Analysis of the Potential Angiogenic Mechanisms of BuShenHuoXue Decoction against Osteonecrosis of the Femoral Head Based on Network Pharmacology and Experimental Validation.

OBJECTIVE: Osteonecrosis of the femoral head (ONFH) is a common orthopedic disease with a high disability rate. The clinical effect of BuShenHuoXue decoction (BSHX) for ONFH is satisfactory. We aimed to elucidate the potential angiogenic mechanisms of BSHX in a rat femoral osteonecrosis model and bone marrow mesenchymal stem cells (BMSCs). METHODS: With in vivo experiments, we established the steroid-induced osteonecrosis of the femoral head (SONFH) model using Sprague-Dawley (SD) rats (8-week-old). The rats were randomly divided into five group of 12 rats each and given the corresponding interventions: control, model (gavaged with 0.9% saline), BSHX low-, medium- and high-dose groups (0.132 3, 0.264 6, and 0.529 2 g/mL BSHX solution by gavage). After 12 weeks, haematoxylin and eosin (H&E) staining was preformed to evaluate rat osteonecrosis. the expression of angiogenic factors (CD31, VEGFA, KDR, VWF) in rat femoral head was detected by immunohistochemistry, qPCR and western blotting. In cell experiment, BMSCs were isolated and cultured in the femoral bone marrow cavity of 4-week-old SD rats. BMSCs were randomly divided into eight groups and intervened with different doses of BSHX-containing serum and glucocorticoids: control group (CG); BSHX low-, medium-, and high-dose groups (CG + 0.661 5, 1.323, and 2.646 g/kg BSHX gavage rat serum); dexamethasone (Dex) group; and Dex + BSHX low-, medium-, and high-dose groups (Dex + 0.661 5, 1.323, and 2.646 g/kg BSHX gavaged rat serum), the effects of BSHX-containing serum on the angiogenic capacity of BMSCs were examined by qPCR and Western blotting. A co-culture system of rat aortic endothelial cells (RAOECs) and BMSCs was then established. Migration and angiogenesis of RAOECs were observed using angiogenesis and transwell assay. Identification of potential targets of BSHX against ONFH was obtained using network pharmacology. RESULTS: BSHX upregulated the expression of CD31, VEGFA, KDR, and VWF in rat femoral head samples and BMSCs (p < 0.05, vs. control group or model group). Different concentrations of BSHX-containing serum significantly ameliorated the inhibition of CD31, VEGFA, KDR and VWF expression by high concentrations of Dex. BSHX-containing serum-induced BMSCs promoted the migration and angiogenesis of RAOECs, reversed to some extent the adverse effect of Dex on microangiogenesis in RAOECs, and increased the number of microangiogenic vessels. Furthermore, we identified VEGFA, COL1A1, COL3A1, and SPP1 as important targets of BSHX against ONFH. CONCLUSION: BSHX upregulated the expression of angiogenic factors in the femoral head tissue of ONFH model rats and promoted the angiogenic capacity of rat RAOECs and BMSCs. This study provides an important basis for the use of BSHX for ONFH prevention and treatment.

Effect of astragaloside IV and salvianolic acid B on antioxidant stress and vascular endothelial protection in the treatment of atherosclerosis based on metabonomics.

Vascular endothelial cells and oxidation reduction system play an important role in the pathogenesis of atherosclerosis (AS). If these conditions are disordered, it will inevitably lead to plaque formation and even rupture. Astragaloside IV (AsIV) and salvianolic acid B (Sal B) are the main active ingredients of Astragalus membranaceus and Salvia miltiorrhiza, respectively, and found to ameliorate vascular endothelial dysfunction and protect against oxidative stress in recent studies. However, it is still unknown if the combination of AsIV and Sal B (AsIV + Sal B) can inhibit the development of plaque through amplifying the protective effect of vascular endothelial cells and anti-oxidative stress effect. To clarify the role of AsIV + Sal B in AS, we observed the efficacy of each group (Control, Model, AsIV, Sal B, and AsIV + Sal B) by biomolecular assays, such as observing the pathological morphology of the aorta by oil red O staining, evaluating the level of oxidative stress and endothelial cells in the serum by the Elisa test, and analyzing the changes of all small molecule metabolites in liver tissue by UPLC-QTOF-MS. Results showed that AsIV, Sal B and AsIV + Sal B decreased the deposition of lipid in the arterial wall, so as to exert the effect of anti-oxidant stress and vascular endothelial protection, where the inhibitory effect of AsIV + Sal B was the most obvious. Metabonomics analysis showed that Sal B regulated the metabolic pathways of arginine and proline. AsIV regulated glycerol metabolism and saturated fatty acid biosynthesis metabolism. AsIV + Sal B is mainly related to the regulation of the citrate cycle (TCA cycle), alanine, aspartic acid, and glutamate metabolism, cysteine, and methionine metabolism. Succinic acid and methionine are synergistic metabolites that exert an enhancing effect when AsIV and Sal B were used in combination. In conclusion, we demonstrated that AsIV acompanied with Sal B can be successfully used for anti-oxidative stress and vascular endothelial protection of AS, and succinic acid and methionine are the synergistic metabolites.

[Efficacy of Hebi Formula Combined Methotrexate on Early Rheumatoid Arthritis Patients with Dis- harmony of Gan and Pi Syndrome and Its Effects on Serum MMP-3 and RANK/RANKL/OPG Expressions].

Objective To observe the efficacy of Hebi Formula (HF) combined Methotrexate (MTX) on early rheumatoid arthritis (RA) patients with disharmony of Gan and Pi syndrome (DGPS) and its effects on matrix metalloproteinase-3 (MMP-3) activator of nuclear factor-KB/receptor activator of nu- clear factor-KB/osteoprotegerin (RANK/RANKL/OPG). Methods Totally 72 early RA patients with DGPS were assigned to the treatment group and the control group according to random digit table, 36 in each group. Patients in the control group took MTX, while those in the treatment group additionally took HF. MTX dose was increased from 7. 5 mg to 12. 5 mg gradually, once per week, and the course of treatment was 24 weeks. Efficacy for Chinese medicine (CM) syndromes, ACR20 improvement rate, laboratory re- lated indices [ rheumatoid factor ( RF ) , erythrocyte sedimentation rate ( ESR ) , C-reactive protein (CRP) , anti-cyclic citrullinated peptide antibody (CCP)], serum levels of MMP-3, OPG, RANKL, and adverse reactions were observed. Results The standard arriving rate of ACR20 was 82. 86% (2935) in the treatment group, higher than that in the control group [51. 52% (173) ;P <0. 05). The effective rate of CM syndrome was 85. 7% (30f35) in the treatment group, higher than that in the control group [63. 6% (21/33) ;P <0. 05). Compared with before treatment in the same group, levels of RF,ESR,CRP,MMP-3, and RANKL decreased, the OPG level increased in the two groups after treatment (P <0. 05, P <0. 01). Compared with the control group, levels of RF, ESR, CRP, and RANKL all decreased with statistical difference (P <0. 01 , P <0. 05). Liver dysfunction occurred in 1 case of the treatment group. Leucopenia occurred in 1 case and liver dysfunction occurred in 2 cases of the control group. Conclusion HF com- bined MTX could improve symptoms of early RA patients with DGPS, and regulate bone destruction in- duced by RANK/RANKL/OPG systems.

[In vitro anti-tumor effect of human dendritic cells vaccine induced by astragalus polysacharin: an experimental study].

OBJECTIVE: To explore the in vitro anti-tumor effect and mechanism of dendritic cell (DC) tumor vaccine induced by astragalus polysacharin (APS). METHODS: Peripheral blood mononuclear cells (PBMCs) isolated from human peripheral blood. DCs obtained from human peripheral blood were cultivated and added with culture solution for in vitro inducing them to immature DCs. On the 5th day of culture, 100 microg/mL (as the final concentration) APS was added to cells in the APS group. DCs were induced to mature in the cytokine groups by adding 20 ng/mL rhTNF-alpha (as the final concentration). Changes of morphology and phenotype of DCs were observed. Mature DCs were sensitized with tumor antigen SGC-7901 and co-cultured with allogeneic T cells. The proliferative function of T lymphocytes was detected by MTT assay. Levels of IL-12 and IFN-gamma in co-cultured supernatant were detected by ELISA. Cytotoxic lymphocytes (CTL) activated by DC were co-cultured with tumor cell SGC-7901. The specific killing capacity of CTL to target cells was detected by LDH release assay. RESULTS: The morphological observation and phenotypic identification of APS induced DCs were in accordance with the characteristics of mature DCs. APS induced mature DCs could stimulate the proliferation of allogeneic T lymphocytes. The proliferation index of T cells increased with increased ratio of stimulator cells to effector cells (P < 0.05). Levels of IL-12 and IFN-gamma in co-culture supernatant significantly increased in a time-dependent manner (P < 0.05). CTL cells activated by sensitization of DCs could significantly kill tumor cells, and the killing effect increased along with increased effector-to-target ratio. CONCLUSION: APS could in vitro induce DCs to mature, promote its antigen-presenting capacity, effectively activate CTLs, and enhance anti-tumor function of the organism.

[Paeonol attenuates oxygen-glucose deprivation injury and inhibits NMDA receptor activation of cultured rat hippocampal neurons].

The purpose of this study is to determine if paeonol can protect hippocampal neurons against injury due to oxygen-glucose deprivation (OGD) injury. The rat neurons were cultured in an OGD environment and the model of OGD injury was established. Paeonol and MK-801, a positive control drug, were added before deprivation. Neuron viability was measured by the reduction of MTT; glutamate was analyzed by amino acid analyzer; binding activity of NMDA receptor was evaluated by liquid scintillation counting and the expression of NMDA receptor NR1 subunit mRNA was semiquantitatively determined by RT-PCR. Compared with OGD injury group, paeonol treatment obviously increased cell survival rate and reduced the binding activity of NMDA receptors and the release of glutamate; and down-regulating the expression of NR1 subunit. These results suggest that paeonol may exhibit its protective effect against OGD injury by the action on NMDA receptor of rats.

Protective effects of paeonol on cultured rat hippocampal neurons against oxygen-glucose deprivation-induced injury.

Mounting evidence has suggested that paeonol possesses plenty of pharmacologic actions. Our research is to determine if paeonol can protect cultured rat hippocampal neurons from oxygen-glucose deprivation(OGD)-induced injury and elucidate the underlying mechanism. We cultivated the rat hippocampal neurons as the object of study and then established the model of oxygen-glucose deprivation. Neuronal viability was measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), while intracellular Ca(2+) concentration was observed by fluorospectrophotometer. The binding force of N-methyl-D-aspartate (NMDA) receptor was evaluated by liquid scintillation counting. Compared with oxygen-glucose deprivation group, paeonol treatment obviously increased cell survival rate and reduced the activity of the binding force of NMDA receptors, reversing the overload of intracellular Ca(2+). These results demonstrate that paeonol protected rat neurons from oxygen-glucose deprivation-induced injury, resulting in alleviating the morphological damage and increasing neuron viability and suggest that paeonol may exhibit its protective effect against oxygen-glucose deprivation-induced injury by targeting on NMDA receptors.