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OBJECTIVE@#To explore the mechanism underlying the hepatoprotective effect of dihydromyricetin (DMY) against lipid accumulation in light of the lipophagy pathway and the inhibitory effect of DMY on HepG2 cell proliferation.@*METHODS@#LO2 cells were cultured in the presence of 10% FBS for 24 h and treated with 100 μg/mL DMY, or exposed to 50% FBS for 24 h followed by treatment with 50, 100, or 200 μg/mL DMY; the cells in recovery group were cultured in 50% FBS for 24 h and then in 10% FBS for another 24 h. Oil red O staining was used to observe the accumulation of lipid droplets in the cells, and the levels of TC, TG, and LDL and activities of AST, ALT and LDH were measured. The expression of LC3 protein was detected using Western blotting. AO staining and transmission electron microscopy were used to determine the numbers of autophagolysosomes and autophagosomes, respectively. The formation of autophagosomes was observed with MDC staining, and the mRNA expression levels of LC3, ATG7, AMPK, mTOR, p62 and Beclin1 were determined with q-PCR. Flow cytometry was performed to analyze the effect of 50, 100, and 200 μg/mL DMY on cell cycle and apoptosis of HepG2 cells; DNA integrity in the treated cells was examined with cell DNA fragmentation test.@*RESULTS@#DMY treatment and pretreatment obviously inhibited lipid accumulation and reduced the levels of TC, TG, LDL and enzyme activities of AST, ALT and LDH in LO2 cells (P < 0.05). In routinely cultured LO2 cells, DMY significantly promoted the formation of autophagosomes and autophagolysosomes and upregulated the expression of LC3 protein. DMY obviously attenuated high FBS-induced inhibition of autophagosome formation in LO2 cells, up- regulated the mRNA levels of LC3, ATG7, Beclin1 and AMPK, and downregulated p62 and mTOR mRNA levels (P < 0.05 or 0.01). In HepG2 cells, DMY caused obvious cell cycle arrest, inhibited cell proliferation, and induced late apoptosis and DNA fragmentation.@*CONCLUSION@#DMY reduces lipid accumulation in LO2 cells by regulating the AMPK/ mTOR-mediated lipophagy pathway and inhibits the proliferation of HepG2 by causing cell cycle arrest and promoting apoptosis.
Subject(s)
Humans , AMP-Activated Protein Kinases/metabolism , Autophagy , Beclin-1 , Cell Proliferation , Flavonols , Hep G2 Cells , Lipids , RNA, Messenger , Signal Transduction , TOR Serine-Threonine Kinases/metabolismABSTRACT
Objective:To assess the anxiolytic effect of Chaimu Anshen granules (CMASG) and investigate its bioactive mechanism. Method:ICR mice were randomly divided into normal group, diazepam group(0.002 g·kg-1),Jieyu Anshen granules group(0.001 4 g·kg-1), high, medium, and low-dose (0.001 98,0.000 99,0.000 495 g·kg-1)Chaimu Anshen granule groups, with 20 mice in each group. To detect the anxiolytic effect of CMASG, mice were intragastrically administered for 4 weeks in the morning, and light-dark box transition test and open field test were performed once the other day. After the behavior tests, blood samples were collected. Six mice of each group were perfused with formalin through heart, and then the brains were fixed for immunohistochemistry test. Hippocampus of the other mice in each group were collected and stored in liquid nitrogen. The content of γ-aminobutyric acid(GABA)and glutamic acid(Glu)in hippocampus and blood samples were detected by enzyme-linked immunosorbent assay (ELISA), and the ratio of GABA/Glu was calculated. The expression of GABAα1 receptor was evaluated by the immunohistochemistry method. To test the hypnosis effect of CMASG, mice were administered intragastrically for 7 days. The sub-threshold dose of pentobarbital sodium in the sleep experiment was tested. Result:Compared with normal group, the light-dark box transitions test demonstrated that low-dose and medium-dose CMASG groups significantly prolonged the duration in light box(PPPPPPPPPPα1 receptor protein in hippocampus showed that the medium-dose CMASG significantly increased the expression of GABAα1 protein. The sub-threshold dose of pentobarbital sodium on sleep experiments confirmed that the medium-dose CMASG significantly increased the rate of sleep in mice. Conclusion:CMASG showed an anxiolytic effect, and its bioactive mechanism was related with the increase of GABA content, and the decrease of Glu content in hippocampus. Furthermore, it increased the expression of GABAα1 protein in hippocampus. The changes in content of GABA and Glu in peripheral blood were positively correlated with the changes in hippocampal tissues, which provided reference for clinical diagnosis. CMASG also exhibited an effect in improvement of sleep.
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Objective:Define traditional Chinese medicine (TCM) therapeutic principles and methods and common prescriptions of benign prostatic hyperplasia (BPH) based on clinical experience of famous doctors. Method:China National Knowledge Infrastructure, China Biology Medicine disc, China Science and Technology Journal Database, Wanfang database were systematically retrieved for literatures of famous doctors in diagnosis and treatment of BPH from January 1, 1997 to December 31, 2018.A literature analysis database was established for integration and analysis of relevant content of TCM therapeutic principles and methods and prescriptions. SPSS 20.0 software was used for statistics to obtain TCM therapeutic principles and methods, common prescriptions and drug distribution regularity of BPH. Result:A total of 109 qualified literatures were included in this study, involving 66 famous doctors and experts, and 9 kinds of BPH therapeutic principles were obtained, including "treatment of both symptoms and root cause of disease", "tonification and purgation in combination", "coordination of Yin and Yang". There are 55 kinds of therapies, including "promoting blood circulation for removing blood stasis", "clearing heat and promoting dampness", "warming and recuperating kidney Yang", "softening and resolving hard mass" and " invigorating kidney Qi". And BPH was mostly diagnosed and treated based on kidney, spleen and lung. Through collection and summarization, totally 38 formulas commonly used in treating BPH obtained. The most commonly used ones were "Guizhi Fuling Wan", "Zishen Tongguan Wan", "Buzhong Yiqi Tang", " Bazheng Tang" and "Jisheng Shenqi Wan". There were 217 commonly used herbs, mainly including " Astragali Radix", "Rehmanniae Radix Praeparata", "Atractylodis Macrocephalae Rhizoma", "Glycyrrhizae Radix et Rhizoma", "Angelicae Sinensis Radix", "Radix Achyranthis Bidentatae", "Peach Kernel", "Pangolin Scales", "Vaccariae Semen", "Salviae Miltiorrhizae Radix et Rhizoma", "Poria", "Rhizoma Alismatis" and "Plantain Seed". Conclusion:The treatment of BPH is based on the principles of "treatment of both symptoms and root cause of disease, and tonification and purgation in combination". The commonly used therapies include prescription for "promoting blood circulation for removing blood stasis", "clearing heat and promoting dampness" and "warming and recuperating kidney Yang". The corresponding prescriptions shall be based on symptoms.
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Objective:Based on the experience of famous doctors, the pattern of syndromes of benign prostatic hyperplasia (BPH) and the distribution of syndrome elements are defined to provide a basis for guiding clinical practice and scientific research. Method:The name list of famous doctors was defined. Relevant literatures on famous doctors' diagnosis and treatment of BPH between January 1, 1997 and December 31, 2018, were systematically retrieved in CNKI, CBM, CQVIP, Wanfang database. According to the inclusion criteria and exclusion criteria, relevant literatures were read, and eligible literatures were included. A literature analysis database was established for analysis and integration of relevant content of traditional Chinese medicine(TCM) syndromes and syndrome elements. SPSS 20.0 software was used for statistics and analysis of BPH TCM syndrome types and distribution characteristics of syndrome elements. Result:This study included a total of 141 eligible documents, involving 92 famous doctors. Based on the doctors' overall discussion of BPH and medical case examples, common types of BPH syndrome included kidney deficiency and blood stasis syndrome, bladder dampness syndrome, kidney Yin deficiency syndrome, kidney Yang deficiency Syndrome, liver Qi stagnation syndrome, main symptom factors of disease location included kidney, bladder, spleen, lung, liver, and main symptom factors of disease nature included blood stasis, Qi deficiency, heat, yang deficiency, and dampness. The characteristics of the symptoms in the medical case were collected and summarized, including frequent urinary symptoms, frequent urination, urinary drip, urgency, nocturia, difficulty in urinating, the tongue is mainly light red, red, dark red, and sputum, the tongue coating is mainly yellow, thin white, thin yellow and white greasy, and the pulse is mainly composed of deep, fine, string and slipping. Conclusion:Based on the experience of famous doctors, the core pathogenesis of BPH is deficient in origin and excessive in superficiality, the kidney deficiency is the root cause, the dampness heat is the symptom, and the compound syndrome is common.