Hasibur Rehman

Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.

Green Tea Polyphenols Stimulate Mitochondrial Biogenesis and Improve Renal Function after Chronic Cyclosporin A Treatment in Rats

Our previous studies showed that an extract from Camellia sinenesis (green tea), which contains several polyphenols, attenuates nephrotoxicity caused by cyclosporine A (CsA). Since polyphenols are stimulators of mitochondrial biogenesis (MB), this study investigated whether stimulation of MB plays a role in green tea polyphenol protection against CsA renal toxicity. Rats were fed a powdered diet containing green tea polyphenolic extract (0.1%) starting 3 days prior to CsA treatment (25 mg/kg, i.g. daily for 3 weeks). CsA alone decreased renal nuclear DNA-encoded oxidative phosphorylation (OXPHOS) protein ATP synthase-β (AS-β) by 42%, mitochondrial DNA (mtDNA)-encoded OXPHOS protein NADH dehydrogenase-3 (ND3) by 87% and their associated mRNAs. Mitochondrial DNA copy number was also decreased by 78% by CsA. Immunohistochemical analysis showed decreased cytochrome c oxidase subunit IV (COX-IV), an OXPHOS protein, in tubular cells. Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α, the master regulator of MB, and mitochondrial transcription factor-A (Tfam), the transcription factor that regulates mtDNA replication and transcription, were 42% and 90% lower, respectively, in the kidneys of CsA-treated than in untreated rats. These results indicate suppression of MB by chronic CsA treatment. Green tea polyphenols alone and following CsA increased AS-β, ND3, COX-IV, mtDNA copy number, PGC-1α mRNA and protein, decreased acetylated PGC-1α, and increased Tfam mRNA and protein. In association with suppressed MB, CsA increased serum creatinine, caused loss of brush border and dilatation of proximal tubules, tubular atrophy, vacuolization, apoptosis, calcification, and increased neutrophil gelatinase-associated lipocalin expression, leukocyte infiltration, and renal fibrosis. Green tea polyphenols markedly attenuated CsA-induced renal injury and improved renal function. Together, these results demonstrate that green tea polyphenols attenuate CsA-induced kidney injury, at least in part, through the stimulation of MB.

Phytochemical, antioxidant and mineral composition of hydroalcoholic extract of chicory (Cichorium intybus L.) leaves.

The phytochemical, antioxidant and mineral composition of hydroalcoholic extract of leaves of Cichorium intybus L., was determined. The leaves were found to possess comparatively higher values of total flavonoids, total phenolic acids. The phytochemical screening confirmed the presence of tannins, saponins, flavonoids, in the leaves of the plant. The leaf extract was found to show comparatively low value of IC50 for 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition. The IC50 value of chicory leaves extract was found to be 67.2 ± 2.6 μg/ml. The extracts were found to contain high amount of mineral elements especially Mg and Zn. Due to good phytochemical and antioxidant composition, C. intybus L., leaves would be an important candidate in pharmaceutical formulations and play an important role in improving the human health by participating in the antioxidant defense system against free radical generation.

Sphingosine kinase-2 inhibition improves mitochondrial function and survival after hepatic ischemia–reperfusion

BACKGROUND & AIMS The mitochondrial permeability transition (MPT) and inflammation play important roles in liver injury caused by ischemia-reperfusion (IR). This study investigated the roles of sphingosine kinase-2 (SK2) in mitochondrial dysfunction and inflammation after hepatic IR. METHODS Mice were gavaged with vehicle or ABC294640 (50 mg/kg), a selective inhibitor of SK2, 1 h before surgery and subjected to 1 h-warm ischemia to ~70% of the liver followed by reperfusion. RESULTS Following IR, hepatic SK2 mRNA and sphingosine-1-phosphate (S1P) levels increased ~25- and 3-fold, respectively. SK2 inhibition blunted S1P production and liver injury by 54-91%, and increased mouse survival from 28% to 100%. At 2 h after reperfusion, mitochondrial depolarization was observed in 74% of viable hepatocytes, and mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. SK2 inhibition decreased mitochondrial depolarization and prevented MPT onset. Inducible nitric oxide synthase, phosphorylated NFκB-p65, TNFα mRNA, and neutrophil infiltration, all increased markedly after hepatic IR, and these increases were blunted by SK2 inhibition. In cultured hepatocytes, anoxia/re-oxygenation resulted in increases of SK2 mRNA, S1P levels, and cell death. SK2 siRNA and ABC294640 each substantially decreased S1P production and cell death in cultured hepatocytes. CONCLUSIONS SK2 plays an important role in mitochondrial dysfunction, inflammation responses, hepatocyte death, and survival after hepatic IR and represents a new target for the treatment of IR injury.

NIM811 (N-Methyl-4-isoleucine Cyclosporine), a Mitochondrial Permeability Transition Inhibitor, Attenuates Cholestatic Liver Injury but Not Fibrosis in Mice

Cholestasis causes hepatocyte death, possibly because of mitochondrial injury. This study investigated whether NIM811 (N-methyl-4-isoleucine cyclosporine), an inhibitor of the mitochondrial permeability transition (MPT), attenuates cholestatic liver injury in vivo. Cholestasis was induced in mice by bile duct ligation (BDL). NIM811 was gavaged (20 mg/kg) before BDL and daily (10 mg/kg) afterward. Mitochondrial depolarization, cell death, and MPT onset were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. After BDL, serum alanine aminotransferase (ALT), hepatic necrosis, and apoptosis all increased. NIM811 decreased ALT, necrosis, and apoptosis by 60 to 86%. In vehicle-treated mice at 6 h after BDL, viable hepatocytes with depolarized mitochondria were 18/high-power field (hpf), and nonviable cells were ∼1/hpf, showing that depolarization preceded necrosis. Calcein entered mitochondria after BDL, indicating MPT onset in vivo. NIM811 decreased depolarization by 72%, prevented calcein entry into mitochondria, and blocked release of cytochrome c. Hepatic tumor necrosis factor α, transforming growth factor-β1, procollagen α1(I) mRNA, α-smooth muscle actin, and Sirius red staining for collagen increased after BDL but were not different in vehicle- and NIM811-treated mice. Taken together, NIM811 decreased cholestatic necrosis and apoptosis but did not block fibrosis, indicating that the MPT plays an important role in cholestatic cell death in vivo.

Inhibiting Survivin Expression Enhances TRAIL-Induced Tumoricidal Activity in Human Hepatocellular Carcinoma Via Cell Cycle Arrest

Human Hepatocellular carcinoma (HCC) cell types exhibit a major resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell death, and the key determinants of mechanisms accounting for TRAIL susceptibility, still remain controversial. Our previous studies showed that overexpression of survivin reduced sensitivity of HCC cells to TRAIL. The aim of this study is to investigate how tumor cells escape TRAIL-mediated surveillance through survivin expression and how to reverse the resistance of TRAIL-inducing apoptosis. Seven tumor cell lines were treated with or without TRAIL protein and antisense oligodeoxynucleotides (ODNs) against survivin in culture. HepG2 and SMMC7721 cells were treated with mimosine, thymidine or nocodazole to synchronize their cell cycle phases and then used to test their sensitivity to TRAIL. In vivo effects of TRAIL plasmid alone or in combination with survivin antisense ODNs on tumor growth were evaluated in a nude mouse hepatoma model of HepG2 cell grafts. Varied levels of survivin mRNA in various cell lines were evaluated and negatively correlated to TRAIL-induced apoptosis. Hepatoma HepG2 and SMMC7721 cells in G1 or S phase are more sensitive to TRAIL than those in G2 phase. Treatment with survivin antisense ODNs caused S phase arrest and significantly enhanced TRAIL-induced apoptosis. TRAIL protein caused G2/M arrest and resulted in an increase of survivin in HepG2 cells. Combined TRAIL plasmid and survivin antisense ODNs significantly supressed the growth of tumor xenografts as compared to TRAIL plamid or antisense ODNs alone during a 4-week of observation. The findings indicate that survivin may play a role in tumor cell resistance to TRAIL-induced apoptosis, at least in part, through cell cycle regulation. Manipulation of survivin expression levels may sensitizes tumor cells to TRAIL-induced apoptosis.

Ischemic preconditioning prevents free radical production and mitochondrial depolarization in small-for-size rat liver grafts.

Background. Ischemic preconditioning (IP) renders tissues more tolerant to subsequent longer episodes of ischemia. This study tested whether IP attenuates injury of small-for-size liver grafts by preventing free radical production and mitochondrial dysfunction. Methods. IP was induced by clamping the portal vein and hepatic artery for 9 min. Livers were harvested 5 min after releasing the clamp. Mitochondrial polarization and cell death were assessed by intravital confocal/multiphoton microscopy of rhodamine 123 (Rh123) and propidium iodide. Free radicals were trapped with &agr;-(4-pyridyl 1-oxide)-N-tert-butylnitrone and measured using electron spin resonance. Results. After quarter-size liver transplantation, alanine aminotransferase, serum bilirubin, necrosis, and apoptosis all increased. IP blocked these increases by more than 58%. 5-Bromo-2′-deoxyuridine labeling and increases of graft weight were only ∼3% and 0.2% in quarter-size grafts without IP, respectively, but increased to 32% and 60% in ischemic-preconditioned grafts, indicating better liver regeneration. Eighteen hours after implantation, viable cells with depolarized mitochondria in quarter-size grafts were 15 per high power field, and dead cells were less than 1 per high power field, indicating that depolarization preceded necrosis. A free radical adduct signal was detected in bile from quarter-size grafts. IP decreased this free radical formation and prevented mitochondrial depolarization. IP did not increase heat shock proteins 10, 27, 32, 60, 70, 72, 75 and Cu/Zn-superoxide dismutase (SOD) but increased heat shock protein-90, a chaperone that facilitates protein import into mitochondria, and mitochondrial Mn-SOD. Conclusion. Taken together, IP decreases injury and improves regeneration of small-for-size liver grafts, possibly by increasing mitochondrial Mn-SOD, thus protecting against free radical production and mitochondrial dysfunction.

Nephrotoxicity and its prevention by taurine in tamoxifen induced oxidative stress in mice

Tamoxifen (TAM) is an anti-neoplastic drug used for the treatment of breast cancer. It decreases the hexose monophosphate shunt and thereby increasing the incidence of oxidative stress in cells leading to tissue injury. The present study was undertaken to investigate modulatory effects of taurine on the nephrotoxicity of TAM with special reference to protection against disruption of nonenzymatic and enzymatic antioxidants. Oxidative stress was measured by renal lipid peroxidation (LPO) level, protein carbonyl (PC) content, reduced glutathione (GSH), activities of phase I and II drug metabolizing and antioxidant enzymes. TAM treatment resulted in a significant (P < 0.001) increase in LPO in kidney tissues as compared to control, while taurine pretreatment showed a significant decrease (P < 0.01) in the LPO in kidneys when compared with the TAM-treated group. Taurine + TAM group animals showed restoration in the level of cytochrome P450 content, activities of glutathione metabolizing enzymes viz., glutathione-S-transferase, glutathione peroxidase, glutathione reductase, glucose-6-phosphate dehydrogenase. Pretreatment of animals with taurine markedly attenuated, PC content, restored the depleted nonenzymatic and enzymatic antioxidants. These results clearly demonstrate the role of oxidative stress, and suggest a protective effect of taurine on TAM-induced nephrotoxicity in mice. Human & Experimental Toxicology (2007) 26 : 509—518

Ameliorating effect of chicory (Chichorium intybus L.) fruit extract against 4-tert-octylphenol induced liver injury and oxidative stress in male rats

The current study was carried out to elucidate the modulating effect of chicory (Cichorium intybus L.) fruit extract (CFR) against 4-tert-OP induced oxidative stress and hepatotoxicity in male rats. Rats were divided into four groups and treated for 8 weeks as follow: group 1: normal control-treated (saline); group 2: chicory fruit extract-treated (100 mg/kg); group 3: 4-tert-OP treated; group 4: 4-tert-OP plus chicory fruit extract. The obtained results revealed that rats which received 4-tert-OP showed a significant increase in liver TBARS and bilirubin, aspartate aminotransferase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP) and gamma-glutamyl transpeptidase (GGTP) activities. While a significant decrease in the levels of GSH, SOD, catalase recorded. On the other hand, CFR extract succeeded to modulate these observed abnormalities resulting from 4-tert-OP as indicated by the reduction of TBARS and the pronounced improvement of the investigated biochemical and antioxidant parameters. Histopathological evidence, together with observed PCNA and DNA fragmentation, supported the detrimental effect of 4-tert-OP and the ameliorating effect of CFR extract on liver toxicity. So, it could be concluded that chicory has a promising role and it worth to be considered as a natural substance for ameliorating the oxidative stress and hepatic injury induced by 4-tert-OP compound.

NIM811 Prevents Mitochondrial Dysfunction, Attenuates Liver Injury and Stimulates Liver Regeneration after Massive Hepatectomy

Background. Massive hepatectomy (MHX) leads to failure of remnant livers. Excessive metabolic burden in remnant livers may cause mitochondrial dysfunction. This study investigated whether blockade of the mitochondrial permeability transition (MPT) with N-methyl-4-isoleucine cyclosporine (NIM811) improves the outcome of MHX. Methods. Mice were gavaged with NIM811 (10 mg/kg before surgery and 5 mg/kg daily afterward) and underwent sham-operation or approximately 90% partial hepatectomy. Results. Serum alanine aminotransferase, necrosis, and apoptosis increased, respectively, to approximately 1200 U/L, 6.1%, and 7% after MHX. NIM811 decreased peak alanine aminotransferase release, necrosis, and apoptosis by 70%, 100%, and 42%, respectively. 5-Bromo-2′-deoxyuridine incorporation, proliferating cell nuclear antigen expression, and the remnant liver weights were all increased significantly by NIM811 treatment, indicating improved liver regeneration. NIM811 also blunted hyperbilirubinemia by 54%, increased serum albumin by 51%, and improved survival from 6% to 40% after MHX. Hepatic mitochondrial depolarization, cell death, and MPT were detected by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. Mitochondrial depolarization occurred in many viable hepatocytes (13 cells/high-power field), and nonviable hepatocytes increased slightly to approximately 1 cell/high-power field at 3 hr after MHX. Entry of calcein into mitochondria after MHX indicated MPT onset. Importantly, NIM811 decreased mitochondria depolarization by more than 60%, blocked MPT onset, and prevented cell death. Decreases of hepatic ATP, mitochondrial cytochrome c release, and caspase-3 activation after MHX were also partially blocked by NIM811. Conclusions. NIM811 minimized liver injury and improved liver regeneration after MHX, at least in part, by preventing MPT onset and subsequent compromised energy supply and proapoptotic cytochrome c release.