One-Pot Hydrothermal Method Preparation of Cerium-Nitrogen-Codoped Carbon Quantum Dots from Waste Longan Nucleus as a Fluorescent Sensor for Sensing Drug Rifampicin.

Currently, the large-scale application of carbon quantum dots (CQDs) is usually limited by their low quantum yield and detection limit. Herein, the abandoned longan nucleus was used as a carbon source to synthesize cerium-nitrogen-codoped carbon quantum dots (Ce/N-CQDs) with strong luminescence intensity. In this work, the fluorescent properties and fluorescent quantum yield of CQDs may be improved by the single cerium-doped carbon quantum dots (Ce-CQDs) and the single nitrogen-doped carbon quantum dots (N-CQDs). Nevertheless, the Ce/N-CQDs exhibited intense fluorescence with a high quantum yield. Compared with CQDs, the quantum yield of Ce/N-CQDs was significantly increased from 5 to 32% and showed high photostability and good water solubility. The Ce/N-CQDs can be used for the direct detection of rifampicin (RFP) in human serum. The concentration demonstrated a good linear relationship in the range of 1.0 × 10-7-9.0 × 10-6 mol/L, with a detection limit of 9.6 × 10-8 mol/L.

Yin-chen Wu-ling powder alleviate cholestatic liver disease: Network pharmacological analysis and experimental validation.

BACKGROUND: Yin-chen Wu-ling Powder (YWP) has potential therapeutic effects on cholestatic liver disease (CLD), however, its active compounds and conceivable mechanism are as yet indistinct. METHODS: The network pharmacology and gene function annotation examined the multiple active ingredients, potential targets, and possible mechanisms of YWP in CLD treatment. Then the molecular docking reassured the reliability of the core compounds including the key genes and farnesoid X receptor (FXR). Finally, The Mdr2-/- mice were used to test the effect and mechanism of YWP against CLD. RESULTS: The network analysis identified nine main active ingredients, including quercetin, capillarisin, eupalitin, isorhamnetin, skrofulein, genkwanin, cerevisterol, gederagenin, and sitosterol. The PPI network predicted the ten hub genes involved were AKT1, MAPK1, MAPK14, IL6, RXRA, ESR1, IL10, NCOA1, CAV1, and EGFR. The KEGG and GO analysis showed that YWP might contribute to CLD treatment through the PI3K/Akt and MAKP signalings to manage pathological reactions, for instance, inflammatory responses. The molecular docking displayed a functional similarity among the core compounds with ursodeoxycholic acid (UDCA) and Obeticholic acid (OCA) on the effects on AKT1, MAPK1, MAPK14, RXRA, and ESR, and the affinity to FXR. In addition, the YWP could significantly attenuate hepatic injury and improve inflammatory response in Mdr2-/- mice. The mechanism exploration showed that YWP mainly decreased inflammatory response by inhibiting AKT/P38MAPK signaling. CONCLUSION: This study firstly revealed the multiple active ingredients, potential targets, and possible mechanism of YWP to treat CLD based on network pharmacology Analysis and molecular docking. YWP could alleviate cholestasis in Mdr2-/- mice by impairing inflammation via inhibiting AKT/P38MAPK Signaling.

[Establishment and Preliminary Analysis of Lung Cancer Cell Line A549 with Stable MAP4 K4 Knockdown].

Objective: To analyze the effect of knocking down MAP4 K4 expression on the proliferation and migration of cancer cells, and to explore its underlining molecular mechanisms. Methods: A stable knockdown MAP4 K4 cell line was constructed and the subcellular localization of the cells was determined with immunofluorescence, cell proliferation assay and cell migration assay. In addition, the effects of down-regulated MAP4 K4 expression were analyzed by examining the difference between the proliferation and migration of cancer cells in the knockdown group and those of the control group. Results: MAP4 K4 was localized in focal adhesion and cell edges in A549 cells. Stable knockdown of MAP4 K4 expression induced cancer cells to grow in clusters and arrested the progression of the cell cycle and cell migration. Further analysis found that knocking down MAP4 K4 expression in A549 cells induced the accumulation of epithelial cell marker E-cadherin, and subsequently, the down-regulation of N-cadherin, a mesenchymal cell marker, thereby disrupting the "cadherin switch" and the epithelial-mesenchymal conversion. Then, the control group and the knockdown group both received the combined treatment of cisplatin at a final concentration of 5 µmol/L and paclitaxel at a final concentration of 20 nmol/L. The stably knocked down MAP4 K4 expressing cells showed significantly enhanced toxicity of chemotherapeutic drugs to cancer cells. Conclusion: The study shows that MAP4 K4 regulates the malignant phenotypes of cancer cells and chemoresistance by regulating "cadherin switch" to promote epithelial-mesenchymal transition in A549 cells.

Mitochondria-Targeted Degradable Nanocomposite Combined with Laser and Ultrasound for Synergistic Tumor Therapies.

Although the development of safe and efficient cancer therapeutic agents is essential, this process remains challenging. In this study, a mitochondria-targeted degradable nanoplatform (PDA-MnO2-IR780) for synergistic photothermal, photodynamic, and sonodynamic tumor treatment was investigated. PDA-MnO2-IR780 exhibits superior photothermal properties owing to the integration of polydopamine, MnO2, and IR780. IR780, a photosensitizer and sonosensitizer, was used for photodynamic therapy and sonodynamic therapy. When PDA-MnO2-IR780 was delivered to the tumor site, MnO2 was decomposed by hydrogen peroxide, producing Mn2+ and oxygen. Meanwhile, alleviating tumor hypoxia promoted the production of reactive oxygen species during photodynamic therapy and sonodynamic therapy. Moreover, large amounts of reactive oxygen species could reduce the expression of heat shock proteins and increase the heat sensitivity of tumor cells, thereby improving the photothermal treatment effect. In turn, hyperthermia caused by photothermal therapy accelerated the production of reactive oxygen species in photodynamic therapy. IR780 selectively accumulation in mitochondria also promoted tumor apoptosis. In this system, the mutual promotion of photothermal therapy and photodynamic therapy/sonodynamic therapy had an enhanced therapeutic effect. Moreover, the responsive degradable characteristic of PDA-MnO2-IR780 in the tumor microenvironment ensured excellent biological safety. These results reveal a great potential of PDA-MnO2-IR780 for safe and highly-efficiency synergistic therapy for cancer.

Bushen Jianpi Formula Combined with Entecavir for the Treatment of HBeAg-Negative Chronic Hepatitis B: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

Background: Bushen Jianpi formula (BSJPF, also known as Lingmao formula) is a traditional Chinese medicine for chronic hepatitis B (CHB). The previous study has suggested that the treatment combination of BSJPF and entecavir (ETV) can achieve a significant loss of hepatitis B e antigen (HBeAg) and a significant decrease in serum level of hepatitis B virus (HBV) DNA in HBeAg-positive CHB patients with mildly elevated alanine aminotransferase. Objective: This study aimed to evaluate the efficacy and safety of BSJPF combined with ETV for treating HBeAg-negative CHB patients. Methods: A total of 640 patients were assigned randomly to the treatment group (receiving BSJPF combined with ETV for 96 weeks) or the control group (receiving a placebo combined with ETV for 96 weeks) in a 1 : 1 ratio. The primary endpoints are the rate of loss of hepatitis B surface antigen (HBsAg). The secondary outcomes included the rate of decrease in the HBsAg concentration to ≥1 lg·IU/mL, the HBV DNA suppression, the decline of the level of covalently closed circular DNA (cccDNA) in the liver, histological improvements, and the rate of ALT normalization. Results: The rate of HBsAg loss in the treatment group was significantly higher than that of the control group (5.5% versus 1.8%, P=0.031). There were 11.1% of patients in the treatment group who recorded a reduction in HBsAg ≥1 lg·IU/mL, which is better than 5.9% of patients in the control group (P=0.043). There was no significant difference between the two groups with regard to the rate of HBV DNA clearance, the reduction in intrahepatic cccDNA, and the rate of ALT normalization (P > 0.05). The rate of liver fibrosis improvement in the treatment group was better than that of the control group (35.5% versus 11.8%, P=0.031), but there was no difference in necroinflammatory improvement (P > 0.05). The adverse events (AEs) were similar between the two groups, except for the abnormal kidney function, with 2.2% in the control group and 0.0% in the treatment group (P=0.028). Conclusion: The combination of BSJPF and ETV can increase the rate of HBsAg loss and the rate of histological fibrosis improvement without serious adverse events in CHB patients. Trial Registration. This trial is registered with ChiCTR-IOR-16009880 on November 16, 2016-retrospectively registered, http://www.chictr.org.cn/showproj.aspx?proj=16836.

Corrigendum to "Immunomodulatory Effects of Combination Therapy with Bushen Formula plus Entecavir for Chronic Hepatitis B Patients".

[This corrects the article DOI: 10.1155/2019/8983903.].

C-C chemokine hepatocellular carcinoma motif ligand 5-deficiency promotes hepatocellular carcinoma progression by affecting B cell recruitment.

OBJECTIVE: To evaluate the expression of C-C motif chemokine ligand 5 (CCL5) in hepatocellular carcinoma (HCC) and to explore its role in regulating the immune microenvironment and the related mechanism in tumor immunity. METHODS: The mRNA expression level of CCL5 in HCC and adjacent non-cancerous tissues was measured by quantitative polymerase chain reaction and the protein expression was examined by immunohistochemistry. Serum CCL5 expression was measured by an enzyme-linked immunosorbent assay (ELISA). C57BL/6 wild-type (WT) and Ccl5-knockout (Ccl5-/- ) mice were utilized to conduct the diethylnitrosamine-induced HCC model. The immune cell population was determined by flow cytometry, and peripheral serum immunoglobulin M (IgM) level was quantified by ELISA. RESULTS: CCL5 expression was low in HCC tissue and peripheral blood compared with adjacent non-cancerous tissues or controls, and its expression was correlated with the overall survival, cancer recurrence and distant metastasis. In the HCC mouse model, liver-to-body weight ratio was of the Ccl5-/- group were higher than that of the WT group. Moreover, compared with the WT mice, the number of B cells in the tumor tissue of the Ccl5-/- mice was lower, while there were no significant differences in the other immune cell populations. Furthermore, serum IgM level of the Ccl5-/- mice was significantly lower than that of the WT mice. CONCLUSION: CCL5 expression is decreased in HCC tissues. CCL5 deficiency reduces B cell recruitment and decreases IgM secretion in HCC, potentially leading to tumor progression.

Toll-like receptor 5-mediated signaling enhances liver regeneration in mice.

BACKGROUND: Toll-like receptor 5 (TLR5)-mediated pathways play critical roles in regulating the hepatic immune response and show hepatoprotective effects in mouse models of hepatic diseases. However, the role of TLR5 in experimental models of liver regeneration has not been reported. This study aimed to investigate the role of TLR5 in partial hepatectomy (PHx)-induced liver regeneration. METHODS: We performed 2/3 PHx in wild-type (WT) mice, TLR5 knockout mice, or TLR5 agonist CBLB502 treated mice, as a model of liver regeneration. Bacterial flagellin content was measured with ELISA, and hepatic TLR5 expression was determined with quantitative PCR analyses and flow cytometry. To study the effects of TLR5 on hepatocyte proliferation, we analyzed bromodeoxyuridine (BrdU) incorporation and proliferating cell nuclear antigen (PCNA) expression with immunohistochemistry (IHC) staining. The effects of TLR5 during the priming phase of liver regeneration were examined with quantitative PCR analyses of immediate early gene mRNA levels, and with Western blotting analysis of hepatic NF-κB and STAT3 activation. Cytokine and growth factor production after PHx were detected with real-time PCR and cytometric bead array (CBA) assays. Oil Red O staining and hepatic lipid concentrations were analyzed to examine the effect of TLR5 on hepatic lipid accumulation after PHx. RESULTS: The bacterial flagellin content in the serum and liver increased, and the hepatic TLR5 expression was significantly up-regulated in WT mice after PHx. TLR5-deficient mice exhibited diminished numbers of BrdU- and PCNA-positive cells, suppressed immediate early gene expression, and decreased cytokine and growth factor production. Moreover, PHx-induced hepatic NF-κB and STAT3 activation was inhibited in Tlr5-/- mice, as compared with WT mice. Consistently, the administration of CBLB502 significantly promoted PHx-mediated hepatocyte proliferation, which was correlated with enhanced production of proinflammatory cytokines and the recruitment of macrophages and neutrophils in the liver. Furthermore, Tlr5-/- mice displayed significantly lower hepatic lipid concentrations and smaller Oil Red O positive areas than those in control mice after PHx. CONCLUSION: We reveal that TLR5 activation contributes to the initial events of liver regeneration after PHx. Our findings demonstrate that TLR5 signaling positively regulates liver regeneration and suggest the potential of TLR5 agonist to promote liver regeneration.

A new fluorescence method for detection of famotidine based on polyethyleneimine-templated Ag nanoclusters.

A rapid, simple, inexpensive fluorescence analysis method for determination of famotidine based on polyethyleneimine (PEI)-capped Ag nanoclusters (PEI-Ag NCs) was developed. The study showed that addition of famotidine could cause efficient quenching of PEI-Ag NC fluorescence, as the presence of famotidine could cause aggregation of Ag NCs and quench its fluorescence. The sensitivity and selectivity of the method were investigated and experimental conditions such as buffer type, pH, temperature, and reaction time were optimized. Under optimized conditions, the results showed a linear profile from 3.7 × 10-8 to 3.7 × 10-5 mol/L, and had a detection limit of 1.6 × 10-9 mol/L (S/N = 3).

Baicalin relieves neuropathic pain by regulating α2-adrenoceptor levels in rats following spinal nerve injury.

In the present study, the ability of baicalin to relieve neuropathic pain due to spinal nerve ligation in rats was explored, and the relationship between baicalin and α2-adrenoceptors (α2-AR) was determined. The neuropathic pain model was established by ligating the L5-L6 spinal nerves in Sprague-Dawley rats. Several α2-AR antagonists were injected into the intramedullary sheath to evaluate the role of baicalin in neuropathic pain. The antagonists included nonselective α2-AR antagonist idazoxan, α2a-AR antagonist BRL 44408, α2b-AR antagonist ARC 239 and α2c-AR antagonist JP 1302. The rats were divided into an untreated control group, saline group, baicalin group and baicalin + α2-AR antagonist groups. Paw withdrawal threshold (PWT) was tested to assess the level of pain felt by the rats. The levels of α2-AR mRNA were tested by reverse transcription-quantitative PCR. Inflammatory factors, including tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-17 and IL-1ß, were analyzed by ELISA. The histopathological changes were assessed by hematoxylin and eosin staining. Flow cytometry was used to examine the percentage of CD4+ peripheral blood mononuclear cells (PBMCs). Compared with the saline group, the PWT value increased after treating with baicalin. However, intrathecal injection of α2-AR antagonist reversed the antinociceptive effects of baicalin. Compared with the saline group, the expression of α2a-AR and α2c-AR mRNA was upregulated significantly in the baicalin group (P<0.05). Levels of α2-AR mRNA were also decreased in the baicalin + idazoxan group compared with the baicalin group (P<0.05). The levels of TNF-α, IL-6, IL-17 and IL-1ß were raised after treatment with baicalin. In addition, baicalin treatment ameliorated the histological damage in the spinal cord. The percentage of CD4+ PBMCs was increased in the saline group compared with the control group (P<0.05). Compared with the baicalin group, the percentage of CD4+ PBMCs was raised after treatment with the α2-AR antagonists. In conclusion, intrathecal injection of baicalin produced an antiallodynic effect in a spinal nerve ligation-induced neuropathic pain model. The mechanism may be related to the regulation of a2-AR expression.

Mechanism of Follicular Helper T Cell Differentiation Regulated by Transcription Factors.

Helping B cells and antibody responses is a major function of CD4+T helper cells. Follicular helper T (Tfh) cells are identified as a subset of CD4+T helper cells, which is specialized in helping B cells in the germinal center reaction. Tfh cells express high levels of CXCR5, PD-1, IL-21, and other characteristic markers. Accumulating evidence has demonstrated that the dysregulation of Tfh cells is involved in infectious, inflammatory, and autoimmune diseases, including lymphocytic choriomeningitis virus (LCMV) infection, inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), IgG4-related disease (IgG4-RD), Sjögren syndrome (SS), and type 1 diabetes (T1D). Activation of subset-specific transcription factors is the essential step for Tfh cell differentiation. The differentiation of Tfh cells is regulated by a complicated network of transcription factors, including positive factors (Bcl6, ATF-3, Batf, IRF4, c-Maf, and so on) and negative factors (Blimp-1, STAT5, IRF8, Bach2, and so on). The current knowledge underlying the molecular mechanisms of Tfh cell differentiation at the transcriptional level is summarized in this paper, which will provide many perspectives to explore the pathogenesis and treatment of the relevant immune diseases.

Long-Term Traditional Chinese Medicine Combined with NA Antiviral Therapy on Cirrhosis Incidence in Chronic Hepatitis B Patients in the Real-World Setting: A Retrospective Study.

OBJECTIVE: To evaluate the impact of long-term Traditional Chinese Medicine (TCM) syndrome differentiation combined with antiviral therapy with Nucleos (t) ide analogues (NAs) on the incidence of cirrhosis in patients with chronic hepatitis B. METHODS: This retrospective cohort study included 521 patients with chronic hepatitis B who underwent a treatment course of ≥3 years from 1998-2019. Of the 521 patients, 261 were defined as TCM users while 260 were TCM nonusers (control group). All the enrolled subjects were followed up until February 2019 to measure the incidence and hazard ratio (HR) of cirrhosis, and the Cox proportional hazards regression model was used to analyze the independent factors affecting the occurrence of cirrhosis. RESULTS: The cumulative incidence of TCM users and nonusers was 6.9% and 13.5%, respectively (P=0.013). Results of the Kaplan-Meier analysis demonstrated that TCM users had a significantly lower cumulative incidence of cirrhosis than TCM nonusers (P=0.011), and TCM users had a significantly lower liver cirrhosis risk than TCM nonusers (adjusted HR = 0.416, 95% CI, 0.231-0.749). The histological evaluation revealed improved fibrosis in 45.0% of TCM users and 11.1% of TCM nonusers (P=0.033). The analysation of the prescriptions including total 119 single Chinese herbs medicinal demonstrated that "replenish qi and fortify the spleen," "clear heat and dispel dampness," and "soothe the liver and regulate qi" are the main treatment methods of TCM for CHB. CONCLUSIONS: Our study demonstrated that long-term TCM use may attenuate liver cirrhosis risk in patients with chronic hepatitis B (CHB).

Ganji Formulation for Patients with Hepatocellular Carcinoma Who Have Undergone Surgery: A Multicenter, Randomized, Double-Blind, Controlled Trial.

Objective. To ascertain the efficacy and safety of Ganji Formulation (GF) for patients with Hepatocellular carcinoma (HCC) who had undergone surgery. Materials and Methods. A total of 262 HCC patients who had undergone liver resection, local ablation, or transcatheter arterial chemoembolization (TACE) were divided randomly into the treatment group and control group. The former was treated with GF and the later with placebo, both for 6 months. The primary endpoint was overall survival (OS). Second endpoints were disease-free survival (DFS) or time to disease progression (TTP). Results. OS of the treatment group was significantly longer than that of the control group (P < 0.05). Subgroup analysis showed that, for patients who received TACE, the TTP was significantly longer in the treatment group than in the control group (P < 0.05). However, for patients who underwent liver resection or local ablation, there was no significant difference in DFS between the two groups (P > 0.05). Conclusion. GF could improve postoperative cumulative survival and prolong the TTP. This clinical trial number is registered with ChiCTR-IOR-15007349.

Immunomodulatory Effects of Combination Therapy with Bushen Formula plus Entecavir for Chronic Hepatitis B Patients.

AIM: To compare the clinical efficacy of the combination therapy with Bushen formula (BSF) plus entecavir (ETV) in naïve chronic hepatitis B (CHB) patients and that in CHB patients with partial virological response to ETV and explore the relevant immunoregulatory mechanism. MATERIALS AND METHODS: Two hundred and twenty CHB patients were enrolled in the historical prospective cohort study. Patients were categorized into a treatment group (T-Group: combination therapy with BSF plus ETV) and a control group (C-Group: ETV). Patients in T-Group and C-Group were grouped into T1/C1 (treatment-naïve patients) and T2/C2 (patients with partial virological response to ETV). Biochemical assessment, viral load quantitation, and HBV markers were tested. Chinese medicine symptom complex score was evaluated and recorded as well. In addition, peripheral blood mononuclear cells were separated from blood samples in 56 patients and 11 healthy donors. The frequencies of Th1, Treg, and dendritic cells (DCs) and expression levels of PD-1/PD-L1 were examined by flow cytometry. RESULTS: In treatment-naïve CHB patients, complete viral suppression rates in HBeAg(-) patients were higher than those in HBeAg(+) patients in both T and C groups. In patients with partial virological response to ETV, the rate of HBsAg decline ≥ 20% in HBeAg(+) patients of T2-Group was higher than that in HBeAg(+) patients of C2-Group. A significant reduction of Chinese medicine symptom complex score was only observed in T-Group. The study of mechanism showed that, compared with healthy controls, Th1 and DC frequencies were decreased in all CHB patients, while Treg frequency was increased only in treatment-naïve patients. In addition, compared with healthy controls, PD-1 expression levels on Th1 and Treg were increased in all patients and PD-L1 expression levels on DCs were increased only in treatment-naïve patients. In treatment-naïve patients, the combination therapy with BSF plus ETV increased Th1 and DC frequencies and decreased Treg frequency, which was correlated with HBsAg decline. In addition, in patients with partial virological response to ETV, the combination therapy downregulated PD-L1 levels on DCs and the frequency of Treg, which was related with HBsAg decline. CONCLUSIONS: In patients with partial virological response to ETV, HBeAg(+) patients tend to achieve ideal effects after the combination therapy with BSF plus ETV, which may correlate with the decrease of Treg frequency and the downregulation of PD-L1 levels on DCs.

Beneficial Effects of Bushen Formula Combined with Enticavir on Chronic Hepatitis B Patients with Suboptimal Response to Enticavir by Regulating B-Cell Differentiation.

BACKGROUND/AIMS: To investigate the clinical effects of the combination therapy with Bushen Formula (BSF) plus enticavir (ETV) on chronic hepatitis B (CHB) patients with suboptimal response to ETV and explore the regulatory mechanisms of BSF on B cells-mediated humoral immunity. METHODS: Sixty-four HBeAg-positive CHB patients with suboptimal response to ETV were enrolled, and were randomly assigned into control group (C-Group, placebo combined with ETV for 12 months) or treatment group (T-Group, BSF combined with ETV for 12 months). Serum samples from 57 treatment-naïve CHB patients and 15 healthy controls were collected. Serum HBV DNA levels were evaluated by real-time PCR. Characteristics of peripheral blood B-cell subtypes were analyzed by flow cytometry. Serum HBV markers and B cell-activating factor (BAFF) levels were detected by ELISA. Chinese medicine symptom complex score was evaluated and recorded. RESULTS: After treatment, the rates of patients with a reduction of HBsAg > 0.5 log10 IU/ml or 1.0 log10 IU/ml and the rates of HBeAg clearance in T-Group were all higher than those in C-group, with no significant intergroup difference. Only in T-Group, Chinese medicine symptom complex score and the frequency of total B cells were significantly decreased, and the frequencies of Bm1, CD24+CD27-switched B cells and plasma cells were markedly increased after treatment compared with those before treatment. Compared with healthy controls, serum BAFF levels in treatment-naïve CHB patients were increased, and there was a significant positive correlation between serum BAFF and HBsAg levels. However, serum BAFF levels did not differ after treatment in T-Group and C-Group. CONCLUSIONS: The combination therapy with BSF plus ETV promotes the reduction of HBsAg level and the clearance of HBeAg in CHB patients with partial response to ETV through regulating the differentiation of B-cell subsets.

Hepatitis B core antigen upregulates B7-H1 on dendritic cells by activating the AKT/ERK/P38 pathway: a possible mechanism of hepatitis B virus persistence.

B7-H1 binding to programmed death-1 may deliver a coinhibitory signal to T cells that is involved in the regulation of T-cell activation and tolerance. B7-H1 plays a key role in dysfunction of dendritic cells (DCs) during chronic HBV infection, but the expression mechanism of B7-H1 remains unclear. One hundred and twenty-nine patients with chronic HBV infection were categorized into either the immune tolerance phase (HBV-IT), the immune clearance phase (HBV-IC), or the inactive carrier phase (HBV-IA). Twenty healthy volunteers were enrolled as controls. Another 16 patients with HBeAg-positive chronic Hepatitis B were enrolled, and entecavir was administrated at 0.5 mg per day for 6 months. The B7-H1 expression level on peripheral DCs was tested by flow cytometry. In vitro, expression levels of B7-H1 and signaling molecules on monocyte-derived DC (MO-DC) induced by recombinant hepatitis B virus C antigen (rhHBcAg) were examined by RT-PCR, flow cytometry, and western blotting, and the apoptosis rate was tested by flow cytometry. The percentages of peripheral DCs and myeloid DCs (mDCs) were decreased and B7-H1 levels were increased in patients compared with controls. Serum HBV-DNA levels were positively correlated with B7-H1 levels on mDCs in patients with HBV-IT. B7-H1 levels on peripheral DCs from patients with chronic hepatitis B decreased after antiviral therapy. In vitro studies demonstrated that the B7-H1 level on MO-DC was upregulated by rhHBcAg, which resulted from the activation of PI3K-AKT, ERK, and P38 signaling pathways, and the percentage of MO-DC was downregulated by rhHBcAg. In addition, rhHBcAg promoted the apoptosis of MO-DC. The data suggest that HBcAg induced B7-H1 upregulation by activating AKT, ERK, and P38 signaling pathways, which inhibited the clearance of HBV-DNA and the reduction of DCs contributed to immune tolerance, which may correlate with apoptosis.

The dynamic changes of circulating invariant natural killer T cells during chronic hepatitis B virus infection.

AIM: The protective role of invariant natural killer T cells (iNKTs) against hepatitis B virus (HBV) infection remains controversial. We sought to clarify the role of peripheral iNKT cells during chronic HBV infection. METHODS: Sixty patients with chronic HBV infection were categorized into an immune tolerance phase (HBV-IT) (n = 16), an immune clearance phase (HBV-IC) (n = 19) and an inactive carrier phase (HBV-IA) (n = 25). Twenty healthy individuals were enrolled as healthy controls. Another 21 HBeAg-positive patients were administrated with entecavir (0.5 mg/day) for 6 months. The percentages of circulating iNKT cells and their IFN-γ and IL-4 expression levels were examined by flow cytometry. The relationships between serum HBV DNA, ALT levels, the percentages of iNKT cells, and their IFN-γ and IL-4 levels were analyzed. RESULTS: Compared to healthy controls, the percentage of iNKT cells decreased in HBV-IT, but increased in HBV-IC and HBV-IA. Circulating IFN-γ-producing iNKT cells gradually increased, whereas IL-4-producing iNKT cells gradually decreased from HBV-IT stage to HBV-IC and HBV-IA stages. The frequency of iNKT cells and their IFN-γ levels were reversely correlated with viral load. The levels of IL-4 expressed by iNKT cells were positively correlated to viral load and the serum ALT levels. After anti-virus therapy, the percentage of IFN-γ-producing iNKT cells increased while the percentage of IL-4-producing iNKT cells decreased. CONCLUSIONS: During chronic HBV infection, the percentages of peripheral iNKT cells and its cytokines expressions of IFN-γ and IL-4 showed dynamic changes. The expression levels of IFN-γ and IL-4 were correlated with the clearance of HBV and liver injury.

[Research on HBV DNA inhibition of plasmid acute infection mouse with betulinic acid].

Betulinic acid is a naturally occurring pentacyclic triterpenoid, which has antiretroviral, antimalarial, and anti-inflammatory properties. The purpose of this study is to investigate the HBV DNA replication inhibition in the mouse model with betulinic acid. Hydrodynamic injection method via the tail vein with the Paywl. 3 plasmid was used to establish the animal mode (n = 15), and the mice were randomly divided into the PBS control group (n = 5), Betulinic acid treatment group (n = 5) and lamivudine control group (n = 5). The day after successful modeling , the mice would have taken Betulinic acid (100 mg x kg(-1)), lamivudine (50 mg x kg(-1)), PBS drugs orally, once daily for 7 days, blood samples were acquired from the orbital venous blood at 3, 5, 7 days after the administering, HBsAg and HBeAg in serum concentration were measured by ELISA and the mice were sacrificed after 7 days, HBV DNA southern detections were used with part of mice livers. The results showed that betulinic acid significantly inhibited the expression of HbsAg in the mice model at the fifth day compared with the control group, and there was no significant differences between the effects of lamivudine and the PBS control group; both the betulinic acid and lamivudine groups had no significant inhibition for the HBeAg expression; the HBV DNA expressions of the liver tissue from the betulinic acid and lamivudine groups were inhibited compared with the control group. Taken together, these results reveal betulinic acid can inhibit the HBsAg expression and replication of the liver HBV DNA in the mouse model.

Lingmao Formula Combined with Entecavir for HBeAg-Positive Chronic Hepatitis B Patients with Mildly Elevated Alanine Aminotransferase: A Multicenter, Randomized, Double-Blind, Placebo-Controlled Trial.

Objective. To determine the efficacy and safety of Lingmao Formula combined with entecavir for HBeAg-positive chronic hepatitis B patients with mildly elevated alanine aminotransferase (ALT). Methods. 301 patients were randomly assigned to receive Lingmao Formula combined with entecavir (treatment group) or placebo combined with entecavir (control group) for 52 weeks. The outcomes of interest included the reduction of serum HBV DNA level, HBeAg loss, HBeAg seroconversion, ALT normalization, and histological improvement. Results. The mean decrease of serum HBV DNA level from baseline and the percentage of patients who had reduction in serum HBV DNA level ≥2 lg copies/mL in treatment group were significantly greater than that in control group (5.5 versus 5.4 lg copies/mL, P = 0.010; 98.5% versus 92.6%, P = 0.019). The percentage of HBeAg loss in treatment group was 22.8%, which was much higher than a percentage of 12.6% in control group (P = 0.038). There was no significant difference between the two groups in histological improvement. Safety was similar in the two groups. Conclusions. The combination of Lingmao Formula with entecavir could result in significant decrease of serum HBV DNA and increase of HBeAg loss for HBeAg-positive chronic hepatitis B patients with mildly elevated ALT without any serious adverse events. Clinical trial registration number is ChiCTR-TRC-09000594.

Beneficial therapeutic effect of Chinese herbal Bushen formula on CHB patients with mildly elevated alanine aminotransferase by down-regulating CD4+CD25+T cells.

ETHNOPHARMACOLOGICAL RELEVANCE: To investigate the clinical effects of the Bushen formula on chronic hepatitis B (CHB) patients with mildly elevated alanine aminotransferase (ALT) (1-2 times ULN) and its relevant immune mechanism. MATERIALS AND METHODS: Fifty CHB patients were enrolled, and they were categorized into two groups according to infection state, including CHB carrier group(24 patients) and mildly elevated ALT (1-2 times ULN) group (26 patients), and 12 healthy volunteers were collected as normal control group. In addition, patients with mildly elevated ALT were taking the Bushen formula for 6 months. Biochemical assessment, viral load quantitation and HBV markers were examined at the Department of Clinical Laboratory, Shanghai Shuguang Hospital. Peripheral blood mononuclear cells were separated from patients and healthy volunteers. T cells were separated from healthy blood, and Hepatitis B core antigen (HBcAg) was added to the culture media. The percentage of CD4+CD25+T cells and the expression levels of IFN-γ and IL-4 were examined by flow cytometry and the expression level of FoxP3 was examined by real-time PCR and flow cytometry. The Chinese medicine symptom complex score was calculated. RESULTS: The percentage of CD4+CD25+T cells and the expression level of Foxp3 in two CHB patient groups were higher than that in the normal control group, and the percentage of CD4+CD25+T cells and the expression level of Foxp3 in the CHB carrier group were highest. The experiment in vitro showed that both the percentage of CD4+CD25+T cells and the expression of Foxp3 were elevated by HBcAg in CD4+T cells separated from normal peripheral blood. In patients with mildly elevated ALT, the serum ALT level was significantly decreased from 81.77±18.24U/L to 64.92±20.60U/L and the serum HBV DNA level was significantly decreased from 6.54±0.95 (log) to 5.15±2.22 (log), and symptom scores were significantly decreased from 6.19±3.99 to 2.77±1.66 after administration of the Bushen formula. In addition, we found that the frequency of CD4+CD25+T cells correlated negatively with the expression level of IFN-γ in CD4+T cells from CHB patients with mildly elevated ALT, and the percentage of CD4+CD25+T cells was decreased from 11.07±4.30% to 8.70±3.49% and the expression level of IFN-γ in CD4+T cells was increased from 13.98±3.25% to 15.85±3.43% after administration of the Bushen formula. CONCLUSION: In CHB patients, the percentage of CD4+CD25+T cells and the expression of Foxp3 in peripheral blood were increased, which can be repeated in HBcAg-stimulated healthy CD4+T cells. The Bushen formula has positive effects on CHB patients with mildly elevated ALT (1-2 times ULN) by reducing serum ALT and HBV DNA levels, which is relevant with the decreasing of the percentage of CD4+CD25+T cells and the increasing of the expression level of IFN-γ in CD4+T cells.