Prohibitin 1 inhibits cell proliferation and induces apoptosis via the p53-mediated mitochondrial pathway in vitro.

BACKGROUND: Prohibitin 1 (PHB1) has been identified as an antiproliferative protein that is highly conserved and ubiquitously expressed, and it participates in a variety of essential cellular functions, including apoptosis, cell cycle regulation, proliferation, and survival. Emerging evidence indicates that PHB1 may play an important role in the progression of hepatocellular carcinoma (HCC). However, the role of PHB1 in HCC is controversial. AIM: To investigate the effects of PHB1 on the proliferation and apoptosis of human HCC cells and the relevant mechanisms in vitro. METHODS: HCC patients and healthy individuals were enrolled in this study according to the inclusion and exclusion criteria; then, PHB1 levels in the sera and liver tissues of these participates were determined using ELISA, RT-PCR, and immunohistochemistry. Human HepG2 and SMMC-7721 cells were transfected with the pEGFP-PHB1 plasmid and PHB1-specific shRNA (shRNA-PHB1) for 24-72 h. Cell proliferation was analysed with an MTT assay. Cell cycle progression and apoptosis were analysed using flow cytometry (FACS). The mRNA and protein expression levels of the cell cycle-related molecules p21, Cyclin A2, Cyclin E1, and CDK2 and the cell apoptosis-related molecules cytochrome C (Cyt C), p53, Bcl-2, Bax, caspase 3, and caspase 9 were measured by real-time PCR and Western blot, respectively. RESULTS: Decreased levels of PHB1 were found in the sera and liver tissues of HCC patients compared to those of healthy individuals, and decreased PHB1 was positively correlated with low differentiation, TNM stage III-IV, and alpha-fetoprotein ≥ 400 µg/L. Overexpression of PHB1 significantly inhibited human HCC cell proliferation in a time-dependent manner. FACS revealed that the overexpression of PHB1 arrested HCC cells in the G0/G1 phase of the cell cycle and induced apoptosis. The proportion of cells in the G0/G1 phase was significantly increased and the proportion of cells in the S phase was decreased in HepG2 cells that were transfected with pEGFP-PHB1 compared with untreated control and empty vector-transfected cells. The percentage of apoptotic HepG2 cells that were transfected with pEGFP-PHB1 was 15.41% ± 1.06%, which was significantly greater than that of apoptotic control cells (3.65% ± 0.85%, P < 0.01) and empty vector-transfected cells (4.21% ± 0.52%, P < 0.01). Similar results were obtained with SMMC-7721 cells. Furthermore, the mRNA and protein expression levels of p53, p21, Bax, caspase 3, and caspase 9 were increased while the mRNA and protein expression levels of Cyclin A2, Cyclin E1, CDK2, and Bcl-2 were decreased when PHB1 was overexpressed in human HCC cells. However, when PHB1 was upregulated in human HCC cells, Cyt C expression levels were increased in the cytosol and decreased in the mitochondria, which indicated that Cyt C had been released into the cytosol. Conversely, these effects were reversed when PHB1 was knocked down. CONCLUSION: PHB1 inhibits human HCC cell viability by arresting the cell cycle and inducing cell apoptosis via activation of the p53-mediated mitochondrial pathway.

VCAN, expressed highly in hepatitis B virus-induced hepatocellular carcinoma, is a potential biomarker for immune checkpoint inhibitors.

BACKGROUND: As a proteoglycan, VCAN exists in the tumor microenvironment and regulates tumor proliferation, invasion, and metastasis, but its role in hepatocellular carcinoma (HCC) has not yet been elucidated. AIM: To investigate the expression and potential mechanism of action of VCAN in HCC. METHODS: Based on The Cancer Genome Atlas Liver Hepatocellular Carcinoma dataset, we explored the correlation between VCAN expression and clinical features, and analyzed the prognosis of patients with high and low VCAN expression. The potential mechanism of action of VCAN was explored by Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes analysis, and gene set enrichment analysis. We also explored immune cell infiltration, immune checkpoint gene expression, and sensitivity of immune checkpoint [programmed cell death protein 1 (PD-1)/cytotoxic T lymphocyte antigen 4 (CTLA4)] inhibitor therapy in patients with different VCAN expression. VCAN mRNA expression and VCAN methylation in peripheral blood were tested in 100 hepatitis B virus (HBV)-related patients (50 HCC and 50 liver cirrhosis). RESULTS: VCAN was highly expressed in HCC tissues, which was associated with a poor prognosis in HCC patients. No significant difference was found in VCAN mRNA expression in blood between patients with HBV-related cirrhosis and those with HCC, but there was a significant difference in VCAN methylation between the two groups. The correlation between VCAN and infiltrations of several different tumor immune cell types (including B cells, CD8+ T cells, and eosinophils) was significantly different. VCAN was strongly related to immune checkpoint gene expression and tumor mutation burden, and could be a biomarker of sensitivity to immune checkpoint (PD1/CTLA4) inhibitors. In addition, VCAN mRNA expression was associated with hepatitis B e antigen, HBV DNA, white blood cells, platelets, cholesterol, and coagulation function. CONCLUSION: High VCAN level could be a possible biomarker for poor prognosis of HCC, and its immunomodulatory mechanism in HCC warrants investigation.

3D printed collagen/silk fibroin scaffolds carrying the secretome of human umbilical mesenchymal stem cells ameliorated neurological dysfunction after spinal cord injury in rats.

Although implantation of biomaterials carrying mesenchymal stem cells (MSCs) is considered as a promising strategy for ameliorating neural function after spinal cord injury (SCI), there are still some challenges including poor cell survival rate, tumorigenicity and ethics concerns. The performance of the secretome derived from MSCs was more stable, and its clinical transformation was more operable. Cytokine antibody array demonstrated that the secretome of MSCs contained 79 proteins among the 174 proteins analyzed. Three-dimensional (3D) printed collagen/silk fibroin scaffolds carrying MSCs secretome improved hindlimb locomotor function according to the Basso-Beattie-Bresnahan scores, the inclined-grid climbing test and electrophysiological analysis. Parallel with locomotor function recovery, 3D printed collagen/silk fibroin scaffolds carrying MSCs secretome could further facilitate nerve fiber regeneration, enhance remyelination and accelerate the establishment of synaptic connections at the injury site compared to 3D printed collagen/silk fibroin scaffolds alone group according to magnetic resonance imaging, diffusion tensor imaging, hematoxylin and eosin staining, Bielschowsky's silver staining, immunofluorescence staining and transmission electron microscopy. These results indicated the implantation of 3D printed collagen/silk fibroin scaffolds carrying MSCs secretome might be a potential treatment for SCI.

Umbilical cord mesenchymal stem cells promote neurological repair after traumatic brain injury through regulating Treg/Th17 balance.

Traumatic brain injury (TBI) is a brain injury resulting from blunt mechanical external forces, which is a crucial public health and socioeconomic problem worldwide. TBI is one of the leading causes of death or disability. The primary injury of TBI is generally irreversible. Secondary injury caused by neuroinflammation could result in exacerbation of patients, which indicated that anti-inflammation and immunomodulatory were necessary for the treatment of TBI. Accumulated evidence reveals that the transplantation of umbilical cord mesenchymal stem cells (UCMSCs) could regulate the microenvironment in vivo and keep a balance of helper T 17(Th17)/ regulatory T cell (Treg). Therefore, it is reasonable to hypothesize that the UCMSCs could repair neurological impairment by maintaining the balance of Th17/Treg after TBI. In the study, we observed the phenomenon of trans-differentiation of T lymphocytes into Th17 cells after TBI. Rats were divided into Sham, TBI, and TBI + UCMSCs groups to explore the effects of the UCMSCs. The results manifested that trans-differentiation of Th17 into Treg was facilitated by UCMSCs, which was followed by promotion of neurological recovery and improvement of learning and memory in TBI rats. Furthermore, UCMSCs decreased the phosphorylation of nuclear factor-kappa B (NF-κB) and increased the expression of mothers against decapentaplegic homolog 3 (Smad3) in vivo and vitro experiments. In conclusion, UCMSCs maintained Th17/Treg balance via the transforming growth factor-ß (TGF-ß)/ Smad3/ NF-κB signaling pathway.

Development of natural products in prevention and treatment of osteoporosis based on GSK-3fJ signaling pathway / 中国药理学通报

Abnormality of glycogen synthase kinase-3 (3 ( GSK- 3(i) triggers a deleterious consequence of disorder of bone me¬tabolism , contributing to osteoporosis.Osteoporosis is character¬ized by bone loss and destruction of bone microstmcture, which often leads to the increase of incidence rate of fracture.Natural products derived from plants can be used as potential dmgs to prevent and treat osteoporosis.In this review, we elaborates on the intimate connection between GSK-3p and bone metabolism.and summarizes natural products of flavonoids, isoflavoncs, pol¬ysaccharides and saponins, phenols, alkaloids and amine deriv¬atives regulating GSK-3(3 pathway in the prevention and treat¬ment of osteoporosis, which may provide scientific evidence for further development of drugs that may effectively prevent and treat osteoporosis.

Integrated printed BDNF/collagen/chitosan scaffolds with low temperature extrusion 3D printer accelerated neural regeneration after spinal cord injury.

Recent studies have shown that 3D printed scaffolds integrated with growth factors can guide the growth of neurites and promote axon regeneration at the injury site. However, heat, organic solvents or cross-linking agents used in conventional 3D printing reduce the biological activity of growth factors. Low temperature 3D printing can incorporate growth factors into the scaffold and maintain their biological activity. In this study, we developed a collagen/chitosan scaffold integrated with brain-derived neurotrophic factor (3D-CC-BDNF) by low temperature extrusion 3D printing as a new type of artificial controlled release system, which could prolong the release of BDNF for the treatment of spinal cord injury (SCI). Eight weeks after the implantation of scaffolds in the transected lesion of T10 of the spinal cord, 3D-CC-BDNF significantly ameliorate locomotor function of the rats. Consistent with the recovery of locomotor function, 3D-CC-BDNF treatment could fill the gap, facilitate nerve fiber regeneration, accelerate the establishment of synaptic connections and enhance remyelination at the injury site.

Erratum to: Integrated printed BDNF/collagen/chitosan scaffolds with low temperature extrusion 3D printer accelerated neural regeneration after spinal cord injury.

[This corrects the article DOI: 10.1093/rb/rbab047.].

Rutaecarpine Ameliorated High Sucrose-Induced Alzheimer's Disease Like Pathological and Cognitive Impairments in Mice.

High sucrose can induce tau hyperphosphorylation and cognitive dysfunction/memory impairment as observed in Alzheimer's disease (AD). Rutaecarpine, a specific (transient receptor potential vanilloid 1 [TRPV1]) agonist, is neuroprotective against high sucrose diet-induced impairment, but detailed mechanisms are still elusive. In this study, we investigated whether rutaecarpine mitigates high sucrose diet-induced pathological alterations and cognitive in AD-like mice. Mice were administered fodder containing 0.01% rutaecarpine and 20% sucrose solution. Our results showed that rutaecarpine significantly attenuated high sucrose diet-induced spatial memory impairment and enhanced synaptic plasticity; rutaecarpine prevented high sucrose diet-induced tau hyperphosphorylation by decreasing glycogen synthase kinase-3ß (GSK-3ß) activity; activation of GSK-3ß reversed the protective effect of rutaecarpine on learning and memory deficits, synaptic plasticity, and tau hyperphosphorylation induced by high-glucose diet significantly, suggesting that GSK-3ß activation is required for high glucose-induced tau hyperphosphorylation. These results demonstrated that rutaecarpine can mitigate high sucrose diet-induced hyperphosphorylation of AD-associated tau protein and cognitive impairment by inhibiting GSK-3ß, which supported that dietary rutaecarpine might have a promising use for therapeutic intervention of AD.

Add-on pegylated interferon augments hepatitis B surface antigen clearance vs continuous nucleos(t)ide analog monotherapy in Chinese patients with chronic hepatitis B and hepatitis B surface antigen ≤ 1500 IU/mL: An observational study.

BACKGROUND: Nucleos(t)ide analog (NA) has shown limited effectiveness against hepatitis B surface antigen (HBsAg) clearance in chronic hepatitis B (CHB) patients. AIM: To evaluate the efficacy and safety of add-on peginterferon α-2a (peg-IFN α-2a) to an ongoing NA regimen in CHB patients. METHODS: In this observational study, 195 CHB patients with HBsAg ≤ 1500 IU/mL, hepatitis B e antigen (HBeAg)-negative (including HBeAg-negative patients or HBeAg-positive patients who achieved HBeAg-negative after antiviral treatment with NA) and hepatitis B virus-deoxyribonucleic acid < 1.0 × 102 IU/mL after over 1 year of NA therapy were enrolled between November 2015 and December 2018 at the Second Affiliated Hospital of Xi'an Jiaotong University, China. Patients were given the choice between receiving either peg-IFN α-2a add-on therapy to an ongoing NA regimen (add-on group, n = 91) or continuous NA monotherapy (monotherapy group, n = 104) after being informed of the benefits and risks of the peg-IFN α-2a therapy. Total therapy duration of peg-IFN α-2a was 48 wk. All patients were followed-up to week 72 (24 wk after discontinuation of peg-IFN α-2a). The primary endpoint was the proportion of patients with HBsAg clearance at week 72. RESULTS: Demographic and baseline characteristics were comparable between the two groups. Intention-to-treatment analysis showed that the HBsAg clearance rate in the add-on group and monotherapy group was 37.4% (34/91) and 1.9% (2/104) at week 72, respectively. The HBsAg seroconversion rate in the add-on group was 29.7% (27/91) at week 72, and no patient in the monotherapy group achieved HBsAg seroconversion at week 72. The HBsAg clearance and seroconversion rates in the add-on group were significantly higher than in the monotherapy group at week 72 (P < 0.001). Younger patients, lower baseline HBsAg concentration, lower HBsAg concentrations at weeks 12 and 24, greater HBsAg decline from baseline to weeks 12 and 24 and the alanine aminotransferase ≥ 2 × upper limit of normal during the first 12 wk of therapy were strong predictors of HBsAg clearance in patients with peg-IFN α-2a add-on treatment. Regarding the safety of the treatment, 4.4% (4/91) of patients in the add-on group discontinued peg-IFN α-2a due to adverse events. No severe adverse events were noted. CONCLUSION: Peg-IFN α-2a as an add-on therapy augments HBsAg clearance in HBeAg-negative CHB patients with HBsAg ≤ 1500 IU/mL after over 1 year of NA therapy.

Dual role of polyamines in heart ischemia/reperfusion injury through regulation of mitochondrial permeability transition pore.

Polyamines (putrescine, spermidine, and spermine) are essential polycations that play important roles in various physiological and pathophysiological processes in mammalian cells. The study was to investigate their role in cardioprotection against ischemia/reperfusion (I/R) injury and the underlying mechanism. Isolated hearts from male Sprague-Dawley rats were Langendorff-perfused and cardiac I/R was achieved by 30 min of global ischemia followed by 120 min of reperfusion. Different concentrations of polyamines (0.1, 1, 10, and 15 µmol/L of putrescine, spermidine, and spermine), cyclosporin A (0.2 µmol/L), or atractyloside (20 µmol/L) were given 10 min before the onset of reperfusion. The hemodynamics were monitored; the lactate dehydrogenase (LDH) levels in the coronary effluent were measured spectrophotometrically; infarct size was determined by the 2,3,5-triphenyltetrazolium chloride staining method; and mitochondrial permeability transition pore (MPTP) opening was determined spectrophotometrically by the Ca2+-induced swelling of isolated cardiac mitochondria. The results showed that compared to I/R alone, 0.1 and 1 µmol/L polyamines treatment improved heart function, reduced LDH release, decreased infarct size, and these effects were inhibited by atractyloside (MPTP activator). In isolated mitochondria from normal rats, 0.1 and 1 µmol/L polyamines treatment inhibited MPTP opening. However, 10 and 15 µmol/L polyamines treatment had the opposite effects, and these effects were inhibited by cyclosporin A (MPTP inhibitor). Our findings showed that polyamines may have either protective or damaging effects on hearts suffering from I/R by inhibiting or activating MPTP opening.

Reduced cardiac ischemia/reperfusion injury by hypothermic reperfusion via activation of transient receptor potential M8 channel.

AIMS: To investigate the cardioprotective effects of hypothermic (25 °C) reperfusion on ischemia/reperfusion injury and the role of transient potential channel M8 (TRPM8) in this process. MAIN METHODS: Western blot and real-time PCR were used to monitor the expression of TRPM8 in myocardium. Myocardial ischemia/reperfusion injury was induced by 30 min of global ischemia followed by 120 min of reperfusion in Langendorff-perfused hearts from Sprague-Dawley rats. The reperfusion was either normothermic (37 °C) or hypothermic (25 °C). Infarct size and left ventricular function were assessed, and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) in the coronary effluent were measured spectrophotometrically, and cardiomyocyte apoptosis was detected by TUNEL assay. The expression of TRPM8, Bcl-2, Bax, cleaved capspase-3, RhoA, and ROCK2 was quantified. KEY FINDINGS: TRPM8 protein and mRNA were expressed in rat myocardium. Hypothermic reperfusion decreased the infarct size, LDH activity, MDA content, apoptosis, and expression of Bax, cleaved caspase-3, RhoA, and ROCK2 compared with normothermic reperfusion. These effects were associated with improved recovery of left ventricular contractility, and were reduced by BCTC, a TRPM8 antagonist. Ischemia/reperfusion injury and the increased expression of Bax, caspase-3, RhoA, and ROCK2 induced by normothermic reperfusion were reduced by Icilin, a TRPM8 agonist. SIGNIFICANCE: Hypothermic reperfusion at 25 °C has cardioprotective effects against ischemia/reperfusion injury via activation of TRPM8 to inhibit the oxidative stress-related RhoA/ROCK2 signal pathway.

Association of gene polymorphisms of pattern-recognition receptor signaling pathway with the risk and severity of hand, foot, and mouth disease caused by enterovirus 71 in Chinese Han population.

Hand, foot, and mouth disease (HFMD) caused by enterovirus 71 (EV71) presents with a wide variety of clinical manifestations. Host immune response is a factor that influences disease susceptibility and severity. We investigated the potential association of gene polymorphisms in the pattern recognition receptor (PRR) pathway with the risk and severity of EV71 infection. A total of 180 EV71 HFMD cases (108 severe case; 72 mild cases) were enrolled. A group of 201 sex- and age-matched children was included as a control. All subjects were genotyped for the most common single-nucleotide polymorphisms (SNPs) in the PRR and the PRR signaling pathway using the SNPscan multiple SNP typing method. Binary logistic regression analysis revealed statistically significant differences in polymorphism of RIG-1 between patients and controls (rs3739674 G vs C: OR = 1.502, 95%CI: 1.120-2.014; rs9695310 G vs C: OR = 1.782, 95%CI: 1.312-2.419). Polymorphisms of RIG-1 rs3739674 (G vs C: OR = 2.047, 95%CI: 1.307-3.205) and TLR3 rs5743305 (A vs T: OR = 0.346, 95%CI: 0.212-0.566) were found to be associated with disease severity. The results indicated that RIG-1 (rs3739674 and rs9695310) polymorphisms are associated with an increased risk of EV71-induced HFMD in Chinese children, whereas RIG-1 rs3739674 and TLR3 rs5743305 polymorphisms are associated with disease severity. These findings support an important role of innate immune mechanism in EV71 infection.

Patients' perceptions of service quality in China: An investigation using the SERVQUAL model.

BACKGROUND AND AIM: The doctor-patient relationship has been a major focus of society. Hospitals' efforts to improve the quality of their medical services have been to reduce the probability of doctor-patient conflicts. In this study, we aimed to determine the gap between expectations and perceptions of service quality according to patients to provide reference data for creating strategies to improve health care quality. METHODS: Twenty-seven hospitals in 15 provinces (municipalities directly beneath the central government) were selected for our survey; we sent out 1,589 questionnaires, of which 1,520 were collected (response rate 95.65%) and 1,303 were valid (85.72% effective recovery rate). Paired t-tests were used to analyze whether there were significant differences between patients' expectations and perceived service quality. A binary logistic regression analysis was used to determine whether there were significant differences in the gap between expectation and perception of service quality according to patients' demographic characteristics. RESULTS: There was a significant difference between the expected and perceived service quality (p < 0.05) according to patients both before and after receiving medical services. Furthermore, the service quality gap of each service dimension was negative. Specifically, the gaps in service quality were as follows: economy, responsiveness, empathy, assurance, reliability, and tangibles. Overall, we can conclude that patients' perceptions of service quality are lower than their expectations. CONCLUSIONS: According to the study results, the quality of health care services as perceived by patients was lower than expected. Hospitals should make adjustments according to the actual situation and should strive to constantly improve the quality of medical services for patients.

Caffeic acid phenethyl ester up-regulates antioxidant levels in hepatic stellate cell line T6 via an Nrf2-mediated mitogen activated protein kinases pathway.

AIM: To investigate the antioxidant effect of caffeic acid phenethyl ester (CAPE) in hepatic stellate cell-T6 (HSC-T6) cells cultured in vitro and the potential mechanisms. METHODS: HSC-T6 cells were cultured in vitro and treated with various concentrations of CAPE for 24, 48 and 72 h, respectively. Cell proliferation was investigated using the MTT assay, and cell ultrastructural alterations were observed by transmission electron microscopy. Flow cytometry was employed to investigate the effects of CAPE on apoptosis and the levels of reactive oxygen species in HSC-T6 cells cultured in vitro. An enzyme immunoassay instrument was used to evaluate antioxidant enzyme expression. The effect on α-smooth muscle actin was shown using immunofluorescence. Gene and protein levels of Nrf2, related factors, and mitogen activated protein kinases (MAPKs), in HSC-T6 cells were detected using RT-PCR and Western blot, respectively. RESULTS: CAPE inhibited the proliferation and activation of HSC-T6 cells cultured in vitro. CAPE increased the antioxidant levels and the translocation of Nrf2 from the cytoplasm to the nucleus in HSC-T6 cells. Moreover, the phosphorylation of MAPKs in cells decreased in response to CAPE. Interestingly, CAPE-induced oxidative stress in the cells was significantly attenuated by pretreatment with MAPKs inhibitors. CONCLUSION: CAPE inhibits cell proliferation and up-regulates the antioxidant levels in HSC-T6 cells partly through the Nrf2-MAPKs signaling pathway.

Guggulsterone induces apoptosis of human hepatocellular carcinoma cells through intrinsic mitochondrial pathway.

AIM: To investigate the effects of guggulsterone on the proliferation and apoptosis of human hepatoma HepG2 cells in vitro and relevant mechanisms. METHODS: Human hepatocellular carcinoma HepG2 cells and normal human liver L-02 cells were treated with different concentrations of guggulsterone (5-100 µmol/L) for 24-72 h. Cell proliferation was tested by MTT assay. Cell cycle and apoptosis were investigated using flow cytometry (FACS). Bcl-2 and Bax mRNA and protein expression was detected by real-time PCR and Western blot, respectively. TGF-ß1, TNF-α, and VEGF contents were determined by ELISA. RESULTS: Guggulsterone significantly inhibited HepG2 cell proliferation in a dose- and time-dependent manner. FACS showed that guggulsterone arrested HepG2 cell cycle at G0/G1 phase. Guggulsterone induced apoptosis was also observed in HepG2 cells, with 24.91% ± 2.41% and 53.03% ± 2.28% of apoptotic cells in response to the treatment with 50 µmol/L and 75 µmol/L guggulsterone, respectively. Bax mRNA and protein expression was significantly increased and Bcl-2 mRNA and protein expression was decreased. ELISA analysis showed that the concentrations of TGF-ß1 and VEGF were significantly decreased and TNF-α concentration was increased. CONCLUSION: Guggulsterone exerts its anticancer effects by inhibiting cell proliferation and inducing apoptosis in HepG2 cells. Guggulsterone induces apoptosis by activation of the intrinsic mitochondrial pathway.

Cytokeratin 8 is increased in hepatitis C virus cells and its ectopic expression induces apoptosis of SMMC7721 cells.

AIM: To investigate cytokeratin 8 (CK8) overexpression during hepatitis C virus (HCV) infection and its pathogenesis, and the effect of ectopic CK8 expression on hepatoma cell lines. METHODS: We successfully established an in vitro HCV cell culture system (HCVcc) to investigate the different expression profiles of CK8 in Huh-7-HCV and Huh-7.5-HCV cells. The expression of CK8 at the mRNA level was determined by real-time polymerase chain reaction (RT-PCR). The expression of CK8 at the protein level was evaluated by Western blotting. We then constructed a eukaryotic expression combination vector containing the coding sequence of human full length CK8 gene. CK8 cDNA was amplified by reverse transcription-PCR and inserted into pEGFP-C1 and the positive clone pEGFP-CK8 was obtained. After confirming the sequence, the recombinant plasmid was transfected into SMMC7721 cells with lipofectamine2000 and CK8 expression was detected using inverted fluorescence microscopy, RT-PCR and Western blotting. Besides, we identified biological function of CK8 on SMMC7721 cells, including cell proliferation, cell cycle and apoptosis detection. RESULTS: RT-PCR showed that the expression level of CK8 in Huh-7-HCV and Huh-7.5-HCV cells was 2.88 and 2.95 times higher than in control cells. Western blot showed that CK8 expression in Huh-7-HCV and Huh-7.5-HCV cells was 2.53 and 3.26 times higher than that in control cells, respectively. We found that CK8 at mRNA and protein levels were both significantly increased in HCVcc. CK8 was up-regulated in SMMC7721 cells. CK8 expression at the mRNA level was significantly upregulated in SMMC7721/pEGFP-CK8 cells. CK8 expression in SMMC7721/ pEGFP-CK8 cells was 2.69 times higher than in SMMC7721 cells, and was 2.64 times higher than in SMMC7721/pEGFP-C1 cells. CK8 expression at the protein level in SMMC7721/pEGFP-CK8 cells was 2.46 times higher than in SMMC7721 cells, and was 2.29 times higher than in SMMC7721/pEGFP-C1 cells. Further analysis demonstrated that forced expression of CK8 slowed cell growth and induced apoptosis of SMMC7721 cells. CONCLUSION: CK8 up-regulation might have a functional role in HCV infection and pathogenesis, and could be a promising target for the treatment of HCV infection.

[Use of parallel factor and two dimensional fluorescence spectroscopy correlation technique for measurement of reaction between MDA and cooked ground meat].

The fluorescence characteristics of oxidation reaction between MDA and cooked ground meat were analyzed by front face three dimensional synchronous fluorescence spectroscopy, parallel factor and two dimensional correlation technique. The results showed that the reaction system has two synchronous fluorescence peaks, one is Ex 292 nm and deltalambda 50 nm, assigned to the fluorescence characteristics of tryptophan residues in proteins; the other is Ex 400 nm, delta 70 nm, corresponding with the fluorescence characteristics of MDA-protein adducts formed during oxidation; The synchronous fluorescence landscape was analyzed using PARAFAC. The loading profiles of 1st and 2nd components had an optimal lambda 50 and 70 nm, respectively. During oxidation reaction, the synchronous fluorescence intensity of tryptophan gradually decreased, while the synchronous fluorescence intensity of MDA-protein adducts gradually increased. Two dimensional correlation synchronous fluorescence spectroscopy technique showed that the variation ratio of fluorescence intensity of tryptophan preceded that of MDA-protein adducts.

Increased expression of chondroitin sulphate proteoglycans in rat hepatocellular carcinoma tissues.

AIM: To investigate the expression of chondroitin sulphate proteoglycans (CSPGs) in rat liver tissues of hepatocellular carcinoma (HCC). METHODS: Thirty male Sprague Dawley rats were randomly divided into two groups: control group (n = 10) and HCC model group (n = 20). Rats in the HCC model groups were intragastrically administrated with 0.2% (w/v) N-diethylnitrosamine (DEN) every 5 d for 16 wk, whereas 0.9% (w/v) normal saline was administered to rats in the control group. After 16 wk from the initiation of experiment, all rats were killed and livers were collected and fixed in 4% (w/v) paraformaldehyde. All tissues were embedded in paraffin and sectioned. Histological staining (hematoxylin and eosin and Toluidine blue) was performed to demonstrate the onset of HCC and the content of sulphated glycosaminoglycan (sGAG). Immunohistochemical staining was performed to investigate the expression of chondroitin sulphate (CS)/dermatan sulphate (DS)-GAG, heparan sulphate (HS)-GAG, keratan sulphate (KS)-GAG in liver tissues. Furthermore, expression and distribution of CSPG family members, including aggrecan, versican, biglycan and decorin in liver tissues, were also immunohistochemically determined. RESULTS: After 16 wk administration of DEN, malignant nodules were observed on the surface of livers from the HCC model group, and their hepatic lobule structures appeared largely disrupted under microscope. Toluidine blue staining demonstrated that there was an significant increase in sGAG content in HCC tissues when compared with that in the normal liver tissues from the control group [0.37 ± 0.05 integrated optical density per stained area (IOD/area) and 0.21 ± 0.01 IOD/area, P < 0.05]. Immunohistochemical studies demonstrated that this increased sGAG in HCC tissues was induced by an elevated expression of CS/DS (0.28 ± 0.02 IOD/area and 0.18 ± 0.02 IOD/area, P < 0.05) and HS (0.30 ± 0.03 IOD/area and 0.17 ± 0.02 IOD/area, P < 0.01) but not KS GAGs in HCC tissues. Further studies thereby were performed to investigate the expression and distribution of several CSPG components in HCC tissues, including aggrecan, versican, biglycan and decorin. Interestingly, there was a distinct distribution pattern for these CSPG components between HCC tissues and the normal tissues. Positive staining of aggrecan, biglycan and decorin was localized in hepatic membrane and/or pericellular matrix in normal liver tissues; however, their expression was mainly observed in the cytoplasm, cell membranes in hepatoma cells and/or pericellular matrix within HCC tissues. Semi-quantitative analysis indicated that there was a higher level of expression of aggrecan (0.43 ± 0.01 and 0.35 ± 0.03, P < 0.05), biglycan (0.32 ± 0.01 and 0.25 ± 0.01, P < 0.001) and decorin (0.29 ± 0.01 and 0.26 ± 0.01, P < 0.05) in HCC tissues compared with that in the normal liver tissues. Very weak versican positive staining was observed in hepatocytes near central vein in normal liver tissues; however there was an intensive versican distribution in fibrosis septa between the hepatoma nodules. Semi-quantitative analysis indicated that the positive rate of versican in hepatoma tissues from the HCC model group was much higher than that in the control group (33.61% and 21.28%, P < 0.05). There was no positive staining in lumican and keratocan, two major KSPGs, in either normal or HCC liver tissues. CONCLUSION: CSPGs play important roles in the onset and progression of HCC, and may provide potential therapeutic targets and clinical biomarkers for this prevalent tumor in humans.

[Establishment of the Chang liver cell line stably overexpressing human UCP2 gene and its effect on mitochondrial membrane potential and reactive oxygen species].

To establish the Chang liver cell line stably overexpressing human uncoupling protein 2 (UCP2) and observe the effect of UCP2 on mitochondrial membrane potential (MMP) and reactive oxygen species (ROS). The Chang liver cell line was transfected with recombinant plasmid containing full-length human UCP2 cDNA (pcDNA3.1-hUCP2) or pcDNA3.1 empty vector. The stable cell line was established by antibiotic screening with Zeocin. UCP2 expression was detected by Western blotting and immunocytochemistry. The UCP2 overexpressing cells were pretreated with genipin at various doses (25, 50 and 100 munol/L). MMP and intracellular ROS were detected by fluorescence spectrophotometry. The total normalized protein content in UCP2 overexpressing cells was 1.6-fold higher than that in unmanipulated normal cells. The fluorescence intensities of Rhodamine123 and DCFH-DA in UCP2 overexpressing Chang liver cells (11.11+/-2.76 and 4.97+/-0.62, respectively) were significantly lower than those in unmanipulated normal cells (15.56+/-2.55, P less than 0.01 and 6.14+/-1.25, P less than 0.05, respectively) and in cells transfected with empty vector (16.11+/-2.93, P less than 0.01 and 6.23+/-1.13, P less than 0.05, respectively). Treatment of UCP2 overexpressing cells with 25, 50 and 100 munol/L genipin caused a dose-dependent increase in fluorescence intensities of Rhodamine123 (14.89+/-2.89, 17.89+/-2.93 and 24.00+/-2.55, respectively, all P less than 0.01) and DCFH-DA (9.16+/-0.78, 10.84+/-1.09 and 11.83+/-1.25, respectively, all P less than 0.01). The Chang liver cell line stably overexpressing UCP2 was established successfully. Using this cell system, UCP2 was found to play a role in mitochondrial function by regulating MMP and ROS.

Telaprevir for chronic hepatitis C with genotype 1: a meta-analysis.

BACKGROUND/AIMS: The examination of HCV virological clearance through several randomized clinical trials of telaprevir in genotype 1 chronic hepatitis C. METHODOLOGY: We analyzed the effect of telaprevir on the end of treatment virological response and the sustained response, and investigated its harmful effect using meta-analysis of 5 randomized controlled trials. RESULTS: Overall analysis revealed a significant effect of telaprevir in both naive patients (RR, 1.32; 95% CI, 1.08-1.60) and previously failed treated patients (p<0.0001). Monotherapy and double therapy seemed to show no effect in naive patients. Triple therapy followed with PegIFN-2a plus ribavirin seemed to be effective in both naive patients and previously failed treated patients. Telaprevir was associated with a significantly higher incidence of serious adverse events (RR, 1.45; 95% CI, 1.00-2.10) and with discontinuation (RR, 2.23; 95% CI, 1.40-3.55) because of adverse events. In naive patients, relapsers and non-responders, the regimen of telaprevir/ PegIFN-2a/ribavirin for 12 weeks followed by PegIFN-2a/ribavirin for 12 weeks (T12PR24) was the optimal regimen regarding to efficiency and duration. CONCLUSIONS: Telaprevir combined with PegIFN-2a plus ribavirin may improve sustained response in genotype 1 chronic hepatitis C. Regimen T12PR24 may be the best regimen in this respect. New randomized controlled trials are required to confirm this meta-analysis.