β-Patchoulene Preconditioning Protects Mice Against Hepatic Ischemia-Reperfusion Injury by Regulating Nrf2/HO-1 Signaling Pathway.

Hepatic ischemia-reperfusion (I/R) injury is one of the main causes of liver dysfunction after the liver resection and transplantation. Hepatic I/R was characterized by the tissue hypoxia during ischemia phase and oxidative stress and immune response during hypoxia-reoxygenation. The objectives of the present study were to determine the protective effects of β-patchoulene (β-PAE), a novel bioactive agent, in a mice model of hepatic I/R injury and to explore its potential mechanisms. A segmental liver warm I/R injury model was performed by occluding the portal vessels for 1 h followed by 6-h reperfusion. Twenty-four mice were randomly divided into three groups: Sham, I/R, and I/R + β-PAE, with eight mice in each group. Mice were intravenously injected with β-PAE (10 mg/kg) or saline 2 h before surgery, and parameters were measured 6 h after designated treatment. Serum aminotransferase, histologic changes, cytokines expression, and apoptosis were determined. The potential effects of β-PAE on macrophage activation and apoptosis were further evaluated in a hypoxia and reperfusion (H/R) model in vitro. Oxidative stress markers (reactive oxygen species production and malondialdehyde) and cytokines expression were measured by commercial kits. Nrf2/HO-1 and NF-ƘB signaling pathways were determined by Western blotting. Finally, blockade of nuclear factor erythroid 2-related factor 2 (Nrf2) with ML385 was used to confirm the involvement of Nrf2/HO-1 pathway in H/R injury. Hepatic I/R induced apparent tissue injury as evidenced by the increased expression of serum aminotransferase, pro-inflammatory mediators production, hepatocellular apoptosis, and necrosis. β-PAE pretreatment protected mice against I/R-induced injury, which was proved by decreased serum aminotransferase and cytokines production, reduced TUNEL-positive cells, and alleviated histopathological lesion. Immunofluorescence staining showed that β-PAE suppressed the M1 polarization of Kupffer cell induced by I/R injury. Moreover, pretreatment with β-PAE suppressed H/R-induced cytokines expression and apoptosis in cultured macrophage. The mechanistic study demonstrated that β-PAE significantly promoted the nuclear Nrf2 translocation and upregulation of HO-1 while downregulating the NF-ƘB signaling pathway in both in vivo and in vitro experiments. Furthermore, blockade of Nrf2 abolished the protective effects of β-PAE on the inhibition of H/R-mediated oxidative stress, inflammatory response, and apoptosis in vitro. β-PAE preconditioning protects mice against hepatic I/R, which was at least in part through the reversing disequilibrium between Nrf2/HO-1 and NF-ƘB pathways. β-PAE might serve as a promising therapeutic agent in the treatment of hepatic I/R injury.

Tao T ，Ye B ，Xu Y ，Wang Y ，Zhu Y ，Tian Y ... -  《-》

Sevoflurane protects the liver from ischemia-reperfusion injury by regulating Nrf2/HO-1 pathway.

We aimed to investigate the role and mechanism of sevoflurane (SEV) preconditioning in liver ischemia-reperfusion (I/R) injury. In vivo, rats were randomly divided into Sham group, I/R rat model group, I/R + SEV group and SEV group. In vitro, hypoxia-reoxygenation (H/R) cell model were established. Hematoxylin-Eosin (H&E) and TUNEL assay were used to evaluate the degree of tissue damage and detect apoptosis in rats, respectively. HO-1, nuclear Nrf2 and cytosolic Nrf2 expressions were detected by immunohistochemical staining, Western blot analysis and quantitative real-time PCR (qRT-PCR), respectively. Contents of Lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) were determined by corresponding kits. Inflammatory factor levels, cell viability, apoptosis were detected by enzyme-linked immunosorbent assay (ELISA), MTT assay, and flow cytometry, respectively.In the I/R group, liver damage was severe, apoptosis-positive cells were increased, HO-1 and nuclear Nrf2 expressions were increased, and cytosolic Nrf2 expression was decreased. After SEV pretreatment, the degree of liver injury and apoptosis in rats were significantly reduced, HO-1 and nuclear Nrf2 expressions were increased significantly, and cytosolic Nrf2 expression was decreased. 4% SEV had the best mitigating effect on H/R-induced liver cell damage, as evidenced by reduced contents of LDH and MDA, decreased inflammatory factors, a lowered apoptosis rate, inhibited ROS production, effectively promoted Nrf2 nucleation, and activated Nrf/HO-1 pathway. ML385 pretreatment significantly inhibited the effect of SEV on hepatocytes.Sevoflurane protects the liver from ischemia-reperfusion injury by regulating the Nrf2/HO-1 pathway.

Ma H ，Yang B ，Yu L ，Gao Y ，Ye X ，Liu Y ，Li Z ，Li H ，Li E ... -  《-》

Salvianolic Acid A Protects Against Oxidative Stress and Apoptosis Induced by Intestinal Ischemia-Reperfusion Injury Through Activation of Nrf2/HO-1 Pathways.

Ischemia-reperfusion (I/R) adversely affects the intestinal mucosa. The major mechanisms of I/R are the generation of reactive oxygen species (ROS) and apoptosis. Salvianolic acid A (SalA) is suggested to be an effective antioxidative and antiapoptotic agent in numerous pathological injuries. The present study investigated the protective role of SalA in I/R of the intestine. Adult male Sprague-Dawley rats were subjected to intestinal I/R injury in vivo. In vitro experiments were performed in IEC-6 cells subjected to hypoxia/ reoxygenation (H/R) stimulation to simulate intestinal I/R. TNF-α, IL-1β, and IL-6 levels were measured using enzyme-linked immunosorbent assay. Malondialdehyde and myeloperoxidase and glutathione peroxidase levels were measured using biochemical analysis. Apoptosis was measured by terminal deoxynucleotidyl transferase mediated dUTP nick-end labeling staining or flow cytometry in vivo and in vitro. The level of reactive oxygen species (ROS) was measured by dichlorodihydrofluorescin diacetate (DCFH-DA) staining. Western blotting was performed to determine the expression of heme oxygenase-1 (HO-1), Nrf2 and proteins associated with apoptosis. The mRNA expressions of Nrf2 and HO-1 were detected by quantitative real-time polymerase chain reaction in vivo and in vitro. Malondialdehyde level and myeloperoxidase and glutathione peroxidase, TNF-α, IL-1β, and IL-6 levels group in intestinal tissue decreased significantly in the SalA pretreatment groups compared to the I/R group. SalA markedly abolished intestinal injury compared to the I/R group. SalA significantly attenuated apoptosis and increased Nrf2/HO-1 expression in vivo and in vitro. However, Nrf2 siRNA treatment partially abrogated the above mentioned effects of SalA in H/R-induced ROS and apoptosis in IEC-6 cells. The present study demonstrated that SalA ameliorated oxidation, inhibited the release of pro-inflammatory cytokines and alleviated apoptosis in I/R-induced injury and that these protective effects may partially occur via regulation of the Nrf2/ HO-1 pathways.

Zu G ，Zhou T ，Che N ，Zhang X ... -  《-》

Veratric acid alleviates liver ischemia/reperfusion injury by activating the Nrf2 signaling pathway.

Oxidative stress following liver ischemia/reperfusion (I/R) is an important pathological mechanism responsible for liver injury. Veratric acid (VA) is a phenolic benzoic acid that has been reported to have antioxidant properties. However, whether VA has protective effects against liver I/R injury remains unclear. In the present study, a mouse liver I/R injury model was established. VA was administered intragastrically for one week before liver I/R. Biochemical indicators, histological analysis, cell apoptosis, oxidative stress, and pathway proteins were tested to evaluate the protective effects of VA on liver I/R injury. Furthermore, a mouse AML12 hepatocyte hypoxia/reoxygenation (H/R) model was used to explore the underlying mechanism. VA alleviated liver I/R injury, as manifested by decreased alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver necrotic area, oxidative stress, and hepatocyte apoptosis. VA pretreatment increased the expression of Nrf2 and its downstream antioxidant proteins heme oxygenase-1 (HO-1) and NADPH quinone oxidoreductase 1 (NQO-1). In addition, VA pretreatment increased AML12 cell activity and decreased oxidative stress; it also decreased the apoptosis induced by H/R. Moreover, the protective effect of VA on hepatocytes was related to the activation of the Nrf2 signaling pathway, and to increases in the Nrf2, HO-1, and NQO-1 protein expression. The inhibition of Nrf2 with ML385 offseted VA-mediated protection in AML12 cells. In conclusion, these results suggest that VA protects the liver from oxidative stress and apoptosis induced by liver I/R injury by activating the Nrf2 signaling pathway.

Yu Q ，Chen S ，Tang H ，Zhang X ，Tao R ，Yan Z ，Shi J ，Guo W ，Zhang S ... -  《-》

Inhibition of PRMT5 Attenuates Oxidative Stress-Induced Pyroptosis via Activation of the Nrf2/HO-1 Signal Pathway in a Mouse Model of Renal Ischemia-Reperfusion Injury.

Extensive evidence has demonstrated that oxidative stress, pyroptosis, and proinflammatory programmed cell death are related to renal ischemia/reperfusion (I/R) injury. However, the underlying mechanism remains to be illustrated. Protein arginine methylation transferase 5 (PRMT5), which mediates arginine methylation involved in the regulation of epigenetics, exhibits a variety of biological functions and essential roles in diseases. The present study investigated the role of PRMT5 in oxidative stress and pyroptosis induced by I/R injury in a mouse model and in a hypoxia/reoxygenation (H/R) model of HK-2 cells. C57 mice were used as an animal model. All mice underwent right nephrectomy, and the left renal pedicles were either clamped or not. Renal I/R injury was induced by ligating the left renal pedicle for 30 min followed by reperfusion for 24 h. HK-2 cells were exposed to normal conditions or stimulation through H/R. EPZ015666(EPZ)-a selective potent chemical inhibitor-and small interfering RNA (siRNA) were administered to suppress the function and expression of PRMT5. The levels of urea nitrogen and creatinine in the serum and renal tissue injury were assessed. Immunohistochemistry, western blotting, and reverse transcription-polymerase chain reaction were used to evaluate pyroptosis-related proteins including nod-like receptor protein-3, ASC, caspase-1, caspase-11, GSDMD-N, and interleukin-1β. Cell apoptosis and cell viability were detected through flow cytometry, and the levels of reactive oxygen species (ROS) and hydrogen peroxide (H2O2) were measured. Ki-67 was used to assess the proliferation of renal tubular epithelium. In addition, the activity of malondialdehyde and superoxide dismutase was determined. I/R or H/R induced an increase in the expression of PRMT5. Inhibition of PRMT5 by EPZ alleviated oxidative stress and I/R- or H/R-induced pyroptosis. In renal tissue, the application of EPZ promoted the proliferation of tubular epithelium. In addition, H/R-induced pyroptosis in HK-2 cells was dependent on oxidative stress in vitro. Administration of either EPZ or siRNA led to decreased expression of pyroptosis-related proteins. Inhibition of PRMT5 also attenuated the I/R- or H/R-induced oxidative stress in vivo and in HK-2 cells, respectively. It also resulted in a distinct decrease in the levels of malondialdehyde and H2O2, and an apparent increase in superoxide dismutase activity in mouse renal tissue. Moreover, it led to a significant decrease in the levels of ROS and H2O2 in HK-2 cells. When activated, NF-E2-related factor/heme oxygenase-1 (Nrf2/HO-1)-a key regulator of various cytoprotective proteins that withstand oxidative damage-can decrease the generation of ROS. Nrf2/HO-1 was downregulated during I/R in tissues and H/R in HK-2 cells, and this effect was reversed by the PRMT5 inhibitor. Furthermore, the expressions of Nrf2 and HO-1 proteins were markedly upregulated by EPZ or siRNA against PRMT5. PRMT5 is involved in ischemia- and hypoxia-induced oxidative stress and pyroptosis in vitro and in vivo. Inhibition of PRMT5 may ameliorate renal I/R injury by suppressing oxidative stress and pyroptosis via the activation of the Nrf2/HO-1 pathway, as well as promoting the proliferation of tubular epithelium. Therefore, PRMT5 may be a promising therapeutic target.

Diao C ，Chen Z ，Qiu T ，Liu H ，Yang Y ，Liu X ，Wu J ，Wang L ... -  《-》