Cyclophilin Research Articles

A homolog of cyclophilin D is expressed in Trypanosoma cruzi and is involved in the oxidative stress\u2013damage response

Mitochondria have an important role in energy production, homeostasis and cell death. The opening of the mitochondrial permeability transition pore (mPTP) is considered one of the key events in apoptosis and necrosis, modulated by cyclophilin D (CyPD), a crucial component of this protein complex. In Trypanosoma cruzi, the protozoan parasite that causes Chagas disease, we have previously described that mitochondrial permeability transition occurs after oxidative stress induction in a cyclosporin A-dependent manner, a well-known cyclophilin inhibitor. In the present work, a mitochondrial parasite cyclophilin, named TcCyP22, which is homolog to the mammalian CyPD was identified. TcCyP22-overexpressing parasites showed an enhanced loss of mitochondrial membrane potential and loss of cell viability when exposed to a hydrogen peroxide stimulus compared with control parasites. Our results describe for the first time in a protozoan parasite that a mitochondrial cyclophilin is a component of the permeability transition pore and is involved in regulated cell death induced by oxidative stress.

Cyclophilin D Acetylation Regulates Cardiac Myocyte Differentiation

INTRODUCTION: Protein acetylation is an important regulator of cellular function. Acetylation of cyclophilin D (CyPD) at lysine 166 increases its ability to open the mitochondria permeability transition pore (PTP), which is an important regulator of mitochondrial metabolism and myocyte differentiation. HYPOTHESIS: Acetylation of CyPD controls PTP activity, mitochondrial structure and function, and myocyte differentiation in the developing heart. METHODS: We use immunoblotting and immunoprecipitation to determine the global protein acetylation state and that of CyPD during embryonic cardiac development in wild type (WT) and CyPD-null mice. We also re-expressed WT and mutant CyPD in CyPD-null embryonic and neonatal cardiac myocyte cultures to determine effects on mitochondrial structure and function and myocyte differentiation. RESULTS: Immunoblotting revealed that global protein acetylation was high in the embryo but fell during later development. In contrast, CyP-D was highly acetylated in the early embryonic heart, but this decreased dramatically during subsequent development. When re-expressed in cultures of CyPD-null myocytes, WT CyPD recapitulated the WT cell phenotype of fragmented mitochondrial structure, low mitochondrial membrane potential (Δψm), and decreased differentiation. Re-expression of a CyPD acetylation mimic mutant (K166Q) had similar or greater effects on these parameters than WT CyPD, while an inactivating (R96G) and de-acetylation mimic (K166R) CyPD mutant did not. CONCLUSION: These data suggest that myocyte differentiation during cardiac development is associated with a decrease in overall protein acetylation, perhaps because of the changes that occur in metabolism as the heart develops. However, specific acetylation of CyPD appears to be particularly important for maturation of mitochondrial function and regulation of cardiac myocyte differentiation.

RNA interference-mediated gene silencing of cyclophilin A enhances the radiosensitivity of PAa human lung adenocarcinoma cells in vitro.

Radiotherapy is currently the major therapeutic strategy for patients with lung cancer. However, radioresistance and various side effects continue to present challenging issues for this form of treatment. A recent study demonstrated that cyclophilin A (CyPA) was overexpressed in non-small cell lung cancer and, therefore, presents a novel potential therapeutic target. In addition, gene-radiotherapy is a novel method for cancer treatment. Therefore, the objective of the present study was to investigate the potential effect of CyPA silencing on radiosensitivity in human lung adenocarcinoma in vitro. The stable CyPA-silencing lung adenocarcinoma (PAa) cell line was generated using lentivirus-mediated small hairpin RNAs. The knockdown of CyPA was determined using fluorescent microscopy and western blot analysis. Cells were irradiated using various doses of cobalt-60 (0, 2, 4, 6 and 8 Gy). The radiosensitizing effects were determined by a clonogenic survival assay. Apoptosis and cell cycle distribution were evaluated using flow cytometry. Silencing of CyPA significantly increased the apoptosis of PAa cells. In addition, the radiosensitivity of cells was markedly enhanced following CyPA silencing. Furthermore, silencing of CyPA, in combination with irradiation, induced G2/M phase cell cycle arrest. Taken together, the data suggest that the silencing of CyPA, combined with radiation therapy, may increase the therapeutic efficacy of lung cancer treatment through regulation of the cell cycle and apoptosis-associated signaling pathways.

Preventing permeability transition pore opening increases mitochondrial maturation, myocyte differentiation and cardiac function in the neonatal mouse heart

In embryonic myocytes, closure of the mitochondrial permeability transition pore (PTP) drives mitochondrial maturation and cardiac myocyte differentiation. Since neonatal cardiac myocytes remain relatively immature, we hypothesized that inducing PTP closure at this age, by inhibiting the PTP regulator, cyclophilin D (CyPD), genetically or with Cyclosporin A (CsA) and NIM811, would increase cardiac function by increasing mitochondrial maturation and myocyte differentiation. Cultured neonatal myocytes or neonatal mice were treated for 5 d with vehicle, CsA or NIM811. Mitochondrial function and structure were measured in vitro. Myocyte differentiation was assessed by immunolabeling for contractile proteins. Cardiac function was determined using echocardiography. The probability of PTP opening was high in WT neonatal myocytes. Treatment with CsA or NIM811 in vitro increased mitochondrial structural complexity and membrane potential, decreased reactive oxygen species levels, and increased myocyte differentiation. WT mice treated with either CsA or NIM811 in vivo for the first 5 d of life had higher ejection fractions. Deleting CyPD had similar effects as CsA and NIM811 on all parameters. It may be feasible to inhibit the PTP using available drugs to increase mitochondrial maturation, myocyte differentiation, and cardiac function in neonates.

The Expression of Cyclophilin A in Ovarian Endometrioma: Its Correlation with Recurrence and Vascularity.

Endometriosis is defined as the presence of functional endometrial tissues outside of the uterine cavity. Ovarian endometrioma is the most common type of endometriosis. It is an estrogen-dependent inflammatory disease that frequently causes infertility and chronic pelvic pain. Cyclophilin A (CyPA) is secreted from various types of cells in response to inflammatory stimuli. Many previous studies have shown that the increased expression and/or heightened plasma levels of CyPA exacerbates inflammation. The aim of this study is to evaluate CyPA immunoreactivity in ovarian endometrioma cyst wall. In this cross-sectional study, CyPA immunoreactivity in endometrial tissue samples obtained from uterine cavity and in endometrioma cyst walls of 44 consecutive women with ovarian endometrioma were compared with control endometrial tissue samples obtained from uterine cavity of 40 women without endometrioma. All endometrioma samples were confirmed via histopathological examination. Finally, the relationship between CyPA immunoreactivity and the clinicopathological findings related to endometrioma were evaluated. The CyPA expression rates in glandular cells, stromal cells, and the capillary endothelium were significantly higher in endometrioma cyst walls of women with ovarian endometrioma than in the control endometrial tissue of women without endometrioma (p = 0.0002, p = 0.0417 and p = 0.0067, respectively). The correlation analysis demonstrated that glandular CyPA expression was correlated with endometrioma recurrence (p = 0.0267). However, stromal and vascular endothelial CyPA expression were correlated with dysmenorrhea recurrence (p = 0.0023 and p = 0.0003, respectively). In conclusion, the increased expression of CyPA in ectopic endometrial tissue is associated with endometrioma recurrences and vascularity.

Development of Novel Therapies for Cardiovascular Diseases by Clinical Application of Basic Research.

Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells, inflammatory cells, activated platelets, and cardiac fibroblasts in response to oxidative stress. Excessive and continuous activation of the RhoA/Rho-kinase system promotes the secretion of CyPA, resulting in the development of multiple cardiovascular diseases. Basigin (Bsg), a transmembrane glycoprotein that activates matrix metalloproteinases, is an extracellular receptor for CyPA that promotes cell proliferation and inflammation. Thus, the CyPA/Bsg system is potentially a novel therapeutic target for cardiovascular diseases. Importantly, plasma CyPA levels are increased in patients with coronary artery disease, abdominal aortic aneurysms, pulmonary hypertension, and heart failure. Moreover, plasma CyPA levels can predict all-cause death in patients with coronary artery disease and pulmonary hypertension. Additionally, plasma soluble Bsg levels are increased and predict all-cause death in patients with heart failure, suggesting that CyPA and Bsg are novel biomarkers for cardiovascular diseases. To discover further novel molecules targeting the CyPA/Bsg system, high-throughput screening of compounds found molecules that ameliorate the development of cardiovascular diseases. In addition to CyPA and Bsg, novel therapeutic targets and their inhibitors for patients with pulmonary arterial hypertension have been recently screened and identified. Ultimately, the final goal is to develop novel biomarkers and medications that will be useful for improving the prognosis and quality of life in patients with cardiovascular diseases.

A 2-year Double-Blind RCT Follow-up Study with Fermented Papaya Preparation (FPP) Modulating Key Markers in Middle-Age Subjects with Clustered Neurodegenerative Disease-Risk Factors.

In recent years a number of studies have reported the significant relationship between metabolic syndrome and neurodegenerative disease. There is accumulating evidence that the interplay of combined genetic and environmental risk factors (from diet to life style to pollutants) to intrinsic age-related oxi-inflammatory changes may be advocated for to explain the pandemic of neurodegenerative diseases. In recent years a specific Fermented Papaya Preparation (FPP) has been shown to significantly affect a number of redox signalling abnormalities in a variety of chronic diseases and as well in aging mechanisms either on experimental and on clinical ground. The aim of the present study was to evaluate FPP use in impending metabolic disease patients with potentially neurodegenerative disease clustered risk factors. The study population consisted of 90 patients aged 45–65 years old, with impending metabolic syndrome and previously selected as to be ApoE4 genotype negative. By applying a RCT, double-blind method, one group received FPP 4.5 g twice a day (the most common dosage utilized in prior clinical studies) while the other received an oral antioxidant cocktail (trans-resveratrol, selenium, vitamin E, vitamin C). Then, after 21 month treatment period, a selected heavy metal chelator was added at the dosage of 3 g/nocte for the final 3 months study treatment. The parameters tested were: routine tests oxidized LDL-cholesterol, anti-oxidised LDL, Cyclophilin-A (CyPA), plasminogen activator inhibitor-1 and CyPA gene expression. From this study it would appear that FPP, unlike the control antioxidant, significantly decreased oxidized-LDL and near normalizing the anti-Ox-LDL/Ox-LDL ratio (p<0.001) although unaffecting the lipid profile per sè. Moreover, only FPP decreased cyclophilin-A plasma level and plasminogen activator-inhibitor (p<0.01) together with downregulating cyclophilin-A gene expression (p<0.01). Insulin resistance was only mildly improved. Heavy metals gut clearance proved to be effectively enhanced by the chelator (p<0.01) and this was not affected by any of the nutraceuticals, nor it added any further benefit to the biological action of FPP.

Cyclophilin A mediates the ox-LDL-induced activation and apoptosis of macrophages via autophagy

BackgroundOxidized low-density lipoprotein (ox-LDL) is the most common inflammatory factor that mediates the activation and apoptosis of macrophages. Cyclophilin A (CyPA) is expressed following oxidative stress, hypoxia, and infection. However, the role of CyPA in the activation and apoptosis of macrophages is unclear. The aims of the study were to determine whether CyPA mediates the ox-LDL-induced activation and apoptosis in RAW264.7 cells and to analyze potential mechanisms. Methods and resultsThrough Western blot and ELISA test, the expression of CyPA induced by ox-LDL is time-dependent in RAW264.7 cells. Gene silencing of CyPA reduced the generation of lipid droplets in the cytoplasm and downregulated the expression of the surface markers of macrophage activation, namely, CD80, CD86, and major histocompatibility complex class 2 antigen. Cell apoptosis is significantly decreased and the level of anti-apoptosis protein bcl-2 is increased in CyPA silent cells compared with the control group. Finally, autophagy-related protein LC3-II/LC3-I ratio level significantly decreased in CyPA silent cells with less autophagosome formation while the blocked autophagy flux was recovered. The differences in the activation and apoptosis between CyPA silent cells and the control cells were inhibited by pre-treatment with class III PI 3-kinase inhibitor 3-MA. ConclusionsThese results indicate that CyPA mediates the ox-LDL-induced activation and apoptosis in RAW264.7 cells by regulating autophagy.

Cyclophilin D over-expression increases mitochondrial complex III activity and accelerates supercomplex formation

Cyclophilin D (CyPD), a mitochondrial matrix protein, has been widely studied for its role in mitochondrial-mediated cell death. Unexpectedly, we previously discovered that overexpression of CyPD in a stable cell line, increased mitochondrial membrane potentials and enhanced cell survival under conditions of oxidative stress. Here, we investigated the underlying mechanisms responsible for these findings. Spectrophotometric measurements in isolated mitochondria revealed that overexpression of CyPD in HEK293 cells increased respiratory chain activity, but only for Complex III (CIII). Acute treatment of mitochondria with the immumosupressant cyclosporine A did not affect CIII activity. Expression levels of the CIII subunits cytochrome b and Rieske-FeS were elevated in HEK293 cells overexpressing CyPD. However, CIII activity was still significantly higher compared to control mitochondria, even when normalized by protein expression. Blue native gel electrophoresis and Western blot assays revealed a molecular interaction of CyPD with CIII and increased levels of supercomplexes in mitochondrial protein extracts. Radiolabeled protein synthesis in mitochondria showed that CIII assembly and formation of supercomplexes containing CIII were significantly faster when CyPD was overexpressed. Taken together, these data indicate that CyPD regulates mitochondrial metabolism, and likely cell survival, by promoting more efficient electrons flow through the respiratory chain via increased supercomplex formation.

Regulated necrosis-related molecule mRNA expression in humans and mice and in murine acute tissue injury and systemic autoimmunity leading to progressive organ damage, and progressive fibrosis.

The species-specific, as well as organ-specific expression of regulated necrosis (RN)-related molecules, is not known. We determined the expression levels of tumour necrosis factor receptor-1 (TNFR1), receptor activated protein kinase (RIPK)1, RIPK3, mixed lineage kinase domain-like (MLKL), CASP8, Fas-associated protein with death domain (FADD), cellular inhibitor of apoptosis protein (CIAP)1, CIAP2, glutathione peroxidase-4 (GPX4), cyclophilin D (CYPD), CASP1, NLRP3 and poly(ADP-ribose) polymerase-1 (PARP1) in human and mouse solid organs. We observed significant differences in expression of these molecules between human and mice. In addition, we characterized their expression profiles in acute as well as persistent tissue injury and chronic tissue remodelling using acute and chronic kidney injury models. We observed that the degree and pattern of induction of RN-related molecules were highly dependent on the trigger and disease pathogenesis. Furthermore, we studied their expression patterns in mice with lupus-like systemic autoimmunity, which revealed that the expression of MLKL, GPX4 and PARP1 significantly increased in the spleen along disease progression and CASP1, RIPK1, RIPK3 and CYPD were higher at the earlier stages but were significantly decreased in the later stages. In contrast, in the kidney, the expression of genes involved in pyroptosis, e.g. NLRP3 and CASP1 were significantly increased and TNFR1, RIPK1, RIPK3, CIAP1/2 and GPX4 were significantly decreased along the progression of lupus nephritis (LN). Thus, the organ- and species-specific expression of RN-related molecules should be considered during designing experiments, interpreting the results as well as extrapolating the conclusions from one species or organ to another species or organ respectively.

Oxidized Low-Density Lipoprotein-Induced Cyclophilin A Secretion Requires ROCK-Dependent Diphosphorylation of Myosin Light Chain

Objective: Accumulation of cyclophilin A (CyPA) within atherosclerotic lesions is thought to be implicated in the progression of atherosclerosis. However, the source of CyPA within atherosclerotic lesions is still unknown. The aim of this study is to determine the role of oxidized low-density lipoproteins (ox-LDL) in vascular smooth muscle cell (VSMC)-derived CyPA secretion and the underlying mechanism. Methods and Results: Abundant CyPA and α-smooth muscle actin (α-SMA) expressed in atherosclerotic lesions was observed in apolipoprotein E-deficient mice. ox-LDL induced CyPA secretion from a primary culture of rat aortic smooth muscle cells in a dose- and time-dependent manner. Sulfosuccinimidyloleate, a CD36 inhibitor, prevented the ox-LDL-induced CyPA secretion. Pre-exposure to either the actin-depolymerizing agent cytochalasin D or the actin-polymerizing agent jasplakinolide inhibited CyPA secretion induced by ox-LDL. Gene silencing of vesicle-associated membrane protein 2 suppressed ox-LDL-induced CyPA secretion. ox-LDL caused the phosphorylation of myosin light chain (MLC). Inhibition of MLC by blebbistatin reversed the secretion of CyPA and the phosphorylation of MLC induced by ox-LDL. MLC kinase inhibitor ML-7 reduced the monophosphorylation of MLC but did not reduce CyPA secretion. Pretreatment with the rho-associated coiled-coil kinase (ROCK) inhibitor Y27632 blocked diphosphorylation of MLC and secretion of CyPA induced by ox-LDL. Conclusions: ox-LDL-induced CyPA secretion requires vesicle transportation, actin remodeling and ROCK-dependent diphosphorylation of MLC. VSMC-derived CyPA induced by ox-LDL may be associated with increased CyPA expression in atherosclerotic lesions.

Cyclosporin A protects against Lead neurotoxicity through inhibiting mitochondrial permeability transition pore opening in nerve cells

Mitochondria play a key role in the process of lead (Pb)-induced impairment in nervous system. To further clarify the underlying mechanism of Pb neurotoxicity, this study was designed to investigate the role of mitochondrial permeability transition (MPT) and cyclophilin D (CyPD), a component of MPT pore (MPTP), in Pb-induced mitochondrial apoptosis in nerve cells. In SH-SY5Y and PC12 cells, Cyclosporin A (CSA), a special inhibitor of CyPD, could alleviate cell death, lactate dehydrogenase (LDH) leakage and adenosine 5 triphosphate (ATP) decrease caused by PbAc. In the following experiments, we found PbAc increased the protein level of CyPD and induced MPT pore (MPTP) opening. When cells were pretreated with CSA to inhibit MPTP opening, the Pb-induced impairment of mitochondrial morphology (swelling and rupture) and the loss of mitochondria were attenuated. In addition, CSA obviously ameliorated the Pb-induced damage of mitochondrial function, such as reactive oxygen species (ROS) boost and mitochondrial membrane potential (MMP) collapse, as well as the release of cytochrome C (Cyto C) and apoptosis-inducing factor (AIF) from mitochondria. These beneficial effects could finally result in cell survival under Pb-exposure conditions. Furthermore, scavenging ROS also significantly abrogated MPTP opening and attenuated Pb neurotoxicity. Therefore, we found that MPT played an important role in Pb-induced mitochondrial damage and, ultimately, cell death. Our results provided a potential strategy for inhibiting PbAc neurotoxicity. However, due to the high Pb concentrations used in this study further investigations at Pb concentrations closer to human exposure are needed to verify the results.

Overexpressed cyclophilin B suppresses aldosterone-induced proximal tubular cell injury both in vitro and in vivo.

The renin-angiotensin-aldosterone system (RAAS) is overactivated in patients with chronic kidney disease. Oxidative stress and endoplasmic reticulum stress (ERS) are two major mechanisms responsible for aldosterone-induced kidney injury. Cyclophilin (CYP) B is a chaperone protein that accelerates the rate of protein folding through its peptidyl-prolyl cis-trans isomerase (PPIase) activity. We report that overexpression of wild-type CYPB attenuated aldosterone-induced oxidative stress (evidenced by reduced production of reactive oxygen species and improved mitochondrial dysfunction), ERS (indicated by reduced expression of the ERS markers glucose-regulated protein 78 [GRP78] and C/-EBP homologous protein [CHOP]), and tubular cell apoptosis in comparison with aldosterone-induced human kidney-2 (HK-2) cells. The in vivo study also yielded similar results. Hence, CYPB performs a crucial function in protecting cells against aldosterone-induced oxidative stress, ERS, and tubular cell injury via its PPIase activity.

Cyclophilin D Is Involved in the Regulation of Autophagy and Affects the Lifespan of P. anserina in Response to Mitochondrial Oxidative Stress.

The mitochondrial permeability transition pore plays a key role in programmed cell death and the induction of autophagy. Opening of the pore is regulated by the mitochondrial peptidyl prolyl-cis, trans-isomerase cyclophilin D (CYPD). Previously it was shown in the aging model organism Podospora anserina that PaCYPD abundance increases during aging and that PaCypD overexpressors are characterized by accelerated aging. Here, we describe a role of PaCYPD in the regulation of autophagy. We found that the accelerated aging phenotype observed in a strain overexpressing PaCypD is not metacaspase-dependent but is accompanied by an increase of general autophagy and mitophagy, the selective autophagic degradation of mitochondria. It thus is linked to what has been defined as “autophagic cell death” or “type II” programmed cell death. Moreover, we found that the previously demonstrated age-related induction of autophagy in wild-type aging depends on the presence of PaCYPD. Deletion of PaCypD leads to a decrease in autophagy in later stages of age and under paraquat-mediated oxidative stress. Finally, we report that PaCYPD is also required for mitohormesis, the beneficial effect of mild mitochondrial stress. Thus, PaCYPD plays a key role in the context-dependent regulation of pathways leading to pro-survival and pro-death effects of autophagy.

Melatonin attenuates angiotensin II-induced cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway.

Melatonin is well known for its cardioprotective effects; however, whether melatonin exerts therapeutic effects on cardiomyocyte hypertrophy remains to be investigated, as do the mechanisms underlying these effects, if they exist. Cyclophilin A (CyPA) and its corresponding receptor, CD147, which exists in a variety of cells, play crucial roles in modulating reactive oxygen species (ROS) production. In this study, we explored the role of the CyPA/CD147 signaling pathway in angiotensin II (Ang II)-induced cardiomyocyte hypertrophy and the protective effects exerted by melatonin against Ang II-induced injury in cultured H9C2 cells. Cyclosporine A, a specific CyPA/CD147 signaling pathway inhibitor, was used to manipulate CyPA/CD147 activity. H9C2 cells were then subjected to Ang II or CyPA treatment in either the absence or presence of melatonin. Our results indicate that Ang II induces cardiomyocyte hypertrophy through the CyPA/CD147 signaling pathway and promotes ROS production, which can be blocked by melatonin pretreatment in a concentration-dependent manner, in cultured H9C2 cells and that CyPA/CD147 signaling pathway inhibition protects against Ang II-induced cardiomyocyte hypertrophy. The protective effects of melatonin against Ang II-induced cardiomyocyte hypertrophy depend at least partially on CyPA/CD147 inhibition.

Inhibition of cyclophilin A suppresses H2O2-enhanced replication of HCMV through the p38 MAPK signaling pathway.

Human cytomegalovirus (HCMV) infection can be accelerated by intracellular and extracellular hydrogen peroxide (H2O2) stimulation, mediated by the activation of the p38 mitogen‐activated protein kinase (MAPK) pathway. However, it remains unknown whether host gene expression is involved in H2O2‐upregulated HCMV replication. Here, we show that the expression of the host gene, cyclophilin A (CyPA), could be facilitated by treatment with H2O2 in a dose‐dependent manner. Experiments with CyPA‐specific siRNA, or with cyclosporine A, an inhibitor of CyPA, confirmed that H2O2‐mediated upregulation of HCMV replication is specifically mediated by upregulation of CyPA expression. Furthermore, depletion or inhibition of CyPA reduced H2O2‐induced p38 activation, consistent with that of H2O2‐upregulated HCMV lytic replication. These results show that H2O2 is capable of activating ROS‐CyPA–p38 MAPK interactions to enhance HCMV replication.

Role of Akt/GSK-3β signaling pathway in isoflurane preconditioning-induced inhibition of mPTP opening during myocardial ischemia-reperfusion in rats

Objective To evaluate the role of serine-threonine kinase(Akt)/glycogen synthase kinase-3 beta(GSK-3β)signaling pathway in isoflurane preconditioning-induced inhibition of mitochondrial permeability transition pore protein(mPTP)opening during myocardial ischemia-reperfusion(I/R)in rats. Methods Ninety-six male Sprague-Dawley rats, aged 3-4 months, weighing 200-250 g, were randomly divided into 4 groups(n=24 each)using a random number table: control group(group C); I/R group; isoflurane preconditioning group(group IPC); Akt inhibitor MK-2206 group(group MK). Myocardial I/R was induced by occlusion of the anterior descending branch of the left coronary artery for 30 min followed by 2 h of reperfusion.In group IPC, 1.5% isoflurane was inhaled for 30 min followed by 45 min washout, and then the model of myocardial I/R injury was established.In group MK, MK-2206 300 μg/kg(in dimethyl sulfoxide)was injected intraperitoneally at 30 min before isoflurane inhalation.At 2 h of reperfusion, 8 rats were selected and sacrificed, and the hearts were removed for determination of myocardial infarct size.At 2 h of reperfusion, 8 rats were selected, and blood samples were collected from the right internal jugular vein for determination of serum cardiac troponin I(cTnI) concentrations.The rats were then sacrificed, and myocardial specimens were obtained for determination of the expression of phosphorylated GSK-3β(p-GSK-3β)in cytoplasm and mitochondria(by Western blot)and co-expression of p-GSK-3β with adenine nucleotide translocator(ANT), voltage-dependent anion channel or cyclophilin D in myocardial tissues(using co-immunoprecipitation). At 2 h of reperfusion, 8 rats were selected and sacrificed, myocardial cells were obtained, and the opening time of mPTP was determined with a laser scanning confocal microscope. Results Compared with group C, the myocardial infarct size and serum cTnI concentrations were significantly increased, and the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated in I/R and IPC groups, the co-expression of p-GSK-3β with ANT was significantly down-regulated, and the opening time of mPTP was shortened in group I/R, and the co-expression of p-GSK-3β with ANT was significantly up-regulated, and the opening time of mPTP was prolonged in group IPC(P 0.05). Compared with group IPC, the myocardial infarct size and serum cTnI concentrations were significantly increased, the expression of p-GSK-3β in cytoplasm and mitochondria was up-regulated, the co-expression of p-GSK-3β with ANT was significantly down-regulated, and the opening time of mPTP was shortened in group MK(P<0.05). No co-expression of p-GSK-3β with voltage-dependent anion channel or cyclophilin D was found in myocardial tissues. Conclusion The mechanism by which isoflurane preconditioning inhibits mPTP opening during myocardial ischemia-reperfusion is partially related to activation of Akt/GSK-3β signaling pathway in rats. Key words: Isoflurane; Ischemic preconditioning; Myocardial reperfusion injury; Mitochondrial membrane transport proteins; Protein-serine-threonine kinases; Glycogen synthase kinase 3

Retraction: Sirtuin-3 deacetylation of cyclophilin D induces dissociation of hexokinase II from the mitochondria.

Retraction: Sirtuin-3 deacetylation of cyclophilin D induces dissociation of hexokinase II from the mitochondria.

Role of cyclophilin D in the treatment of clear cell renal cell carcinoma by sorafenib

Objective To investigate the relationship between Cyclophilin D (CypD) and occurrence and development of clear cell-renal cell carcinoma (ccRCC), and explore the action mechanism of CypD in the treatment of cRCC with sorafenib. Methods We used immunohistochemical analysis to compare CypD expression in human ccRCC tissues (n=53) and adjacent non-cancerous tissues (ANCT, n=34), and the mRNA and protein expression differences in HK-2, A498 and 786-O cell lines were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting respectively. The protein expression of CypD was observed in 786-O cell line treated with sorafenib. Results According to the CypD immunoreactive intensity, the immunoreactivity score of CypD in the ANCT samples was 7.1±0.2, which was significantly higher than that of the ccRCC samples (1.6±0.4, P<0.01). As ompared with HK-2 cells, the expression of CypD mRNA and protein was significantly downregulated in 786-O and A498 cells [75% and 69% (mRNA); 77% and 65% (protein), P<0.01]. The CypD protein expression in 786-O cells treated with sorafenib (50 μmol/L) for 24 h was increased by 63%. Conclusion CypD is negatively correlated with the occurrence and development of ccRCC. Key words: Cyclophilin D; Clear cell-renal cell carcinoma; Sorafenib

Mechanism of Cyclophilin A in tumor

Cyclophilin A (CypA) is found to be highly expressed in different kinds of tumor cells, which could regulate the occurrence and development of many kinds of tumor through multiple signal transduction pathways such as inducing the formation of inflammatory carcinoma, accelerating the transcription cycle of tumor cells, promoting the invasion and metastasis of tumor cells, inhibiting the apoptosis of tumor cells and reducing the sensitivity of tumor cells to chemotherapy drugs. It suggests that CypA might be considered as a kind of oncogene, which is expected to be a novel target for tumor treatment. Key words: Neoplasms; Cyclophilin A; Molecular mechanisms of pharmacological action