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Juvenile zebrafish were used to screen the active components of Lycii Fructus for improving osteoporosis. The screening results were further verified by zebrafish adult osteoporosis model and the action mechanism was explored. Prednisolone was used as the inducer to build osteoporosis models of juvenile and adult zebrafish, and 9 groups of samples of different extracts and chemical parts of Lycii Fructus were given. Alizarin red staining was applied for observing the scale matrix mineralization and bone resorption. The activities of osteoblasts and osteoclasts were detected using alkaline phosphatase (ALP) and tartrate resistant acid phosphatase (TRAP/TRACP) staining. The expressions of bone metabolism-related genes alp, osteoprotectin (opn), osteoblast specific transcription factor (sp7), cathepsin K (ctsk), tracp, and Runt family transcription factor 2b (runx2b) in each group were determined using quantitative polymerase chain reaction. The results showed that all components of Lycii Fructus improved the formation area of the first vertebrae, the staining light density value, and the number of vertebrae joints in juvenile zebrafish and the Lycium barbarum polysaccharide (LBP) treatment group exerted the best effect. In addition, LBP prevented the formation of bone resorption lacunae in zebrafish scales, increased ALP activity, decreased TRAP activity, up-regulated the alp, sp7, and opn genes, and lowered the expressions of ctsk and tracp genes. In conclusion, LBP regulated the activity of osteoblasts and osteoclasts, reduced bone resorption, promoted bone formation and enhanced bone density, which might be the main anti-osteoporosis active fraction of Lycii Fructus. This study provided modern scientific evidence for the scientific connotation of the traditional effect of "strengthening bones and muscles" of Lycii Fructus, provided the reference for the evaluation of the anti-osteoporosis activity of traditional Chinese medicine based on zebrafish adult model, and provided beneficial enlightenment for the bone health needs of the aging society population.
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To study the osteoprotective effect of 1,2,3,4,6-pentyl-O-galloyl-beta-D-glucose (PGG) its anti-osteoblast apoptosis related mechanism was investigated. A model of zebrafish osteoporosis induced by prednisolone (Pred, 25 μmol·L-1) was established in vivo, and calcein staining was used to detect the effect of PGG on the bone area of zebrafish. Bone marrow mesenchymal stem cells were cultured in vitro, and the number of calcified nodules was observed by alizarin red staining, and the relevant indexes of osteoblast differentiation runt-related transcription factor 2 (Runx 2), osteocalcin (OCN) mRNA level were detected by qRT-PCR. The osteoblast cell line MC3T3-E1 cells was cultured in vitro, and 400 μmol·L-1 hydrogen peroxide (H2O2) was used to intervene the injury to detect the effect of PGG on osteoblasts under oxidative stress. The effect of PGG on osteoblast activity was detected by MTT assay. The effect of PGG on apoptosis was observed by Hoechst 33342 staining. Western blot was used to detect the expression of Bcl-2, Bax, nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1). DCFH-DA fluorescence staining for detection of reactive oxygen species (ROS) levels. JC-1 staining was used to detect mitochondrial membrane potential levels. The results showed that PGG could significantly increase the vertebral area of the zebrafish model when compared with the model group. On the 14 th day of osteoblast differentiation, the number of calcified nodules in the PGG group was significantly increased when compared with the control group and the mRNA levels of Runx 2 and OCN were also significantly increased. In addition, under oxidative stress, PGG could increase osteoblast viability, significantly reduce the number of apoptotic cells, and increase the ratio of Bcl-2/Bax. Fluorescence staining results show that PGG decreased intracellular ROS fluorescence density and increased mitochondrial membrane potential. Western blot data showed that PGG could promote the expression of Nrf2 in the nuclear and enhance the expression of downstream protein HO-1. In conclusion, PGG could improve osteoporosis in zebrafish, and this effect may be related to the regulation of Nrf2/HO-1 signaling pathway to improve mitochondrial dysfunction, anti-oxidative stress in osteoblast apoptosis and promote bone formation. This study provides new ideas and clues for the discovery of anti-osteoporosis drugs.
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This study aimed to investigate the mechanisms of Yu-Ping-Feng-San (YPFS) on attenuating allergic inflammation in the initial stage of atopic dermatitis (AD). AD mouse model was established with fluorescein isothiocyanate (FITC) sensitization and elicitation. Epithelial barrier structure was observed with transmission electron microscope. The populations of dendritic cells (DCs) and group 2 innate lymphoid cells (ILC2s) were detected by flow cytometry. Human immortalized keratinocyte (HaCaT) cells were stimulated with Poly(I:C)/TNF-α in vitro to assessthymic stromal lymphopoietin (TSLP), interleukin (IL)-33 and nuclear factor-κB (NF-κB) levels or expressions by immunofluorescence, enzyme linked immunosorbent assay (ELISA) and western blot. In the initial stage of AD, ear swelling and infiltration of inflammatory cells in ear tissues were markedly attenuated with YPFS treatments. The damaged structures of ear epithelium and the increased levels of Th2-cytokines induced by FITC were significantly rescued in YPFS-treated mice. The production of pro-allergic cytokines, TSLP and IL-33, as well as the cell populations of their target cells DCs and ILC2s were decreased in AD model, respectively. Likewise, the levels of TSLP and IL-33 in Poly(I:C)/TNF-α-stimulated HaCaT cells showed the same results. Lower levels of p-NF-κB were detected with YPFS treatment, and the expressions of TSLP and IL-33 could be further decreased with inhibiting of NF-κB. Therefore, YPFS attenuates allergic inflammation in the initial stage of AD probably through regulating NF-κB-TSLP/IL-33 pathway, which may provide a novel effective target for the prevention and treatment of allergic diseases.
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AIM To study the reproductive toxicity of Triptolide,one of the main components of Tripterygium wilfordii Hook.f,on male Caenorhabditis elegans,and its potential mechanism.METHODS Expose L4 larva of him-5 nematodes to 48 h 0.02,0.2,2.0 mg/L of Triptolide.The effect of Triptolide on the fertility of male C.elegans was assessed by crossing test,and the relevant mechanism was explored through measuring spermatids morphology development,spermatids activation,sperm motility and the relative mRNA expression levels of the related spermatogenesis genes.RESULTS Compared with the solvent control group,the 48 h Triptolide treatment induced significantly decreased brood size in 2.0 mg/L Triptolide group,significant reduction in spermatid diameter,spermatid cross-sectional area,rates of spermatid activation,rates of motile sperm in 0.2 and 2.0 mg/L Triptolide groups,and decreased expression levels of spe-10 in 0.02-2.0 mg/L Triptolide groups,decreased spe-15 in 0.2-2.0 mg/LTriptolide groups,and decreased fer-1 and folt-1 in 2.0 mg/L Triptolide groups as well.CONCLUSION Triptolide decreases expression levels of the related genes in spermatogenesis,contributes to an array of indicators inhibition,the spermatids morphology development,the sperm morphology and function development,which leads to decreased fertility of male C.elegans.Therefore,spermatogenesis damage is one of the main pathway that Triptolide adversely affects the fertility.
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To evaluate the effect and mechanism of aerial parts of Salvia miltiorrhiza(SM) on high sugar-induced Drosophila melanogaster metabolic disorder model. The levels of glucose, triglyceride and protein in SM were detected; nymphosis time was recorded, and the reliability of metabolic disorder model as well as the mechanism of aerial parts of SM were evaluated based on metabonomics. The results showed that the levels of glucose and triglyceride in model group were significantly higher than those in normal control group(<0.05). As compared with the model group, the glucose level was significantly decreased in gliclazide(GLZ) group, SM medium(SM-M) and high(SM-H) dose groups(<0.05, <0.01); the triglyceride level was significantly decreased in GLZ group and SM-H group(<0.05, <0.01). By principal component analysis(PCA) and partial least squares discriminant analysis(PLS-DA), the metabolic level of model ones was recovered to a certain degree after intervention by aerial parts of SM. Seventeen marker compounds and four major metabolic pathways were obtained by screening differential metabolites, comparing literature and retrieving the database. The aerial parts of SM may regulate glycolipid metabolism through the impact on histidine metabolism, glycerophospholipid metabolism, pentose and glucuronate interconversions, cysteine and methionine metabolism and glycerolipid metabolism. Extract from aerial parts of SM can regulate the glycolipid metabolism of D. melanogaster metabolic disorder model and make it return to normal condition. This paper provides reference for the value discovery and resource utilization of the aerial parts of S. miltiorrhiza.
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The common fruit fly, Drosophila melanogaster, is a well studied and tractable genetic model organism for exploring molecular mechanisms of human diseases in biological science. The considerable work of Drosophila has promoted the comprehension of relative protein expressions and signaling pathways associated with pathological and physiological activities. Meanwhile, various strains of transgenic drosophila with diverse genetic features have been established. These fly strains can be applied into bioactivity evaluation and drug screen as an emerging human disease model. The development of Chinese medicine has been seriously restricted by lacking of techniques and methods in activity evaluation. D. melanogaster, because of its many distinguishing features, such as rapid reproduction, short life cycle, rich strains, entirety action, highly correlated with human and other characteristics, has become a desirable choice to study Chinese medicine which has complicated composition. Here, progress of researches based on flies in disease models and their application in drug evaluating were reviewed, including aging, neurodegenerative diseases, metabolic disorders and diabetes, sleep disorder, intestinal immunity, reproduction, cancer and cardiac function.
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<p><b>OBJECTIVE</b>To observe the effect of Yupingfeng San (YPFS) against OVA-induced allergic asthma in mice.</p><p><b>METHOD</b>Mice were injected with OVA to establish the allergic asthma model. They were abdominally injected with 20 microg OVA on day 0 and 14, and inhaled aerosol 0.5% OVA solution for 20 min for seven days. The blank control group was administrated with equal volume of saline. YPFS groups with different doses were administrated intragastrically with YPFS every day, with the crude drug dosage of 3.25, 6.5, 13 g x kg(-1), respectively. The model group and control group were administrated with equal volume of saline. The positive control group was given intraperitoneally injected with 1 mg x kg(-1) DEX since aerosol inhalation. Blood was drawn after the last OVA aerosol inhalation to count the number of Eosnophils (Eos) in blood and detect IgE in serum; BALF was collected to count the number of cells and classify; right lung tissues were evenly grinded to detect cytokines IL-4 and IFN-gamma, and left upper lung lobes were collected for pathologic histology.</p><p><b>RESULT</b>The level of Eos and IgE in serum increased significantly in the model group, and a large number of Eos were detected in BALF. Histopathological changes in lung showed bronchial serous exudation, tubular epithelial cells exfoliation, tube narrowing, widened alveolar septum, and bronchial periarterial lymphocytes infiltration. Homogenate of lung tissues showed increase of IL-4, and decrease in IFN-gamma/IL-4 ratio. YPFS groups with different doses displayed decrease of Eos in blood and BALF and IgE content in serum, and relief of pathologic changes in above models. Meanwhile, IL-4 content in homogenate of lung tissues decreased, with the increase in IFN-gamma/IL-4 ratio.</p><p><b>CONCLUSION</b>YPFS shows the inhibitory effects on OVA-induced allergic asthma, involving down regulation of Eos and IgE levels in blood of asthma mice, and infiltration of inflammatory cells in lung tissues. Meanwhile, it can reduce IL-4 in lung homogenates, increase IFN-gamma/IL-4, and inhibits Th2 polarization.</p>
Subject(s)
Animals , Humans , Male , Mice , Asthma , Drug Therapy , Allergy and Immunology , Disease Models, Animal , Drugs, Chinese Herbal , Eosinophils , Allergy and Immunology , Interferon-gamma , Allergy and Immunology , Interleukin-4 , Allergy and Immunology , Mice, Inbred BALB C , OvalbuminABSTRACT
<p><b>OBJECTIVE</b>To establish an evaluation system for animal model with gynecological disease characterized by Qi stagnation and blood stasis syndrome, in order to disprove syndrome characteristics of the model by classic clinical prescriptions, and evaluate the specificity and reliability of the model with macroscopic biological signs and symptoms.</p><p><b>METHOD</b>The model characterized by Qi stagnation and blood stasis syndrome was established by injecting adrenaline into female SD rats and conducting unpredictable chronic stimulus such as reversal of day and night, swimming in cold water, thermal stimulation in oven, noise and tail suspension for two weeks. They were also orally administered with Xiangfu Siwu Tang, Shaofu Zhuyu Tang and positive control drug aspirin in groups. A comprehensive evaluation was conducted for the model on the basis of haemorheology, four blood coagulation indexes, four diagnostic information (digital imaging of tongue, paw and tail, temperature, weight, ingestion, electrocardiograph, and open filed test), and syndrome rating.</p><p><b>RESULT</b>Compared with the normal group, the model group showed obvious changes in haemorheology, four blood coagulation indexes, animal behavior, weight, ingestion, syndrome rating and heart rate. Their tongue and paw pictures were analyzed with Photoshop 7.0, showing significant difference in red, green and blue percentage composition from the normal group. Groups given aspirin and Xiangfu Siwu Tang showed notable changes in haemorheology, four blood coagulation indexes, animal behavior, weight, ingestion, heart rate, syndrome rating, and red, green and blue percentage composition in tongue and paw pictures, whereas the group given Shaofu Zhuyu Tang showed no remarkable improvement.</p><p><b>CONCLUSION</b>The evaluation system for the animal model with gynecological disease characterized by Qi stagnation and blood stasis syndrome is established to provide reference for studies on the evaluation system for qi stagnation and blood stasis syndrome models.</p>
Subject(s)
Animals , Female , Rats , Diagnosis, Differential , Disease Models, Animal , Gynecology , Hemostasis , Medicine, Chinese Traditional , Qi , SyndromeABSTRACT
Objective: To explore the effects of Danggui Shaoyao Powder (DSP) on in vivo uterine contractions and vasomotor factors of rats, and to investigate the mechanism of DSP for healing primary dysmenorrhea. Methods: Female SD rats were randomly divided into model, Verapamil (100 μg/kg, positive control), low-, mid-, and high-dose DSP (4, 8, and 16 g/kg) groups, and the rats were administered once daily for 7 d. The rats were sc administered with diethylstilbestrol (4 mg/kg) on day 7, on the next day the in vivo uterine contraction of rats was observed before and after oxytocin treatment under anesthesia condition, and the levels of nitric oxide (NO) and endothelin (ET) in uterine homogenate were determined. Results: DSP increased the average muscle tension of uterine contraction of normal rats obverously, but inhibited the oxytocin-induced uterine contraction significantly. The changes of the uterine contraction frequency, amplitude, average muscle tension, and uterine vigour of rats before and after oxytocin processing were significantly inhibited. DSP increased the NO level in the uterine of the experimental animals after oxytocin treatment and reduced the level of ET. The NO/ET ratio increased significantly. Conclusion: DSP could inhibit the in vivo uterine contraction, regulate the vasomotor substances, and improve the uterine blood supplement, resulting in the beneficial effect on treating primary dysmenorrhea.
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<p><b>OBJECTIVE</b>To evaluate the effect of Taohong Siwu Tang and its fractions on hotplate-induced pain in mice, acetic acid-induced writhing response, dysmenorrheal model and isolated uterine contraction in vitro in mice, and discuss material basis of effect sites.</p><p><b>METHOD</b>The samples of fractions were prepared by macroporous adsorptive resins (TH-1-TH-15). In the whole animal experiment, the hotplate-induced pain mice model was established to observe the effect of the samples on pain threshold in mice; the acetic acid-induced writhing response mice model was built to observe the effect of the samples on the writhing response in mice; the mice dysmenorrheal model was established to observe the effect of the samples on the writhing response, and calcium ion (Ca2+) and nitric oxide (NO) levels in uterine tissue of mice. In the isolated uterus contraction experiment, the oxytocin-induced isolated uterus contraction mice model was established to observe the effect of the samples on the isolated uterus contraction index. HPLC-DAD method was adopted for the content determination of effect sites.</p><p><b>RESULT</b>According to the evaluation of the integration geological effect, beside TH-2 and TH-4, other three active fractions (TH-9, TH-10 and TH-11) extracted from Taohong Siwu Tang are the main effect sites. Their chemical components were analyzed and identified as monoterpene glycosides, phthalides, organic acids, etc.</p><p><b>CONCLUSION</b>The effect sites of Taohong Siwu Tang on dysmenorrhea are TH-9, TH-10 and TH-11, which are 30% - 50% active fractions obtained from water-soluble small-molecular fractions by gradient elution using ethanol through macroporous absorption resin. Compared with TH-10 and TH-11, TH-9 shows stronger effect, which may be related to the type and content of chemical components it contains.</p>
Subject(s)
Animals , Female , Mice , Chromatography, High Pressure Liquid , Drugs, Chinese Herbal , Therapeutic Uses , Dysmenorrhea , Drug Therapy , Medicine, Chinese Traditional , Mice, Inbred ICRABSTRACT
<p><b>OBJECTIVE</b>To study the effect of TCTS on endometriosis rats.</p><p><b>METHOD</b>On the model of surgical induced rat endometriosis, weight and pathological changes of endometrial transplant and serum hormones were observed.</p><p><b>RESULT</b>Weight of endometrial transplants was reduced by TCTS 11.2 g.kg-1 and 22.5 g.kg-1 and transplants of TCTS treated rat showed poorly developed epithelium, thinner stroma, fewer stromal cells and glands. At the same time elevated serum E2, FSH and LH were reduced by TCTS.</p><p><b>CONCLUSION</b>TCTS can inhibit the growth of endometrial transplants, which is related with serum hormone, especially E2.</p>