Silybin nanoparticles for liver cancer: development, optimization and in vitro - in vivo evaluation.

PURPOSE: Hepatocellular carcinoma (HCC) is a most common liver malignancy. The objective of this study was to prepare silybin nanoparticles (NPs) and optimize the prepared nanoparticles using central composite rotatable design-response surface methodology. METHODS: HCC was induced in rats by supplementing 100 mg/L of diethylnitrosamine (DENA) in drinking water for 8 weeks. Saline, silybin 30 mg/kg body weight and nanoformulation of silybin equivalent to silybin dose were administered orally to 3 groups of 6 animals each. Anticancer activity was evaluated by counting the liver nodules, and H & E staining analysis of tissue sections. RESULTS: The results showed that silybin NPs under optimized conditions gave rise to the entrapment efficiency (EE) of 88%, drug loading (DL) of 15%, mean diameter of 216 nm of the NPs prepared and zeta potential value of -15 mV. In rats treated with silybin NPs, the number of neoplastic nodules was significantly lower, the animals did not exhibit decrease in mean body weight, the number of liver nodules was reduced by >93% with significantly high localization in the liver. CONCLUSION: Orally administered silybin NPs showed improved efficacy and safety compared to silybin for the treatment of HCC in rats.

[Comparative analysis between diatom nitric acid digestion method and plankton 16S rDNA PCR method].

OBJECTIVE: To compare and explore the application value of diatom nitric acid digestion method and plankton 16S rDNA PCR method for drowning identification. METHODS: Forty drowning cases from 2010 to 2011 were collected from Department of Forensic Medicine of Wenzhou Medical University. Samples including lung, kidney, liver and field water from each case were tested with diatom nitric acid digestion method and plankton 16S rDNA PCR method, respectively. The Diatom nitric acid digestion method and plankton 16S rDNA PCR method required 20 g and 2 g of each organ, and 15 mL and 1.5 mL of field water, respectively. The inspection time and detection rate were compared between the two methods. RESULTS: Diatom nitric acid digestion method mainly detected two species of diatoms, Centriae and Pennatae, while plankton 16S rDNA PCR method amplified a length of 162 bp band. The average inspection time of each case of the Diatom nitric acid digestion method was (95.30 +/- 2.78) min less than (325.33 +/- 14.18) min of plankton 16S rDNA PCR method (P < 0.05). The detection rates of two methods for field water and lung were both 100%. For liver and kidney, the detection rate of plankton 16S rDNA PCR method was both 80%, higher than 40% and 30% of diatom nitric acid digestion method (P < 0.05), respectively. CONCLUSION: The laboratory testing method needs to be appropriately selected according to the specific circumstances in the forensic appraisal of drowning. Compared with diatom nitric acid digestion method, plankton 16S rDNA PCR method has practice values with such advantages as less quantity of samples, huge information and high specificity.

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.

[Effects of tillage types on soil aggregate distribution and stability in irrigated sierozem of Gansu Yellow River irrigation area, China]. / 耕作方式对甘肃引黄灌区灌耕灰钙土团聚体分布及稳定性的影响.

A total of six treatments, including continuous conventional tillage (CT), rotary tillage (RT), subsoiling (ST), no-tillage (NT), conventional-no tillage (CT-NT) and subsoiling-no tillage (ST-NT), were conducted to examine the effects of different tillage types on soil aggregates distribution and stability of irrigated sierozem on continuous 8-year-tillage maize fields in the Gansu Yellow River irrigated area in 2014-2017. The results showed that the aggregation and stability of large aggregates in 0-40 cm soil layer were increased by NT and ST-NT treatments, while the size distribution and stability in plough layer were significantly decreased by CT and RT treatments due to strong soil disturbance. Compared with RT, the mechanical stability of aggregates under dry sieving NT was the best. The contents of >0.25 mm aggregate (R0.25), mean weight diameter (MWD) and geometric mean diameter (GMD) increased by 5.8%, 8.0%, and 13.0%, respectively, and fractal dimension (D) decreased by 3.6%. The water-stable aggregates in ST-NT was the best, with R0.25, MWD and GMD increased by 55.3%, 15.1% and 8.7%, respectively, and D value decreased by 0.8%. The percentage of aggregate destruction (PAD) and unstable aggregate index (ELT) of NT and ST-NT treatments were the lowest. PAD was reduced by 5.9% and 7.7% compared with RT, ELT was reduced by 5.8% and 7.2%, respectively. All the results indicated that the subsoiling-notillage (ST-NT) rotation mode was more conducive to the enhancement of soil aggregate content and stability and consistent with the local farmers operating habits, which would be an ideal tillage method and had certain application value for the sustainable agricultural development in this area.

[Effects of subsoiling and straw returning on soil physical properties and maize production in Yellow River irrigation area of Gansu, China]. / 深松和秸秆还田对甘肃引黄灌区土壤物理性状和玉米生产的影响.

A field experiment was conducted to examine the effects of subsoiling 35 cm with maize straw returning, subsoiling 35 cm without maize straw returning, and rotary tillage without maize straw returning on soil compaction, soil bulk density, soil infiltration, soil water content in 0-100 cm depth, nutrients uptake and production of maize on sierozem in the Gansu Yellow River irrigated area in 2015-2017. Compared with subsoiling 35 cm without maize straw returning and rotary tillage without maize straw returning, subsoiling 35 cm with maize straw returning significantly decreased the soil compaction and soil density in 0-40 cm depth. Compared with that in 2015 (before experiment), soil compaction and soil bulk density in subsoiling 35 cm with straw returning was decreased by 42.6% and 7.0%, respectively, after harvest in 2017. Compared with other treatments, subsoiling 35 cm with straw returning had the lowest variation of soil compaction (6.1%) and soil bulk density (3.2%) in 0-40 cm depth before sowing and after harvest in 2016 and 2017. The soil infiltration rate in subsoiling 35 cm with straw returning was significantly improved by 33.6% compared with rotary tillage without maize straw returning. Subsoiling 35 cm with straw retention could significantly increase soil water content and decrease water variation in 0-100 cm soil depth in spring (before maize sowing) and autumn (after maize harvest). Compared with rotary tillage without maize straw returning, water storage in subsoiling 35 cm with straw retention was increased by 15.5% and 5.6% in spring and autumn, respectively. The water use efficiency was enhanced by 32.4%. Furthermore, subsoiling 35 cm with straw retention could increase maize economic yield and biomass yield by 25.6% and 33.3%, compared with rotary tillage without straw retention. Subsoilng and straw retention could promote nutrient absorption, with N, P2O5 and K2O uptake increased by 49.6%, 51.5% and 37.6%, compared with rotary tillage. Overall, our results suggested that subsoiling 35 cm straw retention could improve soil characteristics, stabilize the phy-sical properties of the plough layer, increase soil water content in the 0-100 cm soil layer, and reduce water variation in spring and autumn. Consequently, it was the best management to promote the water and nutrient utilization of maize and achieve high yield. Our findings could provide theoretical basis for further research on the construction technology of the plough layer in Gansu irrigation area.

[The expression of BDNF and PSD-95 in hippocampal CA1 region of morphine-withdrawn rat with different dependent times].

OBJECTIVE: To observe the expression of brain-derived neurotrophic factor (BDNF) and postsynaptic density-95 (PSD-95) in hippocampal CA1 region of rat with morphine dependence for different times and withdrawn for 1 week, and investigate the influence of that morphine dependence is withdrawn on rat hippocampal CA1 area. METHODS: Animal models of rats with morphine withdrawal for 1 week and different morphine dependent times(1 week, 2 weeks, 4 weeks) were established. The expression of BDNF and PSD-95 in hippocampal CA1 were identified with RT-PCR. RESULTS: The expression of BDNF and PSD-95 in hippocampal CA1 decreased in the withdrawn group with morphine dependence for 1 week as compared with that in normal saline (NS) group (P < 0.01), and it increased in the withdrawn group with morphine dependence for 2 weeks as compared with that in morphine-dependent group for 1 week (P < 0.05) but still decreased as compared with that in NS group (P < 0.01), and it decreased in the withdrawn group with morphine dependence for 4 weeks as compared with the other three groups (P < 0.01). CONCLUSION: The expression of BDNF and PSD-95 in hippocampal CA1 decreases in morphine-depended rats withdrawn for 1 week. Morphine withdrawal has a manifest harm in hippocampal CA1 region of rat.

Fish consumption doesn't reduce the risk of hepatocellular carcinoma.

BACKGROUND: Several observational studies have investigated the association between fish consumption and the risk of hepatocellular carcinoma (HCC), however, the results were inconsistent. Hence, we performed a meta-analysis of observational studies to evaluate the effect of fish consumption on HCC risk. METHODS: A systematic search was performed using the Pubmed, Embase, and Cochrane Library Central database for case-control and coshort studies that assessed fish intake and HCC risk. Fixed-effect and random-effect models were used to estimate summary relative risks (RR) and the corresponding 95% confidence intervals (CIs). Subgroup analyses and sensitivity analysis were also performed. RESULTS: Nine case-control studies and three cohort studies were included, involving a total of 1,071,458 participants and 2,627 HCC cases. Meta-analysis showed that there was no association between fish consumption and a significant reduction in HCC incidence (RR = 0.85, 95% CI [0.62, 1.17]). In our subgroup analyses, the result was substantially affected by adjustment for hepatic viruses' infection status. Sensitivity analysis confirmed the stability of results. Furthermore, there was no evidence of publication bias as suggested by Begg's P value (P = 0.411) and Egger's (P = 0.596) test. CONCLUSIONS: In conclusion, our results do not support a significant inverse association of fish consumption with HCC risk. More in-depth studies are warranted to report more detailed results, including stratified results by fish types, preparation methods, and gender.

A meta-analysis of the correlation between the HLA-DRB1*03 allele and chronic hepatitis B in the Han Chinese population.

OBJECTIVE: This study sought to use a meta-analysis approach to comprehensively evaluate correlations between the human leukocyte antigen-DR beta 1 (HLA-DRB1)*03 allele and chronic hepatitis B (CHB) in the Han Chinese population. METHODS: The China Biomedical Literature database (CBMdisc), the Chongqing VIP database (VIP), and the PubMed database were searched. Using the inclusion and exclusion criteria of this study, all relevant case-control studies retrieved in these searches that satisfied the conditions of this investigation were collected. Review Manager (RevMan) 5.2 software was used to conduct a meta-analysis on the results of these studies. RESULTS: There were 9 publications that satisfied the inclusion criteria. These publications included a total of 970 cases in the CHB group and 1185 cases in the normal control group. Egger's test revealed no significant publication bias. A comprehensive analysis indicated that the pooled odds ratio (OR) value was 1.94 with a 95% confidence interval (CI) of 1.23-3.06 (Z=2.84, p=0.004); these findings suggested that in the Han Chinese population, the HLA-DRB1*03 allele is a susceptibility allele related to the occurrence of CHB. CONCLUSION: There is a statistically significant correlation between the HLA-DRB1*03 allele and the occurrence of CHB in the Han Chinese population, and the HLA-DRB1*03 allele may be a susceptibility allele for this disease.

Genetic characterization and serological prevalence of swine hepatitis E virus in Shandong province, China.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is classified into four major genotypes (1 to 4) and swine is the main natural reservoir for genotypes 3 and 4. In this study, a total of 106 bile samples from a slaughterhouse in the Shandong province of China were tested for the partial ORF2 gene of HEV by RT-nPCR to determine the virus genotypes, and two indirect ELISA were developed for the detection of swine HEV specific IgM and IgG antibodies in 980 serum samples from 24 farms, in order to investigate the seroprevalence. Thirty-two out of 106 (30.2%) bile samples were positive for HEV and a high degree of partial ORF2 sequence similarity (86.8-100%) was observed among 20 samples. The viral sequences belonged to genotype 4, subtypes 4a and 4d. One complete genome sequence of a subtype 4d HEV was further determined and characterized. The seroprevalence of HEV IgG and IgM antibodies was 100% (24/24) and 41.7% (10/24) for herds, and 66.4% (651/980) and 1.6% (16/980) for the individual pigs, respectively. These results suggested a high prevalence of genotype 4 of swine HEV infection both in swine farms and at the slaughterhouse in Shandong province, which further raise public-health concerns for zoonosis and pork safety.

Characterization of antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian hepatitis E virus.

Avian hepatitis E virus (HEV) is an emerging virus associated with the big liver and spleen disease or hepatitis-splenomegaly syndrome in chickens and subclinical infections by the virus are also common. The complete genome of avian HEV contains three open-reading frames (ORFs) in which ORF2 protein is part of virus particles and thus contains primary epitopes. Antigenic epitopes of avian HEV ORF2 protein have been described but those associated with the ORF3 have not. To analyze the antigenic domains and epitopes in the ORF3 protein of a Chinese isolate of avian HEV (CaHEV), we generated a series of antigens comprised of the complete ORF3 and also five truncated overlapping ORF3 peptides. The antibodies used in this study were mouse antisera and monoclonal antibodies against ORF3, positive chicken sera from Specific Pathogen Free chickens experimentally infected with CaHEV and clinical chicken sera. Using these antigens and antibodies, we identified three antigenic domains at amino acids (aa) 1-28, 55-74 and 75-88 in which aa 75-88 was a dominant domain. The dominant domain contained at least two major epitopes since field chickens infected with avian HEV produced antibodies against the domain and epitopes. These results provide useful information for future development of immunoassays for the diagnosis of avian HEV infection.

[Correlationship between chemokines and oxidative stress in chronic hepatitis B].

OBJECTIVE: The aim of this study is to investigate the possible associations of chemokines IP-10, Rantes and oxidative stress in chronic hepatits B (CHB). METHODS: 70 CHB patients and 10 healthy controls were enrolled in the study. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of IFN-gamma-inducible protein-10 (IP-10) and regulated on activation normal T-cell-expressed and secreted (Rantes) and oxidative stress parameters (glutathione, GSH; glutathione disulfide, GSSG). Correlationship were analyzed by Spearman's rank correlation. RESULT: The levels of IP-10 and Rantes were higher in CHB patients than healthy controls, and strong positive associations were found between IP-10/Rantes and alanine aminotransferase (ALT). The levels of GSH and GSH/GSSG were lower in CHB patients than healthy controls, and GSH and GSH/GSSG were negatively correlated with ALT. The levels of IP-10 and Rantes were negatively correlated with GSH and GSH/GSSG respectively. CONCLUSION: Strong associations were found between chemokines and oxidative stress which participated in the pathogenesis of CHB.

[Genotypes distribution of hepatitis C virus through multi-center, large sample studies among chronic hepatitis C patients in Chinese Han population].

OBJECTIVE: To understand the distribution of hepatitis C virus (HCV) genotypes in China. METHODS: A national multi-center, large sample epidemiological survey was carried out and certified by an international third-party testing organization. Internationally accepted method for sequencing analysis of viral genotype was used. RESULTS: Patients were those suffered from the 4 major genotypes of chronic hepatitis C, including nine subtypes, which were dominated by genotype 1 (69.6%), with type 1b in particular (accounting for 68.38%). Among the rare gene-6, we saw more subtypes of 6a. In the south, central, and north areas, there was no significant difference seen between the south and the north areas in the distribution of the genotype 1. However, the rate of gene type 2a increased gradually from south to north. In terms of less common type of gene 3b and gene-6, they were seen mainly in the southern provinces. CONCLUSION: In China, Patients with chronic hepatitis C had 4 HCV genotypes, with 9 subtypes. The rarely seen genotypes 3b and 6 were mainly distributed in the southern provinces.

Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China.

Six porcine epidemic diarrhea viruses (PEDVs) were isolated from the fecal samples of piglets infected with PEDV in 2006 in China. The membrane (M) protein genes of six PEDV isolates were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), then cloned, sequenced, and compared with each other as well as those ten PEDV reference strains. The M protein genes of six Chinese PEDV isolates consisted of 692 nucleotides containing a single open reading frame (ORF) of 681 nucleotides, which encoded a 226aa-long peptide. The conserved intergenic motif (ATAAAC), as previously recognized in Br1/87, was found in the 5 nucleotides upstream of the initiator ATG of M protein genes of six Chinese PEDV isolates. The hexamer motif was also found in CV777, JMe2, LZC, and QH. The M protein of six isolates had three main transmembrane domains (aa20-38, aa43-65, aa75-97). The M protein of one isolate, CH/IMT/06, had one potential glycosylation site, but those of the other five isolates had two. The glycosylation sequence Asn-Phe-Thr was highly conserved in the M proteins of six PEDV isolates. The six PEDV isolates showed nucleotide sequence homology between 98.8 and 100% and deduced amino acid sequence homology between 98.2 and 100% with each other. The nucleotide and amino acid identity of M protein genes between the six PEDV isolates and ten reference PEDV strains varied from 97.2 to 99.4% and 96.9 to 100%, respectively. On the basis of the phylogenetic relationship of M protein genes, six Chinese PEDV isolates composed of a separate cluster including one Chinese strain JS-2004-02, however, not including the Chinese strain LJB/03. These results demonstrated that there was a new genotype of PEDV prevailing in China.