[Analysis of efficacy and factors influencing sequential combination therapy with tenofovir alafenamide fumarate after treatment with entecavir in chronic hepatitis B patients with low-level viremia].

Objective: To observe the efficacy and factors influencing sequential or combined tenofovir alafenamide fumarate (TAF) after treatment with entecavir (ETV) in patients with chronic hepatitis B (CHB) with low-level viremia (LLV). Methods: 126 CHB cases treated with ETV antiviral therapy in the Department of Infectious Diseases of the First Affiliated Hospital of Nanchang University from January 2020-September 2022 were retrospectively collected. Patients were divided into a complete virologic response (CVR) group (n = 84) and a low-level viremia (LLV) group (n = 42) according to the HBV DNA level during treatment. Clinical characteristics and laboratory indicators of the two groups at baseline and 48 weeks were analyzed by univariate analysis. Patients in the LLV group were divided into three groups according to their continued antiviral treatment regimen until 96 weeks: continued use of ETV as a control group; replacement of TAF as a sequential group; and combination of ETV and TAF as a combined group. The data of the three groups of patients were analyzed by one-way analysis of variance for 48 weeks. HBV DNA negative conversion rate, HBeAg negative conversion rate, alanine aminotransferase (ALT), creatinine (Cr), and liver stiffness test (LSM) were compared among the three groups after 96 weeks of antiviral treatment. Multivariate logistic regression was used to analyze the independent factors influencing the occurrence of HBV DNA non-negative conversion in LLV patients at 96 weeks. Receiver operating characteristic curve (ROC) was used to evaluate the effectiveness of predicting the occurrence of HBV DNA non-negative conversion in LLV patients at 96 weeks. Kaplan-Meier was used to analyze the cumulative negative rate of DNA in LLV patients, and the Log-Rank test was used for comparison. HBV DNA and HBV DNA negative conversion rates during treatment were observed dynamically. Results: Univariate analysis showed statistically significant differences in age, BMI, HBeAg positivity rate, HBV DNA, HBsAg, ALT, AST, and LSM at baseline between the CVR group and the LLV group (P < 0.05). Univariate analysis of variance revealed no statistically significant difference among the three groups of LLV patients at 48 weeks (P > 0.05). HBV-DNA negative conversion rate in the sequential group and the combination group was significantly higher than that in the control group after 96 weeks of treatment (88.89% vs. 41.18%, 85.71% vs. 41.18%, χ (2) = 10.404, P = 0.006). HBeAg negative conversion rate was higher than that of the control group, with no statistically significant difference (P > 0.05).Compared with the control group, ALT, Cr, and LSM in the sequential group and the combined group were equally improved to varying degrees, with a statistically significant difference (P < 0.05). Subsequent use of ETV and HBV DNA at 48 weeks were independent risk factors for HBV DNA positivity at 96 weeks in LLV patients (P < 0.05). The AUC of HBV DNA at 48 weeks was 0.735 (95%CI: 0.578 ~ 0.891), the cut-off value was 2.63 log(10) IU/ml, and the sensitivity and specificity were 76.90% and 72.40%, respectively. DNA conversion rate was significantly lower in LLV patients receiving 48-week ETV and 48-week HBV DNA≥2.63 log10 IU/mL than in patients receiving sequential or combined TAF and 48-week HBV DNA < 2.63 log(10) IU/mL. HBV DNA negative conversion rates in the sequential group and combined group at 72 weeks, 84 weeks, and 96 weeks were higher than those in the control group during the period from 48 weeks to 96 weeks of continuous treatment, and the differences were statistically significant (P < 0.05). Conclusion: Sequential or combined TAF antiviral therapy could more effectively improve the 96-week CVR rate, as well as hepatic and renal function, and alleviate the degree of hepatic fibrosis in CHB patients with LLV following ETV treatment. Subsequent use of ETV and HBV DNA load at 48 weeks were independent predictors of HBV DNA positivity at 96 weeks in LLV patients.

[The possible mechanisms of simvastatin on apoptosis of lung adenocarcinoma cells].

Objective: To explore the possible mechanisms of simvastatin-induced apoptosis in lung adenocarcinoma cells. Methods: The experiment was divided into control group (vehicle treated A549 cells), different concentrations (10, 20, 40, 80 mg/L) simvastatin group (simvastatin treated with different concentrations of A549 cells), aspartate specific proteinase (caspase) inhibitor (Z-VAD-FMK) group (50 µmol/L Z-VAD-FMK treated A549 cells), 40 mg/L simvastatin combined with Z-VAD-FMK group (40 mg/L simvastatin combined with 50 µmol/L Z-VAD-FMK co-treated A549 cells), interleukin-6 (IL-6) group (IL-6 acts on A549 cells) and different concentrations (10, 20, 40 mg/L) simvastatin combined with IL-6 group (simvastatin combined with IL-6 act on A549 cells). Cell counting kit-8 (CCK8) method was used to detect the effect on survival rate of lung adenocarcinoma A549 cells; Flow cytometry was used to detect the effect of simvastatin on A549 cell cycle; Mitochondrial membrane potential-1 (JC-1) fluorescent probe was wsed to detect the effect of simvastatin on mitochondrial membrane potential (MMP); Flow-type phosphatidl serine protein antibody Annexin V/propidium iodide (Annexin V-FITC/PI) double staining method was used to detect the effect of simvastatin on A549 cell apoptosis; CCK8 method was used to detect the effect of Z-VAD-FMK on the survival rate of A549 cells; TdT-mediated 2'-deoxyuridine 5'-triphosphate (dUTP) nick end labeling (TUNEL) method was used to detect the effect of Z-VAD-FMK on simvastatin-induced apoptosis in A549 cells; Western blot method was used to detect the effect of simvastatin on the expression levels of Janus kinase 2 and activation of signal transducers and activators of transcription 3 (JAK2/STAT3) pathway-related proteins phosphorylated JAK2 (p-JAK2), JAK2, phosphorylated STAT3 (p-STAT3), and STAT3 before and after the activator IL-6 of JAK2/STAT3 pathway acted on A549 cells. Results: The survival rates of A549 cells in the 20-80 mg/L simvastatin-treated groups were significantly lower than that in the control group (all P<0.05), and gradually decreased with the increase of the concentration of the simvastatin and the extension of the action time. The cells in the G(0)/G(1) phase of the simvastatin group were significantly higher than those in the control group, and the cells in the G(2)/M phase were significantly lower than those in the control group (all P<0.01). The MMP of the treatment group with different concentrations of simvastatin was significantly lower than that of the control group (all P<0.05). The apoptosis rate of the 20 mg/L and 40 mg/L simvastatin-treated group was significantly higher than that of the control group (both P<0.01). The cell survival rate of the 40 mg/L simvastatin group and the 40 mg/L simvastatin combined with Z-VAD-FMK group were (52.2±2.7)% and (57.5±3.8)%, respectively, were lower than that of the control group (100.0±2.7)% (both P<0.01). But the difference between 40 mg/L simvastatin group and the simvastatin combined with Z-VAD-FMK group was not statistically significant (P>0.05). The cell numbers with positive fluorescent staining in the 40 mg/L simvastatin group were significantly more than those in the control group, but the cell numbers with positive fluorescent staining in the 40 mg/L simvastatin combined with Z-VAD-FMK group had no statistical significance compared with the simvastatin group (P>0.05). The specific value of p-JAK2/JAK2 and p-STAT3/STAT3 protein relative expressions in the simvastatin-treated group (20, 40 mg/L) were significantly lower than that in the control group, respectively (both P<0.05). The specific value of p-JAK2/JAK2 and p-STAT3/STAT3 protein relative expressions in IL-6 group were significantly higher than those in control group (both P<0.05), the specific value of p-JAK2/JAK2 and p-STAT3/STAT3 protein relative expressions in simvastatin (20, 40 mg/L) combined with IL-6 groups were lower than those in IL-6 group (all P<0.05), respectively. Conclusion: Simvastatin can induce the apoptosis of A549 cells through a non-caspase-dependent mitochondrial apoptosis pathway, which may be achieved by inhibiting the JAK2/STAT3 pathway.