Impact of Fuzheng Huayu tablet on antiviral effect of entecavir in patients with hepatitis B cirrhosis.

BACKGROUND: Fuzheng Huayu tablet is a traditional Chinese medicine (TCM) used for the treatment of liver fibrosis and cirrhosis. However, whether the combination with Fuzheng Huayu tablet could affect the antiviral efficacy of nucleos(t)ide remains a concern. The objective of this trial was to explore the impact of Fuzheng Huayu tablet on antiviral effect of entecavir in patients with hepatitis B cirrhosis. METHODS: A prospective, randomized control trial was conducted. Patients with compensated hepatitis B cirrhosis were randomly divided into the treatment group (entecavir capsule plus Fuzheng Huayu tablet) and the control group (entecavir capsule plus simulant of Fuzheng Huayu), and followed up for 48 weeks. The dynamic changes of HBV DNA load, the rate of serological conversion of HBeAg, liver function, renal function and liver stiffness measurement (LSM) were monitored. The general clinical data and adverse events were also recorded. RESULTS: There was no significant difference in the rate of virological response and cumulative virological response between the treatment group and the control group (P > 0.05). After 48 weeks of treatment, the HBeAg seroconversion rate, biochemical response rate and LSM value were 21.05% and 4.76% (P = 0.164), 86.96% and 65.96% (P = 0.017), 9.5 kpa and 10.6 kpa (P = 0.827) in the treatment group and the control group, respectively. No serious adverse events related to the study therapy occurred during the trial. CONCLUSIONS: The antiviral entecavir combined with Fuzheng Huayu tablet did not affect the antiviral efficacy of entecavir, but could improve the rate of biochemical response, and had a tendency to improve the rate of serological conversion of HBeAg and liver fibrosis in patients with hepatitis B cirrhosis. Fuzheng Huayu tablet is clinically safe for patients with hepatitis B cirrhosis.

Transcriptomic analysis of flower induction for long-day pitaya by supplementary lighting in short-day winter season.

BACKGROUND: Pitayas are currently attracting considerable interest as a tropical fruit with numerous health benefits. However, as a long-day plant, pitaya plants cannot flower in the winter season from November to April in Hainan, China. To harvest pitayas with high economic value in the winter season, it is necessary to provide supplementary lighting at night to induce flowering. To further explore the molecular regulating mechanisms of flower induction in pitaya plants exposed to supplementary lighting, we used de novo RNA sequencing-based transcriptomic analysis for four stages of pitaya plants subjected to light induction. RESULTS: We assembled 68,113 unigenes in total, comprising 29,782 unigenes with functional annotations in the NR database, 20,716 annotations in SwissProt, 18,088 annotations in KOG, and 11,059 annotations in KEGG. Comparisons between different samples revealed different numbers of significantly differentially expressed genes (DEGs). A number of DEGs involved in energy metabolism-related processes and plant hormone signaling were detected. Moreover, we identified many CONSTANS-LIKE, FLOWERING LOCUS T, and other DEGs involved in the direct regulation of flowering including CDF and TCP, which function as typical transcription factor genes in the flowering process. At the transcriptomic level, we verified 13 DEGs with different functions in the time-course response to light-induced flowering by quantitative reverse-transcription PCR analysis. CONCLUSIONS: The identified DEGs may include some key genes controlling the pitaya floral-induction network, the flower induction and development is very complicated, and it involves photoperiod perception and different phytohormone signaling. These findings will increase our understanding to the molecular mechanism of floral regulation of long-day pitaya plants in short-day winter season induced by supplementary lighting.

Molecular characterization and expression analysis of pitaya (Hylocereus polyrhizus) HpLRR genes in response to Neoscytalidium dimidiatum infection.

BACKGROUND: Canker disease caused by Neoscytalidium dimidiatum is a devastating disease resulting in a major loss to the pitaya industry. However, resistance proteins in plants play crucial roles to against pathogen infection. Among resistance proteins, the leucine-rich repeat (LRR) protein is a major family that plays crucial roles in plant growth, development, and biotic and abiotic stress responses, especially in disease defense. RESULTS: In the present study, a transcriptomics analysis identified a total of 272 LRR genes, 233 of which had coding sequences (CDSs), in the plant pitaya (Hylocereus polyrhizus) in response to fungal Neoscytalidium dimidiatum infection. These genes were divided into various subgroups based on specific domains and phylogenetic analysis. Molecular characterization, functional annotation of proteins, and an expression analysis of the LRR genes were conducted. Additionally, four LRR genes (CL445.Contig4_All, Unigene28_All, CL28.Contig2_All, and Unigene2712_All, which were selected because they had the four longest CDSs were further assessed using quantitative reverse transcription PCR (qRT-PCR) at different fungal infection stages in different pitaya species (Hylocereus polyrhizus and Hylocereus undatus), in different pitaya tissues, and after treatment with salicylic acid (SA), methyl jasmonate (MeJA), and abscisic acid (ABA) hormones. The associated protein functions and roles in signaling pathways were identified. CONCLUSIONS: This study provides a comprehensive overview of the HpLRR family genes at transcriptional level in pitaya in response to N. dimidiatum infection, it will be helpful to understand the molecular mechanism of pitaya canker disease, and lay a strong foundation for further research.

MicroRNA-29b-3p prevents Schistosoma japonicum-induced liver fibrosis by targeting COL1A1 and COL3A1.

Schistosomiasis is one of the world's major public health problems in terms of morbidity and mortality, causing granulomatous inflammation and cumulative fibrosis. This study explored in vivo and vitro effects of miR-29b-3p in granulomatous liver fibrosis by targeting COL1A1 and COL3A1 in Schistosoma japonicum infection. Thirty male Balb/c mice were assigned to normal control and model (percutaneous infection of cercariae of S. japonicum) groups. NIH-3T3 mouse embryonic fibroblasts were designated into blank, NC, miR-29b-3p mimic, TGF-ß1, TGF-ß1 + NC, and TGF-ß1 + miR-29b-3p mimic groups. HE and Masson staining were employed to observe the pathological changes and collagenous fibrosis. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was determined by immunohistochemistry. The RT-qPCR, Western blotting and immunofluorescence staining were conducted to determine expression of miR-29b-3p, COL1A1, and COL3A1. CCK-8 assay and flow cytometry were performed to evaluate viability and apoptosis. The relative expression of miR-29b-3p decreased in the model group. The model group showed marked fibrosis in liver tissues. The expression of α-SMA, COL1A1, COL3A1, TIMP-1 was higher in the model group than that in the normal control group. Dual luciferase reporter gene assay revealed that miR-29b-3p directly targeted COL1A1 and COL3A1. Compared with the blank, NC, TGF-ß1 and TGF-ß1 + NC groups, the miR-29b-3p mimic group exhibited up-regulated expression of miR-29b-3p and MMP-9 but down-regulated expression of TIMP-1, HSP47, α-SMA, COL1A1, and COL3A1; while lower cell viability but higher apoptosis rate showed. It indicated that miR-29b-3p prevents S. japonicum-induced liver fibrosis by inhibiting COL1A1 and COL3A1.

[Construction of HSP47-shRNA and its impact on HSP47 at cellular level].

OBJECTIVE: To construct a short hairpin RNA (shRNA) against HSP47 gene, assess the expression level of HSP47 gene in NIH/3T3 cells, and observe the influence on cell function. METHODS: The HSP47-shRNA sequence presented at the downstream of the U6 promoter. The shRNA expression constructs were created using PCR- based methods. The PCR product was digested with Nhe I/Hind Ill and ligated into pGCsi/U6/Neo vector to produce HSP47-pGCsi-U6-shRNA (HSP47-1-pGCsi-U6-shRNA, HSP47-2-pGCsi-U6-shRNA and HSP47-3-pGCsi-U6-shRNA). The non-interference vector and non-related interference vector served as control. The vectors were transfected into NIH/3T3 fibroblast cells by liposome mediated gene transfection method. Transfection efficiency and fluorescence intensity were determined by fluorescence microscopy at 12, 24, 48, and 72 hours after transfection, respectively. Cells were collected before transfection, and at 24, and 48 hours post-transfection, respectively, HSP47 mRNA and protein expression levels were assessed by real-time PCR and Western blotting. The mRNA expression of TGF-pl, collagen types I and Ill in NIH/3T3 cells, and TGF-beta1 levels in cell culture supernatant were determined. RESULTS: HSP47-shRNA vector was transfected into NIH/3T3 cells by liposome-mediated transfection. The transfection efficiency in each HSP47-shRNA plasmid interference group was about 60.0%, and there is no statistical difference among the interference groups (P> 0.05). A small amount of green fluorescent cells were found at 12 h post-transfection. The number of green fluorescent cells increased with the transfection time, and reached strongest at 72 h after transfection. shRNA interference significantly inhabited HSP47 expression in NIHI/3T3 cells. At 24 h after transfection with HSP47-1-shRNA, the inhibition effect was the strongest, and the relative silence efficiency of HSP47 mRNA was (25.83?1.79)%, lower than that of control group and non-related group (P<0.05). Collagen synthesis and secretion by NIH/3T3 cells reduced significantly at 24 and 48 hours post-transfection with HSP47-1-shRNA; and there was a significant difference between HSP47-1-shRNA intervention group and non-related controls. After transfection for 24 and 48 h, mRNA expression of collagen types I and IlI decreased to (56.52 +/- 1.64)% and (53.48 +/- 2.54)%, (54.17 +/- 2.63)% and (50.21 +/- 2.34)%, respectively, significantly lower than that of the control group and non-related group (P<0.05); however, no significant difference was found among the interference groups (P>0.05). In each HSP47-shRNA plasmid interference group, TGF-p1 mRNA expression was the lowest at 24 h post-transfection. The relative mRNA expression level was (63.23?2.18)%, (64.5+3.17)%, and (75.19 +/- 4.20)% in HSP47-1-shRNA, HSP47-2-shRNA, HSP47-3-shRNA groups (P>0.05), respectively, lower than that of the control group and non-related group (P<0.01). At 24 and 48 h post-transfection, TGF-P131 expression was the lowest at 24 h post-transfection, and the relative expression level in HSP47-1-shRNA, HSP47-2-shRNA, HSP47-3-shRNA groups was (51.79 +/- 3.12)%, (66.67 +/- 2.13)%, and (69.61 +/- 3.65)%, respectively. Compared with control group, the expression of TGF-beta1 in HSP47-1-shRNA and HSP47-2-shRNA2 groups was significant inhibited, but there was no significantly difference between control group and HSP47-3-shRNA group (P>0.05). CONCLUSION: HSP47-shRNA. interference plasmid is constructed. HSP47-shRNA effectively inhibits protein expression of HSP47, and results in changes of cell function.

Effect of Anti-HBs on Mortality Among Resolved HBV Infection: a Population-Based Prospective Cohort Study.

INTRODUCTION: Surveillance programs after hepatitis B surface antigen (HBsAg) loss are not yet well established, and the role of hepatitis B surface antibodies (anti-HBs) remains controversial. We aimed to evaluate the risk factors for increased mortality and the association between anti-HBs and all-cause and cause-specific mortality in a representative US (United States) population of patients with resolved HBV (Hepatitis B virus) infections. METHODS: Data were taken from the US National Health and Nutrition Examination Survey (NHANES) 1999-2018. A total of 3455 US adults with resolved HBV infection [defined as hepatitis B surface antigen (HBsAg) negative/anti-hepatitis B core antigen (anti-HBc) positive] were enrolled in this study. The primary outcome measures were all-cause and cause-specific mortality from baseline until 31 December 2019. RESULTS: During a mean follow-up of 10.3 years, 741 deaths occurred. Age, race, marital status, smoking status, physical activity level, and presence of cirrhosis, diabetes, cardiovascular diseases, chronic obstructive pulmonary diseases, cancer, and anti-HBs were significant factors for increased mortality, and a nomogram tool was developed and validated for the risk stratification of mortality. Compared with participants who were anti-HBs positive, those who were anti-HBs negative had a 23% (hazard ratio 1.23, 95% CI 1.02-1.46) higher risk of all-cause mortality in NHANES 1999-2018. For cause-specific mortality, the fully adjusted hazard ratios of participants who were anti-HBs negative were 0.71 (95% CI 0.48-1.06) for heart disease, 1.44 (95% CI 1.01-2.05) for cancer, and 1.44 (95% CI 1.13-1.83) for other conditions, compared to those of participants who were anti-HBs positive. CONCLUSIONS: Among US adults with resolved HBV infections, anti-HBs-negative status was associated with an increased risk of death from all causes and cancer, implying that the role of anti-HBs in resolved HBV infection should not be ignored. On the public health level, more rigorous surveillance was needed for populations of individuals who were isolated anti-HBc positive.

MicroRNA-29a-3p Prevents Drug-Induced Acute Liver Failure through Inflammation-Related Pyroptosis Inhibition.

OBJECTIVE: Little is known about the role of microRNA-29a-3p (miR-29a-3p) in inflammation-related pyroptosis, especially in drug-induced acute liver failure (DIALF). This study aimed to identify the relationship between miR-29a-3p and inflammation-related pyroptosis in DIALF and confirm its underlying mechanisms. METHODS: Thioacetamide (TAA)- and acetaminophen (APAP)-induced ALF mouse models were established, and human samples were collected. The expression levels of miR-29a-3p and inflammation and pyroptosis markers were measured by quantitative real-time polymerase chain reaction (qRT-PCR), Western blotting, or immunochemical staining in miR-29a-3p knock-in transgenic mouse (MIR29A(KI/KI)) DIALF models. In addition, RNA sequencing was conducted to explore the mechanisms. RESULTS: MiR-29a-3p levels were decreased in TAA- and APAP-induced DIALF models. MiR-29a-3p prevented DIALF caused by TAA and APAP. RNA sequencing and further experiments showed that the protective effect of miR-29a-3p on DIALF was mainly achieved through inhibition of inflammation-related pyroptosis, and the inhibition was dependent on activation of the PI3K/AKT pathway. In addition, miR-29a-3p levels were reduced, and pyroptosis was activated in both peripheral blood mononuclear cells and liver tissues of DIALF patients. CONCLUSION: The study supports the idea that miR-29a-3p inhibits pyroptosis by activating the PI3K/AKT pathway to prevent DIALF. MiR-29a-3p may be a promising therapeutic target for DIALF.

[Expression of transforming growth factor-beta1 and connective tissue growing factor in schistosomal hepatic fibrosis of mice].

OBJECTIVE: To establish the model of hepatic fibrosis in mice infected with Schistosoma japonicum and observe the expression of transforming growth factor-beta1 (TGF-beta1) and connective tissue growing factor (CTGF) in mice model. METHODS: Thirty BALB/c mice were randomly assigned to control group and model group. Each mouse of model group was infected with (30 +/- 1) S. japonicum cercariae through the abdominal skin. Serum samples were collected at 4, 6, 8, 10, and 12 weeks after infection, and were analyzed for the levels of ALT and AST. Pathological changes and proliferation of hepatic collagen fibers in liver tissue were observed after HE staining and Masson staining. Immunohistochemistry and real-time PCR were used to detect the protein and mRNA expression of CTGF and TGF-pl. RESULTS: 6-12 weeks after infection, there was significant difference in ALT and AST between model group and control group (P43.05). At 12th week, ALT [(173.53 +/- 31.12) U/I] and AST [(301.00 +/- 34.87) U/LI in model group were higher than those in control group [(42.00 +/- 3.53) and 96.58 +/- 11.26) U/L] . In model group, egg granulomas formed in the liver, and the formation of hepatic fibrosis was significant in portal areas, and there was tree-like hepatic fibrosis around the portal vein branch. 8 weeks after infection, hepatic fibrosis area in mice of model group increased considerably, and there was significant difference in percentage of positive area of collagen between 12th week [(23.83 +/- 1.68) %] and control group [(1.23 +/- 0.14) %] (P < 0.05). 10 and 12 weeks after infection, the percentage of positive area of TGF-beta1 [(22.34 +/- 2.58)% and (25.82 +/- 3.01) %] and CTGF [(1 l.32 +/- 2.44)% and (14.51 +/- 2.05) %] was higher respectively than that of the control [(2.56 +/- 0.87)%, and (1.09 +/- 0.73)% (P < 0.05). 6, 8, 10, and 12 weeks after infection, both TGF-beta1 and CTGF mRNA increased gradually, higher than that in control group (P < 0.05). 10 weeks after infection, TGF-beta1 mRNA relative transcription level was the highest (0.0721 +/- 0.0187) and it was 0.0089 +/- 0.0037 in control group. CTGF mRNA relative transcription level reached the highest value (0.1136 +/- 0.0365) in 12 weeks after infection, while it was 0.0293 +/- 0.0184 in control group. CTGF mRNA expression was positively correlated with the duration of infection (r = 0.927, NO.05). CONCLUSION: The area and cell types of TGFb1 positive expression is the same as that of CTGF in liver tissue of schistosome infected mice (BALB/c). CTGF mRNA expression is significantly related to the duration of infection, but it is not the case for TGFbl.

Prevalence of Cirrhosis/Advanced Fibrosis Among HBsAg-Negative and HBcAb-Positive US Adults: A Nationwide Population-Based Study.

INTRODUCTION: Evaluation of cirrhosis appears to be easily overlooked in the clinic for the HBsAg-negative (hepatitis B surface antigen-negative) and HBcAb-positive (hepatitis B core antibody-positive) population. Herein, we determine the prevalence of cirrhosis/advanced fibrosis among HBsAg-negative/HBcAb-positive US adults. METHODS: Data came from the National Health and Nutrition Examination Survey (NHANES) 2001-2018. A total of 3115 HBsAg-negative/HBcAb-positive US adults were enrolled in this study. We assessed cirrhosis by using the Fibrosis-4 (FIB-4) and aspartate aminotransferase to platelet ratio index (APRI) score. RESULTS: Out of 50,201 NHANES adults, 45,087 were tested for HBcAb/HBsAg, of whom 3115 met the inclusion criteria (HBsAg-negative/HBcAb-positive with available data for FIB-4/APRI). The weighted proportion of HBsAg-negative/HBcAb-positive among US adults was 4.46% (95% CI 4.17-4.75%), affecting 9.87 million US adults. According to the results of the FIB-4, the weighted prevalence of cirrhosis/advanced fibrosis among HBsAg-negative/HBcAb-positive US adults was 3.76% (95% CI 2.80-4.72%), which corresponds to 371,112 (95% CI 276,360-465,864) HBsAg-negative/HBcAb-positive American adults who had already developed cirrhosis. Among those, cirrhosis/advanced fibrosis in the HBsAb-negative (hepatitis B surface antibody) group (6.28%, 95% CI 4.10-8.45%) was significantly higher than in the HBsAb-positive group (3.08%, 95% CI 2.07-4.08%). Results were similar when APRI was used. CONCLUSION: According to the FIB-4, 3.76% of HBsAg-negative and HBcAb-positive US adults had cirrhosis/advanced fibrosis, much higher than in the general population of the USA. Our data highlight the importance of cirrhosis screening in the HBsAg-negative/HBcAb-positive population to prevent advanced liver disease, especially in those who are HBsAb-negative.

Efficacy and safety of Fuzheng Huayu tablet on persistent advanced liver fibrosis following 2 years entecavir treatment: A single arm clinical objective performance criteria trial.

ETHNOPHARMACOLOGICAL RELEVANCE: Antiviral therapy can alleviate liver fibrosis in chronic hepatitis B, but it has a limited effect on advanced liver fibrosis/cirrhosis. Traditional Chinese medicine (TCM), particularly FuZheng HuaYu (FZHY) tablet, appears to have an antifibrotic effect, but its improving resolution of hepatitis b virus (HBV) -associated advanced fibrosis and experienced anti-viral treatment has not been investigated. AIM OF THE STUDY: To observe the safety and efficacy of adjunctive FZHY on the HBV-associated cirrhosis patients who received 2 years of entecavir but still with advanced fibrosis. METHODS: An open-label, multicentre, single arm trial. 251 patients were included and treated with TCM consisted of FZHY tablets 1.6 g and granules, three times a day in addition to entecavir 0.5 mg daily for an additional 48 weeks. Primary outcome was regression of fibrosis (the proportion of patients with a 1-point decrease in the Ishak liver fibrosis score from baseline to week 48). RESULTS: Fibrosis regression occurred in 94 of 184 patients with paired liver biopsy (51.09%, 95% CI: 43.9～58.0). In 132 compensated cirrhosis patients (Ishak score ≥5), 56.06% (74/132, 95% CI: 47.5～64.2) showed fibrosis regression and reached the goal of 54% (15% more than entecavir mono-therapy). 10 patients occurred adverse reaction, most of them were mild, and all recovered or achieved remission. CONCLUSIONS: The combination therapy of FZHY, TCM granules and ETV could regress the liver fibrosis in the patients with HBV cirrhosis, who experienced 2 years of ETV treatment, and it is safe and well tolerated.

[KCTD9, a novel potassium channel related gene, was highly expressed in hepatic NK cells and T cells of fulminant hepatitis mice induced by MHV-3].

OBJECTIVE: To explore the mechanisms of a novel potassium channel gene named KCTD9 (potassium channel tetramerization domain containing 9) in model of fulminant viral hepatitis induced by murine hepatitis virus 3 (MHV-3). METHODS: 78 BALB/cJ mice(6 male) were randomly and equally assigned to two groups, model group of fulminant viral hepatitis induced by MHV3 and its control. 75 C3H/HeJ female mice were done into two groups, 39 for model group of chronic hepatitis induced by MHV3, 36 for control. Various samples including spleen, liver and lymphocytes from mice of two model groups and the controls were examined for KCTD9 expression by real time quantitative PCR and Immunohistochemistry. Independent-samples T test or one-way ANOVA were carried out in different groups. RESULTS: Increased expressions of KCTD9 mRNA was observed in livers of both model mice of fulminant viral hepatitis and chronic hepatitis. Compared with the control mice, the expressions of KCTD9 mRNA were up-regulated by 577.1-, 8.8-, 59.4- and 10.8-fold in hepatic NK cells, CD4+ T cells, CD8+ T cells and splenic NK cells respectively in model mice of fulminant viral hepatitis 48 hr post MHV-3 infection, whereas down-regulation by 43% and 69% in splenic CD4 + T cells and CD8+ T cells were found respectively. In contrast, in model mice of chronic viral hepatitis the expressions of KCTD9 mRNA were down-regulated by 71% and 51% in hepatic CD4+ T cells and NK cells, respectively. The expression of KCTD9 protein was mainly evidenced in infiltrative mononuclear cells of liver as shown by immunohistochemistry. Basal expression was also investigated and showed constitutive expression of KCTD9 in brain, thymus and other organs in BALB/cJ mice. CONCLUSION: A novel potassium channel gene KCTD9 was highly expressed in hepatic NK cells and T cells of fulminant hepatitis mice induced by MHV-3.

CAPRL Scoring System for Prediction of 30-day Mortality in 949 Patients with Coronavirus Disease 2019 in Wuhan, China: A Retrospective, Observational Study.

Background: Coronavirus disease 2019 (COVID-19) is a serious and even lethal respiratory illness. The mortality of critically ill patients with COVID-19, especially short term mortality, is considerable. It is crucial and urgent to develop risk models that can predict the mortality risks of patients with COVID-19 at an early stage, which is helpful to guide clinicians in making appropriate decisions and optimizing the allocation of hospital resoureces. Methods: In this retrospective observational study, we enrolled 949 adult patients with laboratory-confirmed COVID-19 admitted to Tongji Hospital in Wuhan between January 28 and February 12, 2020. Demographic, clinical and laboratory data were collected and analyzed. A multivariable Cox proportional hazard regression analysis was performed to calculate hazard ratios and 95% confidence interval for assessing the risk factors for 30-day mortality. Results: The 30-day mortality was 11.8% (112 of 949 patients). Forty-nine point nine percent (474) patients had one or more comorbidities, with hypertension being the most common (359 [37.8%] patients), followed by diabetes (169 [17.8%] patients) and coronary heart disease (89 [9.4%] patients). Age above 50 years, respiratory rate above 30 beats per minute, white blood cell count of more than10 × 109/L, neutrophil count of more than 7 × 109/L, lymphocyte count of less than 0.8 × 109/L, platelet count of less than 100 × 109/L, lactate dehydrogenase of more than 400 U/L and high-sensitivity C-reactive protein of more than 50 mg/L were independent risk factors associated with 30-day mortality in patients with COVID-19. A predictive CAPRL score was proposed integrating independent risk factors. The 30-day mortality were 0% (0 of 156), 1.8% (8 of 434), 12.9% (26 of 201), 43.0% (55 of 128), and 76.7% (23 of 30) for patients with 0, 1, 2, 3, ≥4 points, respectively. Conclusions: We designed an easy-to-use clinically predictive tool for assessing 30-day mortality risk of COVID-19. It can accurately stratify hospitalized patients with COVID-19 into relevant risk categories and could provide guidance to make further clinical decisions.

[Clinical study on treatment of severe hepatitis with removing dampness and purgative method].

OBJECTIVE: To evaluate the clinical effect of removing dampness and purgative (RDP) method in treating acute, subacute and chronic severe hepatitis. METHODS: One hundred and twenty cases of severe hepatitis were randomly assigned to 2 groups, 60 patients in the control group were treated with routine Western medicine, 60 patients in the treatment group were treated with the same Western medicine plus Chinese medicine prescribed based on RDP principle orally and/or via enema. Fourteen days of treatment constituted one therapeutic course, and patients were treated for 3 courses. Changes of clinical symptoms and signs, complication occurrence, liver function, serum markers of hepatitis B virus, and some biological indexes were observed and compared. The case fatality rate was compared after a 6-month follow-up. RESULTS: The total effective rate and marked improving rate in the treatment group was 71.7% (43/60 cases) and 48.3% (29/60 cases) respectively, while those in the control group, 51.7% (31/60 cases) and 20.0% (12/60 cases) respectively, showing significant difference between the two groups (P < 0.05). After treatment, the clinical symptoms and signs were relieved and complications were reduced in the treatment group, showing marked improvement as compared with that in the control group (P < 0.05). ALT, AST, TBil, quantitative titer of HBV-DNA and HBeAg decreased markedly, and ALB, prothrom-base activity (PTA) and total cholesterol (TC) increased significantly in both groups after treatment (P < 0.01). Significant difference was found in AST, TBil, PTA and quantitative titer of HBV-DNA between the two groups (P < 0.05, P < 0.01). In the 6-month follow-up, the case fatality rate was 23.3% (14/60 cases) in the treatment group, significantly lower than that in the control group (P < 0.05), which was 41.6% (25/60 case) CONCLUSION: RDP treatment is helpful to improve the prognosis of patients with severe hepatitis, it is one of the effective measures for enhancing the efficacy of comprehensive treatment.

[Increased expression of KCTD9, a novel potassium channel related gene, correlates with disease severity in patients with viral hepatitis B].

OBJECTIVE: Studies have shown that potassium channel plays a pivotal role in T cell activation. The expression of potassium channel gene KCTD9 was evidenced being highly upregulated in patients with severe hepatitis B (SHB). To understand this phenomenon further, tissue and cellular expression profiles of KCTD9 were investigated in patients with SHB. METHODS: A rabbit peptide polyclonal antibody was prepared. Various samples including peripheral blood mononuclear cells (PBMCs); livers from patients with SHB or mild chronic hepatitis B, were examined for KCTD9 expression by quantitative real time PCR and immunohistochemistry staining (IHC). Confocal microscopy was used to illustrate the localizations of the expressions. RESULTS: Increased expression of KCTD9 was observed in PBMC in over 35.7% of the patients with SHB when compared with that of patients with mild chronic hepatitis B. In all patients, the relative value of increased KCTD9 mRNA was positively correlated with alanine aminotransferase, aspartate aminotransferase, total bilirubin and direct bilirubin but negatively with serum albumin. The expression was mainly located in hepatocytes, bile duct epithelial cells, Kupffer cells and inflammatory cells, and in the cytoplasm of PBMCs from the healthy individuals and patients with mild chronic hepatitis B, whereas in both cytoplasm and nuclei in those from patients with SHB. CONCLUSION: The increased expression of potassium channel gene KCTD9 correlates with disease severity in patients with viral hepatitis B.

[Dynamic expressions of IL-22 and hepatic stellate cells senescence in mice infected with Schistosoma japonicum].

OBJECTIVE: To investigate the dynamic expressions of interleukin-22 (IL-22) , Interleukin-22 receptor 1 (IL-22R1), and hepatic stellate cells (HSC) senescence in mice with Schistosoma japonicum infection. METHODS: A murine model of S. japonicum infection was established and the serum samples and liver tissues were collected 4, 6, 8, 12 weeks post-infection. The serum samples were detected for the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). The pathological changes and proliferation of hepatic collagen fibers in the liver tissue were observed after HE staining and Masson staining. The HSC senescence in fibrotic livers was determined by the detection of senescence-associated beta-galactosidase (SA-beta-Gal). Sandwich ELISA was used to measure the expressions of IL-22, and Real-time PCR was used to test the mRNA levels of IL-22 and IL-22R1. The control group without S. japonicum infection was set up. RESULTS: The serum levels of ALT and AST significantly increased 8 weeks and 12 weeks after the infection (vs. 0 week, all P < 0.05). The level of IL-22 increased 4 weeks and 6 weeks after the infection (vs. 0 week, both P < 0.05), but reduced 8 weeks post-infection, and was even lower 12 weeks post-infection (vs. 4 weeks and 6 weeks, both P < 0.01). Being consistent with the dynamic expression of IL-22 protein, the mRNA expression of IL-22 began to increase 4 weeks and reached the peak 6 weeks after the infection (vs. 0 week, both P < 0.05), and continuously declined 8 weeks and 12 weeks post-infections (vs. 6 weeks, both P< 0.05). The increase of the expression of IL-22R1 mRNA was correlated with the progression of fibrosis, and the peak was in 12 weeks post-infections (vs. 0 week and 6 weeks, both P < 0.05). The number of senescence-associated beta-galactosidase-positive HSCs was reduced with the decreasing expression of IL-22 in the advanced liver fibrosis. CONCLUSION: IL-22 and IL-22R1 are involved in the pathogenesis of schistosomiasis liver fibrosis. As an inflammation factor, IL-22 significantly increases in the early stage of fibrosis. The expression of IL-22 decreases in the late stage of fibrosis, which may contribute to HSC senescence and restrict liver fibrosis.

Involvement of heat shock protein 47 in Schistosoma japonicum-induced hepatic fibrosis in mice.

Chronic infection with the blood fluke Schistosoma japonicum is associated with both liver cirrhosis and liver cancer. Previously, heat shock protein 47, a collagen-specific molecular chaperone, was shown to play a critical role in the maturation of procollagen. However, less is known about the role of heat shock protein 47 in S. japonicum-induced hepatic fibrosis. We therefore investigated the expression of heat shock protein 47 in S. japonicum-induced liver fibrosis and attempted to determine whether inhibition of heat shock protein 47 could have beneficial effects on fibrosis in vitro and in vivo. In this study, we found that the expression of heat shock protein 47 was significantly increased in patients with Schistosoma-induced fibrosis, as well as in rodent models. Immunohistochemistry revealed heat shock protein 47-positive cells were found in the periphery of egg granulomas. Administration of heat shock protein 47-targeted short hairpin (sh)RNA remarkably reduced heat shock protein 47 expression and collagen deposition in NIH3T3 cells and liver tissue of S. japonicum-infected mice. Life-table analysis revealed a dose-dependent prolongation of survival rates with the treatment of heat shock protein 47-shRNA in murine fibrosis models. Moreover, serum alanine aminotransferase and aspartate transaminase activity, splenomegaly, spleen weight index and portal hypertension were also measured, which showed improvement with the anti-fibrosis treatment. The fibrosis-related parameters assessed were expressions of Col1a1, Col3a1, TGF-ß1, CTGF, IL-13, IL-17, MMP-9, TIMP-1 and PAI-1 in the liver. This study demonstrated that heat shock protein 47-targeted shRNA directly reduced collagen production of mouse liver fibrosis associated with S. japonicum. We conclude that heat shock protein 47 plays an essential role in S. japonicum-induced hepatic fibrosis in mice and may be a potential target for ameliorating the hepatic fibrosis caused by this parasite.

Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea.

Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I­IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II­III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein.Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7­78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.

[Protective actions of acupoint magnetic medicated plaster therapy on hepatic function in the patient of cirrhosis after hepatitis].

OBJECTIVE: To observe the therapeutic effect of acupoint magnetic medicated plaster therapy on cirrhosis after hepatitis. METHODS: One hundred and twenty patients with cirrhosis after hepatitis were randomly divided into 2 groups. The control group (n=60) were treated by the hepatic protective therapy (diammonium glyeyrrhiznate, Silymarin, compound Tanshin, vitamin E), and the treatment group were treated by the liver-protective therapy and acupoint magnetic medicated plaster therapy, for 2-6 therapeutic courses. Clinical symptoms, hepatic function, serum markers of hepatitis B virus and indexes of hepatic fibrosis were investigated. RESULTS: The markedly effective rate and the total effective rate were 65.0% and 95.0% in the treatment group, and 43.3% and 91.7% in the control group, and the serum indexes of hepatic fibrosis decreased signficantly, with significantly diferences (all P < 0.05). CONCLUSION: Acupoint magnetic medicated plaster therapy can improve clinical symptoms, rapidly restore hepatic function, decrease serum indexes of hepatic fibrosis in the patient of cirrhosis after hepatitis.