Chemical and biological assessment of Angelica herbal decoction: Comparison of different preparations during historical applications

The commonly used Angelica herbal decoction today is Danggui Buxue Tang (DBT), which is a dietary supplement in treating menopausal irregularity in women, i.e. to nourish "Qi" and to enrich "Blood". According to historical record, many herbal decoctions were also named DBT, but the most popular formulation of DBT was written in Jin dynasty (1247 AD) of China, which contained Astragali Radix (AR) and Angelicae Sinensis Radix (ASR) with a weight ratio of 5:1. However, at least two other Angelica herbal decoctions recorded as DBT were prescribed in Song (1155 AD) and Qing dynasties (1687 AD). Although AR and ASR are still the major components in the DBT herbal decoctions, they are slightly varied in the herb composition. In order to reveal the efficiency of different Angelica herbal decoctions, the chemical and biological properties of three DBT herbal extracts were compared. Significantly, the highest amounts of AR-derived astragaloside III, astragaloside IV, calycosin and formononetin and ASR-derived ferulic acid were found in DBT described in 1247 AD: this preparation showed stronger activities in osteogenic, estrogenic and erythropoetic effects than the other two DBT. The current results supported the difference of three DBT in chemical and biological properties, which could be a result of different herbal combinations. For the first time, this study supports the popularity of DBT described in 1247 AD.     

Activation of autophagy by FOXO3 regulates redox homeostasis during osteogenic differentiation

Bone remodeling is a continuous physiological process that requires constant generation of new osteoblasts from mesenchymal stem cells (MSCs). Differentiation of MSCs to osteoblast requires a metabolic switch from glycolysis to increased mitochondrial respiration to ensure the sufficient energy supply to complete this process. As a consequence of this increased mitochondrial metabolism, the levels of endogenous reactive oxygen species (ROS) rise. In the current study we analyzed the role of forkhead box O3 (FOXO3) in the control of ROS levels in human MSCs (hMSCs) during osteogenic differentiation. Treatment of hMSCs with H₂O₂ induced FOXO3 phosphorylation at Ser294 and nuclear translocation. This ROS-mediated activation of FOXO3 was dependent on mitogen-activated protein kinase 8 (MAPK8/JNK) activity. Upon FOXO3 downregulation, osteoblastic differentiation was impaired and hMSCs lost their ability to control elevated ROS levels. Our results also demonstrate that in response to elevated ROS levels, FOXO3 induces autophagy in hMSCs. In line with this, impairment of autophagy by autophagy-related 7 (ATG7) knockdown resulted in a reduced capacity of hMSCs to regulate elevated ROS levels, together with a reduced osteoblast differentiation. Taken together our findings are consistent with a model where in hMSCs, FOXO3 is required to induce autophagy and thereby reduce elevated ROS levels resulting from the increased mitochondrial respiration during osteoblast differentiation. These new molecular insights provide an important contribution to our better understanding of bone physiology.     

The sulfur-fumigation reduces chemical composition and biological properties of Angelicae sinensis radix

Angelica Sinensis Radix (roots of Angelica sinensis; ASR) is a popular herbal supplement in China for promoting blood circulation. Today, sulfur-fumigation is commonly used to treat ASR as a means of pest control; however, the studies of sulfur-fumigation on the safety and efficacy of ASR are very limited. Here, we elucidated the destructive roles of sulfur-fumigation on ASR by chemical and biological assessments. After sulfur-fumigation, the chemicals in ASR were significantly lost. The biological activities of anti-platelet aggregation, induction of NO production and estrogenic properties were compared between the water extracts of non-fumigated and sulfur-fumigated ASR. In all cases, the sulfur-fumigation significantly reduced the biological properties of ASR. In addition, application of water extract deriving from sulfur-fumigated ASR showed toxicity to cultured MCF-7 cells. In order to ensure the safety and to achieve the best therapeutic effect, it is recommended that sulfur-fumigation is an unacceptable approach for processing herbal materials.     

Enhanced Bone Tissue Regeneration by Porous Gelatin Composites Loaded with the Chinese Herbal Decoction Danggui Buxue Tang

Danggui Buxue Tang (DBT) is a traditional Chinese herbal decoction containing Radix Astragali and Radix Angelicae sinensis. Pharmacological results indicate that DBT can stimulate bone cell proliferation and differentiation. The aim of the study was to investigate the efficacy of adding DBT to bone substitutes on bone regeneration following bone injury. DBT was incorporated into porous composites (GGT) made from genipin-crosslinked gelatin and β-triclacium phosphates as bone substitutes (GGTDBT). The biological response of mouse calvarial bone to these composites was evaluated by in vivo imaging systems (IVIS), micro-computed tomography (micro-CT), and histology analysis. IVIS images revealed a stronger fluorescent signal in GGTDBT-treated defect than in GGT-treated defect at 8 weeks after implantation. Micro-CT analysis demonstrated that the level of repair from week 4 to 8 increased from 42.1% to 71.2% at the sites treated with GGTDBT, while that increased from 33.2% to 54.1% at GGT-treated sites. These findings suggest that the GGTDBT stimulates the innate regenerative capacity of bone, supporting their use in bone tissue regeneration.     

Guava fruit extract and its triterpene constituents have osteoanabolic effect: Stimulation of osteoblast differentiation by activation of mitochondrial respiration via the Wnt/β-catenin signaling

The aim of this study was to evaluate the skeletal effect of guava triterpene-enriched extract (GE) in rats and identify osteogenic compounds thereof, and determine their modes of action. In growing female rats, GE at 250 mg/kg dose increased parameters of peak bone mass including femur length, bone mineral density (BMD) and biomechanical strength, suggesting that GE promoted modeling-directed bone growth. GE also stimulated bone regeneration at the site of bone injury. In adult osteopenic rats (osteopenia induced by ovariectomy, OVX) GE completely restored the lost bones at both axial and appendicular sites, suggesting a strong osteoanabolic effect. Serum metabolomics studies showed changes in several metabolites (some of which are related to bone metabolism) in OVX compared with ovary-intact control and GE treatment to OVX rats reversed those. Out of six abundantly present triterpenes in GE, ursolic acid (UA) and 2α-hydroxy ursolic acid (2α-UA) induced osteogenic differentiation in vitro as did GE by activating Wnt/β-catenin pathway assessed by phosphorylation of GSK-3β. Over-expressing of constitutively active GSK-3β (caGSK-3β) in osteoblasts abolished the differentiation-promoting effect of GE, UA and 2α-UA. All three increased both glycolysis and mitochondrial respiration but only rotenone (inhibitor of mitochondrial electron transfer) and not 2-deoxyglucose (to block glycolysis) inhibited osteoblast differentiation. In addition, caGSK-3β over-expression attenuated the enhanced mitochondrial respiration caused by GE, UA and 2α-UA. We conclude that GE has osteoanabolic effect which is contributed by UA and 2α-UA, and involve activation of canonical Wnt signaling which in turn modulates cellular energy metabolism leading to osteoblast differentiation.     

A Chinese herbal decoction, Danggui Buxue Tang, activates extracellular signal-regulated kinase in cultured T-lymphocytes

Danggui Buxue Tang (DBT) is prepared from Radix Astragali and Radix Angelicae Sinensis. This Chinese herbal decoction has been shown to stimulate the proliferation of T-lymphocytes; however, the action mechanism of this stimulation has not been revealed. In cultured T-lymphocytes, application of DBT markedly induced the cell proliferation, the release of interleukin-2, -6 and -10, as well as the phosphorylation of extracellular signal-regulated kinases (ERK). The pre-treatment of ERK inhibitor blocked the DBT-induced immune responses. In addition, the polysaccharide-enriched fraction of DBT showed marked responses on the cultured T-lymphocytes suggesting the important role of DBT polysaccharide in triggering such immune responses.     

Advanced oxidation protein products induce pre‐osteoblast apoptosis through a nicotinamide adenine dinucleotide phosphate oxidase‐dependent,mitogen‐activated protein kinases‐mediated intrinsic apoptosis pathway

Osteoblast apoptosis contributes to age‐related bone loss. Advanced oxidation protein products (AOPPs) are recognized as the markers of oxidative stress and potent inducers of apoptosis. We have demonstrated that AOPP accumulation was correlated with age‐related bone loss. However, the effect of AOPPs on the osteoblast apoptosis still remains unknown. Exposure of osteoblastic MC3T3‐E1 cells to AOPPs caused the excessive generation of reactive oxygen species (ROS) by activating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Increased ROS induced phosphorylation of mitogen‐activated protein kinases (MAPKs), which subsequently triggered intrinsic apoptosis pathway by inducing mitochondrial dysfunction, endoplasmic reticulum stress, and Ca²⁺ overload and eventually leads to apoptosis. Chronic AOPP loading in aged Sprague‐Dawley rats induced osteoblast apoptosis and activated NADPH oxidase signaling cascade, in combination with accelerated bone loss and deteriorated bone microstructure. Our study suggests that AOPPs induce osteoblast apoptosis by the NADPH oxidase‐dependent, MAPK‐mediated intrinsic apoptosis pathway.     

Hydrogen sulfide attenuates homocysteine-induced osteoblast dysfunction by inhibiting mitochondrial toxicity

Homocysteine (Hcy) is detrimental to bone health in a mouse model of diet-induced hyperhomocysteinemia (HHcy). However, little is known about Hcy-mediated osteoblast dysfunction via mitochondrial oxidative damage. Hydrogen sulfide (H₂S) has potent antioxidant, anti-inflammatory, and antiapoptotic effects. In this study, we hypothesized that the H₂S mediated recovery of osteoblast dysfunction by maintaining mitochondrial biogenesis in Hcy-treated osteoblast cultures in vitro. MC3T3-E1 osteoblastic cells were exposed to Hcy treatment in the presence or absence of an H₂S donor (NaHS). Cell viability, osteogenic differentiation, reactive oxygen species (ROS) production were determined. Mitochondrial DNA copy number, adenosine triphosphate (ATP) production, and oxygen consumption were also measured. Our results demonstrated that administration of Hcy increases the intracellular Hcy level and decreases intracellular H₂S level and expression of the cystathionine β-synthase/Cystathionine γ-lyase system, thereby inhibiting osteogenic differentiation. Pretreatment with NaHS attenuated Hcy-induced mitochondrial toxicity (production of total ROS and mito-ROS, ratio of mitochondrial fission (DRP-1)/fusion (Mfn-2)) and restored ATP production and mitochondrial DNA copy numbers as well as oxygen consumption in the osteoblast as compared with the control, indicating its protective effects against Hcy-induced mitochondrial toxicity. In addition, NaHS also decreased the release of cytochrome c from the mitochondria to the cytosol, which induces cell apoptosis. Finally, flow cytometry confirmed that NaHS can rescue cells from apoptosis induced by Hcy. Our studies strongly suggest that NaHS has beneficial effects on mitochondrial toxicity, and could be developed as a potential therapeutic agent against HHcy-induced mitochondrial dysfunction in cultured osteoblasts in vitro.     

Methylglyoxal induces oxidative stress and mitochondrial dysfunction in osteoblastic MC3T3-E1 cells

Abstract

Methylglyoxal is a reactive dicarbonyl compound produced by glycolytic processing and identified as a precursor of advanced glycation end products. The elevated methylglyoxal levels in patients with diabetes are believed to contribute to diabetic complications, including bone defects. The objective of this study was to evaluate the effect of methylglyoxal on the function of osteoblastic MC3T3-E1 cells. The data indicated that methylglyoxal decreased osteoblast differentiation and induced osteoblast cytotoxicity. Pretreatment of MC3T3-E1 cells with aminoguanidine (a carbonyl scavenger), Trolox (an antioxidant), and cyclosporin A (a blocker of the mitochondrial permeability transition pore) prevented methylglyoxal-induced cytotoxicity in MC3T3-E1 cells. However, BAPTA/AM (an intracellular Ca²⁺ chelator) and dantrolene (an inhibitor of endoplasmic reticulum Ca²⁺ release) did not reverse the cytotoxic effect of methylglyoxal. Methylglyoxal increased the formation of intracellular reactive oxygen species, mitochondrial superoxide, and cardiolipin peroxidation in osteoblastic MC3T3-E1 cells. Methylglyoxal also decreased the mitochondrial membrane potential and intracellular ATP and nitric oxide levels, suggesting that carbonyl stress-induced loss of mitochondrial integrity contributes to the cytotoxicity of methylglyoxal. Furthermore, the results demonstrated that methylglyoxal induced protein adduct formation, inactivation of glyoxalase I, and activation of glyoxalase II. Aminoguanidine reversed all aforementioned effects of methylglyoxal. Taken together, these data support the notion that high methylglyoxal concentrations have detrimental effects on osteoblasts through a mechanism involving oxidative stress and mitochondrial dysfunction.     

CD73-generated adenosine promotes osteoblast differentiation

CD731 is a GPI-anchored cell surface protein with ecto-5'-nucleotidase enzyme activity that plays a crucial role in adenosine production. While the roles of adenosine receptors (AR) on osteoblasts and osteoclasts have been unveiled to some extent, the roles of CD73 and CD73-generated adenosine in bone tissue are largely unknown. To address this issue, we first analyzed the bone phenotype of CD73-deficient (cd73(-/-)) mice. The mutant male mice showed osteopenia, with significant decreases of osteoblastic markers. Levels of osteoclastic markers were, however, comparable to those of wild-type mice. A series of in vitro studies revealed that CD73 deficiency resulted in impairment in osteoblast differentiation but not in the number of osteoblast progenitors. In addition, over expression of CD73 on MC3T3-E1 cells resulted in enhanced osteoblastic differentiation. Moreover, MC3T3-E1 cells expressed adenosine A(2A) receptors (A(2A)AR) and A(2B) receptors (A(2B)AR) and expression of these receptors increased with osteoblastic differentiation. Enhanced expression of osteocalcin (OC) and bone sialoprotein (BSP) observed in MC3T3-E1 cells over expressing CD73 were suppressed by treatment with an A(2B)AR antagonist but not with an A(2A) AR antagonist. Collectively, our results indicate that CD73 generated adenosine positively regulates osteoblast differentiation via A(2B)AR signaling.     

Pleiotropic activity of lysophosphatidic acid in bone metastasis

Bone is a common metastatic site for solid cancers. Bone homeostasis is tightly regulated by intimate cross-talks between osteoblast (bone forming cells) and osteoclasts (bone resorbing cells). Once in the bone microenvironment, metastatic cells do not alter bone directly but instead perturb the physiological balance of the bone remodeling process controlled by bone cells. Tumor cells produce growth factors and cytokines stimulating either osteoclast activity leading to osteolytic lesions or osteoblast function resulting in osteoblastic metastases. Growth factors, released from the resorbed bone matrix or throughout osteoblastic bone formation, sustain tumor growth. Therefore, bone metastases are the sites of vicious cycles wherein tumor growth and bone metabolism sustain each other. Lysophosphatidic acid (LPA) promotes the growth of primary tumors and metastatic dissemination of cancer cells. We have shown that by acting on cancer cells via the contribution of blood platelets and the LPA-producing enzyme Autotaxin (ATX), LPA promotes the progression of osteolytic bone metastases in animal models. In the light of recent reports it would appear that the role of LPA in the context of bone metastases is complex involving multiple sources of lipid combined with direct and indirect effects on target cells. This review will present our current knowledge on the LPA/ATX axis involvement in osteolytic and osteoblastic skeletal metastases and will discuss the potential activity of LPA upstream and downstream metastasis seeding of cancer cells to bone as well as its implication in cancer induced bone pain. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.     

Effects of gonadal and adrenal androgens in a novel androgen-responsive human osteoblastic cell line

While androgens have important skeletal effects, the mechanism(s) of androgen action on bone remain unclear. Current osteoblast models to study androgen effects have several limitations, including the presence of heterogeneous cell populations. In this study, we examined the effects of androgens on the proliferation and differentiation of a novel human fetal osteoblastic cell line (hFOB/AR-6) that expresses a mature osteoblast phenotype and a physiological number (∼4,000/nucleus) of androgen receptors (AR). Treatment with 5α-dihydrotestosterone (5α-DHT) inhibited the proliferation of hFOB/AR-6 cells in a dose-dependent fashion, while it had no effect on the proliferation of hFOB cells, which express low levels of AR (<200/nucleus). In hFOB/AR-6 cells, co-treatment with the specific AR antagonist, hydroxyflutamide abolished 5α-DHT-induced growth inhibition. Steady-state levels of transforming growth factor-β₁ (TGF-β₁) and TGF-β-induced early gene (TIEG) mRNA decreased after treatment of hFOB/AR-6 cells with 5α-DHT, suggesting a role for the TGF-β₁-TIEG pathway in mediating 5α-DHT-induced growth inhibition of hFOB/AR-6 cells. In support of this, co-treatment of hFOB/AR-6 cells with TGF-β₁ (40 pg/ml) reversed the 5α-DHT-induced growth inhibition, whereas TGF-β₁ alone at this dose had no effect on hFOB/AR-6 cell proliferation. Furthermore, treatment of hFOB/AR-6 cells with 5α-DHT and testosterone (10⁻⁸ M) inhibited basal and 1,25-(OH)₂D₃-induced alkaline phosphatase (ALP) activity and type I collagen synthesis without affecting osteocalcin production. Thus, in this fetal osteoblast cell line expressing a physiological number of AR, androgens decrease proliferation and the expression of markers associated with osteoblast differentiation. These studies suggest that the potential anabolic effect of androgens on bone may not be mediated at the level of the mature osteoblast. J. Cell. Biochem. 71:96–108, 1998. © 1998 Wiley-Liss, Inc.     

Discovery of chemical markers for identifying species,growth mode and production area of Astragali Radix by using ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry

BackgroundAstragali Radix (AR) is a well-known Chinese herbal medicine. The quality of AR can be affected by many factors such as species, growth mode and production area, but there are still no chemical markers to distinguish it.PurposeTo explore chemical markers for improving the quality assessment of AR and discover chemical markers for identifying species, growth mode and production area of AR.MethodsA highly sensitive, efficient and accurate method based on ultra-high performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QQQ-MS/MS) for simultaneous quantitative determination of 14 major chemical components (five flavonoids and nine triterpene saponins) in 94 batches of AR from China, Republic of Korea and Germany was developed for the first time. To explore chemical markers and assess changes in the contents of 14 compounds in the 94 batches of AR samples from different regions, hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed.ResultsAstragaloside III was not only an important chemical marker for distinguishing two species of AR, i.e.: Astragalus mongholicus and A. membranaceus, but also a potential chemical marker for the classification of cultivated and semi-wild AR. In addition, in the batches of cultivated AR, the content of isoastragaloside II and cyclocephaloside II were greater in batches from the region of Shaanxi Province than that of other Provinces in China, but the content of calycosin-7-O-β-D-glucoside and astragaloside IV, which are the quality control markers of AR required by the Chinese Pharmacopoeia, were higher than that of other Provinces in China. In addition, the content of calycosin-7-O-β-D-glucoside, ononin, calycosin and astragaloside I could be used to identify samples of AR collected from China, Republic of Korea and Germany.ConclusionThis UHPLC-QQQ-MS/MS method could be applied to the quantitative evaluation of AR and could be an important and meaningful reference to develop chemical markers for quality control of AR.     

Age-related changes in bone formation,osteoblastic cell proliferation,and differentiation during postnatal osteogenesis in human calvaria

We have determined the age-related changes in the growth characteristics and expression of the osteoblast phenotype in human calvaria osteoblastic cells in relation with histologic indices of bone formation during postnatal calvaria osteogenesis. Histomorphometric analysis of normal calvaria samples obtained from 36 children, aged 3 to 18 months, showed an age-related decrease in the extent of bone surface covered with osteoblasts and newly synthesized collagen, demonstrating a progressive decline in bone formation during postnatal calvaria osteogenesis. Immunohistochemical analysis showed expression of type I collagen, bone sialoprotein, and osteonectin in the matrix and osteoblasts, with no apparent age-related change during postnatal calvaria osteogenesis. Cells isolated from human calvaria displayed characteristics of the osteoblast phenotype including alkaline phosphatase (ALP) activity, osteocalcin (OC) production, expression of bone matrix proteins, and responsiveness to calciotropic hormones. The growth of human calvaria osteoblastic cells was high at 3 months of age and decreased with age, as assessed by (³H)-thymidine incorporation into DNA. Thus, the age-related decrease in bone formation is associated with a decline in osteoblastic cell proliferation during human calvaria osteogenesis. In contrast, ALP activity and OC production increased with age in basal conditions and in response to 1,25(OH)₂, vitamin D₃, suggesting a reciprocal relationship between cell growth and expression of phenotypic markers during human postnatal osteogenesis. Finally, we found that human calvaria osteoblastic cells isolated from young individuals with high bone formation activity in vivo and high growth potential in vitro had the ability to form calcified nodular bone-like structures in vitro in the presence of ascorbic acid and β-glycerophosphate, providing a new model to study human osteogenesis in vitro. J. Cell. Biochem. 64:128–139. © 1997 Wiley-Liss, Inc.     

Effects of the dichloromethane fraction of Dipsaci Radix on the osteoblastic differentiation of human alveolar bone marrow-derived mesenchymal stem cells

Dipsaci Radix is the dried root of Dipsacus asper Wall. It has been used in Korean herbal medicine to treat bone fractures. In this study, we examined the effect of the dichloromethane fraction of Dipsaci Radix (DR(DM)) on the osteoblastic differentiation of human alveolar bone marrow-derived MSCs (ABM-MSCs). The ABM-MSCs were isolated from healthy subjects and cultured in vitro, followed by phenotypic characterization. They showed a fibroblast-like morphology and expressed CD29, CD44, CD73, and CD105, but not CD34. Calcified nodules were generated in response to both dexamethasone (DEX) and DR(DM). There was a significant increase in the alkaline phosphatase (ALP) activity and protein expression of bone sialoprotein (BSP) and osteocalcin (OC) in response to DEX and DR(DM) as compared to control. These results provide evidence for the osteogenic potential of cultured ABM-MSCs in response to DR(DM). Also, an active single compound was additionally isolated from DR(DM). The single compound (hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside) also significantly increased ALP activity and the level of protein expression of BSP and OC. These results highlight the possible clinical applications of DR(DM) and hederagenin 3-O-(2-O-acetyl)-α-L-arabinopyranoside in bone regeneration.     

Uncovering the mechanism of Astragali Radix against nephrotic syndrome by intergrating lipidomics and network pharmacology

BackgroundAstragali Radix (AR), a common Traditional Chinese Medicine (TCM), is commonly used for treating nephrotic syndrome (NS) in China. At present, the research on the efficacy of AR against NS is relative clearly, but there are fewer researches on the mechanism.PurposeThe aim of this study was to evaluate the potential beneficial effects of AR in an adriamycin-induced nephropathy rat model, as well as investigate the possible mechanisms of action and potential lipid biomarkers.MethodsIn this work, a rat model of NS was established by two injections of ADR (3.5 + 1 mg/kg) into the tail vein. The potential metabolites and targets involved in the anti-NS effects of AR were predicted by lipidomics coupled with the network pharmacology approach, and the crucial metabolite and protein were further validated by western blotting and ELISA.ResultsThe results showed that 22 metabolites such as l-carnitine, LysoPC (20:3), and SM (d18:1/16:0) were associated with renal injury. Moreover, SMPD1, CPT1A and LCAT were predicted as lipids linked targets of AR against NS, whilst glycerophospholipid, sphingolipid and fatty acids metabolism were involved as key pathways of AR against NS. Besides, AR could play a critical role in NS by improving oxidative stress, inhibiting apoptosis and reducing inflammation. Interestingly, our results indicated that key metabolite l-carnitine and target CPT1 were one of the important metabolites and targets for AR to exert anti-NS effects.ConclusionIn summary, this study offered a new understanding of the protection mechanism of AR against NS by network pharmacology and lipidomic method.