Surface-Engineered Polygonatum Sibiricum Polysaccharide CaCO3 Microparticles as Novel Vaccine Adjuvants to Enhance Immune Response.

Micro- and nanoparticles delivery systems have been widely studied as vaccine adjuvants to enhance immunogenicity and sustain long-term immune responses. Polygonatum sibiricum polysaccharide (PSP) has been widely studied as an immunoregulator in improving immune responses. In this study, we synthesized and characterized cationic modified calcium carbonate (CaCO3) microparticles loaded with PSP (PEI-PSP-CaCO3, CTAB-PSP-CaCO3), studied the immune responses elicited by PEI-PSP-CaCO3 and CTAB-PSP-CaCO3 carrying ovalbumin (OVA). Our results demonstrated that PEI-PSP-CaCO3 significantly enhanced the secretion of IgG and cytokines (IL-4, IL-6, IFN-γ, and TNF-α) in vaccinated mice. Additionally, PEI-PSP-CaCO3 induced the activation of dendritic cells (DCs), T cells, and germinal center (GC) B cells in draining lymph nodes (dLNs). It also enhanced lymphocyte proliferation, increased the ratio of CD4+/CD8+ T cells, and elevated the frequency of CD3+ CD69+ T cells in spleen lymphocytes. Therefore, PEI-PSP-CaCO3 microparticles induced a stronger cellular and humoral immune response and could be potentially useful as a vaccine delivery and adjuvant system.

Diagnostic and prognostic value of serum Chitinase 3-like protein 1 in hepatocellular carcinoma.

The serum Chitinase 3-like protein 1 (CHI3L1) protein level can distinguish the stages of liver fibrosis to a great extent. However, the diagnostic and prognostic significance of serum CHI3L1 in hepatocellular carcinoma (HCC) is not clarified. To evaluate the diagnostic and prognostic value of CHI3L1 in HCC, a total of 128 HCC patients treated in the HwaMei Hospital, University of Chinese Academy of Sciences, from December 2018 to April 2020 were collected retrospectively. Matched age and gender subjects, 40 patients with liver cirrhosis, 40 patients with chronic hepatitis, and 40 healthy subjects were enrolled in the control group. The relevant clinical laboratory and examination data and the overall survival time (OS) of the HCC patients were collected. The serum CHI3L1 expression level is related to α-fetoprotein (AFP), tumor-node-metastasis (TNM) stage, maximum tumor diameter, liver cirrhosis, and HCC patient's OS (p < 0.05). The area under the curve (AUC) of CHI3L1 was 0.7875 with the cutoff value of 91.36 ng/ml. Combining the serum CHI3L1 and α-fetoprotein (AFP) by a binary logistic regression model can increase the diagnostic sensitivity to 97.5%. Multivariate Cox regression analysis indicated that CHI3L1 is an independent prognostic factor in patients with HCC.

Incidence and risk factors of anti-tuberculosis drug induced liver injury (DILI): Large cohort study involving 4652 Chinese adult tuberculosis patients.

BACKGROUND AND AIMS: Anti-tuberculosis drugs remain as an important cause of drug-induced liver injury (DILI) worldwide. Adverse drug reactions reduce the effectiveness of treatment. We aimed to determine the incidence and risk factors associated with anti-tuberculosis DILI (ATDILI). METHODS: Using established criteria and causality assessment methods, risk factors for ATDILI were identified in a contemporary cohort and validated in another cohort prospectively. Independent determinants of ATDILI were identified using Cox regression analysis. RESULTS: In the derivation cohort (n = 3155), 170 (5.4%) developed ATDILI of which 27 (15.9%) developed jaundice; 9(5.3%) developed acute liver failure (ALF) and 3 died. Among HBsAg positive patients, 11/27 (40.7%) of ATDILI developed after 3 months of starting treatment. In addition, of 218 (6.9%) who developed raised alanine transferase (ALT) levels ≥3 times upper limit normal, 193 (88.5%) resolved and 25 (11.4%) progressed to DILI. Age (HR = 1.014, 95% CI: 1.005-1.023), baseline ALT (HR = 1.014, 95% CI: 1.003-1.024), haemoglobin (HR = 1.011, 95% CI: 1.002-1.020) and HBsAg positivity (HR = 1.516, 95% CI: 1.004-2.290) were independent risk factors for DILI. In the second cohort (n = 1497) of which 85 (5.7%) developed ATDILI. Age (HR = 1.029, 95% CI: 1.003-1.056), baseline AST (HR = 1.036, 95% CI: 1.010-1.062), previous TB treatment (HR = 3.894, 95% CI: 1.304-11.625) and active drinking (HR = 3.624, 95% CI: 1.147-11.454) were risk factors for developing jaundice. CONCLUSION: Elevation of ALT of ≥3 × ULN during anti-TB treatment resolves in the vast majority without developing serious consequences. In two cohorts involving 4652 patients, incidence of ALF and death because of ATDILI are low. Age, baseline ALT, haemoglobin and HBsAg positivity are risk factors for the development of DILI and these inform monitoring and management of these patients.

Risk factors for prolonged virus shedding of respiratory tract and fecal in adults with severe acute respiratory syndrome coronavirus-2 infection.

BACKGROUND: The dynamic alteration and comparative study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA shedding pattern during treatment are limited. This study explores the potential risk factors influencing prolonged viral shedding in COVID-19. METHODS: A total of 126 COVID-19 patients were enrolled in this retrospective longitudinal study. A multivariate logistic regression analysis was carried out to estimate the potential risk factors. RESULTS: 38.1% (48/126) cases presented prolonged respiratory tract viral shedding, and 30 (23.8%) cases presented prolonged rectal swab viral shedding. Obesity (OR, 3.31; 95% CI, 1.08-10.09), positive rectal swab (OR, 3.43; 95% CI, 1.53-7.7), treatment by lopinavir/ritonavir with chloroquine phosphate (OR, 2.5; 95% CI, 1.04-6.03), the interval from onset to antiviral treatment more than 7 days (OR, 2.26; 95% CI, 1.04-4.93), lower CD4+ T cell (OR, 0.92; 95% CI, 0.86-0.99) and higher NK cells (OR, 1.11; 95% CI, 1.02-1.20) were significantly associated with prolonged respiratory tract viral shedding. CD3-CD56+ NK cells (OR, 0.87; 95% CI, 0.76-0.99) were related with prolonged fecal shedding. CONCLUSIONS: Obesity, delayed antiviral treatment, and positive SARS-CoV-2 for stool were independent risk factors for prolonged SARS-CoV-2 RNA shedding of the respiratory tract. A combination of LPV/r and abidol as the initial antiviral regimen was effective in shortening the duration of viral shedding compared with LPV/r combined with chloroquine phosphate. CD4+ T cell and NK cells were significantly associated with prolonged viral shedding, and further studies are to be warranted to determine the mechanism of immunomodulatory response in virus clearance.

The clinical significance of serum chitinase 3-like 1 in hepatitis B-related chronic liver diseases.

AIM: In the present study, we purposed to determine serum chitinase 3-like 1 (CHI3L1) expression characteristics in chronic liver diseases monoinfected with hepatitis B virus and analyze its diagnostic value in liver fibrosis. METHODS: A total of 467 chronic hepatitis B (CHB) patients, 312 liver cirrhosis (LC) patients, and 104 hepatocellular carcinoma (HCC) patients at our institution were enrolled, and clinical indicators were analyzed. RESULTS: Our data have shown that the expression level of serum CHI3L1 was steadily increased from CHB to LC to HCC (P < .001). Serum CHI3L1 expression levels were positively associated with liver stiffness measurement (LSM), fibrosis-4 (FIB-4) index, aspartate aminotransferase-to-platelet ratio index (APRI), and HCC stage. The receiver operating characteristic (ROC) curve proved that serum CHI3L1 was superior to other noninvasive methods (LSM, FIB-4, and APRI) with an area under the ROC curve (AUC) of 0.97 in diagnosing significant fibrosis. CONCLUSIONS: Serum CHI3L1 harbors significant clinical value in chronic liver diseases infected with hepatitis B virus, especially in the diagnosis of fibrosis.

Clinical features of COVID-19 convalescent patients with re-positive nucleic acid detection.

BACKGROUND: Coronavirus disease 2019 (COVID-19) is a pandemic that has rapidly spread worldwide. Increasingly, confirmed patients being discharged according to the current diagnosis and treatment protocols, follow-up of convalescent patients is important to knowing about the outcome. METHODS: A retrospective study was performed among 98 convalescent patients with COVID-19 in a single medical center. The clinical features of patients during their hospitalization and 2-week postdischarge quarantine were collected. RESULTS: Among the 98 COVID-19 convalescent patients, 17 (17.3%) were detected positive severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid during 2-week postdischarge quarantine. The median time from discharge to SARS-CoV-2 nucleic acid re-positive was 4 days (IQR, 3-8.5).The median time from symptoms onset to final respiratory SARS-CoV-2 detection of negative result was significantly longer in re-positive group (34 days [IQR, 29.5-42.5]) than in non-re-positive group (19 days [IQR, 16-26]). On the other hand, the levels of CD3-CD56 + NK cells during hospitalization and 2-week postdischarge were higher in re-positive group than in non-re-positive group (repeated measures ANOVA, P = .018). However, only one case in re-positive group showed exudative lesion recurrence in pulmonary computed tomography (CT) with recurred symptoms. CONCLUSION: It is still possible for convalescent patients to show positive for SARS-CoV-2 nucleic acid detection, but most of the re-positive patients showed no deterioration in pulmonary CT findings. Continuous quarantine and close follow-up for convalescent patients are necessary to prevent possible relapse and spread of the disease to some extent.

Ribavirin augments doxorubicin's efficacy in human hepatocellular carcinoma through inhibiting doxorubicin-induced eIF4E activation.

Activation of eukaryotic translation initiation factor 4E (eIF4E) is a cellular survival mechanism in response to chemotherapy in cancers. In this work, we demonstrate that targeting eIF4E by ribavirin sensitizes hepatocellular carcinoma (HCC) cell response to doxorubicin. Ribavirin inhibits growth and survival of HCC cells, and to a greater extent than in normal liver cells. Its combination with doxorubicin achieves greater efficacy than single drug in vitro and in vivo. Ribavirin suppresses phosphorylation of molecules involved in Akt/mTOR/eIF4E pathway. Overexpression of the phosphomimetic form (S209D) but not the nonphosphorylatable form (S209A) eIF4E significantly reverses the inhibitory effects of ribavirin. Interestingly, doxorubicin significantly increases p-eIF4E(S209) level in a dose- and time-dependent manner, suggesting that doxorubicin induces eIF4E activation in HCC cells. In addition, eIF4E activation induced by doxorubicin in HCC cells is inhibited by ribavirin. Our work demonstrates the greater efficacy of ribavirin and doxorubicin combination and its underlying mechanisms.

Decreased expression of hsa_circ_0003570 in hepatocellular carcinoma and its clinical significance.

BACKGROUND: Circular RNAs (circRNAs) constitute a class of non-coding RNAs recently discovered to be widespread and abundant in mammalian cells. However, the expression features of most of circRNAs in hepatocellular carcinoma (HCC) are unraveled. In this study, we focused on hsa_circ_0003570, which was found to be down-regulated in HCC tissues in our previous microarray screening. METHODS: The hsa_circ_0003570 levels in HCC cell lines, HepG2, SMMC-7721, MHCC97L, MHCC97H, and HCCLM3, and human normal hepatic cell line L02 were detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Then, its levels in 107 paired HCC tissues and adjacent non-tumor tissues, 60 liver biopsy samples from patients with chronic liver diseases were detected by qRT-PCR. The receiver operating characteristic curve (ROC) was used to evaluate the diagnostic value of hsa_circ_0003570 for HCC. RESULTS: Hsa_circ_0003570 was not only first found down-regulated in HCC cell lines (P<.001) but also in HCC tissues (P<.001). Moreover, hsa_circ_0003570 was gradually decreased from chronic hepatitis (CH), to liver cirrhosis (LC) and to HCC tissues (P<.01). Its expression levels were significantly correlated with tumor diameter (P=.035), differentiation (P=.013), microvascular invasion (P=.045), Barcelona Clinic Liver Cancer stages (P=.011), tumor-node-metastasis stages (P=.016), and serum alpha-fetoprotein levels (P=.031). The ROC curve demonstrated that hsa_circ_0003570 had poor performance for differentiating HCC from LC and CH, but had relatively good performance for differentiating LC from CH. CONCLUSIONS: These results indicated that hsa_circ_0003570 expression levels were associated with HCC clinicopathological characteristics.

Antibiotic tigecycline enhances cisplatin activity against human hepatocellular carcinoma through inducing mitochondrial dysfunction and oxidative damage.

Targeting mitochondrial metabolism has been recently demonstrated to be a promising therapeutic strategy for the treatment of various cancer. In this work, we demonstrate that antibiotic tigecycline is selectively against hepatocellular carcinoma (HCC) through inducing mitochondrial dysfunction and oxidative damage. Tigecycline is more effective in inhibiting proliferation and inducing apoptosis of HCC than normal liver cells. Importantly, tigecycline significantly enhances the inhibitory effects of chemotherapeutic drug cisplatin in HCC in vitro and in vivo. Mechanistically, tigecycline specifically inhibits mitochondrial translation as shown by the decreased protein levels of Cox-1 and -2 but not Cox-4 or Grp78, and increased mRNA levels of Cox-1 and -2 but not Cox-4 in HCC cells exposed to tigecycline. In addition, tigecycline significantly induces mitochondrial dysfunction in HCC cells via decreasing mitochondrial membrane potential, complex I and IV activities, mitochondrial respiration and ATP levels. Tigecycline also increases levels of mitochondrial superoxide, hydrogen peroxide and ROS levels. Consistent with oxidative stress, oxidative damage on DNA, protein and lipid are also observed in tigecycline-treated cells. Importantly, antioxidant N-acetyl-l-cysteine (NAC) reverses the effects of tigecycline, suggesting that oxidative stress is required for the action of tigecycline in HCC cells. We further show that HCC cells have higher level of mitochondrial biogenesis than normal liver cells which might explain the different sensitivity to tigecycline between HCC and normal liver cells. Our work is the first to demonstrate that tigecycline is a promising candidate for HCC treatment and highlight the therapeutic value of targeting mitochondrial metabolism in HCC.

Nucleos(t)ide analogs improve long-term prognosis in patients with chronic hepatitis B-associated liver failure.

AIM: Chronic hepatitis B-associated liver failure (CHB-LF) is associated with high mortality. Antiviral therapy with nucleoside and nucleotide analogs (NUCs) has been reported to improve the short-term prognosis of patients with CHB-LF. However, the long-term effects of the therapy remain unclear. We undertook a cohort study to investigate the long-term effect of NUC-based antiviral therapy in patients with CHB-LF. METHODS: A total of 976 patients with CHB-LF were enrolled between January 2001 and December 2009 at the Liver Disease Center of Ningbo No. 2 Hospital (Ningbo, China). The patients were divided into the NUC treatment group (n = 412) and control group (n = 564). The propensity score matching method was used to match the patients between the two groups to equilibrate the covariates. Survival analysis was carried out using the matched samples. The Cox proportional hazard model was used for the analysis of prognostic factors. RESULTS: After propensity matching, 262 pairs were successfully matched. No statistically significant difference was observed in the baseline characteristics of the matching pairs (P > 0.05). The long-term survival rate and survival duration of the NUC treatment group were higher than that of the control group (P < 0.05). Gender, age, Model for End-stage Liver Disease values, cholinesterase levels, white blood cell counts, hepatic encephalopathy, concomitant infection, and treatment with NUCs were found to be the independent factors associated with long-term prognosis. CONCLUSION: Antiviral therapy with NUCs may reduce the mortality rate and improve the long-term prognosis of patients with CHB-LF.

Therapeutic Effect of Gypenosides on Nonalcoholic Steatohepatitis via Regulating Hepatic Lipogenesis and Fatty Acid Oxidation.

Nonalcoholic steatohepatitis (NASH) is the most frequent cause of liver dysfunction and a common global problem. Gypenosides can decrease pathological modifications of high-fat diet-induced rat atherosclerosis; however, its effect and mechanism on NASH remain unclear. In this study, rats were randomly divided into normal control and model groups. Model rats were fed with a high-fat diet and treated with gypenosides, rosiglitazone, or water for 6 weeks. We found that liver tissues showed significant hepatic steatosis and vacuolar degeneration with significantly higher triglyceride (TG), free fatty acid (FFA) and malonyl CoA, serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) activities in model group versus normal control group (p<0.01). Liver tissue mRNA and protein levels of sterol regulatory element binding protein-1c (SREBP-1c), carbohydrate response element binding protein (ChREBP), acetyl-CoA carboxylase (ACCase), and stearoyl CoA desaturase enzyme 1 (SCD1) were significantly increased, while the carnitine palmitoyl transferase-1 (CPT-1) level was significantly decreased in the model group versus the normal control group (p<0.01). Pathological changes of hepatic steatosis; body weight and liver wet weight; liver tissue TG, FFA and malonyl CoA concentrations; serum ALT, AST and GGT activities; liver tissue mRNA and protein levels of SREBP-1c, ChREBP, ACCase, and SCD-1 were significantly decreased; protein and mRNA levels of CPT-1 were significantly increased in the gypenosides group versus model group (p<0.01). In conclusion, gypenosides has therapeutic effect on NASH through regulating key transcriptional factors and lipogenic enzymes involved in fatty acid oxidation during hepatic lipogenesis.

Hepatoma cell-derived leptin downregulates the immunosuppressive function of regulatory T-cells to enhance the anti-tumor activity of CD8+ T-cells.

The adaptive immune response against hepatocellular carcinoma (HCC) could be a therapeutic target to restrain HCC initiation and growth. The interactions between hepatoma cells and immune cells modify the anti-tumor immunity to influence hepatoma cell survival. To explore the potential interplay between hepatoma cells and anti-HCC T-cells, we conducted a HCC induction mouse model to analyze the phenotypic and functional alterations of T-cell subsets. We found that both hepatoma tissues and hepatoma cell lines substantially produced higher leptin, which is an adipokine usually expressed in fat tissue, than normal liver tissue or hepatocytes. We also found that regulatory T-cells (Tregs), effector CD4(+) T-cells and CD8(+) T-cells upregulated expression of leptin receptor (LEPR) in spleens and livers after HCC induction. In vitro study showed that macrophages and dendritic cells isolated from HCC livers upregulated LEPR expression on T-cells. Leptin inhibited Treg activation and function in vitro, demonstrated by lower expression of TGF-ß, IL-10, CTLA4 and GITR in Tregs, as wells weaker suppression of CD8(+) T-cell proliferation and production of cytotoxic mediators. In addition, silencing LEPR in Tregs favored tumor growth in a hepatoma cell line allograft model. Taken together, our study suggests that hepatoma cells could enhance anti-HCC immunity through secreting leptin to down-regulate Treg activity and subsequently promote CD8(+) T-cell response.

Acute-on-chronic liver failure precipitated by hepatic injury is distinct from that precipitated by extrahepatic insults.

UNLABELLED: Patients with acute-on-chronic liver failure (ACLF) represent a heterogeneous population. The aim of the study is to identify distinct groups according to the etiologies of precipitating events. A total of 405 ACLF patients were identified from 1,361 patients with cirrhosis with acute decompensation and categorized according to the types of acute insults. Clinical characteristics and prognosis between the hepatic group and extrahepatic group were compared, and the performance of prognostic models was tested in different groups. Two distinct groups (hepatic-ACLF and extrahepatic-ACLF) were identified among the ACLF population. Hepatic-ACLF was precipitated by hepatic insults and had relatively well-compensated cirrhosis with frequent liver and coagulation failure. In contrast, extrahepatic-ACLF was exclusively precipitated by extrahepatic insults, characterized by more severe underlying cirrhosis and high occurrence of extrahepatic organ failures (kidney, cerebral, circulation, and respiratory systems). Both groups had comparably high short-term mortality (28-day transplant-free mortality: 48.3% vs. 50.7%; P = 0.22); however, the extra-hepatic-ACLF group had significantly higher 90-day and 1-year mortality (90-day: 58.9% vs. 68.3%, P = 0.035; 1-year: 63.9% vs. 74.6%, P = 0.019). In hepatic-ACLF group, the integrated Model for End-Stage Liver Disease (iMELD) score had the highest area under the receiver operating characteristic curve (auROC = 0.787) among various prognostic models in predicting 28-day mortality, whereas CLIF-Consortium scores for ACLF patients (CLIF-C-ACLF) had the highest predictive value in the other group (auROC = 0.779). CONCLUSIONS: ACLF precipitated by hepatic insults is distinct from ACLF precipitated by extrahepatic insults in clinical presentation and prognosis. The iMELD score may be a better predictor for hepatic-ACLF short-term prognosis, whereas CLIF-C-ACLF may be better for extrahepatic-ACLF patients.

Outcomes of Interferon/Ribavirin Therapy in Patients with HCV Defined by Expression of Plasma Soluble Human Leukocyte Antigen-G but Not IL-37.

BACKGROUND Chronic hepatitis C virus (HCV) infection leads to life-threatening complications worldwide. Immunomodulation signals the response to virus clearance. The immune-suppressive molecule human leukocyte antigen-G (HLA-G) has been shown to function in inhibiting both innate and adaptive immune responses. The objective of this study was to investigate the expression of HLA-G and IL-37 in sustained virological response (SVR) and non-SVR HCV-positive patients before and after complete treatment with a combination of pegylated interferon (IFN) and ribavirin (RBV). MATERIAL AND METHODS Our study included 132 chronic hepatitis C patents who received combined therapy with IFN-a and RBV. Both SVR and non-SVR patients were included. The end-of-treatment response was defined as undetectable HCV RNA at week 48. Patients with end-of-treatment response were detected by HCV RNA at 24 weeks after therapy. The expression levels of HLA-G and IL-37 at the end and 24 weeks after treatment were detected by ELISA. RESULTS Plasma HLA-G and IL-37 were significantly increased in HCV-infected patients compared with healthy individuals before treatment. Furthermore, HLA-G in SVR patients was noticeably decreased after treatment, while HLA-G in non-SVR patients had no changes after treatment. Additionally, both in SVR and non-SVR patients, the expression of IL-37 was remarkably reduced compared with baseline after treatment. CONCLUSIONS These findings suggest that elevation of HLA-G and IL-37 in HCV may play an important role in response to combined therapy with IFN-a and RBV. Monitoring the expression of HLA-G during therapy could contribute to adjusting the treatment program of HCV-infected patients.

Increased delayed mortality in patients with acute-on-chronic liver failure who have prior decompensation.

BACKGROUND AND AIM: Patients with acute-on-chronic liver failure (ACLF) represent a complex population with differential prognosis. The aim of the study was to categorize ACLF according to the severity of underlying chronic liver diseases. METHODS: A total of 540 ACLF patients were recruited, including 127 with prior decompensated cirrhosis and 413 without prior decompensation (PD) including 193 with underlying chronic hepatitis and 220 with prior compensated cirrhosis. The clinical characteristics and prognosis of subgroups were compared. Cox proportional hazard model and multinominal logistic regression analysis were performed to identify significant prognostic parameters. RESULTS: The 28-day, 3-month and 1-year survival of ACLF patients with or without PD were 58.9% versus 61.4%, 36.2 versus 52.5%, and 29.1% versus 49.6%, respectively. On multinominal logistic regression analysis or time-to-death analysis by Cox proportional hazard model, PD was significantly associated with post-28-day mortality but not within-28-day mortality. On multivariate time-to-death analysis, older age, high international normalized ratio (INR) and serum bilirubin, low levels of serum sodium and platelet count, and presence of hepatic encephalopathy (HE), upper gastrointestinal bleeding, and respiratory or circulation dysfunction were predictors of within-28-day mortality in patients without PD, whereas the risk factors in patients with PD were high INR, creatinine, presence of HE, and respiratory or circulation dysfunction. CONCLUSION: ACLF patients with or without PD had comparable short-term prognosis but differential 1-year mortality. ACLF patients with PD were distinct from those without PD in age, types of acute insults, severity of hepatic damage, and distribution of complications, and the former group was characterized by increased delayed mortality.

Anti-hepatoma cells function of luteolin through inducing apoptosis and cell cycle arrest.

The aim of this study is to explore the apoptotic induction and cell cycle arrest function of luteolin on the liver cancer cells and the related mechanism. The liver cancer cell line SMMC-7721, BEL-7402, and normal liver cells HL-7702 were treated with different concentrations of luteolin. Cell proliferation ability was tested. Morphological changes of the apoptotic cells were observed under inverted fluorescence microscope after Hoechst33342 staining. We investigated the effect of luteolin on cell cycling and apoptosis with flow cytometry. The mitochondrial membrane potential changes were analyzed after JC-1 staining. Caspases-3 and Bcl-2 family proteins expression were analyzed by real-time PCR. Cell proliferation of SMMC-7721 and BEL-7402 were inhibited by luteolin, and the inhibition was dose-time-dependent. Luteolin could arrest the cells at G1/S stage, reduce mitochondrial membrane potential, and induce higher apoptosis rate and the typical apoptotic morphological changes of the liver carcinoma cells. Q-RT-PCR results also showed that luteolin increased Bax and caspase-3 expression significantly and upregulated Bcl-2 expression in a dose-dependent manner in liver carcinoma cells. However, the normal liver cells HL-7702 was almost not affected by luteolin treatment. Luteolin can inhibit SMMC-7721 and BEL-7402 cell proliferation in a time- and dose-dependent manner. And the mechanism maybe through arresting cell cycle at phase G1/S, enhancing Bax level, reducing anti-apoptotic protein Bcl-2 level, resulting in activating caspase-3 enzyme and decrease of mitochondrial membrane potential, and finally leading to cell apoptosis.

[Effect of siRNA-mediated silencing of Notch2 on proliferation of the HepG2 human hepatocellular carcinoma cells].

OBJECTIVE: To explore the effect of silencing the Notch2 gene by small interfering (si)RNA on the proliferation of the HepG2 human hepatocellular carcinoma (HCC) cells. METHODS: Notch2-siRNA was transfected as a liposomal formulation into HepG2 cells. The non-HCC cell lines SG07901 (gastric cancer) and SW620 (colon cancer) were used as controls. The mRNA expression of Notch2 and Hesl were detected by RTPCR, and the protein expression of Notch2 was detected by western blotting. The proliferation of transfected HepG2 cells was assessed by the cell counting kit-8 (CCK8) colorimetric assay. RESULTS: The untransfected HepG2 cells showed significantly upregulated transcript expression of Notch2, and not of Notch1, Notch3 or Notch4, compared to the other non-HCC cell lines. Following transfection of Noteh2-siRNA into HepG2 cells, the mRNA expression of Notch2 and Hes1 and the protein expression of Notch2 were significantly decreased. The rales of proliferation inhibition in HepG2 following transfection of Notch2-siRNA showed an increasing time-related trend, with 2.64% ± 1620% at 12 h, 38.34% ± 8.80% at 24 h, 70.05% ± 7.80% at 48 h, 70.78% ± 10.00% at 72 h, and 74.22% ± 4.80% at 96 h.The inhibition rate at 24 h of transfection was significantly different from that of the groups of control cells. CONCLUSION: Notch2 is upregulated in the common HCC cultured cell line HepG2. siRNA-mediated silencing of Notch2 exerts inhibition effects on HepG2 proliferation, suggesting the potential for this approach as targeted therapy for treating HCC.

The trade-off of Vibrio parahaemolyticus between bacteriophage resistance and growth competitiveness.

Vibrio parahaemolyticus is a food-borne pathogen, which is often isolated from various seafood products. In this study, two kinds of bacteriophages was isolated from the offshore sediments samples. The anti-phage mutant strain were obtained after seventeen rounds of co-culture of Vibrio parahaemolyticus and mixed bacteriophage, multigroup sequencing was carried out on spontaneous the anti-phage mutant strain and the wild-type strain. We used the Sanger sequencing to verify the accuracy of the mutation sites. Biolog GEN III MicroPlates were used to evaluate the metabolic capacity of wild-type strains and the anti-phage mutant strain. In this study, we found that with flaG gene (slight homology to N terminus of multiple flagellins) mutated, making the bacteriophage unable to absorb to the cell surface of the host. And, the growth competitiveness of the anti-phage mutant strain is lower than the wild-type strain. These results indicated that the fitness cost, including loss of the growth competitiveness, constitutes a barrier to the prevalence of these defense mechanisms. And the selection pressure on different anti-phage strategies depends on the trade-off between mortality imposed by bacteriophages and fitness cost of the defense strategy under the given environmental conditions. In conclusion, this study provides valuable insights into the phage-host interaction and phage resistance in Vibrio parahaemolyticus. Our study provided knowledge for the evolutionary adaption of bacteria against the bacteriophage, which could add more information to understand the phage resistance mechanism before applying in the industry.

[Study on the relationship between hepatitis B virus genotypes and the effect of polyethylene glycol-interferon-alpha therapy on HBeAg-positive chronic hepatitis B].

OBJECTIVE: To investigate the efficacy of polyethylene glycol (PEG)-interferon α (PEG-IFNα) in treating HBeAg-positive chronic hepatitis B (CHB) and explore the relationship between hepatitis B virus (HBV) genotypes and the effect of interferon α (IFNα) therapy. METHODS: A total of 199 CHB patients with known genotypes were given subcutaneous injection of PEG-IFNα-2a or PEG-IFNα-2b once a week for 48 weeks, with another 24 weeks follow up. The seroconversion of HBeAg influenced by HBV genotypes were analyzed after discontinuation of treatment. RESULTS: In local area, genotype C was the major genotype [64.32% (128/199) ]. Except serum ALT and AST level, the differences in gender, age, liver inflammation, degree of liver fibrosis, HBeAg level and HBV DNA level between genotype B and C were not statistically significant (all P > 0.05). The seroconversion rate of HBeAg in patients with genotype B at early stage of therapy (3 months) was significantly higher than that of patients with genotype C [26.76% (19/71) vs 10.16% (13/128), χ(2) = 9.330, P = 0.002]. While at the end of follow-up, seroconversion rate of HBeAg in patients with genotype B (followed up for 6 months) was higher than that of patients with genotype C [39.44% (28/71) vs 30.47% (39/128)], but the difference was not statistically significant (χ(2) = 1.645, P = 0.200). By univariate analysis based on log-rank test, the time of HBeAg seroconversion in patients with genotype B was much earlier than that of genotype C [(13.99 ± 0.67) months vs (15.47 ± 0.41) months], but the difference was not statistically significant (P = 0.150). CONCLUSIONS: The seroconversion rate of HBeAg in patients with genotype B treated with PEG-IFNα was significantly higher than that of genotype C in early stage of therapy (3 months), while similar at the end of therapy.

[Liver histopathological features influencing HBeAg seroconversion in patients with HBeAg-positive chronic hepatitis B responding to Peg-IFN treatment].

OBJECTIVE: To investigate the therapeutic efficiency of antiviral treatment with pegylated-interferon (Peg-IFN) for hepatitis B e antigen (HBeAg)-positive chronic hepatitis B (CHB) and to explore whether liver histopathological features or other factors influence the HBeAg seroconversion treatment response. METHODS: Eighty HBeAg-positive CHB patients with diagnosis confirmed by liver puncture were treated with Peg-IFN(2a or 2b)body weight dose, once weekly). At treatment week 48, the rate of HBeAg seroconversion was determined and used to analyze the influence of liver histopathological features (liver biopsy assessment of: inflammation, graded G0 to G4; fibrosis stage, graded S0 to S4), sex, age, differential levels (pre-treatment baseline vs. week 48 post-treatment) of serum alanine transferase (ALT), and HBV DNA, by binary logistic analysis. RESULTS: At week 48, the overall rate of HBeAg seroconversion was 30.0%. The rate of HBeAg seroconversion gradually advanced with increased liver inflammation (X2 = 8.435, P = 0.015): 9.09% of the 22 patients with G1; 31.58% of the 38 patients with G2; 47.30% of the 19 patients with G3; the one patient with G4. In contrast, the rate of HBeAg seroconversion showed a much weaker association with liver fibrosis (X2 = 5.917, P = 0.116). Only baseline HBeAg level, and no other baseline index, was significantly different between the patients who achieved HBeAg seroconversion and those who did not. Liver inflammation and baseline HBeAg level were identified as influencing factors of HbeAg seroconversion in response to Peg-IFN treatment. CONCLUSION: Peg-IFN therapy induces a higher rate of HBeAg seroconversion in HBeAg-positive CHB patients with severe liver inflammation; histological analysis of pre-treatment liver biopsies may help to identify patients most likely to benefit from the antiviral regimen.