Molecular mechanism of Cigu Xiaozhi formula interfering with HSC-T6 cell activation by regulating HIF-1α signaling pathway based on computer aided drug design / 药学学报

In this study, we investigated the effect of Cigu Xiaozhi formula on HSC-T6 activity in hypoxic microenvironment based on network pharmacology and computer-aided drug design, and predicted and verified its possible targets and related signaling pathways. The potential active components and targets of Cigu Xiaozhi formula were screened by searching Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP), Encyclopaedia of Traditional Chinese Medicine (ETCM) and Bioinformatics Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine (BATMAN-TCM) databases, and the liver fibrosis related targets retrieved from Gene Cards and Pharm GK database were integrated to obtain the potential targets of Cigu Xiaozhi formula in the treatment of liver fibrosis. GO enrichment analysis and KEGG signaling pathway enrichment analysis were performed on Omic Share platform, and Cytoscape software was used to construct the "potential active ingredient-key target-pathway" network. The active components and target proteins were subjected to molecular docking analysis by Auto Dock software. According to the results of molecular dynamics simulation and binding free energy calculation, the top 5 active components with degree were scored. The active components stigmasterol and β-sitosterol were subjected to molecular docking. CoCl2 was used to induce HSC-T6 cells to construct hypoxia model in vitro. The cell viability was detected by CCK-8 assay, and the optimal time and concentration of hypoxia model of HSC-T6 cells was determined to be 100 µmol·L-1 CoCl2 for 24 h. Under hypoxia condition, HSC-T6 cells were activated, the wound healing rate was significantly increased, and the fluorescence signal of activation marker protein α-smooth muscle actin (α-SMA) was significantly enhanced. However, 6% drug-containing serum could inhibit the activation of HSC-T6 cells, and the wound healing rate was significantly decreased, and the fluorescence signal of α-SMA was significantly weakened. Further studies showed that the expressions of hypoxia-inducible factor-1α (HIF-1α), α-SMA and key proteins of Hedgehog (Hh) signaling pathway in HSC-T6 cells were up-regulated under hypoxia, while the expressions of HIF-1α, α-SMA, Patched-1 (Ptch-1) and glioma related oncogene homology-1 (Gli-1) were down-regulated in 6% drug-containing serum group, the YC-1 group and the cyclopamine group. These results indicated that HIF-1α and Hh signaling pathways were involved in the activation of HSC-T6 cells, and the traditional Chinese medicine Cigu Xiaozhi formula could inhibit the activation of HSC-T6 cells, and the mechanism may be related to the inhibition of HIF-1α expression and the blocking of Hh signaling pathway. In conclusion, Cigu Xiaozhi formula can inhibit the activation of HSC-T6 cells by directly acting on HIF-1α and Hh signaling pathway, and exert an anti-hepatic fibrosis effect. The animal experimental protocol has been reviewed and approved by Laboratory Animal Ethics Committee of Gansu University of Chinese Medicine, in compliance with the Institutional Animal Care Guidelines.

Establishment of Acquired Aplastic Anemia SD Rat Model / 中国实验血液学杂志

OBJECTIVE@#To establish a stable and rapidly rat model acquired aplastic anemia.@*METHODS@#The SD rats were exposed to Csγ-ray at 3.5 and 4.0 Gy ( 91 cGy/min), and intraperitoneally injected with CTX at 35 mg/( kg·d) and CHL at 45 mg/( kg·d) in d 4, 6 and 8 after irradiation; the WBC, platelet and reticulocyte counts in peripheral blood, the smears and nucleated cells counts of bone marrow were observed.@*RESULTS@#The levels of peripheral blood 3-lineage cells of SD rats treated with Csγ-irradiation combined with cyclophosphamide and chloramphenicol were significantly reduced, among which white blood cells, platelets and reticulocytes decreased rapidly, and the number of bone marrow nucleated cells decreased significantly; bone marrow pathological sections showed severe reduction of hematopoietic cells, and the non-hematopoietic cells such as fat cells increased, and a serve or lightly reduction of bone marrow cells were found.@*CONCLUSION@#The rat model established by Csγ-ray irradiation combined with cyclophosphamide and chloramphenicol meets the clinical characteristics of aplastic anemia, and this study provides a stable rat model for the study of new therapeutic drugs for acquired aplastic anemia.

Establishment of Cs γ ray Combined with Cyclophosphamide and Chloramphenicol-Mediated Mouse Model of Acquired Aplastic Anemia / 中国实验血液学杂志

OBJECTIVE@#To improve and establish the mouse model with aplastic anemia (AA) mediated by Cs γ-ray irradiation combined with cyclophosphamide (CTX) and chloramphenicol (CHL) injection,so as to provide a stable model for studying the pathogenesis and treatment of AA .@*METHODS@#The BALB/c mice were exposed to Cs γ-ray of 3-5 Gy(91 cGy/min) and were intraperitoneally injected with CTX of 25 mg/(kg.d) and CHL of 62.5 mg/(kg.d) at D 4,5 and 6 after irradiation; the WBC, platelet and reticulocyte counts in peripheral blood as well as the mucleated cell count in bone marrow and bone marrow smears were detected .@*RESULTS@#The 3-lineage cells in peripheral blood of BALB/c mouse model with acquired AA were rapidly reduced, especially WBC, platelet and reticulocyte counts were lowest at D 14,the 3-lineage cells in peripheral blood were still severely reduced at D 28; the nucleated cell count in bone marrow significantly dcreased,the bone marrow hyperplasia was reduced or severely reduced; the pathological sections of bone marrow showed the severe reduction of hematopoietic cells and the increased of non-hematopoietic cells such as fat cells.@*CONCLUSION@#The mouse model with acquired AA has been established by Cs γ-ray irradiation combined with CTX and CHL injection. All detection indicators of this model reach to diagnostic criteria for acquired AA,therefore this mouse model may be used as the model for study of pathogenesis and treatment of acquired AA.

Effect of Diosgenin on Expressions of PPARγ, C/EBPα and Adipokine Secretion in Aplastic Anemia Mice / 中国实验方剂学杂志

Objective: To observe the effect of diosgenin on aplastic anemia (AA) mice and peroxisome proliferator activated receptor γ (PPARγ), CCAAT-enhancer binding protein α (C/EBPα), Adiponectin, Leptin, in order to discuss the potential mechanism of bone marrow mesenchymal stem cells in the process of adipemia. Method: BALB/c mice were randomly divided into control group and model group. The model group was established by 60Coy irradiation combined with tail vein infusion with lymphatic suspension cells of DBA/2 mice. After successful evaluation of the model, the mice were randomly divided into 6 groups:model group, low, medium and high-dose diosgenin groups (37.44,74.88,149.76 mg·kg-1·d-1), cyclosporine group (23.5 mg·kg-1·d-1), and tripterygium glycoside group (9.36 mg·kg-1·d-1), and given corresponding drugs by gavage for 14 days. After the intervention, the peripheral blood of mice in each group was detected, and bone marrow smears were collected to evaluate the proliferation of bone marrow. Bone marrow mesenchymal stem cells (BMMSCs) were isolated and cultured by adherent method and induced by adipogenesis. The mRNA and protein expressions of PPARγ, C/EBPα, Adiponectin, Leptin in BMMSCs were detected by quantitative real-time fluorescent quantitative polymerase chain reaction(Real-time PCR) and Western blot. Result: The white blood cell (WBC), hemoglobin (HGB) and blood platelet (PLT) in peripheral blood of model group were significantly lower than those of normal group (PPγ, C/EBPα, Adiponectin and Leptin in BMMSCs of the model group increased significantly (Pγ, C/EBPα, Adiponectin and Leptin in the middle-dose group diosgenin decreased obviously, which was better than those of Tripterygium glycoside group (P0.05). Conclusion: Diosgenin can promote the recovery of peripheral blood in aplastic anemia mice and improve the hematopoiesis of bone marrow. Diosgenin can reduce the expressions of PPARγ and C/EBPα, the formation of adipocytes and the secretion of Adiponectin and Leptin in adipocytes, and effectively inhibit the process of adipose tissue derived from bone BMMSCs.