Efficacy of the herbal pair, Radix Achyranthis Bidentatae and Eucommiae Cortex, in preventing glucocorticoid-induced osteoporosis in the zebrafish model / 中西医结合学报

OBJECTIVE@#In traditional Chinese medicine, the herbal pair, Radix Achyranthis Bidentatae (RAB) and Eucommiae Cortex (EC), is widely used to treat osteoporosis. Herein, we determined whether this herbal pair can be used to ameliorate glucocorticoid (GC)-induced osteoporosis (GIOP) and find its optimal dosage in zebrafish.@*METHODS@#The characteristics of the aqueous extract of RAB and EC were separately characterized using high-performance liquid chromatography. Osteoporosis was induced in 5-day post-fertilization zebrafish larvae by exposing them to 10 μmol/L dexamethasone (Dex) for 96 h. Seven combinations of different ratios of RAB and EC were co-administered. Treatment efficacy was determined by calculating zebrafish vertebral area and sum brightness, via alizarin red staining, and by detecting alkaline phosphatase (ALP) activity. Multiple regression analysis was conducted to test the optimal dosage ratio.@*RESULTS@#According to the Chinese Pharmacopoeia (2015), β-ecdysone (β-Ecd) is a major bioactive marker in RAB extract, while pinoresinol diglucoside (PDG) is the major marker in EC extract. Both of β-Ecd and PDG content values aligned with the Chinese Pharmacopoeia standards. Treatment with 10 μmol/L Dex reduced zebrafish vertebral area, sum brightness, and ALP activity, but RAB and EC attenuated these effects. Combining 50 µg/mL RAB and 50 µg/mL EC was optimal for preventing GIOP in zebrafish. Reverse transcription-quantitative polymerase chain reaction was used to evaluate the mRNA expression of osteogenesis-related genes. A treatment of 10 μmol/L Dex decreased runt-related transcription factor 2 (Runx2), osteogenic protein-1 (OP-1), bone γ-carboxyglutamic acid-containing protein (BGLAP), and β-catenin levels. This effect was counteracted by RAB and EC co-treatment (P < 0.05). Additionally, the effect of using the two herbal extracts together was better than single-herb treatments separately. These results demonstrated that RAB and EC preserve osteoblast function in the presence of GC. The best mass ratio was 1:1.@*CONCLUSION@#RAB and EC herbal pair could ameliorate GC-induced effects in zebrafish, with 1:1 as the optimal dosage ratio.

Network Pharmacology-based Analysis on Mechanism of Aconiti Lateralis Radix Praeparata and Epimedii Folium in Treatment of Chronic Heart Failure / 中国实验方剂学杂志

Objective:To explore the mechanism of the prescription consisting Aconiti Lateralis Radix Praeparata and Epimedii Folium in the treatment of chronic heart failure （CHF） based on network pharmacology，followed by verification in H9c2 myocardial cells with hypoxia-reoxygenation injury <italic>in vitro</italic> and in zebrafish with vascular endothelial growth factor receptor （VEGFR） tyrosine kinase inhibitor Ⅱ （VRI） -induced vascular insufficiency. Method:The active ingredients in Aconiti Lateralis Radix Praeparata and Epimedii Folium were searched from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP），the corresponding target genes from the Universal Protein Resource （UniProt）， and the CHF-related targets from Online Mendelian Inheritance in Man （OMIM） and GeneCards. Both the active ingredient-potential target network and the active ingredient-CHF-related target network were generated using Cytoscape 3.6.1， followed by the protein-protein interaction （PPI） network construction and Gene Ontology （GO） and Kyoto Encyclopedia of Genes and Genome （KEGG） enrichment analysis based on MetaScape. H9c2 myocardial cells exposed to hypoxia-reoxygenation were selected for determining the proliferation-promoting effect by methyl thiazolyl tetrazolium （MTT） assay. The protein expression of B-cell lymphoma-2（Bcl-2）， Bcl-2-associated X protein（Bax），cysteinyl aspartate-specific protease-3（Caspase-3）， protein kinase B（PKB/Akt），phosphorylated protein kinase B（p-Akt），phosphorylated extracellular signal-regulated kinases 1/2 （p-ERK1/2），extracellular signal-regulated kinase 1/2 （ERK1/2）， and poly adenosine diphosphate ribose polymerase（PARP）was detected by Western blotting. The efficacy of the prescription in promoting angiogenesis was verified in a zebrafish model of VRI-induced vascular injury. Result:There were 28 active ingredients for the prescription， 209 corresponding targets， 1 296 CHF-related targets， and 94 common gene targets shared by the prescription and CHF. PPI network clustering suggested that Aconiti Lateralis Radix Praeparata and Epimedii Folium alleviated CHF by interfering with cell differentiation and metabolism and angiogenesis. GO analysis revealed that CHF relief was achieved via the intervention in such biological processes as cell migration，vascular development， and angiogenesis. Pharmacodynamic experiments verified that Epimedii Folium （10 mg·L^-1） alone and the prescription （10 mg·L^-1）both enhanced the proliferation of H9c2 myocardial cells under the hypoxia-reoxygenation condition （<italic>P</italic><0.05），while the latter also increased the expression of Bcl-2，Bcl-2/Bax， and PARP （<italic>P</italic><0.05） and reduced the expression of Caspase-3， Akt， and ERK （<italic>P</italic><0.05）. The prescription at the concentrations of 0.3 and 0.1 g·L^-1 promoted angiogenesis （<italic>P</italic><0.05）. Conclusion:Aconiti Lateralis Radix Praeparata and Epimedii Folium exert the therapeutic effect against CHF via multiple ingredients，multiple targets， and multiple channels. Such combination promotes the proliferation of H9c2 myocardial cells under hypoxic condition and protects zebrafish from vascular injury by up-regulating the expression of Bcl-2 and PARP，increasing Bcl-2/Bax ratio，and down-regulating the expression of Caspase-3，Akt， and ERK.