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Objective:To observe the influence of NOD-like receptor thermal protein domain associated protein 6 (NLRP6) on hepatic ischemia-reperfusion injury (IRI), and elucidate the related mechanism.Methods:Thirty C57BL/6 mice with body weight of (18.80±1.99) g, were divided randomly into 5 groups, with 6 mice in each group: the mice that experienced only exploratory laparotomy were Sham group; that only underwent an operation to establish a hepatic IRI model were IRI group; that were treated with tail intravenous injection of clodronate (Clo) liposomes before the establishment of hepatic IRI model were Clo group; that received tail intravenous injection of clodronate liposomes and transfusion of bone marrow derived macrophages (BMDM) before the operation were Clo+ BMDM group; that received preoperative tail intravenous injection of clodronate liposomes and transfusion of BMDM with NLRP6 knockdown were Clo+ NLRP6-knockdown group. Real time quantitative polymerase chain reaction analysis (RT-PCR) and Western blot were performed to analyze the expressions of pyroptosis related proteins and factors. Simulate a hypoxia/reoxygenation (H/R) model in vitro, and set up experimental groups: lipopolysaccharide (LPS) + adenosine triphosphate (ATP), LPS+ ATP+ NLRP6-knockdown, H/R, and H/R+ NLRP6-knockdown. The changes of expressions of pyroptosis related proteins and factors were detected by RT-PCR and Western blot. Expression of NF-κB in vivo and in vitro was measured.Results:Compared with those in Sham group, protein expressions of NLRP6, NLRP3, Caspase-1, gasdermin D (GSDMD), IL-1β and IL-18 were remarkably increased in IRI group, but the levels of these proteins were dramatically decreased in Clo group with the exhaustion of macrophages in comparison with in IRI group, which were significantly different statistically (all P<0.05). The levels of these proteins were enhanced again in Clo+ BMDM group with the reconstruction of macrophages in contrast to those in Clo group, while the enhancements were more obvious in Clo+ NLRP6-knockdown group comparing to those in Clo+ BMDM group, with significant differences (all P<0.05). In vitro, pyroptosis rate for LPS+ ATP group was (16.39±1.06)%, which was lower than (27.34±2.79)% for LPS+ ATP+ NLRP6-knockdown group, with a statistical significance ( P<0.05). Meanwhile, pyroptosis rate for H/R group was (20.59±5.66)%, also much more reduced than (37.76±2.00)% for H/R+ NLRP6-knockdown group ( P<0.05). Expressions of NLRP3, Caspase-1, GSDMD, IL-1β, IL-18 and NF-κB p65 in LPS+ ATP+ NLRP6-knockdown group were more elevated than in LPS+ ATP group, and these indices were also more enhanced in H/R+ NLRP6-knockdown group than which in H/R group. Compared to the Sham group, expression of NF-κB p65 significantly increased in IRI group, which was reversed in Clo group, but enhanced again in Clo+ BMDM group and reached a peak in Clo+ NLRP6-knockdown group. Conclusions:Macrophage plays a critical role in immune response to hepatic IRI, wherein NLRP6 functions specifically. NLRP6 acts to suppress inflammation during hepatic IRI through regulating macrophage pyroptosis via inhibiting NF-κB.
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Objective:To investigate the expression of long non-coding RNA 00665 (LINC00665) in hepatocellular carcinoma (HCC) and its regulatory effect on angiogenesis of hepatocellular carcinoma cells and its potential molecular mechanism.Methods:HCC tissues and corresponding paracancerous tissues of 100 patients with HCC in the First Affiliated Hospital of Nanjing Medical University from May 2016 to April 2017 were collected, and the survival prognosis was compared. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression of LINC00665 in HCC tissues and cells. The effect of LINC00665 overexpressed Hep-3B cells on the angiogenesis of human umbilical vein endothelial cells (HUVEC) was examined by tube formation assay and chick chorioallantoic membrane assay. Bioinformatics database predicted the downstream microRNA (miRNA) and target genes of LINC00665, and the relationship between LINC00665, miR-126-5p and vascular endothelial growth factor A (VEGFA) was verified by RT-qPCR, Western blot and dual-luciferase reporter gene assay.Results:The expression level of LINC00665 in HCC (6.5±2.8) was significantly higher than that in paracancer tissues (4.8±3.1), the difference was statistically significant ( t=4.12, P<0.001). According to the median LINC00665 expression level of 100 patients with HCC, the cumulative survival rate of LINC00665 high expression group ( n=50) was lower than that of LINC00665 low expression group ( n=50), and the difference was statistically significant (χ 2=3.79, P=0.008). After co-culture with LINC00665 group (Hep-3B cells overexpressing LINC00665), the length of HUVEC cell tubule formation was (596.0±22.3) μm, and the number of HUVEC cell tubules was (36.3±4.5), which were both higher than NC group with the tubule formation length (127.0±13.5) μm and the number (9.3±1.5) of HUVEC cells co-cultured with Hep-3B cells of control group, and the differences were statistically significant ( t=31.15, 9.82, P<0.001, P=0.001). The chick chorioallantoic membrane assay results were similar to tube formation assay. Western blot detected that the relative expression of VEGFA in LINC00665 group was higher than that in NC group, the difference was statistically significant ( t=7.15, P<0.001). StarBase and DIANA database were used to predict and screen LINC00665 downstream miR-126-5p. StarBase database was used to predict the binding sites of LINC00665/miR-126-5p/VEGFA axis. In dual-luciferase reporter gene assay, the fluorescence intensity of LINC00665 and VEGFA vector co-transfected with miR-126-5p mimics decreased. Conclusion:LINC00665 is highly expressed in HCC and is associated with poor prognosis in patients with HCC. LINC00665 promotes angiogenesis of HCC by regulating the miR-126-5p/VEGFA axis.
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Objective To detect the expression of miR-122 in hepatocellular carcinoma (HCC) cells and tissues,and to explore the role and the underlying mechanisms of miR-122 in HCC cells on invasion and migration.Methods Real-time quantitative polymerase chain reaction of analysis was used to examine the expression of miR-132 in HCC cell lines,a normal liver cell line,HCC tissues and adjacent non-tumor tissues.Over express or inhibit miR-122 by transfection.The invasion and migration of HCC cells were analyzed by Transwell assays.Meanwhile,related mechanism proteins were detected by western blot,including insulin like growth factor 1 receptor(IGF-1 R),E-cadherin,vimentin.Results The expression of miR-122 was decreased in HCC tissue samples compared with the adjacent non-tumor tissues[(20.5 ± 4.2) × 10-4 vs.(30.3 ± 5.6) × 10-4],especially in HCC tissue samples with HBV [(25.6 ± 3.5) × 10-4 vs.(19.1 ±3.2) × 10-4].The same results were observed in HCC cell lines.MHCC-97H cells exhibited lowest level of miR-122 expression[(33.7 ± 1.3) × 10-3],whereas SMMC-7721 exhibited the highest level of miR-122 expression [(65.3 ± 1.3) × 10-3].MiR-122 over-expression suppressed the invasion and migration of MHCC-97H[(218.7±24.0) vs.(418.0 ±23.4),(392.7±12.8) vs.(706.6±19.8)].Knocking down miR-122 promoted the invasion and migration of SMMC-7721 [(233.0 ± 14.4) vs.(145.0-±8.0),(561.3 ±9.6) vs.(218.0 ± 11.3)].Western blot revealed that miR-122 suppressed the expression of IGF-1R,Vimentin and facilitated the expression of E-cadherin.Conclusions The study indicates that miR-122 is downregulated in HCC,especially in HCC tissue samples with HBV.MiR-122 can suppress invasion and migration of hepatocellular carcinoma by regulating IGF-1R and epithelial mesenchymal transition,and may provide a new therapeutic option for HCC.
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Objective To study enhanced recovery after surgery (ERAS) in pancreaticoduodenectomy.Methods A case-control study was conducted on 56 patients who underwent pancreaticoduodenectomy in our hospital from May 2012 to December 2016.These patients were divided into two groups:25 patients received ERAS management (the ERAS group) and 31 patients traditional perioperative management (the control group).The data on postoperative pancreatic leakage,bile leakage,postoperative bleeding,delayed gastric emptying,postoperative intestinal function recovery,hospitalization stay,medical cost and readmission rate within 90 days between the two groups were compared.Results The rate of delayed gastric emptying,postoperative intestinal function recovery,hospitalization stay and medical cost were significantly better in the ERAS group than the control group (all P ＜ 0.05).There were no significant differences in the rates of pancreatic leakage,bile leakage,postoperative bleeding,and readmission within 90 days between the two groups (all P ＞ 0.05).Conclusions Perioperative ERAS in pancreaticoduodenectomy was safe and efficacious.It improved recovery of patients and reduced hospital stay and expenses.
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Objective To investigate the value of diffusion weighted magnetie resonance imaging and apparent diffusion coefficient (ADC) value in the diagnosis of pancreatic cancer.MethodsThe clinical data of 36 patients with pancreatie cancer who were admitted to the Wuxi No.2 People's Hospital from March 2009 to June 2011 were retrospectively analyzed,and the clinical data of 30 healthy volunteers were collected.All candidates received diffusion weighted magnetie resonance imaging examination.The signal intensity ratios ( SIRs ) of the cancer and the pericancerous tissues in the T1 weighted-images (T1WI),T2 weighted-images (T2WI)and diffusion weighted-images (DWI) were compared by using one-way analysis of variance.The ADC values of the cancer and the paricancerous tissues were compared using the paired t test.The differences of the ADC values of the cancer and pericancerous tissues compared with those of healthy individuals were analyzed using an independent sample t test.ResultsThe accuracy rate of preoperative magnetic resonance imaging examination was 92％.The relative SIRs were 0.203 ± 0.190 in the T2 WI,0.399 ± 0.201 in the T1 WI and 0.579 ± 0.291 in the DWI,respectively,with no significant differences across the 3 kinds of images (F=5.92,6.15,6.83,P ＜ 0.05 ),while SIRs of the T1 WI and DWI were significantly higher than that of the T2 WI ( P ＜ 0.05 ).There was no significant difference in SIRs when comparing T1 WI and DWI ( P ＞ 0.05 ).The mean ADC values of the cancer and pericancerous tissues of the pancreatic cancer patients and the pancreatic tissues of healthy individuals were (1.40±0.24) ×10 3 mm2/s,(1.71 ±0.10) ×10-3 mm2/s and (1.73±0.30) ×10-3 mm2/s,respectively,with significant differences across the 3 modes of images (t =10.54,12.08,P ＜ 0.05).ConclusionDWI can show high quality images of the lesions,and ADC value is helpful in the diagnosis of pancreatic cancer.
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Objective To investigate the protective effect of edaravone against ischemiareperfusion injury in small-for-size rat liver grafts and its possible mechanisms. Methods 40 % small-for-size rat liver transplantation model was established by using modified two-cuff technique, adult male SD rats were used as donors and recipients, and 16 recipient rats were randomly divided into two groups (8 cases in each group), saline control group (control group) and edaravone treatment group (ED group). In the ED group, 3 mg/kg edaravone was given intravenously via penile vein 30 min before transplantation in the recipients. The same amount of saline was given in the control group at the same time points. Serum hepatic function (AST and ALT) and histopathological changes were analyzed; the contents of MDA and SOD, and hepatic myeloperoxidase (MPO) activity in liver grafts after 6 h were determined; and TNF-α levels at 6th h after reperfusion were measured by using enzyme-linked immunosorbent assay (ELISA method). Results As compared with control group,serum AST and ALT levels were significantly reduced at the 6th h after reperfusion in ED group (AST: 825. 50 5±72. 87 vs 1188. 03 ± 124. 04; ALT. 687. 40 5±72. 21 vs 988. 66 ± 91.07, P<0. 01 ).The content of MDA was lower and SOD level was higher in ED group significantly than in control group (P<0. 01). As compared with control group, hepatic TNF-α levels and MPO activity at the 6th h after reperfusion were significantly decreased in ED group (P<0. 01 ). Histopathological analysis revealed disruption of lobular architecture, apparent hepatocelluar degeneration accompanied by focal necrosis, significant edema, congestion and inflammatory cell infiltration in periportal area at the 6th h after reperfusion in control group, but minimal liver damage was observed in ED group. Conclusion Edaravone could ameliorate early ischemia-reperfusion injury in small-for-size liver grafts significantly.The protective mechanisms were mediated in part by increasing antioxidant ability, inhibiting lipid peroxidation, and down-regulating inflammatory reaction.
