from 43 to 69 years old with an average of(57.3±6.5) years old and disease duration ranged from 1.5 to 5.0 years with an average of(3.8±1.7) years, and there were 8 cases in gradeⅠ, 13 cases in gradeⅡ, and 9 cases in grade Ⅲ according to Kellgren-Lawrence Grade, PRP 5 ml was injected into knee joint on the first day of No1, 3 week together for 2 times. In the observation group, there were 15 cases including 7 males and 8 females, aged from 45 to 70 years old with an average of (56.7±6.2) years old and disease duration ranged from 1.8 to 5.7 years with an average of (4.0±1.8) years, there were 4 cases in gradeⅠ, 9 cases in gradeⅡand 4 cases in grade Ⅲ according to the Kellgren-Lawrence Grade, PRP 5 ml were injected into knee joints that the time and frequency were the same as those in the control group, and at the same time Bushen Chushi decoction orally were taken 1 dose per day with a total of 28 doses. All patients were treated for four weeks. Visual analogue scale(VAS) and Lequesne MG score before and after treatment were used to evaluate improvement of knee pain and joint function. The TGF-ß1 and Smad-1 levels in serum were measured before and after treatment in two groups. The incidence of complications in two groups was observed. RESULTS: All patients were followed up for 26 to 30 days with an average of (28.0±0.6) days. There was no significant difference in VAS and knee Lequesne MG scores between two groups before treatment(P>0.05). The scores of VAS and knee Lequesne MG on the first day after treatment in both groups were lower than those before treatment(P<0.05). The VAS and knee Lequesne MG scores in observation group were lower than those in control group(P<0.05) on the first day after treatment. The TGF-ß1 level in serum after treatment were higher significantly than that before treatment in two groups(P<0.05). After treatment, TGF-ß1 level in serum in observation group were lower than those in control group with statistically significant differences(P<0.05). The Smad-1 levels in serum after treatment in observation group were higher significantly than that in control group(P<0.05). The levels of Smad-1 were not statistically significant between before and after treatment(P>0.05). There was no significant difference in postopertaive complications between two groups (P>0.05). CONCLUSION: The efficacy of Bushen Chushi decoction combined with PRP in treatment of early and middle KOA is better than that of PRP injection alone. The combined treatment could reduce TGF-ß1 level and increase Smad-1 level in serum, which may be a mechanism to inhibit inflammation and alleviate cartilage degeneration to some extent.

The Effect of Apple-Derived Nanovesicles on the Osteoblastogenesis of Osteoblastic MC3T3-E1 Cells.

Osteoporosis is characterized by low bone mass and elevated structural deterioration of the bone tissue, resulting in bone weakness with an increased risk of fracture. Considering biological activities of various phytochemicals extracted from apples, we herein demonstrated the potential antiosteoporotic effects of apple-derived nanovesicles (apple NVs) using osteoblastic MC3T3-E1 cells. Apple NVs significantly stimulated the growth of MC3T3-E1 cells. The cellular alkaline phosphatase (ALP) activity was significantly upregulated in the 5 µg/mL apple NVs-treated group. In addition, the concentrarion of mineralized nodules was significantly increased in the apple NVs-treated groups. Furthermore, apple NVs increased the expression of the genes and proteins associated with osteoblast growth and differentiation, such as Runx2, ALP, OPN, and BMP2/4, which further activated ERK- and JNK-related mitogen-activated protein kinase signaling. These results demonstrate that apple NVs have a potential to prevent osteoporosis by promoting osteoblastogenesis in osteoblastic MC3T3-E1 cells through regulating the BMP2/Smad1 pathways.

Effect of Bushen Chushi() decoction combined with platelet-rich plasma on regulating TGF-β1 and Smad-1 expression in serum of knee osteoarthritis patient / 中国骨伤

OBJECTIVE@#To investigate the effect of Bushen Chushi decoction combined with platelet-rich plasma(PRP) to treat knee osteoarthritis(KOA) in early and middle stage and its regulation on TGF-β1 and Smad-1 expression in serum.@*METHODS@#Total of 45 patients with KOA in early and middle stage from May 2020 to April 2022 were treated and divided into control group and observation group. In control group, there were 30 patients including 12 males and 18 females, aged from 43 to 69 years old with an average of(57.3±6.5) years old and disease duration ranged from 1.5 to 5.0 years with an average of(3.8±1.7) years, and there were 8 cases in gradeⅠ, 13 cases in gradeⅡ, and 9 cases in grade Ⅲ according to Kellgren-Lawrence Grade, PRP 5 ml was injected into knee joint on the first day of No1, 3 week together for 2 times. In the observation group, there were 15 cases including 7 males and 8 females, aged from 45 to 70 years old with an average of (56.7±6.2) years old and disease duration ranged from 1.8 to 5.7 years with an average of (4.0±1.8) years, there were 4 cases in gradeⅠ, 9 cases in gradeⅡand 4 cases in grade Ⅲ according to the Kellgren-Lawrence Grade, PRP 5 ml were injected into knee joints that the time and frequency were the same as those in the control group, and at the same time Bushen Chushi decoction orally were taken 1 dose per day with a total of 28 doses. All patients were treated for four weeks. Visual analogue scale(VAS) and Lequesne MG score before and after treatment were used to evaluate improvement of knee pain and joint function. The TGF-β1 and Smad-1 levels in serum were measured before and after treatment in two groups. The incidence of complications in two groups was observed.@*RESULTS@#All patients were followed up for 26 to 30 days with an average of (28.0±0.6) days. There was no significant difference in VAS and knee Lequesne MG scores between two groups before treatment(P>0.05). The scores of VAS and knee Lequesne MG on the first day after treatment in both groups were lower than those before treatment(P<0.05). The VAS and knee Lequesne MG scores in observation group were lower than those in control group(P<0.05) on the first day after treatment. The TGF-β1 level in serum after treatment were higher significantly than that before treatment in two groups(P<0.05). After treatment, TGF-β1 level in serum in observation group were lower than those in control group with statistically significant differences(P<0.05). The Smad-1 levels in serum after treatment in observation group were higher significantly than that in control group(P<0.05). The levels of Smad-1 were not statistically significant between before and after treatment(P>0.05). There was no significant difference in postopertaive complications between two groups (P>0.05).@*CONCLUSION@#The efficacy of Bushen Chushi decoction combined with PRP in treatment of early and middle KOA is better than that of PRP injection alone. The combined treatment could reduce TGF-β1 level and increase Smad-1 level in serum, which may be a mechanism to inhibit inflammation and alleviate cartilage degeneration to some extent.

Curcuminoids Inhibit Angiogenic Behaviors of Human Umbilical Vein Endothelial Cells via Endoglin/Smad1 Signaling.

BACKGROUND: Angiogenesis is primarily attributed to the excessive proliferation and migration of endothelial cells. Targeting the vascular endothelial growth factor (VEGF) is therefore significant in anti-angiogenic therapy. Although these treatments have not reached clinical expectations, the upregulation of alternative angiogenic pathways (endoglin/Smad1) may play a critical role in drug (VEGF-neutralizing agents) resistance. Enhanced endoglin expression following a VEGF-neutralizing therapy (semaxanib®) was noted in patients. Treatment with an endoglin-targeting antibody augmented VEGF expression in human umbilical vein endothelial cells (HUVECs). Therefore, approaches that inhibit both the androgen and VEGF pathways enhance the HUVECs cytotoxicity and reverse semaxanib resistance. The purpose of this study was to find natural-occurring compounds that inhibited the endoglin-targeting pathway. METHODS: Curcuminoids targeting endoglin were recognized from two thousand compounds in the Traditional Chinese Medicine Database@Taiwan (TCM Database@Taiwan) using Discovery Studio 4.5. RESULTS: Our results, obtained using cytotoxicity, migration/invasion, and flow cytometry assays, showed that curcumin (Cur) and demethoxycurcumin (DMC) reduced angiogenesis. In addition, Cur and DMC downregulated endoglin/pSmad1 phosphorylation. CONCLUSIONS: The study first showed that Cur and DMC demonstrated antiangiogenic activity via the inhibition of endoglin/Smad1 signaling. Synergistic effects of curcuminoids (i.e., curcumin and DMC) and semaxanib on HUVECs were found. This might be attributed to endoglin/pSmad1 downregulation in HUVECs. Combination treatment with curcuminoids and a semaxanib is therefore expected to reverse semaxanib resistance.

Asiaticoside attenuates bleomycin-induced pulmonary fibrosis in A2aR-/- mice by promoting the BMP7/Smad1/5 signaling pathway.

Idiopathic Pulmonary fibrosis(PF)is a chronic progressive disease, which is a lack of effective treatment,and the pathogenesis of IPF is not fully elucidated. Asiaticoside(AS) is isolated from Centella asiatica and has the effect of promoting scar healing and reducing scar formation. However,its possible role in idiopathic pulmonary fibrosis remains unclear. Adenosine A2A receptor (A2AR) is reported a protective factor in pulmonary fibrosis, and the bone morphogenetic protein 7 (BMP7) signaling pathway plays a crucial role in fibrosis in multiple organs. But the impact of A2AR on the BMP7 pathway has not yet been reported. Therefore, we hypothesized AS may promote the expression of A2AR, and then influence the BMP7/Smad1/5 pathway to alleviate pulmonary fibrosis. A2AR-/- mice and wild-type (WT) mice were administered bleomycin (BLM) by intratracheal injection. AS (50 mg/kg/d) was given daily for 28 days. AS reduced collagen deposition in lung tissue, interstitial lung inflammation. Furthermore, AS promoted A2AR expression and BMP7 pathway. Collectively, AS may attenuate BLM-induced pulmonary fibrosis by upregulating the BMP7 signaling pathway through A2AR.

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.

Baohuoside-I suppresses cell proliferation and migration by up-regulating miR-144 in melanoma.

CONTEXT: Baohuoside-I was reported to induce apoptosis in non-small-cell lung cancer and inhibit the growth of multiple myeloma cells. The antitumour potential of baohuoside-I has not been demonstrated in melanoma yet. OBJECTIVE: To investigate the potential antitumour activity of baohuoside-I against melanoma and elucidate its underlying molecular mechanism. MATERIALS AND METHODS: Cell viability was evaluated by MTT assay. The malignant invasion capacity was measured with trans-well assay. The relative expression change of microRNAs was profiled with microarray. TargetScan was utilized for prediction of target gene of miR-144. Regulatory effect of miR-144 on SMAD1 was determined by dual luciferase reporter assay. Endogenous SMAD1 protein in response to ectopic expression of miR-144 was determined by immunoblotting. Xenograft mice were employed to evaluate antitumour potential of baohuoside-I (25 mg/kg by tail intravenous injection every two days) in vivo. RESULTS: Baohuoside-I significantly inhibited proliferation (45 ± 4% reduction in M14 and 35 ± 3% reduction in MV3 at 24 h) and migration (70 ± 4% reduction in M14 and 72 ± 3% reduction in MV3) in melanoma cells. Mechanistically, baohuoside-I up-regulated miR-144 expression levels (3 ± 0.2-fold). Silence of miR-144 reversed the inhibition of baohuoside-I in melanoma. We have identified that SMAD1 was the novel target of miR-144. Moreover, baohuoside-I suppressed melanoma in vivo (52 ± 8% reduction in xenograft tumour size at day 20). CONCLUSIONS: Our data suggested significant antitumour potential of baohuoside-I against melanoma both in vitro and in vivo, which warrants further laboratory investigation and clinical trial.

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.

Different Degrees of Iodine Deficiency Inhibit Differentiation of Cerebellar Granular Cells in Rat Offspring, via BMP-Smad1/5/8 Signaling.

Iodine deficiency (ID) during development results in dysfunction of the central nervous system (CNS) and affects psychomotor and motor function. It is worth noting that maternal mild and marginal ID tends to be the most common reason of preventable neurodevelopmental impairment, via a mechanism that has not been elucidated. Therefore, our aim was to study the effects of developmental mild and marginal ID on the differentiation of cerebellar granule cells (GCs) and investigate the activation of BMP-Smad1/5/8 signaling, which is crucial for the development and differentiation of cerebellum. Three developmental rat models were created by feeding dam rats with a diet deficient in iodine and deionized water supplemented with potassium iodide. Our results showed that different degrees of ID inhibited and delayed the differentiation of cerebellar GCs on postnatal day (PN) 7, PN14, and PN21. Moreover, mild and severe ID reduced the expression of BMP2 and p-Smad1/5/8, and increased the levels of Id2 on PN7, PN14, and PN21. However, marginal ID rarely altered expression of these proteins in the offspring. Our study supports the hypothesis that mild and severe ID during development inhibits the differentiation of cerebellar GCs, which may be ascribed to the down-regulation of BMP-Smad1/5/8 signaling and the overexpression of Id2. Furthermore, it was speculated that maternal marginal ID rarely affected the differentiation of cerebellar GCs in the offspring.

Bone Morphogenetic Protein-2 Induces Donor-Dependent Osteogenic and Adipogenic Differentiation in Human Adipose Stem Cells.

UNLABELLED: Bone morphogenetic protein-2 (BMP-2) is a growth factor used to stimulate bone regeneration in clinical applications. However, there are contradicting reports on the functionality of BMP-2 in human adipose stem cells (hASCs), which are frequently used in tissue engineering. In this study, we analyzed the effects of BMP-2 on SMAD1/5 signaling, proliferation, and differentiation in hASCs. Our results indicated that BMP-2 induced dose-dependent (25-100 ng/ml) activation of SMAD signaling. Furthermore, the cell proliferation analysis revealed that BMP-2 (100 ng/ml) consistently decreased the proliferation in all the cell lines studied. However, the analysis of the differentiation potential revealed that BMP-2 (100 ng/ml) exhibited a donor-dependent dual role, inducing both osteogenic and adipogenic differentiation in hASCs. The quantitative alkaline phosphatase (qALP) activity and mineralization levels were clearly enhanced in particular donor cell lines by BMP-2 stimulus. On the contrary, in other cell lines, qALP and mineralization levels were diminished and the lipid formation was enhanced. The current study also suggests that hASCs have accelerated biochemical responsiveness to BMP-2 stimulus in human serum-supplemented culture medium compared with fetal bovine serum. The production origin of the BMP-2 growth factor is also important for its response: BMP-2 produced in mammalian cells enhanced signaling and differentiation responses compared with BMP-2 produced in Escherichia coli. These results explain the existing contradiction in the reported BMP-2 studies and indicate the variability in the functional end mechanism of BMP-2-stimulated hASCs. SIGNIFICANCE: This study examined how bone morphogenetic protein-2 (BMP-2) modulates the SMAD signaling mechanism and the proliferation and differentiation outcome of human adipose stem cells (hASCs) derived from several donors. The results indicate that BMP-2 triggers molecular SMAD signaling mechanisms in hASCs and regulates differentiation processes in human serum-culture conditions. Importantly, BMP-2 has dual activity, inducing osteogenic and adipogenic differentiation, subject to hASC donor line studied. These findings explain contradictory previous results and highlight the importance of further studies to understand how signaling pathways guide mesenchymal stem cell functions at the molecular level.