Exploring the multicomponent synergy mechanism of Zuogui Wan on postmenopausal osteoporosis by a systems pharmacology strategy.

OBJECTIVE: To explore the multi-component synergistic mechanism of Zuogui Wan (, ZGW) in treating postmenopausal osteoporosis (PMOP). METHODS: The main components and target genes of ZGW were screened via the Traditional Chinese Medicine Systems Pharmacology (TCMSP). In addition, the target gene sets of PMOP were derived from the GeneCards and Online Mendelian Inheritance in Man databases. The search tool for recurring instances of neighbouring genes (STRING) 11.0 software was used to analyze the interaction among intersecting genes. Cytoscape 3.6.1 software and the Matthews correlation coefficient (MCC) algorithm were used to screen the core genes. Fifty Sprague-Dawley female rats were randomly divided into the sham-operated (Sham) group and the four ovariectomized (OVX) subgroups. Rats subjected to Sham or OVX were administered with the vehicle (OVX, 1 mL water/100 g weight), 17ß-estradiol (E2, 50 µg·kg-1·d-1), and lyophilized powder of ZGW at a low dose of 2.3 (ZGW-L) and high dose of 4.6 (ZGW-H) g·kg-1·d-1 for three months. The bone density and bone strength were assessed using dual-energy X-ray and three-point bending tests, respectively. Furthermore, enzyme-linked immun-osorbent assay, Hematoxylin-eosin staining, and western blot analysis were used to determine the potential pharmacological mechanisms of action of ZGW in PMOP. RESULTS: A total of 117 active compounds of ZGW were screened from the TCMSP. Furthermore, 108 intersecting genes of drugs and diseases were identified. Using STRING software and the MCC algorithm, ten core genes, including C-X-C chemokine living 8 (CXCL8), C-C chemokine receptor type 2 (CCR2), alpha-2a active receptor (ADRA2A), melatonin receptor type 1B (MTNR1B), and amyloid-beta A4 protein (APP), were identified. The anti-osteoporosis regulation network of ZGW was constructed using the Cytoscape software. The animal experiments demonstrated that ZGW groups significantly reduced the serum levels of ß-C-terminal telopeptide of type I collagen (ß-CTX) and increased serum levels of bone-specific alkaline phosphatase (BALP) (P < 0.05, P < 0.01). The OVX group exhibited a significant decrease in bone mineral density and bone strength compared with the Sham group (P < 0.01). Moreover, treatment with ZGW resulted in increased trabecular thickness, improved arrangement of trabecular structure, and reduced empty bone lacunae. Furthermore, treatment with ZGW significantly increased the protein expression of CXCL8, ADRA2A, and CCR2 (P < 0.05, P < 0.01), and significantly decreased the protein expression of Runx2 (P < 0.01). Furthermore, the ZGW and E2 groups demonstrated significantly increased BMD (P < 0.05, P < 0.01), improved bone strength (P < 0.05, P < 0.01), reduced expression of CXCL8, ADRA2A, and CCR2, and increased runt-related transcription factor 2 levels in bone tissue (P < 0.05, P < 0.01) compared with the OVX group. However, there were no significant differences in MTNR1B and APP expression among the groups. CONCLUSION: ZGW shows synergistic mechanisms in PMOP through multiple components, targets, and pathways.

Yiqihuoxue recipe induces differentiation of rat bone marrow mesenchymal stem cells towards neurons in vitro / 中国组织工程研究

BACKGROUND: Previous studies have demonstrated that Yiqihuoxue recipe (Buyanghuanwu decoction) can inhibit cell apoptosis and antagonize free radical injury in cerebral ischemia/reperfusion models. However, there have been few reports regarding the effects of Yiqihuoxue recipe on stem cell differentiation. OBJECTIVE: To observe the effects of Yiqihuoxue recipe on the differentiation of rat bone marrow mesenchymal stem cells (MSCs) towards neurons. DESIGN, TIME AND SETTING: The present controlled observational expedment based on cells was performed at the Henan University of Traditional Chinese Medicine, i.e., the Third-Grade Laboratory of the State Administration of Traditional Chinese Medicine between March 2006 and February 2008.MATERIALS: Twenty clean Sprague-Dawley (SD) rats, aged 1-2 months, weighing 100-150 g, of either gender, were included in this study. The Chinese medicine compound Yiqihuoxue recipe was made in the Manufacturing Laboratory of the First Affiliated Hospital of Henan College of Traditional Chinese Medicine as follows. 60 g Danggui (Radix Angelicae Sinensis), 30 g Taoren(Semen Persicae), 30 g Chuanxiong(Szechwan Lovage Rhizome) were selected and mixed in 720 mL water for extract volatile oil for later use. The remaining gruffs and extract were supplemented with 1 200 g Huangqi(Radix Astragali), 60 g Chishao (Radix Paeoniae Rubra), 30 g Dilong(Lumbricus), 30 g Honghua(Flos Carthami) before 920 mL water was added. After I hour of decoction and filtering, the gruffs were decocted for another 1 hour after adding 8 640 mL water. Then all decocted liquid was merged and condensed to 600 mL and mixed with 3 mL of volatile oil and tween-80. After high-temperature and high-pressure stedlization, the products were preserved for future use. 1 mL of drug was equal to 2.4 g raw herbs. METHODS: Bone marrow mononuclear cells were isolated by density gradient centrifugation. After culture, bone marrow MSCs were amplified and purified. Passage 10 cell suspension was inoculated into a 40 mm-diameter plastic culture dish. Dulbecco's modified eagle's medium supplemented with 0.1 volume fraction of fetal bovine serum and basic fibroblast growth factors was added for 24 hours of culture when adherent cells reached 60%-90% confluence in each group. Thereafter, the "Yiqihuoxue" group was incubated for 5 hours using medium containing DMSO, butylated hydroxyanisole, 13-mercaptoethanol, and Yiqihuoxue recipe; simultaneously, the control group was treated for 5 hours with fluid containing DMSO, butylated hydroxyanisole, and β- mercaptoethanol.MAIN OUTCOME MEASURES: Identification of bone marrow MSCs by flow cytometry; Detection of Nestin-, and neuron specific enolase(NSE)-, and glial fibrillary acidic protein(GFAP)-positive expression by immunohistochemistry. RESULTS: Flow cytometery results demonstrated that cell surface marker CD90 and CD106 expression was positive, while CD45 and CD34 expression was negative. Immunohistochemistry results showed that the numbers of Nestin-positive cells (1 and 3 hours after induction) and NSE- and GFAP-positive cells (5 hours after induction) were significantly greater in the Yiqihuoxue group than in the control group (P < 0.01). CONCLUSION: Yiqihuoxue recipe can induce the differentiation of bone marrow MSCs towards neurons in vitro.