Structural identification of an polysaccharide isolated from Epimedium brevicornum and its beneficial effect on promoting osteogenesis in osteoblasts induced by high glucose.

AIM: Diabetes osteoporosis (DOP) is a chronic bone metabolic disease induced by diabetes, whose morbidity continues to increase. Epimedium brevicornum Maxim (EB), a popular Chinese traditional medicine, has been used to treat bone diseases in China for thousands of years. But its material basis and specific mechanism of action are not clear. METHODS: Epimedium brevicornum crude polysaccharide (EPE) is the main component, in this research the characterized the structure of EBPC1 purified from EPE was detected and its effects on cell proliferation, differentiation, and cytoskeletal in osteoblasts induced by high glucose. RESULTS: The molecular weight of EBPC1 was 10.5 kDa. It was mainly comprised of glucose and galactose, and the backbone of EBPC1 was→4)-α-D-Galp-(1→4)-α-D-Galp-(1→6)-ß-D-Galp-(1→6)-ß-D-Galp-(1→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→. The results from in vitro experiments revealed that EBPC1 significantly increased alkaline phosphatase (ALP) activity and mineralized nodule formation in primary osteoblasts, also significantly up-regulated expression of Alp mRNA and Runx2 mRNA in the presence of EBPC1 pretreatment. Moreover, EBPC1 modulated apoptosis via the regulation of Bax/Bcl2. CONCLUSION: These results indicate that EBPC1 treatment can promote osteogenesis during DOP, which can ameliorate apoptosis by regulating Bax/Bcl2 and accelerating osteogenesis in osteoblasts.

Evaluation of the mechanism of Rujiling capsules in the treatment of hyperplasia of mammary glands based on network pharmacology and molecular docking.

OBJECTIVE: The present study aimed to elucidate the molecular network mechanism of the Rujiling capsule in the treatment of hyperplasia of mammary glands through network pharmacology and molecular docking. MATERIALS AND METHODS: TCMSP and TCMID databases were screened for the active components and their action targets of the Rujiling capsule, whereas the disease targets of hyperplasia of mammary glands were searched in GeneCard and DisGeNET databases. Venny software was employed to identify the common targets of drugs and diseases. Cytoscape software was used to construct the network pharmacological diagram of "drug-active components-target" and the intersection targets were subjected to protein-protein interaction analysis by STRING platform and Cytoscape software. The DAVID database was exploited for gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of the intersection target. After that, the key target genes with a degree value greater than the median were verified with the active components in molecular docking. RESULTS: A total of 691 drug targets, 251 disease targets, and 108 intersection targets were obtained after retrieval and screening. Among the 686 items enriched by GO included 522 biological processes, 110 molecular functions, and 54 cellular components. At the same time, 114 signal pathways were enriched by KEGG. The results of molecular docking revealed that the docking energies of main active components and some core targets were all <-5 kcal/mol. CONCLUSION: Henceforth, highlighted the role of the Rujiling capsule in the treatment of hyperplasia of mammary glands through multiple components, multiple targets, and multiple signal pathways.

Regulatory effects of bio-intensity electric field on transformation of human skin fibroblasts / 中华烧伤杂志

Objective: To investigate the regulatory effects of bio-intensity electric field on the transformation of human skin fibroblasts (HSFs). Methods: The experimental research methods were used. HSFs were collected and divided into 200 mV/mm electric field group treated with 200 mV/mm electric field for 6 h and simulated electric field group placed in the electric field device without electricity for 6 h. Changes in morphology and arrangement of cells were observed in the living cell workstation; the number of cells at 0 and 6 h of treatment was recorded, and the rate of change in cell number was calculated; the direction of cell movement, movement velocity, and trajectory velocity within 3 h were observed and calculated (the number of samples was 34 in the simulated electric field group and 30 in 200 mV/mm electric field group in the aforementioned experiments); the protein expression of α-smooth muscle actin (α-SMA) in cells after 3 h of treatment was detected by immunofluorescence method (the number of sample was 3). HSFs were collected and divided into simulated electric field group placed in the electric field device without electricity for 3 h, and 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group which were treated with electric fields of corresponding intensities for 3 h. Besides, HSFs were divided into simulated electric field group placed in the electric field device without electricity for 6 h, and electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group treated with 200 mV/mm electric field for corresponding time. The protein expressions of α-SMA and proliferating cell nuclear antigen (PCNA) were detected by Western blotting (the number of sample was 3). Data were statistically analyzed with Mann-Whitney U test, one-way analysis of variance, independent sample t test, and least significant difference test. Results: After 6 h of treatment, compared with that in simulated electric field group, the cells in 200 mV/mm electric field group were elongated in shape and locally adhered; the cells in simulated electric field group were randomly arranged, while the cells in 200 mV/mm electric field group were arranged in a regular longitudinal direction; the change rates in the number of cells in the two groups were similar (P>0.05). Within 3 h of treatment, the cells in 200 mV/mm electric field group had an obvious tendency to move toward the positive electrode, and the cells in simulated electric field group moved around the origin; compared with those in simulated electric field group, the movement velocity and trajectory velocity of the cells in 200 mV/mm electric field group were increased significantly (with Z values of -5.33 and -5.41, respectively, P<0.01), and the directionality was significantly enhanced (Z=-4.39, P<0.01). After 3 h of treatment, the protein expression of α-SMA of cells in 200 mV/mm electric field group was significantly higher than that in simulated electric field group (t=-9.81, P<0.01). After 3 h of treatment, the protein expressions of α-SMA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were 1.195±0.057, 1.606±0.041, and 1.616±0.039, respectively, which were significantly more than 0.649±0.028 in simulated electric field group (P<0.01). Compared with that in 100 mV/mm electric field group, the protein expressions of α-SMA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly increased (P<0.01). The protein expressions of α-SMA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were 0.730±0.032, 1.561±0.031, and 1.553±0.045, respectively, significantly more than 0.464±0.020 in simulated electric field group (P<0.01). Compared with that in electric field treatment 1 h group, the protein expressions of α-SMA in electric field treatment 3 h group and electric field treatment 6 h group were significantly increased (P<0.01). After 3 h of treatment, compared with that in simulated electric field group, the protein expressions of PCNA of cells in 100 mV/mm electric field group, 200 mV/mm electric field group, and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 100 mV/mm electric field group, the protein expressions of PCNA of cells in 200 mV/mm electric field group and 400 mV/mm electric field group were significantly decreased (P<0.05 or P<0.01); compared with that in 200 mV/mm electric field group, the protein expression of PCNA of cells in 400 mV/mm electric field group was significantly decreased (P<0.01). Compared with that in simulated electric field group, the protein expressions of PCNA of cells in electric field treatment 1 h group, electric field treatment 3 h group, and electric field treatment 6 h group were significantly decreased (P<0.01); compared with that in electric field treatment 1 h group, the protein expressions of PCNA of cells in electric field treatment 3 h group and electric field treatment 6 h group were significantly decreased (P<0.05 or P<0.01); compared with that in electric field treatment 3 h group, the protein expression of PCNA of cells in electric field treatment 6 h group was significantly decreased (P<0.01). Conclusions: The bio-intensity electric field can induce the migration of HSFs and promote the transformation of fibroblasts to myofibroblasts, and the transformation displays certain dependence on the time and intensity of electric field.

Comparative Transcriptomic Analyses of Developing Melanocortin Neurons Reveal New Regulators for the Anorexigenic Neuron Identity.

Despite their opposing actions on food intake, POMC and NPY/AgRP neurons in the arcuate nucleus of the hypothalamus (ARH) are derived from the same progenitors that give rise to ARH neurons. However, the mechanism whereby common neuronal precursors subsequently adopt either the anorexigenic (POMC) or the orexigenic (NPY/AgRP) identity remains elusive. We hypothesize that POMC and NPY/AgRP cell fates are specified and maintained by distinct intrinsic factors. In search of them, we profiled the transcriptomes of developing POMC and NPY/AgRP neurons in mice. Moreover, cell-type-specific transcriptomic analyses revealed transcription regulators that are selectively enriched in either population, but whose developmental functions are unknown in these neurons. Among them, we found the expression of the PR domain-containing factor 12 (Prdm12) was enriched in POMC neurons but absent in NPY/AgRP neurons. To study the role of Prdm12 in vivo, we developed and characterized a floxed Prdm12 allele. Selective ablation of Prdm12 in embryonic POMC neurons led to significantly reduced Pomc expression as well as early-onset obesity in mice of either sex that recapitulates symptoms of human POMC deficiency. Interestingly, however, specific deletion of Prdm12 in adult POMC neurons showed that it is no longer required for Pomc expression or energy balance. Collectively, these findings establish a critical role for Prdm12 in the anorexigenic neuron identity and suggest that it acts developmentally to program body weight homeostasis. Finally, the combination of cell-type-specific genomic and genetic analyses provides a means to dissect cellular and functional diversity in the hypothalamus whose neurodevelopment remains poorly studied.SIGNIFICANCE STATEMENT POMC and NPY/AgRP neurons are derived from the same hypothalamic progenitors but have opposing effects on food intake. We profiled the transcriptomes of genetically labeled POMC and NPY/AgRP neurons in the developing mouse hypothalamus to decipher the transcriptional codes behind the versus orexigenic neuron identity. Our analyses revealed 29 transcription regulators that are selectively enriched in one of the two populations. We generated new mouse genetic models to selective ablate one of POMC-neuron enriched transcription factors Prdm12 in developing and adult POMC neurons. Our studies establish a previously unrecognized role for Prdm12 in the anorexigenic neuron identity and suggest that it acts developmentally to program body weight homeostasis.

[Basic pathogenesis of asthenia of healthy energy and blood stasis in liver cirrhosis studied by serum proteomics].

OBJECTIVE: To study the basic pathogenesis of "asthenia of healthy energy and blood stasis" in liver cirrhosis studied by Chinese syndromes and serum proteomics. METHODS: The information of four methods of examinations and serum samples were collected from 44 cases of male cirrhotic patients and 17 cases of healthy male volunteers. The different syndrome groups were summarized according to syndrome differentiation and frequency analysis using the patient's information of four methods of examinations. The serum proteins were isolated by magnetic beads and detected by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The proteins expressed differently between cirrhotic patients of different syndrome types and healthy volunteers were analyzed by statistical analysis software (product of Bruker Corporation ClinProTools 2.1 software). The diagnosis model was established by QC algorithm. RESULTS: The liver cirrhosis syndrome with the appearance frequency of more than 30% was sequenced from high to low as fatigue, listlessness, spider telangiectasia, liver palms, anorexia, bleeding from the nose, the gum or the subcutaneous tissue, the abdominal distention, shortness of breath while moving, dim facial complexion, pricking pain of the flank, weak waist and knees, dull pain in the flank, burning sensation of five centers, or low fever, hectic fever, and night sweat. The cases belonging to Child-Pugh A in the seventeen patients of the Pi-qi asthenia syndrome group accounted for 64.7%. The cases belonging to Child-Pugh C in the twelve patients of the Gan-Shen yin deficiency syndrome group accounted for 66.7%. The cases belonging to Child-Pugh A were similar to the cases belonging to Child-Pugh C in the fifteen patients of the blood stasis syndrome group, being more than 40%. Such syndromes as spider telangiectasia, liver palms, shortness of breath while moving, burning sensation of five centers, or low fever, hectic fever, and night sweat, varicose vein of the abdominal wall, and edema of lower extremities appeared more frequently in Child-Pugh C than in Child-Pugh A (all P < 0.05). The characteristic protein expression peak with mass-to-charge ratio of 4642.81, 4963.91, 5247.8, 5805.95, 6305.27, and 12447.7 in the Pi-qi asthenia syndrome diagnosis model were chosen. The former five peaks could be found in Child-Pugh A and Child-Pugh C. The protein expression peak with mass-to-charge ratio of 9 290. 3 was the characteristic protein expression peak in the Gan-Shen yin deficiency syndrome diagnosis model. The protein expression peak with mass-to-charge ratio of 9290.06 and 7 768. 29 were down-regulated in the Gan-Shen yin deficiency syndrome group compared with the other two syndromes groups. The protein expression peaks 9290.3 and 7768.29 were included in the diagnosis model of hepatitis B cirrhosis. They did not appear in Child-Pugh A, while they were gradually down-regulated in Child-Pugh B and Child-Pugh C. Of the other seventeen protein expression peaks in patients of the Gan-Shen yin deficiency syndrome, eight expressed in Child-Pugh A. The protein expression peaks 4964.55 and 5806.83 that expressed both in Child-Pugh A and Child-Pugh C constituted the characteristic protein peaks of the hepatitis B cirrhosis blood stasis diagnosis model. The diagnosis model of the Pi-qi asthenia syndrome was established with the sensitivity of 100% and the specificity of 82.35%. The diagnosis model of the Gan-Shen yin deficiency syndrome was established with the sensitivity of 100% and the specificity of 94.12%. The diagnosis model of the blood stasis syndrome was established with the sensitivity of 100% and the specificity of 100%. CONCLUSIONS: Asthenia of healthy energy and blood stasis was the basic pathogenesis during the whole process of liver cirrhosis. Asthenia of healthy energy covers Pi-qi asthenia and Gan-Shen yin deficiency. Gan-Shen yin deficiency was obvious in the compensation stage of liver cirrhosis, but it has manifested in this stage. So early treatment was necessary.

[Effects of recipes for replenishing qi and activating blood on senescence related gene expressions in the liver of aging rats].

OBJECTIVE: To observe the effects of recipes for replenishing qi and activating blood on p16, p21, proliferating cell nuclear antigen (PCNA), cyclin D1 and cyclin E gene expressions in the liver of aging rats. METHODS: A recipe for replenishing qi and a recipe for activating blood were administered to aging rats respectively, and the effects of the above recipes on the expressions of senescence related genes (p16, p21, PCNA, cyclin D1 and cyclin E) were examined by RT-PCR and Western blotting methods. RESULTS: The expressions of p16, p21 and cyclin D1 mRNAs and proteins in the liver of the untreated aging rats were up-regulated, while the expressions of PCNA and cyclin E mRNAs and proteins decreased. As compared with the untreated aging rats, both recipes could down-regulate the expressions of cyclin D1 mRNA and protein and up-regulate the expressions of cyclin E mRNA and protein, but had no obvious effects on the expressions of mRNAs and proteins of p16, p21 and PCNA. CONCLUSION: Recipes for replenishing qi and activating blood can improve the liver cell proliferation of aging rats via down-regulating the expressions of cyclin D1 mRNA and protein and up-regulating the expressions of cyclin E mRNA and protein.

[Comparative study on the regulatory effects on senescence related cell cycle gene expression by TCM principles of tonifying shen, invigorating pi benefiting qi, and activating blood circulation].

OBJECTIVE: To compare the effects of TCM therapeutic principles of tonifying Shen (TS), benefiting Qi (BQ), invigorating Pi (IP) and activating blood circulation (ABC) herbs in regulating the gene expression in senescence related cell cycle. METHODS: Drug sera containing TCM herbs of the above-mentioned principles were used to treat the aged human diploid fibroblast cell line 2BS. The effect of TCM on the senescence related cell cycle and its related gene expression (P16INK4, Cyclin D1 and PCNA) were examined by means of cell proliferative doublings, flow cytometry, RT-PCR and Western blot analysis. RESULTS: TCM herbs of TS and BQ could improve the cell cycle, down-regulate the P16 and Cyclin D1 mRNA/protein expression, up-regulate PCNA mRNA/protein expression, while TCM herbs of IP and ABC showed insignificant effect on these indexes. CONCLUSION: TCM herbs of TS and BQ have effect in improving cell cycle, it may be achieved through promoting the P16 pathway of gene expression.