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Objective To investigate the effect and related mechanism of microRNA (miR)-494 on the hepatic ischemia-reperfusion injury (HIRI). Methods Twenty-four male SD rats were randomly divided into four groups (n=6 in each group). In the sham operation group, abdominal surgery without hepatic ischemia-reperfusion was performed. In the HIRI group, partial liver ischemia was performed for 60 min, followed by 6 h perfusion. In the HIRI+agomir-miR-494 group, intraperitoneal injection of agomir-miR-494 (20 μL) was daily given within preoperative 7 d. In HIRI+agomir-NC group, an equivalent quantity of agomir-NC was daily injected intraperitoneally within preoperative 7 d. The expression level of miR-494 messenger RNA(mRNA) in the liver tissues in each group was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression levels of liver injury and oxidative stress related indexes were measured by relevant kits. The histopathological changes of the liver in each group were observed. The quantity of apoptotic cells and cytoplasmic histone-related DNA fragments in the liver tissues of rats was detected by relevant kits. The expression levels of the proteins related to the phosphatidylinositol-3-kinase(PI3K)/protein kinase(AKT) signaling pathway were measured by Western blot. Results The expression level of miR-494 mRNA in the rat liver tissues in the HIRI+agomir-miR-494 group was significantly higher than that in the HIRI+agomir-NC group (P < 0.01). The levels of the serum liver injury and oxidative stress related indexes in the HIRI+agomir-miR-494 group were significantly lower than those in the HIRI+agomir-NC group (all P < 0.01). Compared with those in the HIRI+agomir-NC group, the quantity of cellular necrosis was significantly reduced, the cell integrity was considerably increased and the quantity of TUNELpositive cells was evidently decreased in the HIRI+agomir-miR-494 group (all P < 0.05). The expression levels of poly ADP-ribose polymerase(PARP), cysteinyl aspartate specific proteinase-3(Caspase-3) and Bax in the HIRI+agomirmiR-494 group were significantly lower than those in the HIRI+agomir-NC group (all P < 0.05). The quantity of DNA fragments in the HIRI+agomir-miR-494 group was significantly less than that in the HIRI+agomir-NC group (P < 0.01). The expression levels of p-AKT, p-mammalian target of rapamycin(mTOR) and p-p70S6K in the HIRI+agomir-miR-494 group were significantly higher than those in the HIRI+agomir-NC group (all P < 0.05). Conclusions miR-494 can alleviate the severity of HIRI in rats by activating the PI3K/AKT signaling pathway.
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Objective To investigate the effects of nicotinamide phosphoribosyl transferase (NAMPT) inhibitor FK866 on polymicrobial sepsis-induced liver injury in mice. Methods Eighty-four healthy male C57BL/6J mice were divided into four groups by random number table method (n = 21): sham group, sepsis-induced liver injury model by cecal ligation and perforation group (CLP group), vehicle+CLP group and FK866+CLP group. FK866 (10 mg/kg) or same volume dimethyl sulfoxide were given intraperitoneally into mice 24, 12 and 0.5 hours prior to CLP in the FK866+CLP group or the vehicle+CLP group, respectively. Fifteen mice in each group were used to observe the 48-hour survival after operation. The remaining 6 mice were sacrificed 20 hours after operation to harvest venous blood and liver tissue samples for index detection. The levels of serum alanine transaminase (ALT) and aspartate aminotransferase (AST) were measured by colorimetry; the levels of serum NAMPT, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were measured by enzyme linked immunosorbent assay (ELISA); the mRNA expressions of TNF-α and IL-6 were measured by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR); the protein expressions of hepatic NAMPT, cytoplasmic IκBα and nuclear factor-κB (NF-κB) were measured by Western Blot. Results Compared with the sham group, the 48-hour survival in the CLP group was significantly decreased; serum and liver NAMPT protein levels were significantly increased, serum ALT, AST, TNF-α, IL-6 levels and mRNA expressions of TNF-α, IL-6 in liver tissue were significantly increased; the expression of cytoplasmic IκBα protein was significantly decreased, and the expression of nuclear NF-κB protein was significantly increased; which indicated that CLP induced NF-κB activation, inflammation and liver injury. There was no significant difference between the vehicle+CLP group and the CLP group. Compared with the vehicle+CLP group, the 48-hour survival in FK866+CLP group was significantly increased (53.33% vs. 26.67%); serum ALT, AST, TNF-α, IL-6 levels and mRNA expressions of TNF-α, IL-6 in liver tissue were significantly decreased [serum ALT (U/L): 128.94±32.48 vs. 237.24±58.61, serum AST (U/L):289.89±68.74 vs.468±82.17, serum TNF-α (pg/L): 65.17±18.74 vs.127.64±48.18, serum IL-6 (ng/L): 31.78±5.23 vs. 60.87±13.12, liver TNF-α mRNA (2-ΔΔCt): 8.37±4.17 vs. 18.24±6.12, liver IL-6 mRNA (2-ΔΔCt): 18.58±7.12 vs.34.24±6.71], the expression of cytoplasmic IκBα protein was significantly increased (IκBα/GAPDH: 0.23±0.03 vs. 0.12±0.04), while expression of nuclear NF-κB protein was significantly decreased (NF-κB/Lamin B1: 0.25±0.04 vs. 0.42±0.05), with statistically significant differences (all P < 0.05). Conclusion NAMPT inhibitor FK866 protects polymicrobial sepsis-induced liver injury via the inhibition of NF-κB activation and inflammation.
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Objective To investigate the effects of baicalein (Bai) on acute lung injury (ALI) induced by intestinal ischemia/reperfusion (I/R) and its mechanism in mice.Methods Twenty-four male C57BL/6J mice were divided into three groups by random number table:namely sham group,I/R group and Bai+I/R group,with 8 mice in each group.Intestinal I/R induced lung injury model was reproduced by clamping superior mesenteric artery for 90 minutes,followed by reperfusion.Bai (100 mg/kg) was intraperitoneally injected 1 hour before ischemic challenge in the Bai+I/Rgroup.The mice in sham group underwent the similar procedure with I/R group but without vascular occlusion.All mice were sacrificed at 4 hours of reperfusion,and blood was collected from inferior vena cava and lung tissues were harvested.Lung tissues were stained with hematoxylin-eosin (HE),and histological changes were examined under light microscope for pathological score.Lung wet/dry (W/D) ratio was calculated.Lung cell apoptosis was determined by TdT-mediated dUTP nick end labeling (TUNEL) technique.Serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6(IL-6) were determined by enzyme-linked immunosorbent assay (ELISA).The mRNA expressions of TNF-α and IL-6 in lung tissues were determined by real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR).The protein expression levels of cytoplasmic inhibitory factor-α of nuclear factor-κB (IκB-α) and nucleus NF-κB were determined by Western Blot.Results Under light microscope,a normal lung tissue structure was shown in the sham group and no evidence of obvious lung injury was found.In the I/R group,the alveolar structure was seriously damaged.The alveolar wall was widened and there was significant interstitial edema and leukocytes infiltration.In the Bai+I/R group,pathological damage was significantly decreased as indicated by reduced lung tissue edema and leukocytes infiltration.Compared with the sham group,the lung pathological scores,W/D ratio and cellular apoptosis in the I/R group were significantly increased.Bothserum TNF-α and IL-6 contents and lung TNF-α and IL-6 mRNA expressions were significantly increased.Furthermore,I/R significantly resulted in a decrease of IκB-α in the cytoplasm and an increase of NF-κB in the nucleus.Notably,Bai treatment significantly attenuated ALI induced by intestinal I/R injury.Compared with the I/R group,the lung pathological scores and W/D ratio in the Bai+I/R group were significantly decreased (lung pathological score:4.59±1.17 vs.6.27±1.34,W/D ratio:3.79±0.28 vs.4.32±0.57),cellular apoptosis was significantly decreased [(4.85 ± 2.47)％ vs.(8.15 ± 2.33)％],both serum TNF-α and IL-6 contents and lung TNF-α and IL-6 mRNA expressions were significantly decreased [serum TNF-α (pg/L):124.18±30.49 vs.167.72 ± 38.65,IL-6 (ng/L):1.65 ± 0.69 vs.2.43 ± 0.57;lung TNF-α mRNA (2-△△Ct:4.75 ± 2.38 vs.7.69 ± 2.32,IL-6 mRNA (2-△△ Ct):16.45 ±4.39 vs.27.69 ± 6.82],additionally,Bai pretreatment significantly increased cytoplasmic IκB-α protein expression (gray value:0.47 ± 0.11 vs.0.27 ± 0.09),while decreased nuclear NF-κB protein expression (gray value:0.57 ± 0.13 vs.1.07 ± 0.14,all P ＜ 0.05).Conclusion Bai could attenuate intestinal I/R injury induced ALI via the inhibition of inflammation and apoptosis.
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<p><b>OBJECTIVE</b>To investigate the clinical value of serum anti-Ku86 in early detection of hepatocellular carcinoma (HCC).</p><p><b>METHODS</b>Expression levels of Ku86 protein in HCC and adjacent normal liver tissues were detected by Western blotting. Serum anti-Ku86 level in 83 patients with early HCC and 124 patients with liver cirrhosis were detected by enzyme-linked immunosorbent assay (ELISA). Chemiluminescence was used to measure the serum level of α-fetoprotein (AFP).</p><p><b>RESULTS</b>Expression of Ku86 protein in HCC was increased when compared with the adjacent normal liver tissues (0.21 ± 0.05 vs. 0.08 ± 0.02, P < 0.01). Serum anti-Ku86 level was significantly elevated in HCC patients compared with that in liver cirrhosis patients (0.47 ± 0.22 vs. 0.22 ± 0.06 Abs at 450 nm, P < 0.01), but there was no significant difference between HBV infection and HCV infection in HCC patients (0.51 ± 0.19 vs. 0.47 ± 0.24, P = 0.267). Of note, serum anti-Ku86 level was significantly decreased after surgical resection of the tumors in the 30 HCC cases tested (P < 0.01). The results of ROC analysis indicated a better performance of anti-Ku86 (0.857) than AFP (0.739) for early detection of HCC. In 83 HCC patients, the positive rate of anti-Ku86 was 61.4% (51/83), significantly higher than that of the AFP positive rate (27.7%, 23/83). The anti-Ku86 level was positive in 37 of 60 HCC cases with negative AFP. Combination assay of AFP and anti-Ku86 could detect 60 of 83 HCC cases (72.3%, 60/83). There was no significant correlation of anti-Ku86 and AFP (r = 0.156, P = 0.161).</p><p><b>CONCLUSIONS</b>Serum anti-Ku86 level is significantly elevated and is not related to HBV and HCV infection in HCC patients. Serum anti-Ku86 antibody may be a potential biomarker for early detection of HCC, and can be used in combination with AFP in clinics.</p>