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Objective:The effect of intermedin (IMD) on ATP-induced activation of inflammatory bodies and pyroptosis of cells and its mechanism were studied using lipopolysaccharide (LPS)-sensitized mouse macrophage line RAW 264.7.Methods:The cells were divided into the control groups, the LPS groups, LPS+IMD groups, and LPS+IMD+LY294002 groups. The expression of interleukin (IL)-1β and IL-18 and the activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammatory cells were detected by real-time PCR and western blotting, and the pyroptosis of cells was detected by propidium iodide (PI) staining. The measurement data was represented by MS± SD, and the inter-group difference was compared with ANOVA calculations, and P<0.05 represented the difference with statistical significance. Results:Compared with the control group [(0.83±0.09) vs (0.49±0.04)], the ratio of phosphorylated phosphatidylinositol-3-kinase, p-PI3K)/phosphatidylinositol-3-kinase (PI3K) (0.44±0.05) and p-Akt/Akt (0.27±0.06) in the LPS group was significantly decreased. The ratios of p-PI3K/PI3K (1.22±0.18) and pAkt/Akt (0.83±0.09) in LPS+IMD group was significantly increased ( F=31.40, P<0.001; F=50.88, P<0.001). Compared with the control group, the mRNA and protein expressions of IL-1β, IL-18 and NLRP3 inflammasome (NLRP3, caspase-1, ASC) in RAW264.7 cells were up-regulated in the LPS group (LPS and ATP). Compared with LPS group, IMD treatment inhibited the expression of inflammatory cytokines IL-1β, IL-18 and NLRP3 inflammasome, which was blocked by LY294002, a blocker of PI3K/Akt pathway. The results of real-time PCR showed that the relative expression of IL-1β mRNA was (1.00±0.11) in the control group, (8.32±0.61) in the LPS group, (8.32±0.55) in the LPS+IMD group, and (7.23±0.41) in the LPS+IMD+LY group ( F=15.42, P<0.001). The relative expression of IL-18 mRNA in the control group was (1.00±0.17), (1.82±0.21) in the LPS group, (1.14±0.15) in the LPS+IMD group, and (1.53±0.11) in the LPS+IMD+LY group respectively ( F=18.16, P<0.001). The relative expression of NLRP3 mRNA in the control group was (1.00±0.13), (2.58±0.18) in the LPS group, (1.07±0.17) in the LPS+IMD group, and (1.33±0.32) in the LPS+IMD+LY group respectively ( F=15.98, P< 0.001); The relative expression of caspase-1 mRNA in the control group was (1.00±0.09), (6.20±0.19) in the LPS group, (3.43±0.06) in the LPS+IMD group, and (5.50±0.45) in the LPS+IMD+LY group respectively ( F=18.39, P<0.001). The relative expression of ASC mRNA in the control group was (1.00±0.21), (4.58±0.48) in the LPS group, (2.07±0.51) in the LPS+IMD group, and (3.33±0.32) in the LPS+IMD+LY group respectively ( F=15.19, P<0.001). Western blotting results showed that the relative expression of IL-1β protein was as follows: (100%) in the control group, [(188±14)%] in the LPS group, [(112±11)%] in the LPS+IMD group, and [(171±27)%] in the LPS+IMD+LY group respectively ( F=21.25, P<0.001). The relative expression of IL-18 protein in the control group was 100%, [(183±16)%] in the LPS group, [(115±19)%] in the LPS+IMD group, and [(179±23)%] in the LPS+IMD+LY group respectively ( F=19.62, P<0.001). The relative expression of NLRP3 protein was 100% in the control group, [(149±15)%] in the LPS group, [(106±10)%] in the LPS+IMD group, and [(144±15)%] in LPS+IMD+LY group respectively ( F=14.35, P<0.001). The relative expression of ASC protein was 100% in the control group, [(188±12)%] in the LPS group, [(110±18)%] in the LPS+IMD group, and [(192±8)%] in the LPS+IMD+LY group ( F=15.79, P<0.001). Conclusion:IMD inhibits the activation of NLRP3 inflammasome and cell pyroptosis by regulating PI3K/Akt activity.
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Objective:To investigate the effects of hepatitis C virus (HCV) NS3/4A protein on proliferation, apoptosis and division of human hepatocarcinoma SMMC-7721 cells.Methods:The recombinant plasmid pLV-Green-NS3/4A and pLV-puro-NS3/4A were produced by homologous recombination method, and then the cells were divided into pLV-Green-NS3/4A or pLV-puro-NS3/4A transfection group and their corresponding control group (transfected with empty vector). The expression of recombinant protein was detected by immunofluorescence staining and Western blotting. Methyl thiazol tetrazolium (MTT) method was used to detect cell proliferation, DAPI staining was used to detect cell apoptosis, and immunofluorescence was used to analyze cell devision.Results:The growth state of SMMC-7721 cells in the pLV-Green-NS3/4A transfection group was worse than that in the corresponding empty vector control group. Compared with the corresponding empty vector control group, the proliferation activity of SMMC-7721 cells in the pLV-Green-NS3/4A transfection group on the 3rd and 4th day was inhibited (both P < 0.05). The apoptosis rates of cells in the pLV-Green-NS3/4A transfection group and the corresponding empty vector control group were (20.3±3.5)% and (5.3±1.5)%, and the difference was statistically significant ( t = -8.57, P < 0.001). Compared with the corresponding empty vector control group, the cells in the pLV-puro-NS3/4A transfection group had more multipolar spindles, and the multipolar division rates of the two groups were (2.33±0.58)% and (6.01±0.99)%, and the difference was statistically significant ( t = -5.50, P = 0.005). Conclusions:HCV NS3/4A can inhibit the proliferation, promote the apoptosis and multipolar division of human hepatocarcinoma SMMC-7721 cells.
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Objective:To investigate the expression of DNA damage repair factor DNA damage-binding protein 1 (DDB1) in hepatocellular carcinoma tissues, and the effect of DDB1 gene silencing on DNA repair and targeted killing in human hepatocellular carcinoma SMMC-7721 cells.Methods:The UALCAN platform was used to perform bioinformatics analysis on the expression of DDB1 in hepatocellular carcinoma tissues (371 cases) and paracancerous tissues (50 cases) in The Cancer Genome Atlas (TCGA) database and the correlation of DDB1 expression with the overall survival of liver cancer patients were analyzed by bioinformatics using the UALCAN platform. SMMC-7721 cells were transfected with small interfering RNA (siRNA) targeting DDB1 and negative control siRNA, which were DDB1 silencing group and negative control group, respectively. X-ray irradiation induced exogenous DNA double strand break (DSB) damage in the two groups of cells. Immunofluorescence staining (γH2AX was used for assessing cellular DSB damage, RPA32s33 and Rad51 were used for assessing homologous recombination repair) and Western blotting (were used to detect the level of RPA32s33 protein) were used to analyze the effect of DDB1 gene silencing on DSB damage repair. Sister chromosome exchange (SCE) experiment was used to analyze the frequency of SCE of homologous recombination of cells in DDB1 silencing group and negative control group. Tetramethylazozolium salt (MTT) method was used to analyze the killing effect of PARP inhibitor olapani (10 μmol/L), cisplatin (1 μg/ml) and olapani combined with cisplatin on SMMC-7721 cells in DDB1 silencing group and negative control group.Results:Bioinformatics analysis showed that the level of DDB1 mRNA in liver cancer tissues was higher than that in paracancerous tissues ( P < 0.001), and the overall survival of patients with high expression of DDB1 was worse than that of patients with low expression of DDB1 ( P = 0.029). When cultured for 4 hours after X-ray irradiation, the number of γH2AX foci in cells of the negative control group had mostly disappeared, and there were still more cells in DDB1 silencing group [(5.1±2.0) per cell vs. (13.4±2.0) per cell, t = -5.08, P = 0.007]. When cultured for 4 hours after X-ray irradiation, the number of RPA32s33 foci in the negative control group [(30.8±5.0) per cell vs. (13.2±1.6) per cell] and the number of Rad51 foci [(19.5±1.8) per cell vs. (8.3±3.3) per cell] were higher than those in the DDB1 silencing group, and the differences between the two groups were statistically significant (both P < 0.01). The frequency of SCE in the negative control group was higher than that in the DDB1 silencing group [(21.2±3.0)% vs. (11.2±1.6)%, t = 5.07, P = 0.007]. MTT assay showed that after olaparib treatment, the survival rate of cells in the DDB1 silencing group was lower than that in the negative control group [(40.3±3.6)% vs. (79.8±1.3)%, t = 17.94, P < 0.001]. When treated with olapani combined with cisplatin, the survival rate of cells in the two groups further decreased, and the survival rate of cells in the DDB1 silencing group was lower than that in the negative control group [(10.2±2.8)% vs. (29.6±3.4)%, t = 7.72, P = 0.002]. When treated with cisplatin alone, there was no significant difference in cell survival between DDB1 silencing group and negative control group [(41.9±5.1)% vs. (49.8±3.3)%, t = 2.71, P = 0.054]. Conclusions:The high expression of DDB1 in hepatocellular carcinoma tissues may be an important factor in the treatment resistance and poor prognosis of hepatocellular carcinoma. Knockdown of DDB1 gene expression can promote the sensitivity of SMMC-7721 cells to PARP inhibitors, and its mechanism may be related to the homologous recombination repair defect of SMMC-7721 cells caused by DDB1 silencing.
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ObjectiveTo investigate the effect and mechanism of Guilu Erxiangao on Alzheimer's disease （AD） model rats induced by hydrocortisone and amyloid β-protein（Aβ） based on the theory of kidney-brain correlation. MethodIntraperitoneal injection of hydrocortisone and intracerebroventricular injection of Aβ were performed to induce AD in rats， and different concentrations of Guilu Erxiangao were used for intervention. The indexes of hippocampus， kidney and adrenal gland were measured， and the spatial learning and memory ability of AD rats was observed by Morris water maze experiment. The levels of testosterone （T） and corticosterone （CORT） in serum samples were determined by enzyme-linked immunosorbent assay （ELISA）. Liquid chromatography-mass spectrometry （LC-MS） was used to collect and analyze the serum metabolic data of model rats. The active components and corresponding targets of Guilu Erxiangao were collected using Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform（TCMSP）， a Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine （BATMAN-TCM） and Traditional Chinese Medicine Integrated Database（TCMID）. GeneCards and Online Mendelian Inheritance in Man （OMIM） were retrieved to obtain AD-related targets， and protein-protein interaction （PPI） network was constructed to perform gene ontology （GO）and Kyoto Encyclopedia of Genes and Genomes （KEGG） enrichment analysis. The expression level of interleukin-6 （IL-6） in the hippocampus of rats was detected by Western blot. ResultCompared with the model group， the low-， medium- and high-dose groups of Guilu Erxiangao exhibited significantly increased hippocampus index， kidney index and adrenal gland index， reduced CORT levels in serum and down-regulated IL-6 levels in hippocampal tissues. According to the results of water maze experiment， as compared with the model group， the platform crossing times of rats was significantly increased in the low- and high-dose groups of Guilu Erxiangao， with evidently prolonged distance traveled in quadrant Ⅲ （%） and time in quadrant Ⅲ （%）. A total of 24 serum differential metabolites associated with AD were identified by LC-MS， and 50 high-frequency common compounds and 187 high-frequency common targets for AD treatment were screened by network pharmacology method. Results demonstrated phosphatidylinositol 3-kinases（PI3K）/protein kinase B（Akt） signaling pathway plays an important role in the complex AD pathological mechanism. ConclusionGuilu Erxiangao can significantly improve the cognitive dysfunction of AD model rats induced by hydrocortisone and Aβ， reduce serum CORT levels and IL-6 levels in hippocampal tissues， and regulate the metabolic level， which provides a reference for its clinical application.
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Objective@#To explore the association between dietary pattern which benefit for normal kidney function and the risk of cognitive decline or impairment in the elderly.@*Methods@#In 2015, subjects aged 60 and over from four counties in the Nutrition and Chronic Disease Family Cohort project, were followed up in 2017. Cognitive function was repeatedly assessed, using the Mini Mental State Evaluation (MMSE) scale. Dietary pattern that benefit for normal kidney function was extracted, using the reduced rank regression method and followed by logistic regression models to explore the associations between scores that showing the kidney function on dietary patterns and the risk of cognitive deterioration and impairment in two years among those who were with normal cognition in 2015.@*Results@#Dietary pattern that benefit for normal kidney function, was characterized by high consumption of cereal, vegetables, legume and fruits but with less meat and soy products. Comparing with the group with lowest score quartile on this dietary pattern, the risk of cognitive deterioration in the highest quartile group was significantly low (P<0.01) in two years, with an odds ratio as 0.57 (95%CI: 0.37-0.85). Linear trend was also obviously visible (P=0.007) in this group. The ones at the highest quartile group among the normal cognition ones in 2015, the risk of cognitive impairment also significantly reduced (P<0.05) in two years time, with an odds ratio as 0.52 (95%CI: 0.29-0.93). Also, linear trend could obviously be seen (P=0.01).@*Conclusion@#Dietary pattern that benefit for normal kidney function was both inversely associated with cognitive deterioration and impairment, in two years.
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OBJECTIVE: To study the preparation and in vitro release profile of compound vitamin microencapsules using cross-linked gelatin as wall material. METHODS: At the presence of hydrogen peroxide, the cross-linked gelatin was prepared with ferulic acid as the cross-linking agent. During this reaction, horseradish peroxidase was used as the catalyst. Influences of reaction conditions on the cross-linking degree were investigated. Compound vitamin microcapsules were prepared by spray-drying technique using the obtained cross-linked gelatin as wall material. The core material was the mixture of thiamine hydrochloride (vitamin B1), riboflavin (vitamin B2), pyridoxine hydrochloride (vitamin B6), folic acid and nicotinamide. The effect of the ratio of wall material to core material on the encapsulation efficiency and loading of the vitamins were investigated. The size and surface morphology of the compound vitamin microcapsules were characterized. The encapsulation efficiency, loading and in vitro release property of the core material were determined by fluorospectrophotometry. RESULTS: A comparatively high cross-linking degree (ca. 10%) of cross-linked gelatin was obtained under the following reaction conditions:temperature of 40°C, pH value of 8.0, gelatin concentration of 9% (W/V), ferulic acid concentration of 40 mmol·L-1 and reaction time of 24 h. The vitamins were embedded by the cross-linked gelatin and the encapsulation efficiency was more than 85%. Scanning electron microscopy (SEM) study showed that the compound vitamin microcapsules had a regular spherical shape but the majority presented rough surfaces or dents. Particle size analysis indicated that the microcapsules had a mean diameter of 15.27 μm. At the ratio of coating material to core material was 10/1 (W/W), the vitamins encapsulated with the cross-linked gelatin released completely in 30 min in simulated gastric fluid, and they released completely in 16 min in simulated intestinal fluid. They released slower than the vitamins encapsulated with the gelatin accordingly. CONCLUSION: Compound vitamin microencapsules with high encapsulation efficiency and sustained release effect can be obtained by spray-drying using cross-linked gelatin as wall material.
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<p><b>BACKGROUND</b>Chronic kidney disease (CKD) is a growing public health problem with well-established risk factors. Other contributing factors, however, remain to be identified. Systemic inflammation in asthma plays a significant role in the development of other diseases. We therefore initiated a study to assess whether the growing prevalence of asthma is associated with an increase in the risk of CKD.</p><p><b>METHODS</b>We conducted a retrospective cohort study using data from 3015 patients with asthma aged 14 years and older who were registered and followed up in Asthma Control Study at the Department of Respiratory Medicine of three medical centers from 2005 to 2011. History, asthma control test (ACT), and asthma stage were used to assess the traits of asthma. CKD was defined as proteinuria and/or reduced estimated glomerular filtration rate (eGFR) (<60 ml×min(-1)×1.73 m(-2)) in two consecutive follow-up surveys. We used logistic regression models, adjusting for age, sex, and other confounding factor to determine associations between the traits of asthma and CKD. Kaplan-Meier curves were used to analyze patient outcomes.</p><p><b>RESULTS</b>A total of 2354 subjects with complete data were recruited for this study with mean age (45.4±10.4) years. After 6 years of follow-up, 9.6% (n = 227) of the analytic cohort developed proteinuria and 3.1% (n = 72) progressed to eGFR <60 ml×min(-1)×1.73 m(-2). The patients with >20 years asthma history, not well-controlled or persistent asthma patients had higher incidence of proteinuria and reduced eGFR compared with patients with ≤20 years asthma history, at least well-controlled or remission asthma, respectively. The multivariable adjusted OR for proteinuria and reduced eGFR in participants with persistent asthma was 1.49; (95% confidence interval (CI) 1.17-1.91) and 2.07 (95% CI 1.34-4.42). Compared to patients with no asthma traits, there was a significant risk (OR, 3.39; 95% CI 1.36-8.73) for those who met all three traits, including asthma history >20 years, not well-controlled and persistent stage, after adjusting for potential confounding factors.</p><p><b>CONCLUSIONS</b>In this retrospective cohort study, we found that persistent asthma was associated with an increased risk of CKD, which was independent of obesity, diabetes, hypertension, and other well-established risk factors. Future studies should be directed to elucidate the mechanisms underlying the association between asthma and CKD.</p>
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Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Asma , Taxa de Filtração Glomerular , Fisiologia , Insuficiência Renal Crônica , Estudos Retrospectivos , Fatores de RiscoRESUMO
<p><b>OBJECTIVE</b>To investigate the time course of changes in the expression of fibroblast activation protein (FAP) during healing of skin scald burns in rats.</p><p><b>METHODS</b>Adult Wistar rats were randomized into two equal groups (n=42) and subject to superficial second degree and deep second degree scald burns on the dorsal skin groups, with 6 normal rats serving as the control group. At 6 h, 12 h, and 1, 3, 7, 14, and 21 days after burns, 6 rats from each group were sacrificed to detect FAP expression by immunohistochemistry and Western blotting.</p><p><b>RESULTS</b>FAP was expressed on the cell membrane and in the cytoplasm of the fibroblasts, especially those around the neovessels. In both burn groups, FAP expression increased significantly at 6 h after burns. In superficial burn group, FAP expression was comparable between 6 and 12 h and between 1 and 3 days (P>0.05), but showed significant differences between the other time points (P<0.05). In deep burn group, FAP expression was comparable between 12 h, 1 day and 3 days (P>0.05) but differed significantly between the other time points (P<0.05). In both burn groups, FAP expression reached the peak level at 7 days followed by a gradual declination. At 21 days after the burns, FAP maintained a significantly higher expression level than the control level (P<0.05).</p><p><b>CONCLUSION</b>The time course of the changes of FAP expression following scald burns suggests an important role of FAP in the healing process of scald burns.</p>
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Animais , Ratos , Queimaduras , Metabolismo , Reabilitação , Face , Gelatinases , Metabolismo , Proteínas de Membrana , Metabolismo , Ratos Wistar , Serina Endopeptidases , Metabolismo , Pele , Metabolismo , CicatrizaçãoRESUMO
ObjectiveTo investigate the anti-tumorigenesis function of rhDCN on the leukemia K562 cells in vitro and analyze the possible mechanism.Methods Exponential phase of K562 cells were transfected with pcDNA3.1(+)-DCN,and PBS,liposome alone,and pcDNA3.1(+) vector were as control groups.Morphology change of K562 cells was detected by Wright stain,and cell proliferation activity was detected by MTT. Cell cycle and apoptosis of K562 cells were assessed by FCM. The expression of apoptosis-related protein,including bcl-xl,Mcl-1 and Bax were detected by Western blot.ResultsWright stain showed that typical apoptotic morphology of K562 cells were observed in DCN transfected group.There were no morphological changes of apoptosis in other groups. MTT method results showed that proliferation inhibition rate of the transfected cells [24 h (16.14±1.08) ％,48 h (14.07±1.01) ％,72 h (20.29±1.19) ％]was higher than that of the other control groups (P ＜ 0.05).FCM results showed that the apoptosis index (20.15±1.31) ％of the DCN transfected group was higher than that of the other groups (P ＜ 0.05),and most of cells arrested in the G0/G1 phase (51.15±0.57) ％ (P ＜ 0.05).Western blot results showed the expression levels of Bax were increased while bcl-xl and Mcl-1 were decreased in pcDNA3.1(+)-DCN/K562 group.ConclusionrhDCN can inhibit the growth of K562 cells and induce the apoptosis.The effect of DCN on bcl-xl,Mcl-1,Bax may play a role in its mechanism.
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Objective To investigate the suppression effect, the apoptosis and TGF-β_1 mRNA expression of rhDCN and dororubicin(ADM) on leukemic K562 cell line. Methods K562 cells in Logarithmic growth phase were divided into Saline group, pcDNA3.1 (+)-DCN group, ADM group, and pcDNA3.1 (+)-DCN-ADM group. Morphology change of cell was detected by Wright stain, cell proliferation activity was assessed by MTT. The apoptosis index of K562 cells was assessed by FCM, and TGF-β_1 mRNA of cell was assessed by RT-PCR. Results Wright stain showed that more pronounced morphological apoptosis changes of K562 cells in combined group. MTT method results showed that the proliferation inhibition rate of the combined group was (61±1.32) % higher than that of individual intervention group [DCN group, (20±1.90) %; ADM group, (47±1.04) %](P <0.05). FCM results showed that the apoptosis index of the combined group was (61.30± 0.9) %, higher than that of Individual intervention group [DCN group, (28.25±1.3) %; ADM group, (31.85± 1.5) %](P <0.05). TGF-β_1 mRNA synthesis of combined group was significantly decreased. Conclusion rhDCN can markedly enhance cytotoxicity of ADM on K562 cells, and the mechanisms of apoptosis may be due to down-regulation of TGF-β_1 mRNA. Specific mechanisms will be further studied.