Comparative Metabolomic Profiling of Hepatocellular Carcinoma Cells Treated with Sorafenib Monotherapy vs. Sorafenib-Everolimus Combination Therapy.

BACKGROUND: Sorafenib-everolimus combination therapy may be more effective than sorafenib monotherapy for hepatocellular carcinoma (HCC). To better understand this effect, we comparatively profiled the metabolite composition of HepG2 cells treated with sorafenib, everolimus, and sorafenib-everolimus combination therapy. MATERIAL AND METHODS: A 2D HRMAS 1H-NMR metabolomic approach was applied to identify the key differential metabolites in 3 experimental groups: sorafenib (5 µM), everolimus (5 µM), and combination therapy (5 µM sorafenib +5 µM everolimus). MetaboAnalyst 3.0 was used to perform pathway analysis. RESULTS: All OPLS-DA models displayed good separation between experimental groups, high-quality goodness of fit (R2), and high-quality goodness of predication (Q2). Sorafenib and everolimus have differential effects with respect to amino acid, methane, pyruvate, pyrimidine, aminoacyl-tRNA biosynthesis, and glycerophospholipid metabolism. The addition of everolimus to sorafenib resulted in differential effects with respect to pyruvate, amino acid, methane, glyoxylate and dicarboxylate, glycolysis or gluconeogenesis, glycerophospholipid, and purine metabolism. CONCLUSIONS: Sorafenib and everolimus have differential effects on HepG2 cells. Sorafenib preferentially affects glycerophospholipid and purine metabolism, while the addition of everolimus preferentially affects pyruvate, amino acid, and glucose metabolism. This phenomenon may explain (in part) the synergistic effects of sorafenib-everolimus combination therapy observed in vivo.

XPD Functions as a Tumor Suppressor and Dysregulates Autophagy in Cultured HepG2 Cells.

BACKGROUND: Recent clinical studies have linked polymorphisms in the xeroderma pigmentosum group D (XPD) gene, a key repair gene involved in nucleotide excision repair, to increased risk of hepatocellular carcinoma (HCC). However, the cellular effects of XPD expression in cultured HCC cells remain largely uncharacterized. Therefore, the aim of this study was to characterize the in vitro cellular effects of XPD expression on the HCC cell line HepG2. MATERIAL AND METHODS: HepG2 cells were transfected as follows to create four experimental groups: pEGFP-N2/XPD plasmid (XPD) group, EGFP-N2 plasmid (N2) control group, lipofectamine™ 2000 (lipid) control group, and non-transfected (CON) control group. An MTT cell proliferation assay, Annexin V-APC apoptosis assay, colony formation assay, scratch wound migration assay, Transwell migration assay, and Western blotting of the autophagic proteins LC3 and p62 were conducted. RESULTS: XPD expression significantly inhibited HepG2 cell proliferation (p<0.05), significantly promoted HepG2 cell apoptosis (p<0.05), significantly inhibited HepG2 colony formation (p<0.05), significantly decreased HepG2 cells' migratory ability (p<0.05), and significantly lowered HepG2 cells' invasive capacity (p<0.05). Western blotting showed that XPD expression significantly increased LC3 expression (p<0.05) and significantly reduced p62 expression (p<0.05). CONCLUSIONS: XPD expression serves as a tumor suppressor and dysregulates autophagic protein degradation in HepG2 cells in vitro. Further in vivo pre-clinical studies and clinical trials are needed to validate XPD's potential as a tumor-suppressive gene therapy.

ADAM33 polymorphisms and susceptibility to allergic rhinitis: a meta-analysis.

Several polymorphisms in a disintegrin and metalloproteinase 33 (ADAM33) have been implicated in susceptibility to allergic rhinitis (AR), but the results are inconclusive. This meta-analysis was aimed to clarify the impact of ADAM33 polymorphisms on AR risk. Pubmed, EMBASE and Cochrane library were searched until 11 October 2013 for eligible studies on seven ADAM33 polymorphisms: T1, T2, S1, S2, V4, Q-1 and T+1. Data were extracted, and pooled odd ratios (ORs) as well as 95 % confidence intervals (CIs) were calculated. Six studies with 1,135 AR patients and 1,565 controls were included. It was found that ADAM33 T1 (AG+GG vs. AA, OR 1.47, 95 % CI 1.23-1.75, I (2) = 94 %; G vs. A, OR 1.53, 95 % CI 1.32-1.78, I (2) = 94 %), T2 (GA+AA vs. GG, OR 1.26, 95 % CI 1.06-1.51, I (2) = 92 %; G vs. A, OR 1.27, 95 % CI 1.08-1.50, I (2) = 92 %), V4 (CG+GG vs. CC OR 1.35, 95 % CI 1.14-1.59, I (2) = 95 %;G vs. C OR 1.28, 95 % CI 1.13-1.44, I (2) = 96 %) and Q-1 (GA+AA vs. GG OR 1.55, 95 % CI 1.24-1.95, I (2) = 74 %; G vs. C OR 1.46, 95 % CI 1.19-1.79, I (2) = 73 %) polymorphisms were significantly associated with AR susceptibility but not S1, S2 and T+1. In Asians, the same result was found. This meta-analysis indicated that ADAM33 T1, T2, V4 and Q-1 polymorphisms may be the risk factors which conferred to AR susceptibility. The differences in ethnicity did not influence the associations obviously. Gene-gene and gene-environment interactions should be investigated in the future.

Associations between PTPN2 polymorphisms and susceptibility to ulcerative colitis and Crohn's disease: a meta-analysis.

OBJECTIVE: Ulcerative colitis (UC) and Crohn's disease (CD) result from an interaction between genetic and environmental factors. Though several polymorphisms have been identified in PTPN2, their roles in the incidence of UC and CD are conflicting. This meta-analysis was aimed to clarify the impact of these polymorphisms on UC and CD risk. METHOD: PubMed, EMBASE, Cochrane Library and CBM were searched until 23 July 2013 for eligible studies on three PTPN2 polymorphisms: rs2542151, rs1893217 and rs7234029. Data were extracted, and pooled odd ratios (ORs) as well as 95 % confidence intervals (95 % CIs) were calculated. CONCLUSION: The meta-analysis indicated that rs2542151, rs1893217 and rs1893217 were associated with increased CD risk, while the former was associated with increased UC risk. The differences in age of onset and ethnic groups may influence the associations. Gene-gene and gene-environment interactions should be investigated in the future. RESULTS: Seventeen studies with 18,308 cases and 20,406 controls were included. Significant associations were found between rs2542151 polymorphism and CD susceptibility (OR = 1.22, 95 % CI, 1.15-1.30, I (2) = 32 %), as well as between rs2542151 and UC susceptibility (OR = 1.16, 95 % CI, 1.07-1.25, I (2) = 39 %). A similar result was found in Caucasians, but not in Asians. Moreover, a significant increase in CD risk for all carriers of the minor allele of rs1893217 (OR = 1.45, 95 % CI, 1.23-1.70, I (2) = 0 %) and rs7234029 (OR = 1.36, 95 % CI, 1.16-1.59, I (2) = 0 %) were found. For children, the rs1893217 polymorphism appeared to confer susceptibility to CD (OR = 1.56, 95 % CI, 1.28-1.89, I (2) = 0 %).

Adiponectin polymorphisms and non-alcoholic fatty liver disease risk: a meta-analysis.

BACKGROUND AND AIM: The adiponectin polymorphism has been implicated in susceptibility to non-alcoholic fatty liver disease (NAFLD), but the results remain inconclusive. The aim of this meta-analysis is to investigate the association between adiponectin polymorphisms and NAFLD risk. METHODS: All eligible case-control studies published up to September 2013 were identified by searching PubMed, Web of Science, and CNKI. Effect sizes of odds ratio (OR) and 95% confidence interval (95% CI) were calculated by using a fixed- or random-effect model. RESULTS: A total of 10 case-control studies were included; of those, there were nine studies (1223 cases and 1580 controls) for +45T>G polymorphism, seven studies (876 cases and 989 controls) for +276G>T polymorphism, and three studies (299 cases and 383 controls) for -11337C>G polymorphism. Overall, a significantly increased risk was found for +45T>G and -11377C>G polymorphism (+45T>G: OR = 1.45, 95% CI: 1.06-2.00 for recessive model, OR = 1.48, 95% CI: 1.07-2.06 for GG vs TT; -11377C>G: OR = 1.52, 95% CI: 1.10-2.09 for dominant model, OR = 3.88, 95% CI: 1.29-11.68 for GG vs CC), while for +276G>T polymorphism, we found a significantly decreased risk between them (OR = 0.65, 95% CI: 0.45-0.94 for recessive model, OR = 0.58, 95% CI: 0.40-0.84 for TT vs GG). In subgroup analysis by ethnicity, significant association was detected among Asians for +276G>T polymorphism, but not for +45T>G polymorphism. Besides, none of the three adiponectin polymorphisms was associated with the serum adiponectin levels. CONCLUSION: This meta-analysis suggests that adiponectin +45T>G and -11377C>G polymorphisms might be a risk factor for NAFLD, while +276G>T polymorphism may be a protective factor for NAFLD among Asians.

Interleukin-4 receptor α-chain polymorphisms and susceptibility to allergic rhinitis: a meta-analysis.

Several polymorphisms in interleukin-4 receptor α-chain (IL-4RA) have been implicated in susceptibility to allergic rhinitis (AR), but the results are inconclusive. This meta-analysis was aimed to clarify the impact of IL-4RA polymorphisms on AR risk. Pubmed, EMBASE and Cochrane Library were searched until 2 October 2013 for eligible studies on IL-4RA polymorphism. Data were extracted, and pooled odd ratios (ORs) as well as 95 % confidence intervals (95 % CIs) were calculated. Ten studies with 1,552 AR patients and 1,473 controls were included. The results indicated that IL4RA Gln551Arg polymorphism was associated with AR susceptibility in Asian (AG vs. AA OR = 1.63, 95 % CI 1.17-2.28, I (2) = 57 %; GG vs. AA, OR = 1.69, 95 % CI 1.00-2.86, I (2) = 7 %; AG + GG vs. AA, OR = 1.68, 95 % CI 1.18-2.39, I (2) = 64 %; GG vs. AG + AA, OR = 1.47, 95 % CI 0.87-2.49, I (2) = 0 %; G vs. A, OR = 1.54, 95 % CI 1.14-2.10, I (2) = 64 %) but not in Caucasian. IL4RA Ile50 Val as well as Ser478Pro polymorphisms were not associated with AR susceptibility both in Asian and in Caucasian. Gene-gene and gene-environment interactions should be investigated in the future.

Geographic differences in psychological symptoms, sleep quality, and quality of life in patients with inflammatory bowel disease: A multicenter study in China.

OBJECTIVE: We aimed to explore the geographic differences in psychological symptoms, sleep quality, and quality of life (QoL) among adult patients with inflammatory bowel disease (IBD). METHODS: A unified questionnaire was developed to collect data on psychological status and QoL of IBD patients from 42 hospitals across 22 provinces, municipalities, and autonomous regions in China's mainland from September 2021 to May 2022. RESULTS: A total of 2478 patients with IBD were surveyed. The proportions of patients with anxiety (28.5% vs 23.1%), depression (32.3% vs 27.8%), and poor QoL (44.8% vs 32.2%) were significantly higher in patients from the northern region compared to the southern region (all P < 0.05). In the western region, the proportions of patients with anxiety (31.9% vs 23.0%), depression (37.7% vs 26.7%), sleep disturbances (64.5% vs 58.5%), and poor QoL (44.9% vs 34.8%) were significantly higher than in the eastern and central regions (all P < 0.01). Patients from inland regions had significantly higher rates of anxiety (27.1% vs 23.3%), depression (32.5% vs 26.0%), sleep disturbance (62.0% vs 57.7%), and poor QoL (43.5% vs 29.9%) compared to those from coastal regions (all P < 0.05). In economically underdeveloped areas, the proportions of patients with depression (33.1% vs 28.5%) and poor QoL (52.0% vs 32.4%) were significantly higher than in economically (relatively) developed areas (both P < 0.05). CONCLUSION: There are significant geographic differences in psychological symptoms, sleep quality, and QoL among Chinese patients with IBD, which might provide valuable insights for global IBD research and clinical practice.

A Nomogram Model for Prediction of Mortality Risk of Patients with Dangerous Upper Gastrointestinal Bleeding: A Two-center Retrospective Study.

OBJECTIVE: This study aimed to establish a nomogram model to predict the mortality risk of patients with dangerous upper gastrointestinal bleeding (DUGIB), and identify high-risk patients who require emergent therapy. METHODS: From January 2020 to April 2022, the clinical data of 256 DUGIB patients who received treatments in the intensive care unit (ICU) were retrospectively collected from Renmin Hospital of Wuhan University (n=179) and the Eastern Campus of Renmin Hospital of Wuhan University (n=77). The 179 patients were treated as the training cohort, and 77 patients as the validation cohort. Logistic regression analysis was used to calculate the independent risk factors, and R packages were used to construct the nomogram model. The prediction accuracy and identification ability were evaluated by the receiver operating characteristic (ROC) curve, C index and calibration curve. The nomogram model was also simultaneously externally validated. Decision curve analysis (DCA) was then used to demonstrate the clinical value of the model. RESULTS: Logistic regression analysis showed that hematemesis, urea nitrogen level, emergency endoscopy, AIMS65, Glasgow Blatchford score and Rockall score were all independent risk factors for DUGIB. The ROC curve analysis indicated the area under curve (AUC) of the training cohort was 0.980 (95%CI: 0.962-0.997), while the AUC of the validation cohort was 0.790 (95%CI:0.685-0.895). The calibration curves were tested for Hosmer-Lemeshow goodness of fit for both training and validation cohorts (P=0.778, P=0.516). CONCLUSION: The developed nomogram is an effective tool for risk stratification, early identification and intervention for DUGIB patients.

Validation of a real-time biliopancreatic endoscopic ultrasonography analytical device in China: a prospective, single-centre, randomised, controlled trial.

BACKGROUND: Endoscopic ultrasonography (EUS) is a key procedure for the diagnosis of biliopancreatic diseases. However, the performance among EUS endoscopists varies greatly and leads to blind spots during the operation, which can impair the health outcomes of patients. We previously developed an artificial intelligence (AI) device that accurately identified EUS standard stations and significantly reduced the difficulty of ultrasonography image interpretation. In this study, we updated the device (named EUS-IREAD) and validated its performance in improving the quality of EUS procedures. METHODS: In this single-centre, randomised, controlled trial, we updated EUS-IREAD so it consisted of five learning models to identify eight EUS stations and 24 anatomical structures. The trial was done at the Renmin Hospital of Wuhan University (Wuhan, China) and included patients aged 18 years or older with suspected biliopancreatic (pancreas and biliopancreatic duct) lesions due to clinical symptoms, radiological findings, or laboratory findings, and with a high risk of pancreatic cancer. Patients were randomly assigned (1:1) by a dedicated research assistant using a computer-generated random number series (with a block size of four) to undergo the EUS procedure with or without the assistance of EUS-IREAD. Endoscopists in the EUS-IREAD-assisted group were required to observe all standard stations and anatomical structures according to the prompts by the AI device. Data collectors, the independent data anaylsis team, and patients were masked to group allocation. The primary outcome was the missed scanning rate of standard stations between the two groups, which was assessed in patients who underwent EUS procedure in accordance with the assigned intervention (per protocol). This trial is registered with ClinicalTrials.gov, NCT05457101. FINDINGS: Between July 9, 2022, and Feb 28, 2023, 290 patients (mean age 55·93 years [SD 14·06], 152 [52%] male, and 138 [48%] female) were randomly assigned and analysed, including 144 in the EUS-IREAD-assisted group and 146 in the control group. The EUS-IREAD-assisted group had a lower missed scanning rate of stations than the control group (4·5% [SD 0·8] vs 14·3% [1·0], -9·8% [95% CI -12·2 to -7·5]; odds ratio 3·6 [95% Cl 2·6 to 4·9]; p<0·0001). No significant adverse event was found during the study. INTERPRETATION: Our study confirms the capability of EUS-IREAD to monitor the blind spots and reduce the missed rate of stations and structures during EUS procedures. The EUS-IREAD has the potential to play an essential part in EUS quality control. FUNDING: Innovation Team Project of Health Commission of Hubei Province and College-enterprise Deepening Reform Project of Wuhan University.

XPD could suppress growth of HepG2.2.15 and down-regulate the expression of hepatitis B virus x protein through P53 pathway.

OBJECTIVES: We investigated the effects of xeroderma pigmentosum D (XPD) on the growth of hepatoma cells and the expressions of P21, Bax, Bcl-2 and Hepatitis B virus X protein (HBx). In addition, we examined whether XPD affected the aforementioned genes via the P53 pathway. METHODS: Human hepatoma cells (HepG2.2.15) were transfected with XPD expression vector, followed by incubation with Pifithrin-α (P53 inhibitor). By using RT-PCR and Western blotting, the expression levels of XPD, P53, phospho-P53 (ser-15), P21, Bax, Bcl-2 and HBx were detected. The cell cycle and the apoptosis rate were examined with flow cytometry, and the cell viability was detected by MTT. RESULTS: Over-expression of XPD up-regulated the expressions of P53, phospho-P53 (ser-15), P21 and Bax but down-regulated the expressions of Bcl-2 and HBx. XPD inhibited the viability of HepG2.2.15 and exacerbated the apoptosis. However, the inhibition of P53 by Pifithrin-α abolished the above-mentioned effects of XPD. CONCLUSION: XPD could suppress growth of hepatoma cells, up-regulate the expressions of P21 and Bax, and down-regulate the expressions of Bcl-2 and HBx through the P53 pathway. There may be mutual influences among XPD, P53 and HBx that co-regulate hepatocarcinogenesis.

Noscapine induces mitochondria-mediated apoptosis in human colon cancer cells in vivo and in vitro.

Noscapine, a phthalide isoquinoline alkaloid derived from opium, has been widely used as a cough suppressant for decades. Noscapine has recently been shown to potentiate the anti-cancer effects of several therapies by inducing apoptosis in various malignant cells without any detectable toxicity in cells or tissues. However, the mechanism by which noscapine induces apoptosis in colon cancer cells remains unclear. The signaling pathways by which noscapine induces apoptosis were investigated in colon cancer cell lines treated with various noscapine concentrations for 72 h, and a dose-dependent inhibition of cell viability was observed. Noscapine effectively inhibited the proliferation of LoVo cells in vitro (IC(50)=75 µM). This cytotoxicity was reflected by cell cycle arrest at G(2)/M and subsequent apoptosis, as indicated by increased chromatin condensation and fragmentation, the upregulation of Bax and cytochrome c (Cyt-c), the downregulation of survivin and Bcl-2, and the activation of caspase-3 and caspase-9. Moreover, in a xenograft tumor model in mice, noscapine injection clearly inhibited tumor growth via the induction of apoptosis, which was demonstrated using a TUNEL assay. These results suggest that noscapine induces apoptosis in colon cancer cells via mitochondrial pathways. Noscapine may be a safe and effective chemotherapeutic agent for the treatment of human colon cancer.

[Inhibitive mechanisms of Pim-3 affecting fulminant hepatic apoptosis].

To investigate the mechanisms of serine/threonine kinase Pim-3 inhibition of fulminant hepatic apoptosis. Thirty-two rats were randomly divided into four groups (n = 8 each): normal controls (A); pretreatment with Ringer's solution (B), vector plasmid (C), or Pim-3 recombinant plasmid (D) by hydrodynamics-based procedure followed by intraperitoneal injections of lipopolysaccharide (LPS) and D-galactosamine (D-GalN) after one day. At 8 h after the LPS/D-GalN injections, liver tissues were collected from all groups of mice and analyzed for cell apoptosis by detecting caspase-3 activity (measured in relative fluorescence units, RFU). Changes in expression of relevant genes were determined by RT-PCR and Western blotting. Caspase-3 activity was induced in response to LPS/D-GalN injection. Pim-3-pretreated rats showed a lower level of caspase-3 activity than the Ringer's-pretreated or vector plasmid-pretreated rats [(141.7+/-13.7)RFU vs. (508.1+/-32.0) or (493.5+/-33.1) RFU; all P less than 0.01]. High expressions of the liver injury marker gene, iNOS, and the apoptosis-induced genes, p53 and Bax, were found after LPS/D-GalN challenge, and were suppressed by exogenous Pim-3 gene injection. In addition, exogenous Pim-3 gene injection induced high expression of the liver anti-apoptosis protein, Bcl-2, but had no effect on Bax protein expression. The Pim-3 gene can block fulminant hepatic apoptosis by affecting the expression of the iNOS liver injury gene and the p53, Bax and Bcl-2 apoptosis-related genes.

Development and validation of a nomogram to predict overall survival in patients with incidental gallbladder cancer: A retrospective cohort study.

Objective: The aim of this study was to develop and validate a nomogram to predict the overall survival of incidental gallbladder cancer. Methods: A total of 383 eligible patients with incidental gallbladder cancer diagnosed in Shanghai Eastern Hepatobiliary Surgery Hospital from 2011 to 2021 were retrospectively included. They were randomly divided into a training cohort (70%) and a validation cohort (30%). Univariate and multivariate analyses and the Akaike information criterion were used to identify variables independently associated with overall survival. A Cox proportional hazards model was used to construct the nomogram. The C-index, area under time-dependent receiver operating characteristic curves and calibration curves were used to evaluate the discrimination and calibration of the nomogram. Results: T stage, N metastasis, peritoneal metastasis, reresection and histology were independent prognostic factors for overall survival. Based on these predictors, a nomogram was successfully established. The C-index of the nomogram in the training cohort and validation cohort was 0.76 and 0.814, respectively. The AUCs of the nomogram in the training cohort were 0.8, 0.819 and 0.815 for predicting OS at 1, 3 and 5 years, respectively, while the AUCs of the nomogram in the validation cohort were 0.846, 0.845 and 0.902 for predicting OS at 1, 3 and 5 years, respectively. Compared with the 8th AJCC staging system, the AUCs of the nomogram in the present study showed a better discriminative ability. Calibration curves for the training and validation cohorts showed excellent agreement between the predicted and observed outcomes at 1, 3 and 5 years. Conclusions: The nomogram in this study showed excellent discrimination and calibration in predicting overall survival in patients with incidental gallbladder cancer. It is useful for physicians to obtain accurate long-term survival information and to help them make optimal treatment and follow-up decisions.

[Mechanism of regulation of hepatoma cell cycle by XPD/P44 subcomplex : an in vitro experiment].

OBJECTIVE: To explore the effects of xeroderma pigmentosum group D(XPD)/P44 subcomplex on the cell cycle of the hepatoma cells. METHODS: Human hematoma cells of the line SMMC-7721 were cultured and transfected with human XPD gene by Lipofectamine and 2 strains with stably transfected plasmid pEGFG-N2 and stably transfected recombinant plasmid pEGFG-N2/XPD were selected. After stably transfection,the antisense oligonucleotides of P44 were added to treat the stably transfected cells. The cells were divided into 6 groups: Group (1) (control group), Group (2) transfected with the blank plasmid pEGFP-N2, Group (3) transfected with the recombinant plasmid pEGFP-N2/XPD, Group (4) transfected with ASODN complementary to the translation initiation site of pEGFP-N2/XPD, Group (5) transfected with antisense oligodeoxynucleotides (ASODN) complementary to the translation terminal site of pEGFP-N2/XPD, and Group (6) transfected with ASODN complementary to the translation exon5 site of pEGFP-N2/XPD. The expression levels of wild-type P44, XPD, cdk7, cdk2, c-myc, and cdc25A were detected by RT-PCR and Western blotting. The cell growth and the cell cycle were examined by MTT and flow cytometry (FCM). RESULTS: The P44 and XPD mRNA expression levels of Group (4) were significantly higher than those of Groups (1) and (2) (both P < 0.01). Western blotting indicated that the changes of P44 and XPD protein expression levels were consistent with those of their mRNAs respectively; while the mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were all decreased. MTF method showed that the hepatoma cells grew slowly, FCM showed that the number of the cells arrested at the G1 stage of Group (3) were higher than those of Groups (1) and (2). After the blockage of P44 gene expression, the expression levels of XPD mRNA and protein were decreased. The XPD mRNA and protein expression levels of Groups (4), (5), and (6) were significantly higher than those of Group (3) (all P < 0.01). The mRNA and protein expression levels of cdk7, cdk2, c-myc, and cdc25A were upregulated. MT method indicated that cells grew fast. FCM showed that the numbers of the cells arrested at the G1 stage of Group (4), (5), and (6) were all lower than that of Group ((3) The expression levels of cell cycle regulatory genes including cdk7, cdk2, c-myc, and cdc25A were markedly decreased,the hepatoma cells grew slowly; after the blockage of P44 gene expression the expression levels of XPD mRNA and protein were decreased, whereas the expression levels of the cell cycle regulatory genes mentioned above were enhanced, and the hepatoma cells grew faster. CONCLUSION: XPD gene inhibits the proliferation and promotes the apoptosis of hepatoma cells. The expression of XPD may be regulated by its molecular partner P44. XPD/P44 subcomplex is involved in the regulation of DNA damage checkpoint.

Mitochondrial pathway mediated by reactive oxygen species involvement in α-hederin-induced apoptosis in hepatocellular carcinoma cells.

AIM: To investigate the antitumor activity of α-hederin in hepatocellular carcinoma (HCC) cells and its underlying mechanisms in vitro and in vivo. METHODS: SMMC-7721, HepG-2 and Huh-7 HCC cells were cultured in vitro and treated with α-hederin (0, 5 µmol/L, 10 µmol/L, 15 µmol/L, 20 µmol/L, 25 µmol/L, 30 µmol/L, 35 µmol/L, 40 µmol/L, 45 µmol/L, 50 µmol/L, 55 µmol/L, or 60 µmol/L) for 12 h, 24 h, or 36 h, and cell viability was then detected by the Cell Counting Kit-8. SMMC-7721 cells were treated with 0, 5 µmol/L, 10 µmol/L, or 20 µmol/L α-hederin for 24 h with or without DL-buthionine-S,R-sulfoximine (2 mmol/L) or N-acetylcysteine (5 mmol/L) pretreatment for 2 h, and additional assays were subsequently performed. Apoptosis was observed after Hoechst staining. Glutathione (GSH) and adenosine triphosphate (ATP) levels were measured using GSH and ATP Assay Kits. Intracellular reactive oxygen species (ROS) levels were determined by measuring the oxidative conversion of 2',7'-dichlorofluorescin diacetate. Disruption of the mitochondrial membrane potential was evaluated using JC-1 staining. The protein levels of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C were detected by western blotting. The antitumor efficacy of α-hederin in vivo was evaluated in a xenograft tumor model. RESULTS: The α-hederin treatment induced apoptosis of HCC cells. The apoptosis rates in the control, low-dose α-hederin (5 µmol/L), mid-dose α-hederin (10 µmol/L) and high-dose α-hederin (20 µmol/L) groups were 0.90% ± 0.26%, 12% ± 2.0%, 21% ± 2.1% and 37% ± 3.8%, respectively (P < 0.05). The α-hederin treatment reduced intracellular GSH and ATP levels, induced ROS, disrupted the mitochondrial membrane potential, increased the protein levels of Bax, cleaved caspase-3, cleaved caspase-9, apoptosis-inducing factor and cytochrome C, and decreased Bcl-2 expression. The α-hederin treatment also inhibited xenograft tumor growth in vivo. CONCLUSION: The α-hederin saponin induces apoptosis of HCC cells via the mitochondrial pathway mediated by increased intracellular ROS and may be an effective treatment for human HCC.

[Protective role of Pim-3 gene in intestinal mucosa damaged by burn or lipopolysaccharide].

OBJECTIVE: To explore the protective role of Pim-3 gene in intestinal mucosa damaged by burn or lipopolysaccharide (LPS). METHODS: (1) Ninety Wistar mice were randomly divided into 3 equal groups: Group A, undergoing 30% grade III burning on the back; Group B, undergoing intraperitoneal injection of LPS; and Group C, undergoing intraperitoneal injection of normal saline. 3, 6, 12, 24, and 48 hours after the treatment 5 rats from each group were killed with their small intestine tissues taken out. RT-PCR was used to detect the mRNA expression of Pim-3, a serine/threonine kinase, occludin, intercellular adhesion molecule (ICAM)-1, and Western blotting was used to detect the protein expression of Pim-3 and occludin. (2) Intestinal endothelial cells (IECs) of newborn Wistar rats were collected, cultured, and divided into 6 groups: Group (1), treated with LPS (endotoxin), Group (2), transfected with blank plasmid pEGFP-N(2) and treated with LPS, Group (3), transfected with recombinant pEGFP-N(2)/Pim-3and treated with LPS, Group (4), as normal control group, Group (5), transfected with blank plasmid pEGFP-N(2), and Group (6), transfected with recombinant plasmid pEGFP-N(2)/Pim-3. Six hours later RT-PCR was used to detect the mRNA expression of Pim-3, ICAM-1, and occludin. The apoptosis of the cells was examined by flow cytometry. RESULTS: (1) In Groups A and B the mRNA expression of Pim-3 began to increase 3 h later, peaked 6 h later, and then gradually decreased. The Pim-3 mRNA expression of Group C, however, remained always at a low level. The ICAM-1 mRNA expression levels of Groups A and B were constantly up-regulated 6 h later, all significantly higher than those of Group C (all P < 0.01). The occludin mRNA expression levels of Groups A and B began to increase 3 hours later, and peaked 12 hours later, all significantly higher than those of Group C (all P < 0.05). (2) The mRNA expression levels of Pim-3 and occludin of Group (6) was significantly higher than those of Groups (4) and (5) (both P < 0.05). However, there was no significant difference in ICAM-1 mRNA expression among Groups (4), (5), and (6). The mRNA expression levels of Pim-3, occluding, and ICAM-1 of Groups (3) were all significantly higher than those of Groups (1) and (2) (all P < 0.05). The mRNA expression levels of ICAM-1 of Groups (1), (2), and (3) were all significantly lower than those of Groups (4), (5), and (6) (all P < 0.05). The apoptotic rates of groups (4), (5), and (6) were all very low. The apoptotic rates of Groups (1) and (2) were 41.3% and 44.80% respectively, both significantly higher than that of Group (3) (36.03%, both P < 0.05). CONCLUSION: Both burning and LPS stimulate endogenous Pim-3 gene expression in small intestine which lasts a short time. Pim-3 gene not only suppresses the apoptosis of IECs, but also strengthens the occludin expression and inhibits the intestinal tract inflammatory reaction induced by LPS. LPS induces ICAM-1 expression, which can be inhibited by Pim-3.

[Construction of a recombinant Pim3-expressing plasmid and expression and activity thereof: experiment with rats].

OBJECTIVE: To construct a plasmid expressing green fluorescent protein (GFP) and gene of Pim-3, a member of the serine/threonine kinase family, and to investigate the in vivo expression of the construct and its effect on cell apoptosis. METHODS: Pim-3 gene was cloned from myocardium tissues of Wistar rat by RT-PCR and subcloned into GFP-expressing plasmid vector pEGFP-N2 by restriction enzyme. The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-ligase and then identified through enzyme digestion and gene sequencing. Thirty-two Wistar rats were randomly divided into 4 equal groups: Group A, as control group; Group B, injected intravenously with Ringer's solution; Group C, injected with blank vector, and Group D, injected with the recombinant plasmid pEGFP-N2/Pim-3. One day later, endotoxin/D-galactoamine (D-GalN) was intraperitoneally injected. 24 hours later the rats were killed. Fluorescence microscopy was used to observe the expression of the reporter gene GFP in the liver tissues. RT-PCR was used to detect the Pim-3 mRNA expression. The hepatic apoptosis was detected by TUNEL assay. The activity of caspase-3 was detected. RESULTS: A 998 bp target cDNA fragment with restriction enzyme sites was amplified and inserted into the multiple clone site of pEGFP-N2 successfully. High expression levels of the target gene Pim-3 and reporter gene GFP were achieved in the rat liver after transfer of the recombinant plasmid. The relative Pim-3 expression level of Group D was 0.49 +/- 0.15, significantly higher than those of Groups A, B, and C (0.06 +/- 0.02, 0, and 0 respectively, all P < 0.01). The apoptotic index of Group D was (4.9 +/- 1.2)%, significantly lower than those of Groups B and C [(72.5 +/- 6.1)% and (69.8 +/- 5.7)% respectively, both P < 0.01]; however, not significantly different from that of Group A [(3.1 +/- 0.7)%]. The activity of caspase-3 of Group D was (76 +/- 27) pmol.min(-1).mg(-1), significantly lower than those of Groups B and C [(147 +/- 55) and (142 +/- 50) pmol.min(-1).mg(-1), respectively, both P < 0.01]; however, not significantly different from that of Group A (60 +/- 15). CONCLUSION: The recombinant plasmid pEGFP-N2/Pim-3 can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis.

Dietary patterns and depression risk: A meta-analysis.

Although some studies have reported potential associations of dietary patterns with depression risk, a consistent perspective hasn't been estimated to date. Therefore, we conducted this meta-analysis to evaluate the relation between dietary patterns and the risk of depression. A literature research was conducted searching MEDLINE and EMBASE databases up to September 2016. In total, 21 studies from ten countries met the inclusion criteria and were included in the present meta-analysis. A dietary pattern characterized by a high intakes of fruit, vegetables, whole grain, fish, olive oil, low-fat dairy and antioxidants and low intakes of animal foods was apparently associated with a decreased risk of depression. A dietary pattern characterized by a high consumption of red and/or processed meat, refined grains, sweets, high-fat dairy products, butter, potatoes and high-fat gravy, and low intakes of fruits and vegetables is associated with an increased risk of depression. The results of this meta-analysis suggest that healthy pattern may decrease the risk of depression, whereas western-style may increase the risk of depression. However, more randomized controlled trails and cohort studies are urgently required to confirm this findings.

[Role of hepatocellular apoptosis and mechanisms of liver injury in lipopolysaccharide-induced acute liver failure in D-galactosamine-sensitized rats].

OBJECTIVE: To investigate the situation of hepatocellular apoptosis in D-galactosamine (D-GalN)-sensitized rats with lipopolysaccharide (LPS)-induced acute liver failure and the mechanisms of liver injury therein. METHODS: Forty eight Wistar rats were randomly divided into 6 equal groups to be injected peritoneally with LPS (50 microg/kg) and D-GalN (300 mg/kg) (treatment groups) or normal saline of the same volume (control groups), and then were killed 6, 24, or 48 hours later. Blood samples were collected from the portal vein or vena cava inferior to detect the contents of serum alanine aminotransferase (ALT), livers were take out to detect the hepatocellular apoptosis by TUNEL assay or ultrastructural observations, and the expressions of iNOS, p53, and p21waf1/cip1 gene were detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The ALT levels of the treatment groups were all significantly higher than those of the corresponding control groups, with the peaks 24 hours after treatment. Transmission electron microscopy showed that apoptotic cells were rare in the control subgroups, but were abundant in the liver tissues of the treatment subgroups. The apoptotic indices of liver cells of the 6, 24, and 48 hours treatment subgroups were 7.3% +/- 1.5%, 71.8% +/- 10.3%, and 68.2% +/- 11.9% respectively, all significantly higher than those of the control groups (2.6% +/- 1.1%, all P < 0.05). The apoptotic index increased gradually along with the time, however, the apoptotic indices of the 24 and 48 hours treatment subgroups were not significantly different (P > 0.05). The mRNA expression levels of iNOS gene of the control subgroups, 6 hours treatment subgroup, 24 hours treatment subgroups, and 48 hours treatment subgroup were 0, 0.53 +/- 0.11, 0.36 +/- 0.08, and 0.15 +/- 0.04 respectively with a significant difference among different subgroups, and with a peak 6 hours after treatment. The p53 expressions of the control subgroups, 6 hours treatment subgroup, 24 hours treatment subgroups, and 48h treatment subgroup were 0.031 +/- 0.006, 0.022 +/- 0.008, 0.49 +/- 0.11, and 0.39 +/- 0.17 respectively, being low in both control subgroups and 6h treatment subgroup and significantly upregulated in the 24 and 48 hours treatment groups. Expression of p21waf1/cip1 was not detected in the control subgroups and 48 hours treatment subgroup, but was found in the 6 hours and 24 hours treatment subgroups, with a peak in the 24 hours treatment subgroup. CONCLUSION: Acute liver failure can be induced by low dose LPS in D-GalN-sensitized rats, which may be associated with the early high expression of iNOS gene; Apoptosis is the important morphological feature in this process.

Liver X receptor-ß improves autism symptoms via downregulation of ß-amyloid expression in cortical neurons.

BACKGROUND: We study the effect of liver X receptor ß (LXRß) on ß-amyloid (Aß) peptide generation and autism behaviors by conducting an animal experiment. METHODS: In autistic mice treated with LXRß agonist T0901317, enzyme linked immunosorbent assay was used to measure Aß in brain tissue homogenates. Western blot was used to detect Aß precursors, Aß degradation and secretase enzymes, and expression of autophagy-related proteins and Ras/Raf/Erkl/2 signaling pathway proteins in brain tissue. Changes in autism spectrum disorder syndromes of the BTBR mice were compared before and after T0901317 treatment. RESULTS: Compared with the control group, autistic mice treated with LXRß agonist T0901317 showed significantly lower Aß level in brain tissue (P < 0.05), significantly higher Aß degradation enzyme (NEP, IDE proteins) levels (all P < 0.05), significantly lower Aß secretase enzyme BACE1 protein level (P < 0.05), and significantly lower Ras, P-C-Raf, C-Raf, P-Mekl/2, P-Erkl/2 protein levels (all P < 0.05). BTBR mice treated with T0901317 showed improvements in repetitive stereotyped behavior, inactivity, wall-facing standing time, self-combing time and center stay time, stayed longer in platform quadrant, and crossed the platform more frequently (all P < 0.05). CONCLUSIONS: LXRß could potentially reduce brain Aß generation by inhibiting Aß production and promoting Aß degradation, thereby increasing the expression of autophagy-related proteins, reducing Ras/Raf/Erkl/2 signaling pathway proteins, and improving autism behaviors.