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ObjectiveTo observe the intervention of modified Sanrentang on the lipopolysaccharide-induced proliferation of rat glomerular mesangial cells， the phosphatidylinositol-3 kinase（PI3K）/protein kinase B（PKB/Akt）/nuclear factor kappa B（NF-κB） signaling pathway， and to investigate its mechanism in improving kidney inflammation in rats with immunoglobulin A nephropathy（IgAN）. MethodThe 18 rats were divided into 3 groups by serum pharmacology method： normal group， high-dose and low-dose （20.70，10.35 g·kg-1·d-1） groups with 6 rats in each group. Modified Sanrentang high- and low-dose groups were intragastric with the corresponding solution of modified Sanrentang， and normal group was intragastric with equal volume of distilled water. After 5 days of intragastric administration， blood samples were collected to prepare drug-containing serum. Rat mesangial （HBZY-1） were divided into five groups of normal group， LPS 10 mg·L-1 in the model group， benazepril（50 μmol·L-1）， modified Sanrentang high- and low-dose group. Preclude the use of methyl thiazolyl tetrazolium（MTT） method detect the proliferation activity of HBZY-1 cells， enzyme-linked immunosorbent assay（ELISA） was used to determine the content of each group type Ⅳ collagen（ColⅣ），Western blot and Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） were used to detect protein and mRNA expression levels of PI3K/Akt/NF-κB signaling pathway. ResultAs compared with the normal group， MTT assay showed that exposure to LPS significantly enhanced the proliferative activity， the ColⅣ was increased significantly of HBZY-1 cells（P<0.01）， p-Akt， p-p65 was increased significantly （P<0.01）. Compared with the model group， the proliferation and ColⅣ of rat chronic glomerulonephritis cells induced by LPS by inhibiting PI3K/Akt/NF-κB signaling pathway（P<0.01）， and the phosphorylation of Akt was significantly inhibited（P<0.01）， the expression levels of NF-κB p65 was reduced in modified Sanrentang high-dose group（P<0.01）. ConclusionModified Sanrentang could inhibit cell proliferation and the content of ColⅣ in rat mesangial cells induced by LPS， and its mechanism might be related to suppression of PI3K/Akt signaling pathway.
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The traditional Chinese medicines(TCM) for activating blood circulation and the TCM for regulating Qi are often used in combination in clinical practice. However, their mechanisms are still unclear. The activity spectrum of targets can fuse the active components, targets and intensity of action, which provides support for the discussion of efficacy targets. The chemical components of common TCM sets for activating blood circulation and regulating Qi, as well as the negative sets not for activating blood circulation and re-gulating Qi were obtained from the database of TCM. By the similarity analysis of chemical components in TCM for activating blood circulation and DrugBank database, the predicted targets of chemical components in TCM for activating blood circulation were obtained, and the similarity value of the two was taken as the activity value of the active components and predicted targets. Then, the component-target activity value was weighted. The activity values of herb acting on the same target were fused to construct activity spectra of targets of the herbs for activating blood circulation, herbs for regulating Qi and negative herbs. The targets whose activity values of activating blood circulation and regulating Qi were higher than those of negative herbs were selected as potential targets of efficacy. Protein-protein interaction networks were constructed for topological, GO and KEGG enrichment analysis to determine the key targets of efficacy of activating blood circulation and regulating Qi. The component-target activity information collected from DrugBank database contained 4 499 compounds, 627 targets and 11 295 action relationships. The activating blood function protein-protein interaction network contained 206 nodes and 1 728 edges, while the regulating Qi function protein-protein interaction network contained 230 nodes and 986 edges. The enrichment analysis of topology, GO and KEGG showed that TCM for activating blood circulation mainly exerted its anti-inflammatory, neuroprotective and angiogenic effects on signaling cascade pathway mediated by VEGF/VEGFR2, ERK signaling pathway, calcium signaling pathway and PI3 K-AKT signaling pathway, and the key targets included mitogen activated protein kinases 3(MAPK3), proto-oncogene tyrosine-protein kinase Src(SRC), mitogen activated protein kinases 1(MAPK1), epidermal growth factor receptor(EGFR), phosphatidylinositol 4,5-bisphosphate 3-kinase catalytic subunit alpha isoform(PIK3 CA), peroxisome proliferators-activated receptor gamma(PPARG), nitric oxide synthase 3(NOS3), prostaglandin G/H synthetase 2(PTGS2), matrix metalloproteinase-9(MMP9), and vascular endothelial growth factor A(VEGFA). TCM for regulating Qi mainly exerted anti-inflammatory and neuroprotective effects by acting on MAPK signaling pathway and PI3 K-AKT signaling pathway, and the key targets included mitogen activated protein kinases 8(MAPK8), SRC, mitogen activated protein kinases 14(MAPK14), and RAC-alpha serine/threonine-protein kinase(AKT1), mitogen activated protein kinases 3(MAPK3). Based on the activity spectrum of targets, the targets of the TCM for activating blood and the targets of the TCM for regulating Qi were analyzed to provide reference for the study of efficacy targets of TCM, and also provide some scientific basis for clinical application.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Medicine, Chinese Traditional , Protein Interaction Maps , Qi , Vascular Endothelial Growth Factor AABSTRACT
Caveolin-1 (Cav-1), a major structural protein of caveolae, is implicated in the vesicular uptake processes of transcytosis and cell signaling. However, its role in modulating protein glycosylation and tumor metastasis remains to be further elucidated. In the present study, it was shown that Cav-1 promotes the expression of O-GlcNAcylation and O-GlcNAc transferase (OGT), and triggers the invasion and metastasis of hepatocellular carcinoma (HCC) cells. The results of RT-qPCR, Western blot and dual lucif-erase reporter assay showed that Cav-1 negatively regulated the expression of transcription factor RUNX2 in HCC. Subsequently, this results in attenuate RUNX2-induced transcription of miR24. miR24 suppresses mouse HCC cells invasion and metastasis via directly targeting Ogt mRNA 3′UTR. This research provides evidence of Cav-1-mediated OGT expression and O-GlcNAc (O-linked N-acetylglucosamine) elevation. These data give insight into a novel mechanism of HCC occurrence and development.
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Objective To evaluate the role of polysaccharide from Phellinus igniarius (PPI) in the improvement of oxidative stress, hepatic granuloma and hepatic fibrosis in Schistosoma japonicum-iniected in mice. Methods The mouse model of schistosomiasis was established by S. japonicum cercariae infection via the abdomen. Balb/c mice were randomly assigned into 5 groups, including the healthy control group (Group A), infection control group (Group B), PPI treatment group (Group C), praziquantel treatment group (Group D) and PPI-praziquantel combination group (Group E), of 10 mice in each group. Each mouse in groups B, C, D and E was infected with (30 ± 2) S. japonicum cercariae. Then, mice in groups D and E were given praziquantel by gavage at a dose of 500 mg/kg for successive two days on day 42 post-infection, while mice in groups C and E were given PPI by gavage at a dose of 400 mg/kg for successive 30 days on day 42 post-infection. Histopathological changes of hepatic tissues were observed using hematoxylin-eosin (HE) staining, and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN) were determined, while the activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), glutathione reductase (GSH-R) and glutathione (GSH) were detected in Mouse liver homogenates. The expression of transforming growth factor-beta (TGF-β) and alpha-smooth muscle actin (α-SMA) was quantified in hepatic tissues using immunohistochemistry, and the Nrf2 and Gsta4 gene expression was quantified using quantitative real-time PCR (qPCR) assay. Results Untreated mice presented typical pathological changes of schistosomal hepatic disorders, while PPI treatment effectively alleviated hepatic egg granulomas and collagen deposition. S. japonicum infection resulted in aggravation of hepatic lipid peroxidation, induction of oxidative stress, elevated serum MDA level and a reduction in the activity of GSH and antioxidant enzymes activities in mice. As compared to infected but untreated mice, PPI treatment suppressed hepatic lipid peroxidation, increased the GSH activity and restored the activity of antioxidant enzymes. In addition, PPI treatment inhibited the TGF-β signaling pathway and up-regulated the Nrf2 and Gsta4 gene expression. Conclusions PPI plays a critical role in the treatment of schistosomiasis-induced hepatic fibrosis. It may improve oxidative stress damages through up-regulating Nrf2 and Gsta4 gene expression, thereby suppressing the development of hepatic egg granulomas and hepatic fibrosis.
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Objective To evaluate the role of polysaccharide from Phellinus igniarius (PPI) in the improvement of oxidative stress, hepatic granuloma and hepatic fibrosis in Schistosoma japonicum-iniected in mice. Methods The mouse model of schistosomiasis was established by S. japonicum cercariae infection via the abdomen. Balb/c mice were randomly assigned into 5 groups, including the healthy control group (Group A), infection control group (Group B), PPI treatment group (Group C), praziquantel treatment group (Group D) and PPI-praziquantel combination group (Group E), of 10 mice in each group. Each mouse in groups B, C, D and E was infected with (30 ± 2) S. japonicum cercariae. Then, mice in groups D and E were given praziquantel by gavage at a dose of 500 mg/kg for successive two days on day 42 post-infection, while mice in groups C and E were given PPI by gavage at a dose of 400 mg/kg for successive 30 days on day 42 post-infection. Histopathological changes of hepatic tissues were observed using hematoxylin-eosin (HE) staining, and serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), hyaluronic acid (HA), laminin (LN) were determined, while the activities of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), glutathione reductase (GSH-R) and glutathione (GSH) were detected in Mouse liver homogenates. The expression of transforming growth factor-beta (TGF-β) and alpha-smooth muscle actin (α-SMA) was quantified in hepatic tissues using immunohistochemistry, and the Nrf2 and Gsta4 gene expression was quantified using quantitative real-time PCR (qPCR) assay. Results Untreated mice presented typical pathological changes of schistosomal hepatic disorders, while PPI treatment effectively alleviated hepatic egg granulomas and collagen deposition. S. japonicum infection resulted in aggravation of hepatic lipid peroxidation, induction of oxidative stress, elevated serum MDA level and a reduction in the activity of GSH and antioxidant enzymes activities in mice. As compared to infected but untreated mice, PPI treatment suppressed hepatic lipid peroxidation, increased the GSH activity and restored the activity of antioxidant enzymes. In addition, PPI treatment inhibited the TGF-β signaling pathway and up-regulated the Nrf2 and Gsta4 gene expression. Conclusions PPI plays a critical role in the treatment of schistosomiasis-induced hepatic fibrosis. It may improve oxidative stress damages through up-regulating Nrf2 and Gsta4 gene expression, thereby suppressing the development of hepatic egg granulomas and hepatic fibrosis.
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<p><b>OBJECTIVE</b>To establish a neonatal Tibet minipig model of hypoxic-ischemic encephalopathy and evaluate the magnetic resonance imaging (MRI) manifestations and pathological findings.</p><p><b>METHODS</b>Six neonatal (1-3 days old) Tibet minipigs were randomized into model group (n=4) and control group (n=2). In model group, hypoxic-ischemic encephalopathy was induced by surgical ligation of the bilateral carotid artery followedimmediately by hypoxic exposure in a hypoxia chamber for 1 h. ESWAN was performed at 2 h, 24 h, 3 days and 5 days after induction of HIE or at 2 h after sham surgery in the control animals to evaluate the brain damage. Conventional MRI scans (T2FLAIR, T2WI, and DWI) were also performed at 24 h after the modeling.</p><p><b>RESULTS</b>In the neostriatum, values of T(2)*-weighted MRI increased and reached the peak level at 3 days post-injury (P<0.05). Subcortical white matter T(2)* values reached the peak level at 24 h (P<0.05). Neostriatum R(2)* values were at the lowest level at 3 days (P<0.05). Magnitude values were significantly increased after the model establishment (P<0.05). DWI showed multiple mild focal high signals in the bifrontal subcortical white matter and bilateral neostriatum; T2FLAIR showed slightly increased signal; T2WI showed no obvious abnormalities. SWI showed dilated medulla veins adjacent to the bilateral lateral ventricles and basal ganglia. In the early stage of HIE, brain pathologies were characterized mainly by edema and venous congestion with occasional focal necrosis and hemosiderin deposition.</p><p><b>CONCLUSION</b>ESWAN sequence is capable of detecting bleeding and brain edema, and T(2)*, R(2)*, and magnitude values can be used to estimate the changes of brain damage following HIE.</p>
Subject(s)
Animals , Disease Models, Animal , Hypoxia-Ischemia, Brain , Diagnosis , Pathology , Magnetic Resonance Imaging , Swine , Swine, Miniature , TibetABSTRACT
<p><b>BACKGROUND AND OBJECTIVE</b>Heparanase-1 (HPA-1) can promote angiogenesis and metastasis of malignant tumors and plays an important role in the genesis and development of tumors. This study was to explore the effects of specific small interfering RNA (siRNA) targeting HPA-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cells.</p><p><b>METHODS</b>The expression of HPA-1 in Hca-F, Hca-P, and Hepa1-6 cells, which have high, low, and no metastatic potential, respectively, was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and enzyme-linked immunosorbent assay (ELISA). After transfection with two specific siRNAs targeting HPA-1, siRNA-1 and siRNA-2, and treatment with heparin, invasiveness of Hca-F cells was observed by Matrigel invasion assay.</p><p><b>RESULTS</b>HPA-1 was negative in Hepa1-6 cells while positive in both Hca-F and Hca-P cells. The expression levels of both HPA-1 mRNA and protein were obviously higher in Hca-F cells than in Hca-P cells. HPA-1 proteins could be secreted into culture supernatant of Hca-F and Hca-P cells, and the amount of secreted HPA-1 detected by Western blot analysis was larger in Hca-F cells than in Hca-P cells (1.34 ± 0.02 vs. 0.60 ± 0.01, P < 0.001), which was consistent with the results of ELISA. Both siRNA-1 and siRNA-2 downregulated the expression of HPA-1 and the siRNA-2 did more efficiently. The number of invasive Hca-F cells treated with siRNA-2 or heparin alone was larger than that of Hca-F cells treated with combination of them (9 ± 1 vs. 4 ± 1, P = 0.013; 15 ± 2 vs. 4 ± 1, P = 0.008), but smaller than that of untreated Hca-F cells (9 ± 1 vs. 22 ± 2, P = 0.006; 15 ± 2 vs. 22 ± 2, P = 0.026).</p><p><b>CONCLUSION</b>The combined application of specific siRNA targeting HPA-1 and heparin is more effective in inhibiting the invasiveness of mouse hepatoma cells.</p>
Subject(s)
Animals , Mice , Cell Line, Tumor , Cell Movement , Down-Regulation , Glucuronidase , Genetics , Bodily Secretions , Heparin , Pharmacology , Liver Neoplasms, Experimental , Metabolism , Pathology , Neoplasm Invasiveness , Neoplasm Metastasis , RNA, Messenger , Metabolism , RNA, Small Interfering , Genetics , Pharmacology , TransfectionABSTRACT
<p><b>OBJECTIVE</b>To investigate the relationship of hemorheological parameters between Tibet mini-pigs, Beagle dogs and human.</p><p><b>METHODS</b>Blood samples were collected from adult Tibet mini-pigs, Beagle dogs and human to detect such hemorheological parameters as the whole blood viscosity (WBV) (high, middle, and low shear rate), PV, HCT, ESR and Fi.</p><p><b>RESULTS</b>The male Tibet mini-pigs had significantly lower WBV (150, 30, 5, and 1 s(-1)) and Fi than the female mini-pigs (P<0.05, 0.01, 0.05, 0.01, and 0.01, respectively). The WBV of male Beagle dogs (150 and 1 s(-1)) was significantly lower that in than female dogs (P<0.05). The WBV of male human subjects (1 s(-1)) and HCT were significantly higher, but ESR significantly lower than those in female human subjects P<0.05, 0.01 and 0.01, respectively). WBV (1 s(-1)), PV, and ESR in Beagle dogs were significantly lower, but HCT and Fi significantly higher than those in Tibet mini-pig and human subjects (P<0.05, 0.01, 0.05, and 0.01, respectively). All the hemorheological parameters were similar between Tibet mini-pigs and human (P<0.05).</p><p><b>CONCLUSION</b>The hemorheological parameters of Tibet mini-pigs are closer to those of human than those of Beagle dogs.</p>
Subject(s)
Adult , Animals , Dogs , Female , Humans , Male , Blood Sedimentation , Blood Viscosity , Hematocrit , Hemorheology , Swine , Swine, MiniatureABSTRACT
<p><b>OBJECTIVE</b>To analyze the mitochondrial DNA (mtDNA) D-loop region sequence variation in Tibet Mini-Pigs in relation to the blood parameters and provide the molecular genetic basis for developing new species of laboratory animals.</p><p><b>METHODS</b>The genomic DNA was extracted from the whole blood samples of 59 Tibet mini-pigs to amplifying the mtDNA D-loop for sequence analysis. Nine physiological and nine biochemical blood parameters of Tibet mini-pigs were measured .</p><p><b>RESULTS</b>Based on the variation of the tandem repeat motif, the mtDNA D-loop region of Tibet mini-pigs was classified into two types, namely type A and B with the percentage of 57.6% and 42.4%, respectively, roughly matching the 3 transform sites (305, 500, 691) at the 5' end. In the 18 blood parameters, only red blood cell count showed significant differences between types A and (P<0.01).</p><p><b>CONCLUSION</b>Based on the sequence variation of the mtDNA D-loop region, Tibet mini-pigs can be divided into two types that show a significant difference in red blood cell count.</p>
Subject(s)
Animals , Base Sequence , DNA, Mitochondrial , Chemistry , Genetics , Hematologic Tests , Polymerase Chain Reaction , Swine , Blood , Genetics , TibetABSTRACT
<p><b>OBJECTIVE</b>To investigate the effects of methanol extract of Celastrus orbiculatu (MECO) on synovial hyperplasia and cartilage erosion and degradation in rheumatoid arthritis (RA), and explore the possible mechanisms to provide clues for new drug development for RA treatment.</p><p><b>METHODS</b>The articular synovium from patients with RA and normal articular cartilage were co-implanted into the back of severe combined immunodeficient (SCID)mice to establish the chimeric model SCID- HuRAg. Four weeks later, the mice were given MECO intragastrically at 30 mg/day, leflunomide at 500 microg/day or distilled water, respectively, for 4 consecutive weeks. After completion of the treatments, the histological scores of the grafts for synovial hyperplasia, cartilage invasion by synoviocyte and cartilage degradation around the chondrocytes were evaluated, and serum level of tumor necrosis factor-alpha (TNF-alpha) was measured with radioimmunoassay. The expression of TNF-alpha mRNA and the cell apoptosis in the synovium were detected with in situ hybridization (ISH) and TUNEL, respectively, and the results were analyzed with the image analysis system.</p><p><b>RESULTS</b>The grafts survived in the mice till the end of experiment. MECO and leflunomide, in comparison with distilled water, significantly lowered the scores for synovial hyperlasia (2.00+/-0.76 and 2.25+/-0.89 vs 3.63+/-0.52), cartilage erosion (1.69+/-0.80 and 2.00+/-1.36 vs 3.75+/-0.53), cartilage degradation (1.88+/-0.83 and 2.13+/-0.83 vs 3.63+/-0.74) and serum TNF-alpha level (0.84+/-0.09 and 0.83+/-0.12 vs 0.99+/-0.11 ng/ml). Cell apoptosis of the synovium increased significantly with MECO and leflunomide treatments, but the expression of TNF-alpha mRNA in the synovium decreased significantly in MECO group.</p><p><b>CONCLUSION</b>MECO can effectively suppress synovial hyperplasia and cartilage erosion and degradation SCID-HuRAg mice by reducing TNF-alpha production in the synovium and promoting synovial apoptosis. MECO can be comparable with leflunomide in their effect, but the former is more effective in suppressing TNF-alpha expression in the synovium.</p>