Inhibition of prohibitin 2 enhances the sensitivity of non-small cell lung cancer cell line A549 to erlotinib / 基础医学与临床

Objective To explore the effects of prohibitin 2(PHB2)on sensitivity of non-small cell lung cancer cell line A549 to erlotinib(Erl)and its potential mechanisms.Methods RACK1-specific small interfering RNA was transfected in A549 cells for knocking-down of PHB2.The effects of PHB2 inhibition on cell proliferation and apop-tosis induced by Erl were observed.The colocalization of microtuble-associated protein light chain 3 alpha(LC3)and mitochondria was visualized by MitoTracker staining and green fluorescent protein-microtuble-associated protein light chain 3 alpha(GFP-LC3)transfection.Cell proliferation was detected by 5-ethynyl-2′-deoxyuridine(EdU)staining.Cell colony formation was evaluated by colony forming assay.Apoptotic index of A549 cells was evaluated by TUNEL.Western blot was used to measure the expressions of PHB2 and LC3Ⅱ.Mitochondrial transmembrane potential,cytochrome c and respiratory chain complexⅠ/Ⅱ/Ⅴactivity were analyzed by the commercially availa-ble kits.Results Compared with the siPHB2 and siCtrl+Erl group,the EdU-positive A549 cells and the number of cell colonies decreased significantly(P＜0.05),while the TUNEL-positive A549 cells increased significantly(P＜0.05)in the siPHB2+Erl group.In addition,compared with the siPHB2 and siCtrl+Erl group,mitochondrial transmembrane potential and respiratory chain complexⅠ/Ⅱ/Ⅴactivity decreased significantly(all P＜0.05)and the levels of cytochrome c increased in mitochondrial fractions(P＜0.05)and decreased in cytosolic fractions(P＜0.05)in the siPHB2+Erl group.Conclusions PHB2 inhibition significantly improves sensitivity of A549 cells to Erl,which may be explained by inhibition of PHB2-mediated mitochondrial autophagy.

Resonant Recognition Model Study for Interactions between SARS CoV 2 and Human Proteins

This study was devoted to the Resonant Recognition Model (RRM) analysis of SARS-CoV-2 proteins and their possible interaction with other human proteins, specifically, SARS CoV replicases and methyl transferases, were tested, via RRM analysis, for possible interactions with host CD4 T receptor proteins and prohibitins which participate in human organism response to viral infections. The following protein sequences were studied: twenty human SARS coronavirus methyltransferase proteins, eight replicase proteins, twenty-one prohibitin proteins, and eleven CD4 -T-cell surface antigens T4 proteins. Results revealed RRM peaks at f1=0.07349 and f2=0.2839. The peak at f1 was also common for interaction between SARS-CoV-2 methyl transferases and human prohibitins, where opposite phase suggest binding between these proteins during viral infection. This interaction was not supported for viral methyltransferase and human CD4 receptors (72.4 o phase shift). Viral replicases exhibited opposite phase interaction with both prohibitins and CD4 receptors. Overall, RRM revealed common RRM frequencies for both replicases and methyl transferases, and added plausibility to interactions between SARSCoV2 methyl transferase and human prohibitin, as well as between SARS Cov2 replicase and human prohibitin and CD4 T-cell receptors(AU)

Este estudio se dedicó al análisis mediante el Modelo de Reconocimiento Resonante (RRM) de las proteínas del SARS-CoV-2 y su posible interacción con otras proteínas humanas, específicamente, fueron analizadas las replicasas de SARS CoV y las metiltransferasas, mediante análisis RRM, para detectar posibles interacciones con las Proteínas del receptor CD4 T y las prohibitinas humanas, las cuales participan en la respuesta del organismo humano a las infecciones virales. Se estudiaron las siguientes secuencias de proteínas: veinte proteínas metiltransferasas del coronavirus del SARS humano, ocho replicasas, veintiuna prohibitinas y once proteínas T4 de antígenos de superficie de células T CD4. Los resultados revelaron picos de RRM en f1 = 0.07349 y f2 = 0.2839. El pico en f1 también fue común para la interacción entre las metiltransferasas del SARS-CoV-2 y las prohibitinas humanas, donde la fase opuesta sugiere la unión entre estas proteínas durante la infección viral. Esta interacción no fue apoyada para la metiltransferasa viral y los receptores CD4 humanos (cambio de fase de 72,4 o). Las réplicas virales exhibieron una interacción de fase opuesta tanto con las prohibitinas como con los receptores CD4. En general, el análisis de RRM reveló frecuencias comunes de RRM para replicasas y metiltransferasas, y apoyó plausibilidad de las interacciones entre la metiltransferasa de SARSCoV2 y la prohibitina humana, así como entre la replicasa de Cov2 del SARS con la prohibitina humana y los receptores de células T CD4(AU)

Prohibitin regulates mTOR pathway via interaction with FKBP8 / 医学前沿

The ability of tumor cells to sustain continuous proliferation is one of the major characteristics of cancer. The activation of oncogenes and the mutation or inactivation of tumor suppressor genes ensure the rapid proliferation of tumor cells. The PI3K-Akt-mTOR axis is one of the most frequently modified signaling pathways whose activation sustains cancer growth. Unsurprisingly, it is also one of the most commonly attempted targets for cancer therapy. FK506 binding protein 8 (FKBP8) is an intrinsic inhibitor of mTOR kinase that also exerts an anti-apoptotic function. We aimed to explain these contradictory aspects of FKBP8 in cancer by identifying a "switch" type regulator. We identified through immunoprecipitation-mass spectrometry-based proteomic analysis that the mitochondrial protein prohibitin 1 (PHB1) specifically interacts with FKBP8. Furthermore, the downregulation of PHB1 inhibited the proliferation of ovarian cancer cells and the mTOR signaling pathway, whereas the FKBP8 level in the mitochondria was substantially reduced. Moreover, concomitant with these changes, the interaction between FKBP8 and mTOR substantially increased in the absence of PHB1. Collectively, our finding highlights PHB1 as a potential regulator of FKBP8 because of its subcellular localization and mTOR regulating role.

Prohibitin (PHB) interacts with AKT in mitochondria to coordinately modulate sperm motility / 亚洲男科学杂志(英文版)

Prohibitin (PHB), an evolutionarily conserved mitochondrial inner membrane protein, is highly expressed in cells that require strong mitochondrial function. Recently, we demonstrated that the deletion of Phb in spermatocytes results in impaired mitochondrial function. In addition, PHB expression in the mitochondrial sheath of human sperm has a significantly negative correlation with mitochondrial reactive oxygen species levels, but a positive one with mitochondrial membrane potential and sperm motility. These results suggest that mitochondrial PHB expression plays a role in sperm motility. However, the mechanism of PHB-mediated regulation of sperm motility remains unknown. Here, we demonstrate for the first time that PHB interacts with protein kinase B (AKT) and exists in a complex with phospho-PHB (pT258) and phospho-AKT in the mitochondrial sheath of murine sperm, as determined using colocalization and coimmunoprecipitation assays. After blocking AKT activity using wortmannin (a phosphatidylinositol 3-kinase [PI3K] inhibitor), murine sperm have significantly ( P < 0.05) decreased levels of phospho-PHB (pT258) and the total and progressive motility. Furthermore, significantly ( P < 0.05) lower levels of phospho-PI3K P85 subunit α+γ (pY199 and pY467) and phospho-AKT (pS473; pT308) are found in sperm from infertile asthenospermic and oligoasthenospermic men compared with normospermic subjects, which suggest a reduced activity of the PI3K/AKT pathway in these infertile subjects. Importantly, these sperm from infertile subjects also have a significantly ( P < 0.05) lower level of phospho-PHB (pT258). Collectively, our findings suggest that the interaction of PHB with AKT in the mitochondrial sheath is critical for sperm motility, where PHB phosphorylation (pT258) level and PI3K/AKT activity are key regulatory factors.

Expression and clinical significance of prohibitin in breast cancer based on bioinformatics / 临床检验杂志

Objective@#To analyze the expression and clinical significance of prohibitin (PHB) in invasive breast cancer (BC) based on high throughput multi-omics databases. @*Methods@#The breast cancer data were downloaded from TCGA and METABRIC databases, and the expressions of PHB in BC tissues and adjacent normal tissues were compared by various bioinformatics tools. The correlations of PHB expression with clinicopathological features and prognosis of BC patients were analyzed, and the interaction and function of PHB protein were predicted. @*Results@#Compared with adjacent normal tissues, PHB was highly expressed in many kinds of cancer tissues, especially in invasive breast cancer where the gene mutation and expression changes of PHB had higher proportion. The expression level of PHB had good diagnostic efficacy for BC (P＜0.01). The expression level of PHB was significantly correlated with the expressions of ER and HER2, PAM50 typing and tumor purity of BC patients (P＜0.05). The survival analysis showed that the high expression of PHB was an independent risk factor of BC (P＜0.01). [STBX]HRAS, KSR1[STBZ] and ARAF interacted with PHB, with significant correlation. The changed expressions of [STBX]HRAS, KSR1[STBZ] and ARAF could be found in BC tissues. @*Conclusion@#The expression of PHB increases in various cancer tissues such as breast cancer, ovarian cancer and so on. The high expression of PHB has significant influence on the prognosis of BC patients. The expression of PHB has good diagnostic efficacy for BC, which may be used as a potential marker for the diagnosis and prognosis of BC.

Sophoral flavones inhibit proliferation of cardiac fibroblasts via regulating the expression of prohibitin / 药学学报

The aim of the present study was to investigate the effect of Sophoral flavones on proliferation of cardiac fibroblasts (CFb) induced by high glucose and its underlying mechanism. Cardiac fibroblasts were exposed to different concentration of D-glucose (15, 25 and 35 mmol·L-1) at different time point (24, 48 and 72 h) in order to determine cell proliferation, and the model group was established by culturing CFb with 25 mmol·L-1D-glucose for 48 h. Sophoral flavones (12.5, 25 and 50 mg·L-1) were employed for intervention. The cell viability was measured by MTT assay, and the levels of transforming growth factor-β1 (TGF-β1), matrix metalloproteinase-2 (MMP-2), collagen Ⅰ and collagen Ⅲ were measured by ELISA. In addition, flow cytometry was employed to detect the cell cycle; while the protein expression of prohibitin (PHB) was observed via immunocytochemistry and Western blot. This animal experiment had been approved by Jilin Medical University Experiment Animal Ethics Review Committee. The results showed that 25 mmol·L-1 glucose could promote the proliferation of CFb; and the contents of TGF-β1, MMP-2, collagen Ⅰ and collagen Ⅲ in the model group were higher than that of control (P<0.05). The number of cells in S and G2 phase increased under high glucose condition. In the model group, PHB translocation occurred at 6 h and protein expression decreased at 48 h (P<0.01). Compared with the model group, 12.5-50 mg·L-1 Sophoral flavones reduced the contents of TGF-β1, MMP-2, collagen Ⅰ and collagen Ⅲ, increased the number of G1 phase cells, and increased the expression of PHB protein at 48 h (P<0.05), with no effect on the nuclear translocation of PHB. These results indicated that Sophoral flavones could prevent the proliferation of CFb induced by high glucose, the mechanism of which may be related to increasing the expression of PHB protein.

The novel long noncoding RNA LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells via its interaction with PHB2 / 亚洲男科学杂志(英文版)

We sought to investigate the underlying mechanism of action of the long noncoding RNA (lncRNA) LOC283070 in the development of androgen independence in prostate cancer. The interactions between LOC283070 and target proteins were investigated by RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays. Subcellular fractionation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the subcellular localization of LOC283070. Western blotting was performed to detect the expression of prohibitin 2 (PHB2). Luciferase activity assays were performed to evaluate the effects of LOC283070 and PHB2 on the androgen receptor (AR) signaling pathway. A methyl thiazolyl tetrazolium (MTT) assay and a growth curve assay were used to test cell viability. Flow cytometry was performed to analyze cell cycles. A transwell assay was employed to test cell migration. We identified PHB2 as an interaction partner of LOC283070 in the pull-down and RIP experiments. Furthermore, we confirmed that the enrichment of LOC283070 with PHB2 in androgen-independent LNCaP (LNCaP-AI) cells was much greater than that in LNCaP cells. Moreover, the expression of PHB2 was not significantly different between the two cell lines, and the expression of LOC283070 in the nuclei of the LNCaP-AI cells was significantly greater than that in the LNCaP cells. In vitro data revealed that PHB2 overexpression significantly inhibited AR activity and cell proliferation and migration and induced accumulation of prostate cancer cells in G0/G1 phase. Moreover, the overexpression of LOC283070 fully abrogated the effects of PHB2 overexpression. In conclusion, we found that LOC283070 can bind to PHB2 located in the nucleus and inhibit its effect, and this is one of the mechanisms by which LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells.

The novel long noncoding RNA LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells via its interaction with PHB2 / 亚洲男科学杂志(英文版)

We sought to investigate the underlying mechanism of action of the long noncoding RNA (lncRNA) LOC283070 in the development of androgen independence in prostate cancer. The interactions between LOC283070 and target proteins were investigated by RNA pull-down and RNA-binding protein immunoprecipitation (RIP) assays. Subcellular fractionation and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were used to detect the subcellular localization of LOC283070. Western blotting was performed to detect the expression of prohibitin 2 (PHB2). Luciferase activity assays were performed to evaluate the effects of LOC283070 and PHB2 on the androgen receptor (AR) signaling pathway. A methyl thiazolyl tetrazolium (MTT) assay and a growth curve assay were used to test cell viability. Flow cytometry was performed to analyze cell cycles. A transwell assay was employed to test cell migration. We identified PHB2 as an interaction partner of LOC283070 in the pull-down and RIP experiments. Furthermore, we confirmed that the enrichment of LOC283070 with PHB2 in androgen-independent LNCaP (LNCaP-AI) cells was much greater than that in LNCaP cells. Moreover, the expression of PHB2 was not significantly different between the two cell lines, and the expression of LOC283070 in the nuclei of the LNCaP-AI cells was significantly greater than that in the LNCaP cells. In vitro data revealed that PHB2 overexpression significantly inhibited AR activity and cell proliferation and migration and induced accumulation of prostate cancer cells in G0/G1 phase. Moreover, the overexpression of LOC283070 fully abrogated the effects of PHB2 overexpression. In conclusion, we found that LOC283070 can bind to PHB2 located in the nucleus and inhibit its effect, and this is one of the mechanisms by which LOC283070 is involved in the transition of LNCaP cells into androgen-independent cells.

Deficiency of Sphingosine-1-Phosphate Reduces the Expression of Prohibitin and Causes β-Cell Impairment via Mitochondrial Dysregulation

BACKGROUND: Emerging evidence suggests that sphingolipids may be involved in type 2 diabetes. However, the exact signaling defect through which disordered sphingolipid metabolism induces β-cell dysfunction remains unknown. The current study demonstrated that sphingosine-1-phosphate (S1P), the product of sphingosine kinase (SphK), is an essential factor for maintaining β-cell function and survival via regulation of mitochondrial action, as mediated by prohibitin (PHB). METHODS: We examined β-cell function and viability, as measured by mitochondrial function, in mouse insulinoma 6 (MIN6) cells in response to manipulation of cellular S1P and PHB levels. RESULTS: Lack of S1P induced by sphingosine kinase inhibitor (SphKi) treatment caused β-cell dysfunction and apoptosis, with repression of mitochondrial function shown by decreases in cellular adenosine triphosphate content, the oxygen consumption rate, the expression of oxidative phosphorylation complexes, the mitochondrial membrane potential, and the expression of key regulators of mitochondrial dynamics (mitochondrial dynamin-like GTPase [OPA1] and mitofusin 1 [MFN1]). Supplementation of S1P led to the recovery of mitochondrial function and greatly improved β-cell function and viability. Knockdown of SphK2 using small interfering RNA induced mitochondrial dysfunction, decreased glucose-stimulated insulin secretion (GSIS), and reduced the expression of PHB, an essential regulator of mitochondrial metabolism. PHB deficiency significantly reduced GSIS and induced mitochondrial dysfunction, and co-treatment with S1P did not reverse these trends. CONCLUSION: Altogether, these data suggest that S1P is an essential factor in the maintenance of β-cell function and survival through its regulation of mitochondrial action and PHB expression.

Prohibitin2 expression and its relation with astrocyte proliferation in rats after traumatic brain injury / 中华神经医学杂志

Objective To research the prohibitin2 (PHB2) expression and cellular localization in the rat brain cortex after traumatic brain injury (TBI),and explore its relationship with astrocyte proliferation and neuron apoptosis.Methods TBI rat models were established by knife injurying the brain.Western blotting was used to detect the PHB2 expression variation trend in the brain cortex.Immunohistochemical method was used to detect the distribution of PHB2 in damaged cerebral cortex,and immunofluorescence was applied to research the PHB2 expression changes and cellular localization in the rat brain cortex after TBI,and explore its relationship with astrocyte proliferation and neuron apoptosis.Results (1) The TBI models were established successfully;12 h after TBI,the PHB2 expression started to increase obviously,and PHB2 expression reached to its peak level 5 d after TBI.(2)The PHB2 expression in the cortex of TBI rats was significantly increased as compared with that in the sham-operated group,control group and contralateral side of TBI rats.(3) PHB2 mainly located at the astrocytes and neurons of the cerebral cortex after TBI.(4) Co-localization was noted in astrocytes and cell proliferation marker PCNA,and in PHB2 and PCNA,which indicated that proliferation of astrocytes existed and PHB2 involved in the proliferation.(5) Co-localization was noted in astrocytes and cell apoptosis marker A-caspase-3,and in PHB2 and A-caspase-3,which indicated that TBI induced cell apoptosis,and PHB2 involved in the apoptosis.Conclusion PHB2 has a high expression in the cerebral cortex of rats after TBI,and these changes are related to astrocyte proliferation and neuronal apoptosis.

Detection of urinary neutrophil gelatinase associated lipocalin and blood prohibitin levels in early diagnosis of children with acute kidney injury / 中华全科医师杂志

Objective To assess the detection of urinary neutrophil gelatinase associated lipocalin (NGAL) and blood prohibitin (PHB) levels in early diagnosis of children with acute kidney injury (AKI).Methods One hundred and twenty children with severe allergic purpura,sepsis,kidney disease or heart disease admitted from June 2011 to June 2013 in our hospital were enrolled,including 60 cases with AKI and 60 cases without AKI;and 60 healthy children were selected as the control group.The urinary NGAL and blood PHB levels were measured with ELISA method.Results At d1 after diagnosis,the urinary NGAL level in severe AKI group [(146.76 ±61.22) μg/L] was higher than that in non-AKI group [(21.79 ± 17.31) μg/L] and control group [(17.42 ± 13.11) μg/L] (t =15.430 and 22.216,P ＜ 0.01).At d2 after diagnosis,the urinary NGAL level in severe AKI group [(82.31 ± 44.76) μg/L] was higher than that in non-severe AKI group [(21.56 ± 28.56) μg/L] (t =8.863,P ＜0.01).At d1 of diagnosis,the blood PHB level in severe AKI group was higher than that in non-severe AKI group [(14.03 ±6.43) vs.(8.01 ± 6.13),t =11.271,P =0.004];blood PHB in severe AKI group was higher than that in non-AKI group [(10.63 ± 4.21) vs.(8.00 ± 4.76),t =7.051,P =0.017].The levels of urinary NGAL and serum PHB gradually decreased over time in children with severe AKI.The area under the ROC curve (AUC) of urinary NGAL and blood PHB for diagnostic of AKI were 0.833 and 0.952 (P ＜ 0.01),respectively.The diagnostic rate of the combination of the two parameters was 100％.Conclusions The diagnostic value of PHB alone or NGAL in AKI still may be improved.The detection of PHB combined with NGAL can make up each other and improve the diagnosis of AKI,which contribute to the early diagnostic of AKI for clinical workers and provide effective intervention measures,and then reduce the mortality.

Role of JAK2/STAT3-regulated prohibitin in cardioprotection of H2 S postconditioning in hypoxia/reoxygenation-treated cardiomyocytes / 中国药理学通报

Aim ToinvestigatewhethertheJAK2/STAT3 signaling pathway regulates prohibitin expres-sion to protect cardiomyocytes against hypoxia/reoxy-genation injury in hydrogen sulfide postconditioning. Methods Primaryculturedcardiomyocytesfromneo-natal rats were divided into 6 groups: control group ( Normal) , hypoxia/reoxygenation group ( H/R ) , hy-drogen sulfide postconditioning group ( NP) , hydrogen sulfide with AG490 group ( N + A ) , AG490 group ( AG) , DMSO group ( DMSO) . The survival percent-age of cardiomyocytes and the release of LDH were tested at pre-hypoxia and reoxygenation 2h. After reox-ygenation, cell apoptosis was detected by flow cytome-try. The expression of t-STAT3, p-SATAT3 and PHB were determined with Western blot analysis. Results No obvious changes were observed among the groups before hypoxia (P <0. 05). After reoxygenation 2h, compared with H/R group, NP group significantly im-proved the survival rate of cardiomyocytes ( P <0. 05 ) , inhibited the release of LDH and the myocardi-al apoptosis ( P <0. 05 ) , meanwhile up-regulated the p-STAT3 and PHB expression. However, AG490 abol-ished the cardioprotection offered by hydrogen sulfide postconditioning and the increase in p-STAT3 and PHB expression.Conclusion Hydrogensulfidepostcondi-tioning may protect cardiomyocytes against hypoxia/reoxygenation injury through the JAK2/STAT3 pathway upregulating the expression of prohibitin.

Effects of uric acid on mitochondrial oxidative damage and apoptosis in human renal tubular epithelial cells / 中华肾脏病杂志

Objective To observe the effects of uric acid (UA) on mitochondrial oxidative damage and apoptosis in renal tubular epithelial cells (HK-2),and investigate the possible mechanism.Methods HK-2 cells were exposed to UA (480 μmol/L,720 μmol/L) for different time (0 h,24 h,48 h)in vitro.The mitochondrial ROS production was detected by MitoSOX staining.The mitochondrial membrane potential was measured by JC-1 staining.The expressions of prohibitin and AIF were examined by Western blotting and irnmunofluorescence cytochemistry.The cell apoptosis was measured by Annexin V-FITC/PI staining.Results The mitochondrial ROS production in HK-2 cells exposed to 480 μ mol/L UA was increased than that of control group at 24 h (P ＜ 0.05),and increased gradually with UA concentration and incubation time increasing,while the mitochondrial membrane potential was reduced at the same time.There were no significant changes in AIF expression and apoptosis rate of HK -2 cells exposed to 480 μmol/L UA for 24 h compared with that of control group (P ＞ 0.05),while both of them were up-regulated when HK-2 cells were exposed to 480 μmol/L UA for 48 h and 720 μmol/L JA for 24 h and 48 h (P ＜ 0.05).The prohibitin expression in HK-2 cells exposed to 480 μmol/L UA was reduced than that of control group at 24 h (P ＜ 0.05),and down-regulated gradually with UA concentration and incubation time increasing.Conclusion Uric acid can induce the mitochondrial ROS production increased,the mitochondrial membrane potential reduced,the prohibitin expression down-regulated and the mitochondrial apoptosis pathway activated in HK-2 cells.

Effect of all-trans retinoic acid on expressions of Prohibitin1 and Prohibitin2 in hypoxic damage of renal tubular epithelial cells / 中华实用儿科临床杂志

Objective To investigate the effect of all-trans retinoic acid(ATRA) on the expressions of Prohibitin1 (PHB1) and Prohi-bitin2 (PHB2) in hypoxic damage of renal tubular epithelial cells (RTEC).Methods Rat proximal tubular epithelial cell line NRK-52E culture was performed in the 37 ℃ 50 mL/L carbon dioxide incubator with mixture medium of fetal bovine serum and double-antibody(100 mL medium plus 5 mL fetal calf serum and 1 mL double-antibody).After 3 times cell propagation,the cells were divided into 3 groups:normal group,model group and ATRA intervention group.The normal group wasn't disposed,and the model group was put into vacuum tank filled with hypoxic gas(950 mL/L nitrogen and 50 mL/L carbon dioxide) to induce a hypoxic damage of RTEC.The ATRA intervention group was added 0.1 μmol/L ATRA and was treated with hypoxic gas as model group.After 24 h and 36 h,the mRNA expressions of PHB1,PHB2 and transforming growth factor-β1 (TGF-β1) were measured by using real-time PCR method,and the protein expressions of PHB1,PHB2 and TGF-β1 were detected by using Western blot method.Results 1.Compared with normal group,NRK-52E cells PHB1,PHB2 protein expressions and mRNA expressions at 2time points(24 h,36 h) were significantly decreased in model group and ATRA intervention group (all P ＜ 0.05),and the longer hypoxia time,the lower expression value.Compared with model group,NRK-52E cells PHB1 and PHB2 protein expressions and mRNA expressions in ATRA intervention group were increased significantly at 2 time points (all P ＜ O.05).2.Compared with normal group,NRK-52E cells TGF-β1 expressions and mRNA expressions at 2 time points(24 h,36 h) were significantly increased in model group and ATRA intervention group(all P ＜ 0.05),and the longer hypoxia time,the higher expression value;Compared with model group,NRK-52E cells TGF-β1 protein expressions and mRNA expressions in ATRA intervention group were decreased significantly at 2 time points(all P ＜ 0.05).Conclusions ATRA can significantly up-regulate the mRNA expressions and protein expressions of PHB1 and PHB2 in hypoxic damage of RTEC,and ATRA may have a protective effect against hypoxic damage.

Anti-oxidation of Tanshinone ⅡA and Prohibitin on Cardiomyocytes / 中草药·英文版

Objective To investigate the anti-apoptotic mechanism of tanshinone ⅡA and the function of prohibitin(PHB)onmyocardial cells apoptosis induced by hydrogen peroxide(H2O2).Methods Myocardial cells were primary culturedneonate rat were cultured in medium with 200 μmol/L H2O2,and the medium was supplemented with tanshinone ⅡA(1 × 10-4 mol/L)in advance for 24 h.PHB in myocardial cells was knocked down by RNA interference,and theexpression level of PHB was determined by Western blotting analysis.Flow cytometric analysis was used to detectapoptosis rate,intracellular calcium concentration([Ca2+]i),and mitochondrial membrane potential(MMP).ResultsH2O2-mediated cell apoptosis resulted in activation of PHB,increasing of[Ca2+]i,and decreasing of MMP.TanshinoneⅡA profoundly inhibited myocardial cell apoptosis induced by H2O2,decreased[Ca2+]i,and increased MMP.Specificsilence of PHB by siRNA down-regulated the expression level of PHB,increased apoptosis rate and[Ca2+]i,and decreasedMMP.Conclusion The results demonstrate that tanshinone ⅡA could attenuate apoptosis induced by H2O2,and theactivation of PHB induced by H2O2 is the major regulatory pathway of cyto-protective gene expression against oxidativestress.

Proteomics technique in screening of differential proteins for liver regeneration after partial hepatectomy in rats / 第二军医大学学报

Objective: To screen for the differentially expressed proteins during rat liver regeneration after partial hepatectomy (PH) by proteomics technique. Methods: Healthy male SD rats were randomly divided into 2 groups: hepatectomy group and sham operated group. The hepatectomy model was produced by 70% PH (n=35) and the sham operated rats (n=5) underwent the same surgical protocol without hepatectomy. Rats were executed at 2, 12, 24, 36, 48, 72 and 168 h after partial hepatectomy (each time 5 rats) and the right lobes were harvested. The total protein was extracted and analyzed by two-dimensional gel electrophoresis and mass spectrometric analysis. The differential proteins were then analyzed by Western blotting. Results: The spots of differential protein began to rise at 2 h after PH and peaked at 36 h after PH. A total of 78 protein spots were identified and 35 significant protein spots were found by mass spectrometric analysis. The 35 protein sports fell into 5 types according to their dynamic changes: 3 up-regulated and 2 down-regulated (with different regulation time periods and amplitudes); and their functions involved oxidative stress response, acute reaction, lipid and energy metabolism, intracellular signaling transduction, cell proliferation, etc., with some having unknown functions. Western blotting analysis showed that the prohibitin protein began to increase 2 h after PH and decreased to the normal levei after 48 h. Conclusion: It is indicated that many proteins and signal transduction pathways participate in the liver regeneration after partial hepatectomy.

Expression and effects of Prohibitin in unilateral ureteral obstruction rats with renal interstitial fibrosis / 临床儿科杂志

Objective To detect and investigate the expression and the effect of Prohibitin (PHB) in rats with renal interstitial fibrosis (RIF) induced by unilateral ureteral obstruction (UUO) .Methods Forty-eight Wistar male rats (6-weeks-old) were randomly assigned into 2 groups,sham-operated and model group.The model group rats were subjected to left ureteral ligation after anesthesia and the sham-operated group rats were subjected to sham operation.Six rats were killed 7,14,21,28 days after operation respectively.The renal tissues were collected.The index of RIF was calculated.The expressions of mRNA and protein of PHB were assayed by real time polymerase chain reaction and immunohistochemistry. Results Compared with sham-operation group,at each time point,the model group had significantly increased index of RIF (P < 0.01) and the obstruction for a longer period showed the higher index; the model group had significantly decreased expression of mRNA and protein of PHB (P < 0.01) and the obstruction for a longer period showed the lower expression; the model group had significantly increased expression of mRNA and protein of TGF-β1 (P < 0.01) and the obstruction for a longer period showed the higher expression.Correlation analysis showed that the index of RIF was negatively correlated with FHB (γ = -0.825) and positively correlated with TGF-β1 (γ = 0.995),while there was a positive correlation between PHB and TGF-β1 (γ = -0.786).Conclusions The lower expression of PHB in renal tissue of UUO rats might suggest that it play an important role in RIF.

Serum levels of prohibitin in normal children and those with nephrotic syndrome / 临床儿科杂志

Objective To detect the serum prohibitin protein(PHB)level in children with renal interstitial damage and analyze the correlation between PHB and renal interstitial fibrosis(RIF). Methods Serum PHB protein levels were determined by Western blot analysis in 36 children with kidney diseases,and 30 healthy children were studied as control. Levels of BUN,Scr,and urinary microprotein series(including ALBU/Cr,NAGU/Cr,IgG U/Cr,α1-MU/Cr)were studied by automatic biochemical analyzer. Renal interstitial damage was semiquantitatively graded according to Katafuchi's method. The correlation between serum levels of serum PHB protein and those of BUN,Scr as well as urine microprotein were analyzed. Results Serum PHB protein was positive in children with diverse kidney diseases however it was negative in the normal controls(P ＜ 0.05). Serum PHB levels were significantly higher in children with proliferative glomerulonephritis than those with non-proliferative glomerulonephritis(P ＜ 0.05). Statistical analysis indicated that serum PHB levels positively correlated with the degree of tubulointerstitial lesions(r ＝ 0.868,P ＜ 0.001)as well as the glomerular injuries(r ＝ 0.753,P ＜ 0.001). And,serum PHB levels were also positively correlated with urinary microprotein including NAG(r ＝ 0.586,P ＜ 0.001)and IgG(r ＝ 0.341,P ＜ 0.001). Conclusions Serum PHB levels were significantly increased in children with kidney diseases and were positively correlated with the degrees of renal interstitial damage,suggesting that PHB might be a potential clinical marker for detecting tubulointerstitial lesions.

Immunohistochemical Stain of Prohibitin in Various Breast Tumors

The diagnosis of breast disease relies primarily on histopathological diagnosis of hematoxylin-eosin stained specimens. Recently, the histopathological diagnosis has been complemented to an extent by analyses of a growing array of immunohistochemical and molecular markers. Prohibitin is an evolutionarily conserved gene with homologues found in organisms ranging from yeast to man. Prohibitin has anti-proliferous activity and available data suggest a role in such diverse processes as normal cell cycle regulation, replicate senescence, cellular immortalization, and the development of sporadic breast tumors. In this study, the prohibitin protein was immunohistochemically stained in representative samples from 10 patients with fibrocystic diseases, 10 with fibroadenomas, 10 with ductal carcinomas in situ, and 33 with infiltrating ductal carcinomas of the breast. There were weaker expressions throughout the tissue in benign breast diseases, but there was stronger staining in the glandular epithelium of breast cancers than with the stromal components. The epithelial and the stromal prohibitin expressions were elevated in carcinomas in situ and in infiltrating ductal carcinomas. However, the expression was most notable in infiltrating ductal carcinomas. There was no correlation between the prohibitin protein and the histologic grade or the TNM stage in breast cancer(p<0.05). These results show that imunohistochemical staining of prohibitin can be used as a diagnostic biomarker in breast cancer.