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Objective:To explore the effect and possible mechanism of normal temperature mechanical perfusion(NMP)plus bone marrow mesenchymal stem cells(BMMSCs)on early endoplasmic reticulum stress(ERs)and cellular apoptosis after donation after cardiac death(DCD)donor liver transplantation.Methods:BMMSCs were isolated and cultured by adherence method.Sixty Sprague-Dawley(SD)rats were randomly divided into five groups of sham operation(sham), static cold storage(SCS), NMP, BMMSC and NMP plus BMMSCs(BP)( n=12 each). Liver tissue and serum sample of each group were harvested at Day 1/7 post-operation.Hematoxylin-eosin(HE)staining was employed for observing pathological changes of liver tissue; TdT-mediated dUTP nick end labeling(TUNNEL)staining for detecting cellular apoptosis; immunohistochemistry for detecting the expression of hepatocyte transcription factor C/EBP homologous protein(CHOP); Western blot for detecting the expressions of GRP-78, p-PERK, ATF4, CHOP and cleaved caspase-3. Results:Compared with SCS group, hepatic injury and inflammation significantly declined in NMP, BMMSC and BP groups and improvement of hepatic injury was the most pronounced in BP group.Cellular apoptosis lessened markedly in BP group at Day 1/7 as compared with SCS group and the difference was statistically significant.The expressions of ERs-related proteins GRP-78, p-PERK and ATF4 spiked in SCS group and the expressions of pro-apoptotic proteins CHOP and cleaved caspase-3 were significantly elevated and declined markedly in BP group.And the difference was statistically significant.Conclusions:BMMSCs plus NMP can significantly improve hepatocyte apoptosis and inflammatory response after DCD donor liver transplantation.And its mechanism may be correlated with suppressing early endoplasmic reticulum stress of hepatocytes.
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Objective:To investigate the effect of heme oxygenase-1 (HO-1) modified bone marrow mesenchymal stem cells (BMMSCs) combined with normothermic machine perfusion (NMP) on the intestinal barrier function in rats with acute rejection of liver transplantation.Methods:Specific pathogen free 2 male Brown Norway (BN) rats (4-5 weeks, 40-60 g) were used to isolat BMMSCs, and HO-1 was infected by adenovirus. Of 24 male Lewis rats (7-8 weeks old, 200-220g) were used as donors, 30 male BN rats (8-9 weeks old, 220-240 g) were used as recipients. Acute rejection models of orthotopic liver transplantation were established in rats using two cuff technique. BN recipient rats were randomly divided into five groups: sham group, abdomen of the mice was open and closed within 30 min; NMP livers were simply mechanically perfused for 4 h; the BMP group were perfused with BMMSCs through the portal vein; the HBP group were perfused with HO-1/BMMSCs through the portal vein; the FK506 livers were mechanically perfused for 4 h and administered intragastrically of tacrolimus daily following surgery, 6 per group, on days 14 after surgery, the relevant indicators were taken and the rejection activity index (RAI) changes were investigated. The changes of intestinal pathological were analyzed by HE staining and transmission electron microscope, the expression levels of zonula occludens-1 (ZO-1) and occludin protein in intestinal tissue were detected by Western blotting, the concentrations of lipopolysaccharide, D-lactic acid and diamine oxidase (DAO) in serum were detected by ELISA.Results:The RAI of HBP group (2.80±0.84) and FK506 group (2.20±0.84) were significantly lower than that of NMP group (7.60±1.14) and BMP group (6.00±1.58), the differences were statistically significant (all P<0.05). The intestinal villi in NMP group were significantly sparse, wrinkled and disorderly arranged while the degree of intestinal injury in BMP group, HBP group and FK506 group were more mitigated. Electron microscope observation showed that the microvilli of intestinal epithelial cells in HBP group were rich and orderly, and the tight junction structure between cells was complete. The protein expression levels of ZO-1 and Occludin in the intestinal tissues of HBP group [(0.87±0.06) (1.28±0.26)] were higher than those of NMP group [(0.41±0.12) (0.27±0.18)] and FK506 group [(0.52±0.15) (0.63±0.22)], the differences were statistically significant (all P<0.05). The concentration of lipopolysaccharide, D-lactic acid and DAO in serum of HBP group was lower than those of NMP group and FK506 group, the differences were statistically significant (all P<0.05). Conclusion:HO-1/BMMSCs combined with NMP protects the intestinal mucosal barrier function of BN rats with acute rejection after liver transplantation.
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Objective:To investigate the role of ferroptosis in bone marrow mesenchymal stem cells (BMMSCs) combine with normothermic machine perfusion (NMP) in repairing steatotic liver donor after cardiac death (DCD) in SD rats.Methods:BMMSCs were derived from SD rats to establish the DCD model of rats steatotic liver. A total of 24 rats were randomly divided into four groups: simple steatotic liver model group (Sham), static cold storage group (SCS), NMP, BMMSCs combine with NMP preservation group (BNMP), and the preservation time was 4 hours. The donor liver function was evaluated by liver structure, liver enzymes and lactic acid content of perfusion fluid, bile secretion and inflammatory cytokines; furthermore, in order to evaluate the occurrence of liver ferroptosis, the content of Fe 2+, malondialdehyde and glutathione (GSH) in liver tissue, as well as the mRNA or protein expression changes of cyclooxygenase-2 (COX-2), prostaglandin-endoperoxide synthase 2 (Ptgs2), glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) were detected. Results:After DCD steatotic donor liver was preserved for 4 hours, the liver injury, pro-inflammatory and anti-inflammatory cytokines expression in the BNMP and NMP groups were better than those in the SCS group. During the machine perfusion preservation period, alanine aminotransferase [(189.0±12.5)U/L vs. (227.7±16.2)U/L], aspartate aminotransferase [(207.3±18.6)U/L vs. (247.0±11.8)U/L] and lactic acid [(2.3±0.3)mmol/L vs. (2.9±0.2)mmol/L] in the BNMP group is lower than those in NMP group, moreover, the amount of hepatic bile secretion in the BNMP group [(1 245.7±46.8) μl vs. (1 014.3±67.9) μl] was more than that in NMP group, the difference was statistically significant (all P<0.05). The content of Fe 2+ and malondialdehyde in the liver tissue of BNMP group was significantly lower than those of SCS and NMP groups, on the contrary, the content of GSH was significantly higher than those of SCS and NMP groups. In addition, in the BNMP group, the mRNA level of Ptgs2 and protein level of COX-2 in the liver were significantly reduced, and expression of GPX4 and FTH1 were significantly higher than those of NMP and SCS groups, the differences were statistically significant (all P<0.05). Conclusion:BMMSCs combine with normothermic machine perfusion can better repair SD rats DCD steatotic donor liver and its mechanism of action may be related to its regulation on liver ferroptosis.
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Dendritic cells are the key cells to regulate immunity. Tolerogenic dendritic cells (Tol-DC) with immunosuppressive function play an important role in inducing and maintaining immune tolerance. In recent years, a class of liver Tol-DC with low level of presenting antigen and inhibition of T cell activation and proliferation has become a research hotspot to reduce the rejection of liver transplantation. At the same time, there are many strategies to induce phenotypic stable Tol-DC, which lays a foundation for its clinical application. This article introduces the current understanding of liver Tol-DC and its mechanism of regulating the immunity of liver transplantation, in order to provide new ideas for immunotherapy of liver transplantation.
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Objective@#To study the effect of benazepril on the expression of nuclear factor E2 related factor 2 (Nrf2), nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) and reactive oxygen species (ROS) concentration in rats with hepatic fibrosis and to explore the possible antifibrotic mechanism of benazepril.@*Methods@#Twenty-two healthy male Sprague-Dawley rats were randomly divided into 3 groups: control group (6 rats), model group (8 rats) and benazepril treatment group (8 rats). Two rats died during modeling and treatment in the model group and the benazepril treatment group, and a model of hepatic fibrosis induced by carbon tetrachloride (CCL4) was established. The rats in benazepril group were given benazepril for 8 weeks by gastric gavage. The assessment of liver tissue damage in each group was measured using conventional hematoxylin-eosin and Masson staining. The mRNA level of Nrf2, NOX4 in liver tissue was detected by RT-PCR, and serum ROS concentration was determined by colorimetry. All data were expressed in mean ± standard deviations, and were analyzed using SPSS21.0 statistical software. The data were compared using one-way analysis of variance, and the LSD-t method was used for pairwise comparison between the two groups. The correlation analysis was performed by Spearman’s correlation analysis.@*Results@#In the liver of the model group, with the aggravation of liver fibrosis the expression of Nrf2mRNA, NOX4 mRNA and ROS concentration were higher than control group [(4.01 ± 3.40), (31.78 ± 3.96), (1.82 ± 0.46) μg/ ml vs. (0.12 ± 0.11), (2.03 ± 0.31), (1.56±0.84) μg/ml, P < 0.05]. After benazepril treatment, NOX4 mRNA expression and ROS concentration were decreased than the model group [(15.93 ± 5.01), (0.78 ± 0.44) μg/ml vs. (31.78 ± 3.96), (1.82 ± 0.46) μg /ml, P < 0.05], while Nrf2 mRNA expression was higher than the model group [(6.69 ± 4.86) vs. (4.01 ± 3.40), P < 0.05]. There was a positive correlation between Nrf2 and NOX4, Nrf2 and ROS, and NOX4 and ROS (r = 0.616, 0.411, 0.802, P < 0.05).@*Conclusion@#Benazepril may exert an anti-hepatic fibrosis effect by activating Nrf2 expression, or may inhibit the ROS-mediated oxidative stress in response to NOX4.
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Objective@#To investigate the effects of angiotensin II type 1 receptor antagonist valsartan on leptin, leptin receptor and collagen in rats with hepatic fibrosis.@*Methods@#Thirty-six male wistar rats were randomly divided into control group, model group and drug-treated group, with 12 rats in each group. Liver fibrosis models were made by subcutaneous injection of carbon tetrachloride on the dorsal of the rats, simultaneously gastric gavage with Valsartan and were killed at the end of 8th week. The degree of liver fibrosis was observed by HE and Masson staining. The serum leptin (LP) and TGFβ1 were determined by ELISA. Liver LP mRNA and leptin receptor mRNA (OB-R mRNA) were detected by RT-PCR. Liver LP, OB-R and collagen I were detected by Western blot. The data of multiple groups were analyzed by one-way analysis variance (ANOVA), and linear correlation was performed between serum LP and TGF β1.@*Results@#After the intervention of valsartan, HE and Masson staining showed that the degree of liver fibrosis was significantly reduced. The levels of serum LP and TGFβ1 in the control group were (18.92 ± 7.10) ng/ml and (9.13 ± 1.58) pg/ml respectively, which were significantly lower than those in the model group (46.92 ± 28.54) ng/ml and (16.39 ± 3.56) pg/ml, And (29.27 ± 7.27) ng/ml and (12.24 ± 2.94) pg/ml in the drug-treated group, respectively. The F values were 7.864 and 20.057 respectively. The P values were < 0.05. The differences were statistically significant. The relative expression levels of LP and OB-R mRNA in the control group were 0.35 ± 0.18 and 0.62 ± 0.18, respectively, which were significantly lower than those in the model group (1.79 ± 1.79 and 1.52 ± 1.44, and drug-treated group 0.48 ± 0.34 and 0.75 ± 0.26, respectively), F values = 6.914,3.894, P values were < 0.05, the differences were statistically significant. The relative expression levels of LP, OB-R and collaten I in liver were 0.71 ± 0.13, 0.81 ± 0.11 and 0.76 ± 0.13 in the model group, 0.97 ± 0.06, 1.04 ± 0.06, and 1.05 ± 0.04 respectively in the drug-treated group and 0.74 ± 0.05, 0.93 ± 0.05 and 0.91 ± 0.05. The F values were 15.425, 13.757 and 19.130 respectively in three groups (P < 0.001), the difference was statistically significant.@*Conclusion@#Valsartan, an angiotensin II type 1 receptor antagonist, can reduce the expression of leptin and leptin receptor, reduce the production of TGFβ1 and collaten I, and play an anti-hepatic fibrosis effect.