Hepatic Ischemia-reperfusion Injury Group Research Articles

Exosome-related gene identification and diagnostic model construction in hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (HIRI) may cause severe hepatic impairment, acute hepatic insufficiency, and multiorgan system collapse. Exosomes can alleviate HIRI. Therefore, this study explored the role of exosomal-related genes (ERGs) in HIRI using bioinformatics to determine the underlying molecular mechanisms and novel diagnostic markers for HIRI. We merged the GSE12720, GSE14951, and GSE15480 datasets obtained from the Gene Expression Omnibus (GEO) database into a combined gene dataset (CGD). CGD was used to identify differentially expressed genes (DEGs) based on a comparison of the HIRI and healthy control cohorts. The impact of these DEGs on HIRI was assessed through gene set enrichment analysis (GSEA) and gene set variation analysis (GSVA). ERGs were retrieved from the GeneCards database and prior studies, and overlapped with the identified DEGs to yield the set of exosome-related differentially expressed genes (ERDEGs). Functional annotations and enrichment pathways of these genes were determined using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. Diagnostic models for HIRI were developed using least absolute shrinkage and selection operator (LASSO) regression and support vector machine (SVM) algorithms. Key genes with diagnostic value were identified from the overlap, and single-sample gene-set enrichment analysis (ssGSEA) was conducted to evaluate the immune infiltration characteristics. A molecular regulatory interaction network was established using Cytoscape software to elucidate the intricate regulatory mechanisms of key genes in HIRI. Finally, exosome score (Es) was obtained using ssGSEA and the HIRI group was divided into the Es_High and Es_Low groups based on the median Es. Gene expression was analyzed to understand the impact of all genes in the CGD on HIRI. Finally, the relative expression levels of the five key genes in the hypoxia-reoxygenation (H/R) model were determined using quantitative real-time PCR (qRT-PCR). A total of 3810 DEGs were identified through differential expression analysis of the CGD, and 61 of these ERDEGs were screened. Based on GO and KEGG enrichment analyses, the ERDEGs were mainly enriched in wound healing, MAPK, protein kinase B signaling, and other pathways. GSEA and GSVA revealed that these genes were mainly enriched in the TP53, MAPK, TGF[Formula: see text], JAK-STAT, MAPK, and NFKB pathways. Five key genes (ANXA1, HNRNPA2B1, ICAM1, PTEN, and THBS1) with diagnostic value were screened using the LASSO regression and SVM algorithms and their molecular interaction network was established using Cytoscape software. Based on ssGSEA, substantial variations were found in the expression of 18 immune cell types among the groups (p < 0.05). Finally, the Es of each HIRI patient was calculated. ERDEGs in the Es_High and Es_Low groups were enriched in the IL18, TP53, MAPK, TGF[Formula: see text], and JAK-STAT pathways. The differential expression of these five key genes in the H/R model was verified using qRT-PCR. Herein, five key genes were identified as potential diagnostic markers. Moreover, the potential impact of these genes on pathways and the regulatory mechanisms of their interaction network in HIRI were revealed. Altogether, our findings may serve as a theoretical foundation for enhancing clinical diagnosis and elucidating underlying pathogeneses.

Bone Marrow Mesenchymal Stem Cell Extracellular Vesicle-derived miR-27b- 3p activates the Wnt/Β-catenin Pathway by Targeting SMAD4 and Aggravates Hepatic Ischemia-reperfusion Injury.

To investigate the roles of extracellular vesicles (EVs) secreted from bone marrow mesenchymal stem cells (BMSCs) and miR-27 (highly expressed in BMSC EVs) in hepatic ischemia‒ reperfusion injury (HIRI). We constructed a HIRI mouse model and pretreated it with an injection of agomir-miR-27-3p, agomir-NC, BMSC-EVs or control normal PBS into the abdominal cavity. Compared with the HIRI group, HIRI mice preinjected with BMSC-EVs had significantly decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels and alleviated liver necrosis (P<0.05). However, compared with HIRI+NC mice, HIRI+miR-27b mice had significantly increased ALT and AST levels, aggravated liver necrosis, and increased apoptosis-related protein expression (P<0.05). The proliferation and apoptosis of AML-12 cells transfected with miR-27 were significantly higher than the proliferation and apoptosis of AML-12 cells in the mimic NC group (P<0.01) after hypoxia induction. SMAD4 was proven to be a miR-27 target gene. Furthermore, compared to HIRI+NC mice, HIRI+miR-27 mice displayed extremely reduced SMAD4 expression and increased levels of wnt1, β-catenin, c-Myc, and Cyclin D1. Our findings reveal the role and mechanism of miR-27 in HIRI and provide novel insights for the prevention and treatment of HIRI; for example, EVs derived from BMSCs transfected with antimiR- 27 might demonstrate better protection against HIRI.

The protective effect of Dogwood preparation on hepatic ischemia–reperfusion injury in mice by down-regulating PTGS2

Hepatic ischemia - reperfusion injury (HIRI) is a major cause of postoperative complications and mortality after hepatobiliary surgery, but there is currently no effective treatment strategy. Dogwood is a valuable Chinese Medicine which helps to protect the liver. Our animal experiments showed that ethanol extract (DD) of Dogwood could significantly lower the levels of Aspartate aminotransferase (AST) and Alanine aminotransferase (ALT) in HIRI mice serum. The findings revealed that DD had protective effect against HIRI in mice. Furthermore, we used network pharmacology to screen 613 target genes corresponding to Loganin, Ursolic acid and Oleanolic acid of the main active components contained in DD and 1117 target genes corresponding to HIRI, with 87 common target genes among them. The STRING database was then used to generate a protein–protein interaction (PPI) dataset of 87 target genes, which was then imported into Cytoscape3.7.2 to generate the top 10 target genes using the MCC function of cytoHubba. Next, using the DAVID online database, 87 target genes were assigned to 35 KEGG pathways. Pathways in cancer had 17 target genes with the highest score. The top 10 target genes identified by MCC function were cross-referenced with 17 target genes identified by Pathways in cancer. HSP90AA1, MAPK3, PTGS2, MMP2, IL2, PPARG, MTOR and IL6 were identified as target genes. In the meantime, transcriptomic sequencing was performed, and a total of 165 differential genes between drug administration group and HIRI group were identified. At last PTGS2 is locked as the hub target gene of DD intervention in HIRI. Following that, immunohistochemistry and real-time fluorescence quantitative PCR were used to verify the results. The results showed that DD protected mice against HIRI by lowering the expression level of PTGS2. In conclusion, DD may protect the mice against HIRI by regulation of PTGS2 expression.

Cordycepin Protects against Hepatic Ischemia/Reperfusion Injury via Inhibiting MAPK/NF-κB Pathway.

Hepatic ischemia/reperfusion injury (HIRI) is a common complication of liver surgery requiring hepatic disconnection, such as hepatectomy and liver transplantation. The aim of this study was to investigate the effects of cordycepin on HIRI and to elucidate the underlying mechanisms. Balb/c mice were randomly divided into six groups: a normal control group, sham group, H-cordycepin group, HIRI group, L-cordycepin (25 mg/kg) + HIRI group, and H-cordycepin (50 mg/kg) + HIRI group. Mice were subjected to I/R, and cordycepin was intragastrically administered for seven consecutive days before surgery. Orbital blood and liver specimens were collected at 6 and 24 h after HIRI. Serum levels of ALT and AST were decreased in the cordycepin pretreatment groups. Notably, cordycepin attenuated the inflammatory response and the production of proapoptosis proteins, while increasing expression of antiapoptosis proteins and decreasing expression of autophagy-linked proteins. Furthermore, cordycepin inhibited activation of the MAPK/NF-κB signaling pathway. Collectively, these results indicate that cordycepin pretreatment ameliorated hepatocyte injury caused by HIRI. As compared with the HIRI group, cordycepin pretreatment mitigated the inflammatory response and inhibited apoptosis and autophagy via regulation of the MAPK/NF-κB signaling pathway.

The Protective Effect of Panax notoginseng Mixture on Hepatic Ischemia/Reperfusion Injury in Mice via Regulating NR3C2, SRC, and GAPDH.

Panax notoginseng mixture (PNM) has the characteristics of multicomponent, multitarget, and multieffect, which can cope with the multidirectional and multidimensional complex pathological process caused by hepatic ischemia/reperfusion injury (HIRI). Our animal experiments showed that PNM composed of notoginseng, dogwood, and white peony root could significantly reduce the level of aspartate transaminase and alanine aminotransferase in the blood of mice with HIRI, indicating that this preparation had a protective effect on HIRI in mice. Therefore, on this basis, the molecular mechanism of PNM intervention in HIRI was further explored by network pharmacology. First, target genes corresponding to active components and HIRI were obtained through databases such as TCMSP, Pharm Mapper, Swiss Target Prediction, GeneCards, and so on. All target genes were standardized by Uniprot database, and a total of 291 target genes with their intersection were obtained. Then, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and biological processes (BPs) of 291 target genes were obtained through the online public platform of DAVID. A total of 177 KEGG pathways and 337 BPs were obtained by setting p < 0.01 and false discovery rate <0.05. The network mapping map of components and disease targets was drawn by Cytoscape, and the top 10 Hub target genes related to HIRI were obtained. At the same time, the String database was used to obtain the protein–protein interaction dataset, which was imported into Cytoscape, and the first 10 Hub target genes were obtained. The Hub target genes obtained by the above two methods were molecular docking with their corresponding small molecule compounds through DockThor online tool. The results showed that the docking of paeoniflorin with glyceraldehyde 3-phosphate dehydrogenase (GAPDH), paeoniflorin and loganin with SRC, ginsenoside Rb1 with NR3C2, ursolic acid and oleanolic acid with IL-6, paeoniflorin docking VEGFA, and MMP9. Finally, NR3C2, SRC, and GAPDH were identified as target genes in this study by referring to relevant literature reports. After verification by immunohistochemical experiments, compared with the sham group, the above three target genes were highly expressed in the HIRI group (p < 0.01). Compared with the HIRI group, the expression of three target genes in the PNM + HIRI group was significantly decreased (p < 0.01). The results showed that PNM could protect mouse HIRI by decreasing the expression of NR3C2, SRC, and GAPDH.

The protective effect of glycyrrhizin on hepatic ischemia-reperfusion injury in rats and possible related signal pathway.

Objective(s):To investigate the protective effect of glycyrrhizin (GL) on hepatic ischemia-reperfusion injury (HIRI).Materials and Methods:Forty SD rats were randomly divided into sham group, HIRI group, GL 100 mg/kg group, and GL 200 mg/kg group. The pathological alterations of liver tissue in each group were observed. The levels of alanine transaminase (ALT), aspartate aminotransferase (AST), endothelin-1 (ET-l), nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) were detected. Western blot was used to detect the expression levels of cytoplasmic protein caspase-3, Bax, Bcl-2, heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor 2 (Nrf2), and nuclear protein Nrf2.Results:Compared with the HIRI group, the levels of AST, ALT, ET-1, TNF-α, IL-1β, and IL-6 in GL groups were lower, serum NO content was higher, MDA content was lower, SOD and GSH-Px activities were significantly increased, apoptosis index was lower (P<0.05), which was more obvious in high-dose GL (200 mg/kg) group. The LC3-II/LC3-I ratio and Beclin-1 protein expression levels in the GL group were significantly lower than the HIRI group, but the expression levels of cytoplasmic protein HO-1 and nuclear protein Nrf2 were significantly higher than those of the HIRI group, which was more obvious in the high-dose GL group (P<0.05).Conclusion:GL has a protective effect on the liver of HIRI rats, and its mechanism may be related to activation of the Nrf2/HO-1 signaling pathway, inhibition of oxidative stress, inflammation, autophagy, and apoptosis.

Role of Remote Ischemic Preconditioning in Hepatic Ischemic Reperfusion Injury.

The effect of remote ischemic preconditioning (RIPC) has been proposed that mediates the protective response in ischemia reperfusion injury (IRI) of various organs. In this study, we investigated the effect of RIPC in hepatic IRI, by assessing biomarker of oxidative stress and inflammatory cytokines. Moreover, we intended to demonstrate any such protective effect through nitric oxide (NO). Twenty-five rats were divided into the 5 groups: (1) Sham; (2) RIPC; (3) hepatic IRI; (4) RIPC + hepatic IRI; (5) C-PTIO, 2-(4-carboxyphenyl)-4,5dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3oxide, + RIPC + hepatic IRI. RIPC downregulated the level of aspartate aminotransferase (AST), alanine aminotransferase (ALT), histologic damage, and activity of Malondialdehyde (MDA). However, there was no significant reduction in the level of tumor necrosis factor-alpha (TNF-α) and nuclear factor kappa B (NF-κB). AST and ALT levels, and hepatic tissue morphology in the C-PTIO group showed a significant improvement compared to those of the RIPC + hepatic IRI group. The application of RIPC before hepatic ischemia downregulated the oxidative stress, not the inflammatory cytokines. Moreover, these protective effect of RIPC would be mediated through the activation of NO as well as anti-oxidant effect.

Exosomes Mediate Hippocampal and Cortical Neuronal Injury Induced by Hepatic Ischemia-Reperfusion Injury through Activating Pyroptosis in Rats.

Background The neuronal injury and cognitive dysfunction after liver transplantation have severe effects on the prognosis and life quality of patients. Accumulating evidence suggests that both exosomes and pyroptosis could participate in hepatic ischemia-reperfusion injury (HIRI) and play key roles in neuronal death. However, the link between exosomes and neuronal pyroptosis in HIRI awaits further investigation. Methods After establishing the HIRI rat models, we primarily studied the role of pyroptosis in hippocampal and cortical neuron injury through detecting NOD-like receptor protein 3 (NLRP3), pro-caspase-1, cleaved-caspase-1, apoptosis-associated speck-like protein containing CARD (ASC), gasdermin D (GSDMD), interleukin-1beta (IL-1β), and interleukin-18 (IL-18) expressions with western blotting, immunohistochemical staining, and enzyme-linked immunosorbent assay (ELISA). Then, we intravenously injected normal male rats with exosomes isolated from the sera of HIRI-challenged rats and pretreated rats with MCC950, a specific inhibitor of NLRP3, and carried out the same assay. We also detected the levels of reactive oxygen species (ROS), superoxide dismutase (SOD), and malondialdehyde (MDA) in the hippocampal and cortical tissues. Results The results indicated that NLRP3 inflammasome and caspase-1-dependent pyroptosis were activated in the hippocampus and cortex of HIRI rats. Furthermore, serum-derived exosomes from HIRI-challenged rats not only had the ability to cross the blood-brain barrier (BBB) but also had the similar effects on neuronal pyroptosis. Moreover, ROS and MDA productions were induced in the HIRI and exosome-challenged groups. In addition, to some degree, MCC950 could alleviate HIRI-mediated hippocampal and cortical neuronal pyroptosis. Conclusion This study experimentally demonstrated that circulating exosomes play a critical role in HIRI-mediated hippocampal and cortical injury through regulating neuronal pyroptosis.

Effect of NLRP3 in liver tissues of mice with hepatic ischemia-reperfusion injury

Objective According investigate the expression of NLRP3 in liver tissues of mice with hepatic ischemia-reperfusion injury (HIRI), to determin the role of NLRP3 in the process of HIRI. Methods Established mice model of partial HIRI. Forty-two male C57BL/6 mice (aged 7 to 8 weeks, weight 20 to 25 g) were respectively divided into 7 groups: no-treatment control group, sham operation group, HIRI groups (2、6、12、24 h) and CY-09 group, 6 mice in each group. The injury of the hepatic tissues in the 7 groups was analyzed based on detecting the levels of alanine transaminase (ALT), aspartate transaminase (AST), interleukin-1β (IL-1β), interleukin-18 (IL-18), tumor necrosis factor-α (TNF-α) by ELISA. HE and TUNEL staining were used to observe the pathological changes of liver tissues after HIRI. Western blotting assay were carried out to detect the expressions of NLRP3 and Caspase-1. Measurement data were expressed as mean±standard deviation (Mean±SD), and one-way variance analysis was used for comparison between groups. If the variance was not uniform, Dunnett C test was used. Results Serum ALT, AST, IL-1β, IL-18 and TNF-α of mice detected in HIRI groups were higher than no-treatment control group and sham operation group at all endpoints (P<0.05). The relative expression of NLRP3 and Caspase-1 in the liver tissues of mice in the HIRI groups were significantly higher than that in the no-treatment control group and sham operation group. Serum ALT, AST, IL-1β, IL-18 and TNF-α of mice detected in CY-09 group were lower than HIRI groups at all endpoints (P<0.05). Less hepatocellular necrosis were exhibited in CY-09 group, comparing to HIRI groups. The hepatocyte apoptosis rate of mice in the CY-09 group was significantly lower than that in the 12 h HIRI group (P<0.05). The relative expression of NLRP3 in the liver tissues of mice in the CY-09 group was significantly lower than that in other groups. The relative expression of Caspase-1 in the liver tissues of mice in the CY-09 group was significantly lower than that in other groups except the no-treatment control group and sham operation group. Conclusions HIRI cause an increase in NLRP3 expression. The inhibition of NLRP3 can reduce HIRI. Key words: Liver diseases; Reperfusion injury; Ischemia; Mice; NLRP3

The value of many non-gaussian distribution models diffusion weighted imaging in evaluating the model of hepatic ischemia reperfusion injury model in rats

Objective To explore the value of multiple non-gaussian distribution models DWI in evaluating hepatic ischemia reperfusion injury (HIRI) model in rats. Methods Forty-two SD rats were divided into 7 groups by random numeric table method. Each group had 6 rats. The 7 groups were 6 h, 12 h, 1 d, 3 d, 7 d, 14 d group after HIRI and control group, respectively. The experimental groups underwent right hepatic portal vein and hepatic artery ligation, and received reperfusion 3 h after operation. MRI scanning (T1WI, T2WI, single b-values DWI and 15 b-values DWI) was performed at 6 h, 12 h, 1 d, 3 d, 7 d and 14 d after reperfusion. The control group underwent sham operation and MRI scanning. According to monoexponential model, biexponential model, threxponential model, stretched-exponential model DWI and diffusion kurtosis imaging, many parametres were obtained and their dynamic changes at each time point were observed. The parameters included standard apparent diffusion coefficient (ADCs), pure diffusion coefficients (D), pseudodiffusion coefficients (D*), perfusion fraction (f), ultra-high apparent diffusion coefficient (ADCu), distributed diffusion coefficient (DDC), water diffusion heterogeneity index (α), mean diffusion coefficient (MD) and mean diffusion kurtosis (MK). One way ANOVA was used to compare the differences of parameters among different groups. Results MRI examination and pathological examination were successfully completed in all rats. The right hepatic lobe in the experimental groups appeared hypointense on T1WI and hyperintense on T2WI relative to control group from 6 h after operation. The infarcted liver lobe in the experimental groups became significantly smaller at 1 week after HIRI and almost disappeared at 2 weeks after HIRI. The findings of DWI at different b-values were consistent with those at T2WI. There were significant differences in parameters among 6 h, 12 h, 1 d, 3 d, 7 d, 14 d groups after HIRI and control group (P<0.05). There were significant differences among 12 h after HIRI, 1 d after HIRI, 3 d after HIRI and 6 h after HIRI, 7 d after HIRI, 14 d after HIRI, control group concerning ADCs values respectively (P<0.05). There were significant differences among 6 h after HIRI, 12 h after HIRI, l d after HIRI, 3 d after HIRI, 7 d after HIRI and 14 d after HIRI, control group concerning D, D*, f, ADCu, α, DDC, MK values respectively (P<0.05). In addition, there were significant differences among 1 d after HIRI, 3 d after HIRI and 6 h after HIRI, 12 h after HIRI, 7 d after HIRI and 14 d after HIRI, control group concerning D values respectively (P<0.05). There were significant differences among 6 h after HIRI, 12 h after HIRI, 1 d after HIRI, 3 d after HIRI and 7 d after HIRI, 14 d after HIRI, control group concerning f values respectively (P<0.05). There were significant differences among 12 h after HIRI and 14 d after HIRI, control group concerning MD values respectively (P<0.05). There were significant differences between 1 d after HIRI and 14 d after HIRI concerning MD values (P<0.05). Conclusion Multiple non-gaussian distribution models DWI is superior to conventional DWI in evaluating HIRI model in rats. Key words: Liver; Ischemia reperfusion injury; Non-gaussian distribution model; Multi b-values; Magnetic resonance imaging

Protective effects of rosiglitazone on hepatic ischemia reperfusion injury in rats

To explore the protective effect of rosiglitazone (RGZ) on hepatic ischemia reperfusion injury (HIRI) and the underlying mechanisms. Methods: A rat model of ischemia-reperfusion injury was established by clamping the left and middle lobe of liver with noninvasive vascular clamp. A total of 30 Sprague-Dawley rats were randomly divided into a sham group, an HIRI group, and a RGZ group (10 rats in each group). Two hours after reperfusion, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, lactate dehydrogenase (LDH) level, malondialdehyde (MDA) content and catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) activities were examined. HE staining was used to observe liver pathological morphology. The liver peroxisome proliferators-activated receptor γ (PPAR-γ), p-PPAR-γ, nuclear factor related factor 2 (Nrf-2), antioxidant response element (ARE), heme oxygenase 1 (HO-1) and quinone oxidoreductase-1 (NQO-1) were detected by Western blot. Results: Compared with the HIRI group, the levels of ALT, AST, LDH and MDA in the RGZ group were significantly decreased (all P<0.05), while the levels of Nrf-2, ARE, HO-1 and NQO-1 in the RGZ group were significantly increased. The hepatic swelling, necrosis and pathological damage were decreased (all P<0.05). In addition, there was no difference in the level of PPAR-γ between the 2 groups (P>0.05). Conclusion: PPAR-γ agonist RGZ can attenuate HIRI, which may be related to activating Nrf2/ARE signaling pathway and enhancement of antioxidant ability.

Effects of propofol pretreatment on myocardial cell apoptosis and SERCA2 expression in rats with hepatic ischemia/reperfusion

IntroductionHepatic ischemia-reperfusion injury is a common pathophysiological process in liver surgery. Whether Propofol can reduce myocardial ischemia-reperfusion injury induced by hepatic ischemia-reperfusion injury in rats, together with related mechanisms, still needs further studies. ObjectiveTo investigate if propofol would protect the myocardial cells from apoptosis with hepatic ischemia-reperfusion injury. MethodsMale Sprague-Dawley rats (n=18) were randomly allocated into three groups: Sham Group (Group S, n=6), Hepatic Ischemia-reperfusion Injury Group (Group IR, n=6) and Propofol Group (Group P, n=6). Group S was only subjected to laparotomy. Group IR was attained by ischemia for 30min and reperfusion for 4h. Group P was subjected identical insult as in Group IR with the administration of propofol started 10min before ischemia with 120mg.kg−1, following by continuous infusion at 20mg.kg−1.h−1. Cell apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay. Endoplasmic reticulum Ca2+-ATPase2 (SERCA2) and cysteine-containing aspartic acid cleaved-caspase3 (cleaved-caspase3) were assayed by western blot and Altimeter polymerase chain reaction. ResultsApoptosis rate was increased, with mRNA and protein of SERCA2 down-regulated and cleaved-caspase3 up-regulated in Group IR compared with Group S (p<0.01). Apoptosis rate was decreased, with mRNA and protein of SERCA2 up-regulated and cleaved-caspase3 down-regulated in Group P compared with Group IR (p<0.01). ConclusionsPropofol can reduce hepatic ischemia-reperfusion injury-induced myocardial cell apoptosis, meanwhile, can up-regulate mRNA and protein of SERCA2 in rats.

Role of short-term starvation in alleviating hepatic ischemia-reperfusion injury in mice and possible mechanism of action

Objective: To investigate the role of short-term starvation (STS) in alleviating hepatic ischemia-reperfusion injury in mice and possible mechanism of action. Methods: Wild-type male C57BL/6 mice aged 8 weeks were randomly divided into 75% hepatic ischemia-reperfusion injury group (IR group), STS+75% hepatic ischemia-reperfusion injury group (STS group), and sirtinol+STS+75% hepatic ischemia-reperfusion injury group (SIR group), using a random number table, and sham-operation groups (IR-Sham group, STS-Sham group, and SIR-Sham group) were also established. The serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured, and the histomorphological changes of the liver were observed, as well as the expression of Sirt1, LC3B, and P62 proteins in liver tissue and the results of LC3B fluorescence staining. An analysis of variance was used for comparison of data between multiple groups, and the t-test was used for comparison of data between two groups. Results: Compared with the IR group, the STS group had significant reductions in the serum levels of ALT (3 152.7 ± 735.6 U/L vs 8 414.2 ± 1 052.2 U/L, P < 0.01) and AST (3 577.0 ± 714.0 U/L vs 10 845.8 ± 1 145.7 U/L, P < 0.01) and significant alleviation of liver pathological injury (Suzuki score: 1.50±0.55 vs 3.50±0.55, P < 0.01). Compared with the STS group, the SIR group had significant increases in the serum levels of ALT (7 002.7 ± 1 485.2 U/L vs 3 152.7 ± 735.6 U/L, P < 0.01) and AST (8 980.7 ± 1 739.1 U/L vs 3 577.0 ± 714.0 U/L, P < 0.01) and significant exacerbation of liver pathological injury (Suzuki score: 3.33 ± 0.52 vs 1.50 ± 0.55, P < 0.01). Compared with the IR group and the IR-Sham group, the STS group and the STS-Sham group had significant increases in the mRNA and protein expression of Sirt1 and the protein expression of LC3B and a significant reduction in the protein expression of P62, as well as a significant increase in the percentage of LC3B-positive cells in liver tissue (22.83% ± 5.19% / 22.17% ± 4.83% vs 10.16% ± 3.06% / 10.83% ± 1.94%, both P < 0.01). Compared with the STS group and the STS-Sham group, the SIR group and the SIR-Sham group had significant reductions in the expression of Sirt1 and LC3B proteins and a significant increase in the expression of P62 protein, as well as a significant reduction in the percentage of LC3B-positive cells in liver tissue (11.83% ± 9.24% / 14.67% ± 4.68% vs 22.83% ± 5.19% / 22.17% ± 4.83%, both P < 0.01). Conclusion: STS can effectively alleviate hepatic ischemia-reperfusion injury, and its protective effect may be associated with increasing the expression of Sirt1, inducing and promoting hepatocyte autophagy, and reducing hepatocyte death.

The influence and mechanism research of bamboo leaf flavone on hepatic ischemia- reperfusion injury in rats

Objective To explore the protective effect of bamboo leaf flavone in hepatic ischemia-reperfusion injury (HIRI). Methods SD rats were divided into the sham group, the HIRI group and the bamboo leaf flavone intervention group. Rats in bamboo leaf flavone intervention group received bamboo leaf flavone (30 mg/kg) by intraperitoneal injection for 14 consecutive days before establishing the model of hepatic ischemia-reperfusion. Rats in the sham group and the HIRI group received the same volume normal saline. The serum alanine aminotransferase (ALT) and aspertate aminotransferase (AST) were detected. The serum IL-1, IL-6 were detected by ELISA. The expression of caspase-3 in liver tissue was detected by the Western Blot. Results Compared to the HIRI group, the ALT (215.31 ± 52.21 U/L vs. 343.11 ± 78.32 U/L), AST (501.59 ± 70.13 U/L vs. 863.97 ± 63.58 U/L), IL-1 (315.30 ± 88.64 U/L vs. 596.85 ± 102.34 U/L), IL-6 (106.75 ± 10.25 U/L vs. 183.64 ± 20.69 U/L) in the bamboo leaf flavone intervention group significantly decreased (P<0.05). Compared to the HIRI group, expression of caspase-3 (0.38 ± 0.05 vs. 0.86 ± 0.09) in the bamboo leaf flavone intervention group significantly decreased (P<0.05). Conclusions Bamboo leaf flavone pretreatment could reduce HIRI and the mechanism may be associated with inhibiting the expression of caspase-3 protein. Key words: Reperfusion injury; Bamboo leaf flavone; Caspase-3; Rats

Sulforaphane reduces apoptosis and oncosis along with protecting liver injury-induced ischemic reperfusion by activating the Nrf2/ARE pathway.

The purpose of this study was to determine the effect of sulforaphane (SFN) on hepatic ischemia reperfusion injury (HIRI) and to explore the underlying mechanisms. The rat model of HIRI was established. Thirty-two rats were randomly divided into sham (A), SFN (B), HIRI (C), and SFN+HIRI (D) groups. Animals in the HIRI and Sham groups were treated with equal volumes of the vehicle of SPF. Liver functions were evaluated by serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Liver samples were collected for histological examination and determination of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and glutathione peroxidase (GSH-Px) levels. Mitochondrial Na(+)-K(+)-ATPase and Ca(2+)-ATPase were measured by colorimetry. Expression levels of NQO1, Nrf2, and HO-1 in liver tissue were detected by western blot analysis. Additionally, oncosis and apoptosis were detected by Annexin V-FITC/PI immunofluorescent flow cytometry analysis. The HIRI group showed a significant increase in serum levels of liver enzymes (ALT, AST) associated with histopathological damage in the liver. Pre-treatment with SFN could reduce the levels of MDA and MPO in liver tissue and improve the activities of SOD, GSH, GSH-Px, and mitochondrial Na(+)-K(+)-ATPase and Ca(2+)-ATPase in liver tissue. Moreover, SFN could still increase the expression of NQO1, Nrf2, and HO-1 in liver tissue and decrease the oncosis and apoptosis of liver cells. Pre-treatment with SFN could attenuate HIRI via the activation of Nrf2/ARE signaling pathways, ameliorate oxidative stress, and maintain the normal activities of Na(+)-K(+)-ATPase and Ca(2+)-ATPase, thus reducing the occurrence of cell oncosis and apoptosis. Therefore, SFN can be considered a potential candidate as an anti-ischemic medication to minimize HIRI.

Role of protein kinase C in reduction of hepatic ischemia-reperfusion injury by CO2 preconditioning in rats

Objective To investigate the role of protein kinase C (PKC) in reduction of hepatic ischemiareperfusion injury by CO2 preconditioning in rats.Methods Forty-eight male Wistar rats,aged 8-10 weeks,weighing 230-270 g,were randomly divided into 3 groups (n =16 each):hepatic ischemia-reperfusion injury group (group HIRI),CO2 preconditioning group (group P),and c helerythrine (CHE,a specific inhibitor of PKC) group (group CHE).The portal vein,hepatic artery and bile duct of the left lateral and median lobes of the liver were occluded for 1 h,followed by 4 h reperfusion in anesthetized rats.The rats inhaled 50％ O2-50％ N2 for 1 h during mechanical ventilation in group HIRI.In P group,the rats inhaled 50％ O2-45％ N2-5％ CO2 for 1 h during mechanical ventilation and then inhaled 50％ O2-50％ N2 and the hepatic ischemia-reperfusion injury was performed 15 min later.In group CHE,CHE 5 mg/kg was injected intraperitoneally at 10 min before mechanical ventilation,and the other procedures were similar to those previously described in P group.Before mechanical ventilation,immediately before ischemia,and at 0,1,2,3 and 4 h of reperfusion,mean arterial pressure (MAP) was recorded and arterial blood samples were obtained for blood gas analysis.At 4 h of reperfusion,the serum aspartate amino transferase (AST) and alanine amino-transferase (ALT) activities and tumor necrosis factor-α (TNF-α) concentration (by ELISA) were determined and hepatic specimens were obtained for detection of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity (by spectrophotometry),and the expression of activated caspase-3 (by immuno-histochemistry) and PKC (by Western blot) in hepatic tissues.Apoptosis index was calculated by using TUNEL.Results Compared with group HIRI,MAP,PaO2 and PaCO2were significantly increased immediately before ischemia and during reperfusion in group P,MAP and PaCO2 were increased during reperfusion and PaO2 was increased immediately before ischemia and during reperfusion in group CHE,the serum ALT and AST activities,TNF-α concentrations,MDA content and apoptosis index were decreased,and the expression of activated caspase-3 was down-regulated in P and CHE groups,and the SOD activity was increased,and the expression of PKC was up-regulated in group P (P ＜ 0.05 or 0.01),and no significant changes were found in the SOD activity and PKC expression in CHE group (P ＞ 0.05).Compared with group P,MAP was significantly increased immediately after onset of reperfusion,while decreased at 1-4 h of reperfusion,PaO2 was decreased immediately before ischemia and during reperfusion,PaCO2 was decreased at 3 h of reperfusion,the serum ALT and AST activities,TNF-α concentrations,MDA content and apoptosis index were increased,and the expression of activated caspase-3 was up-regulated,and the expression of PKC was downregulated in group CHE (P ＜ 0.05).Conclusion PKC is involved in reduction of hepatic ischemia-reperfusion injury by CO2 preconditioning in rats. Key words: Carbon dioxide; Protein kinase C; Reperfusion injury; Liver

Effects and mechanisms of store-operated calcium channel blockade on hepatic ischemia-reperfusion injury in rats

To further investigate the important role of store-operated calcium channels (SOCs) in rat hepatocytes and to explore the effects of SOC blockers on hepatic ischemia-reperfusion injury (HIRI). Using freshly isolated hepatocytes from a rat model of HIRI (and controls), we measured cytosolic free Ca(2+) concentration (by calcium imaging), net Ca(2+) fluxes (by a non-invasive micro-test technique), the SOC current (I(SOC); by whole-cell patch-clamp recording), and taurocholate secretion [by high-performance liquid chromatography and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays]. Ca(2+) oscillations and net Ca(2+) fluxes mediated by Ca(2+) entry via SOCs were observed in rat hepatocytes. I(SOC) was significantly higher in HIRI groups than in controls (57.0 ± 7.5 pA vs 31.6 ± 2.7 pA, P < 0.05) and was inhibited by La(3+). Taurocholate secretion by hepatocytes into culture supernatant was distinctly lower in HIRI hepatocytes than in controls, an effect reversed by SOC blockers. SOCs are pivotal in HIRI. SOC blockers protected against HIRI and assisted the recovery of secretory function in hepatocytes. Thus, they are likely to become a novel class of effective drugs for prevention or therapy of HIRI patients in the future.