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ObjectiveThis study aimed to investigate the effects of eugenol on inhibiting the inflammatory activation of human umbilical cord mesenchymal stem cells （HUC-MSCs） and the pro-inflammatory phenotype of hepatic stellate cells （HSCs） in liver fibrosis， and to explore their underlying mechanisms. MethodsHUC-MSCs were cultured and identified in vitro， and the toxicity of eugenol to HUC-MSCs was evaluated by MTT method. The effect of eugenol on the migration ability of HUC-MSCs was investigated by in vitro scratch test. The expression of α-SMA， COL1A1， Smad2/3 and p-Smad2/3 of LX-2 cells activated by TGF-β1 treated with EU-MSCs-CM and MSCs-CM were detected by WB assay. EU-MSCs-CM and MSCs-CM treated THP-1 macrophages stimulated with Lipopolysaccharide （LPS） were analyzed for the expression of surface markers CD11b， CD86， and CD206 by flow cytometry. Additionally， the expression of pro-inflammatory genes TNF-α， IL-1β， and IL-6 in THP-1 macrophages was detected by qPCR. ResultsThe results of MTT method showed that the viability of the cells remained above 90% after 24 h and 48 h treatment at 0， 7.5， 15 μg/mL. In vitro scratches showed that eugenol treatment enhanced HUC-MSCs migration. WB results showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment significantly inhibited the expression of α-SMA， COL1A1， Smad2/3， and p-Smad2/3 of activated HSCs. Flow cytometry showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment had a more significant inhibitory effect on CD86， a M1-type polarization marker in THP-1 macrophages. The results of qPCR experiment showed that compared with MSCs-CM treatment， EU-MSCs-CM treatment more significantly inhibited the expressions of TNF-α， IL-1β and IL-6 of THP-1 macrophage proinflammatory genes. ConclusionsEugenol enhances the inhibitory effect of HUC-MSCs on inflammatory activation of HSCs， possibly by regulating TGF-β1/Smads signaling pathway. It also enhances the inhibitory effect of HUC-MSCs on the pro-inflammatory phenotype of macrophages. Proinflammatory macrophages can promote inflammatory activation of HSCs.
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BACKGROUND: iRegene collagen sponge exhibits stable physical and chemical properties, and has passed the test by the State Food and Drug Administration of China. OBJECTIVE: To study the hemostatic effect and the biocompatibility of the iRegene collagen sponge on a liver wound by means of rat models. METHODS: Liver trauma bleeding models were made in 21 Sprague-Dawley rats. These model rats were randomized into three groups (n=7 per group): experimental group with implantation and external application of iRegene collagen sponge; positive control group with implantation of medical collagen sponge and external application of iRegene collagen sponge; blank control group with external application of medical gauze. The bleeding time and amount on the liver wounds were observed. Histological observation on the liver wound was performed at 7, 14, 28 days after intervention. RESULTS AND CONCLUSION: The bleeding time was shorter in the experimental group than the positive control group (P ≤ 0.05). Beyond that, there was no difference in the bleeding amount and time among the three groups. Histological findings on the liver wound showed that the iRegene collagen sponge in the experimental group was completely wrapped with fibrous connective tissues and began to degrade at 7 days after intervention, the Inflammatory cell infiltration mainly occurred in neutrophils, and new capillaries were observed in peripheral connective tissues; at 14 days after intervention, the fibrous connective tissues became remarkably thickened, the number of neurophils was reduced, and the number of macrophages was increased; at 28 days after intervention, the iRegene collagen sponge degraded completely, most of the liver tissues recovered, and there were macrophages, monocytes, fibroblasts and capillaries in the inflammatory connective tissues adjacent to a part of liver tissues. Similar findings were observed in the positive control group. In the blank control group, there were obvious connective tissues on the wound and red blood cells in the liver sinus, and occasionally liver tissue bleeding and vacuolar degeneration were visible; at 28 days after intervention, there were thickened connective tissues on the wound, red blood cells in the liver sinus and reversed hepatic stellate cells. To conclude, the iRegene collagen sponge possesses effective hemostatic effects on liver wounds and shows good histocompatibility.
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BACKGROUND: iRegene collagen sponge exhibits stable physical and chemical properties, and has passed the test by the State Food and Drug Administration of China. OBJECTIVE: To study the hemostatic effect and the biocompatibility of the iRegene collagen sponge on a liver wound by means of rat models. METHODS: Liver trauma bleeding models were made in 21 Sprague-Dawley rats. These model rats were randomized into three groups (n=7 per group): experimental group with implantation and external application of iRegene collagen sponge; positive control group with implantation of medical collagen sponge and external application of iRegene collagen sponge; blank control group with external application of medical gauze. The bleeding time and amount on the liver wounds were observed. Histological observation on the liver wound was performed at 7, 14, 28 days after intervention. RESULTS AND CONCLUSION: The bleeding time was shorter in the experimental group than the positive control group (P ≤ 0.05). Beyond that, there was no difference in the bleeding amount and time among the three groups. Histological findings on the liver wound showed that the iRegene collagen sponge in the experimental group was completely wrapped with fibrous connective tissues and began to degrade at 7 days after intervention, the Inflammatory cell infiltration mainly occurred in neutrophils, and new capillaries were observed in peripheral connective tissues; at 14 days after intervention, the fibrous connective tissues became remarkably thickened, the number of neurophils was reduced, and the number of macrophages was increased; at 28 days after intervention, the iRegene collagen sponge degraded completely, most of the liver tissues recovered, and there were macrophages, monocytes, fibroblasts and capillaries in the inflammatory connective tissues adjacent to a part of liver tissues. Similar findings were observed in the positive control group. In the blank control group, there were obvious connective tissues on the wound and red blood cells in the liver sinus, and occasionally liver tissue bleeding and vacuolar degeneration were visible; at 28 days after intervention, there were thickened connective tissues on the wound, red blood cells in the liver sinus and reversed hepatic stellate cells. To conclude, the iRegene collagen sponge possesses effective hemostatic effects on liver wounds and shows good histocompatibility.
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Objective To evaluate the sensitivity to 5 clinically commonly used anticancer drugs in vivo using the zebrafish xenotransplantation models of human lung cancer,stomach cancer,and liver cancer cells,respectively. Methods Zebrafish xenotransplantation models of A549 lung cancer cells,SGC-7901 stomach cancer cells and HepG2 liver cancer cells were established. The xenograft models of A549 cells were treated with three different doses of cis-platinum, paclitaxel, vinorelbine, endostar and bevacizumab, respectively. The SGC-7901 model was treated with three concentrations or doses of paclitaxel, irinotecan, hydroxyurea, cis-platinum and 5-fluorouracil, respectively. And the HepG2 model was treated with three concentrations or doses of adriamycin,gemcitabine,hydroxyurea,cis-platinum and 5-fluorouracil. The tumors were analyzed and quantified in vivo by fluorescence microscopy,and the inhibition rates of tumor growth with each drug were calculated and compared with the model control group for statistical significance. Results All of the tested anticancer drugs showed inhibitory effect on tumor cells in the zebrafish xenograft models with statistical significance in a dose-dependent manner. During the drug sensitivity test,the inhibition rate of bevacizumab on A549 lung cancer cells decreased in the order(65%)> cis-platinum(55%)> vinorelbine(40%)> endostar(39%)>paclitaxel(27%). As for the SGC-7901 stomach cancer cells, the tumor growth inhibition rate decreased in the order hydroxyurea(46%)> 5-FU(31%)= irinotecan(31%)> paclitaxel(26%)> cis-platinum(24%). And the therapeutic effect of cis-platinum on the HepG2 liver cancer cells decreased in the order(64%)> hydroxyurea(56%)>gemcitabine(46%)> adriamycin(45%)> 5-FU(38%). Conclusions Zebrafish xenotransplantation models of cancer cells are suitable for in vivo sensitivity test of anticancer drugs.
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Objective:To investigate the expression of immunoglobulin A (IgA) in human mesangial cells (HMCs).Methods:The HMCs were cultured.The subcellular location of IgA was detected by immunofluorescence staining;the transcripts of Ig α,Ig κ and Ig λ constant region were detected by reverse transcription-polymerase chain reaction (RT-PCR) and further analyzed by DNA sequencing.The expressions of Igαt and Ig λ were detected at transcription level by Western blot after the cytoplasmic protein extraction.The culture supernatant was collected to explore whether IgA could be secreted out of the cell and the protein was further analyzed by mass spectrometry after being purified by affinity chromatography with jacalin-sepharose.The results of DNA sequencing and mass spectrometry were aligned with the mRNA and amino acid sequences in the National Center of Biotechnology Information (NCBI) data-base.Results:By immunofluorescence staining,we detected the presence of IgA heavy chain Ig α,light chain,both Ig κ and Ig λ in expressions of transcripts of Ig α1,Ig α2,Ig κ and Ig λ in the HMCs and the alignment of the sequences of the RT-PCR products with those of the Ig Cα1,Ig Cα2,Ig κ and Ig λ mRNA in the NCBI database exhibited that the similarities were 99％,97％,98％ and 97％,respectively.Western blot showed Ig α and Ig λ expressions in the cell lysate and secretion of Ig α1 and Ig α2 heavy chains in cell culture supernatant.To further explore the protein that secreted into the supernatant,after supernatant affinity chromatography with jacalin-sepharose,the proteins were separated by sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) and the band approximating to 65 000 was cut and sent to mass spectrometry.The results were aligned with the amino acid sequences of Ig α1 and Ig α2 constant region in NCBI database,showing that amino acids between No.52 and No.104,amino acids between No.154 and No.221,amino acids between No.276 and No.327 from Ig Cα1 and amino acids between No.52 and No.113,amino acids between No.151 and No.204,amino acids between No.251 and No.314 from Ig Cα2 were the same with those derived from B cells.Conclusion:Our findings suggested that HMCs could synthesize and secret IgA.
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Objective To investigate impacts of chronic hepatitis B virus (HBV) co-infection on immune responses and clinical manifestations in acute infection of dengue virus type 1.Methods The serum levels of interferon (IFN)a,IFNβ,IFNγ,interleukin (IL)-10 and tumor necrosis factor (TNF)α of 310 dengue serotype 1 (DENV1)-infected patients were assessed by enzyme linked immunosorbent assay (ELISA),among which 8％ (25/310) were chronic HBV co-infection.Meanwhile,serum samples from 41 healthy adults and 47 chronic HBV infected subjects were recruited as controls.Comparisons among groups were done by one factor analysis of variance and correlation analysis of results was done by spearman rank correlation test.Results The serum level of IFN-α [(95.1 ± 279.3) pg/mL] was significantly higher than IFN-β[(2.8 ± 16.2) pg/mL] during acute dengue infection,while IFN-α level [(86.5±358.1) pg/mL] reduced in patients with HBV co-infection.The secretion kinetics of IFNα,IFNγ (pro-inflammatory cytokine) and IL-10 (anti-inflammatory cytokine)were analyzed.The medians of IFNα,IFNβ and IL-10 level were elevated to peak on day 2,day 3-4and day 6 after fever onset,respectively.Additionally,IFNα levels in patients with only dengue infection were negatively correlated with the platelet counts and serum alanine aminotransferase levels (r=-0.2327,0.2122,both P＜0.01).Conclusions Chronic HBV co-infection alters human immune responses elicited by acute dengue viral infection.Moreover,IFNα secretion may be associated with hemorrhagic tendency,while protective against inflammatory damage of liver.
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<p><b>OBJECTIVE</b>To study the role of apoptosis and the expression of apoptosis-related genes Fas ligand (FasL), Fas, caspase-3 and caspase-8 in an animal model of non-alcoholic steatohepatitis (NASH).</p><p><b>METHODS</b>An experimental progressive NASH model was established by feeding male C57BL6/J mice with a high fat, methionine-choline deficient (MCD-) diet for two days, five days, ten days, three weeks and eight weeks. Control mice were fed methionine-choline supplemented (MCD+) diet. Hepatic steatosis, inflammation and fibrosis were graded by examining their H and E stained liver sections. Hepatocyte apoptosis was detected by TUNEL assay. Expressions of mRNA and protein of FasL, Fas and caspase-8 were performed by quantitative real time RT-PCR and Western blot. Caspase-3 activity assay was conducted using ApoAlert caspase-3 assay kit.</p><p><b>RESULTS</b>In MCD- mice, minimal hepatic steatosis was observed at day 5, and by day 10, mild steatosis with inflammatory infiltration was found. Severe steatohepatitis was noted at week 3, and fibrosis at week 8. TUNEL assay showed that apoptotic index in MCD- group was higher than that in MCD+ group at week 3 (15.59%+/-4.87% vs 5.17%+/-3.19%, P less than 0.05) and at week 8 (11.29%+/-3.22% vs 5.41%+/-1.54%, P less than 0.05). Compared to MCD+ group, the expression of FasL was dramatically increased on day 10 and in week 3 in MCD- mice both at the mRNA and protein levels (P less than 0.05 and P less than 0.01). Expression of Fas mRNA was up-regulated in weeks 3 and 8 (P less than 0.01), and expression of Fas in protein level was higher at week 8 (P less than 0.01) in MCD- group. Expression of caspase-8 significantly increased at the mRNA level at week 3 and week 8 (P less than 0.01 and P less than 0.05 respectively) and at the protein level at week 8 (P less than 0.05) in MCD- group. In all of the time points except for day 5, caspase-3 activities were significantly more enhanced in MCD- group than that in MCD+ group (P less than 0.05).</p><p><b>CONCLUSIONS</b>In our experimental NASH model, hepatic apoptosis was frequently detected. Increased apoptosis was probably attributable to up-regulation of apoptosis-related genes, such as FasL/Fas system, and activation of the caspase pathway. These changes may provoke hepatic apoptosis and the development of inflammation and fibrosis.</p>
Asunto(s)
Animales , Masculino , Ratones , Apoptosis , Caspasa 3 , Metabolismo , Caspasa 8 , Metabolismo , Proteína Ligando Fas , Metabolismo , Hígado Graso , Genética , Metabolismo , Patología , Hepatocitos , Metabolismo , Hígado , Patología , Ratones Endogámicos C57BLRESUMEN
0.05) for the index of renal function(Cr-s,UA,UREA).The patients treated by general low protein diet had worse nutrition state and worse compliance than those before treatment.Better nutrition state and better compliance were found in personal diet group.Conclusion: The personal diet model should be used to the patients basing on the different conditions.