To examine the effect of Gankang Suppository on duck hepatitis B virus (DHBV), the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum. The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups: a Gankang Suppository treatment group, an acyclovir (ACV) group and a DHBV model group (control), with each group having 16 animals. All the animals were given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medication and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day 14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values (log) of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment (P=0.0092, P=0.0070, P=0.0080, respectively). Compared with DHBV model control group, the ALT level was significantly decreased (P=0.0020, P=0.0019, respectively) on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment (P=0.0298). Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal (P=0.0016). Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7 days after the withdrawal as compared with model control group (P=0.0282, P=0.0084, P=0.0195, respectively). It is concluded that Gankang Suppository can effectively suppress DHBV replication, reduce the levels of serum ALT and AST and improve hepatic histology.

To study the effect of endotoxin on liver apoptosis, L02 liver cells were cultured and passaged in vitro, and then stimulated by endotoxin at 10 mg/mL for 4, 8, 16 and 24 h respectively. Liver apoptosis was flow cytometrically and fluorescently detected. Immunohistochemistry was used to detect the delivery of smac and caspase9. The delivery of liver cell smac and the activity of caspase3 were measured by caspase3 assay kit. The hepatic failure models of rats were established by using D-galactosamine. The blood serum and liver tissues were collected for the detection of the liver function, the level of endotoxin and the activity of caspase3 by using chromogenic substrate limulus amebocyte lysate method (LAL) and caspase3 active assay kit. The expression of smac and caspase9 in liver cells was detected by Western blotting. With in vitro study, the L02 cells stimulated by LPS condensed into conglobation and formed apoptotic bodies. After those cells were stained by hoechst, the apoptotic cells displayed blue color under the fluorescent microscope. The apoptosis rate was increased over time and the apoptosis was mainly of advanced stage. Meanwhile, the rate of smac delivery and activity of caspase9 and caspase3 were increased on L02 cell membrane. In vivo, hepatic failure and obvious endotoxemia were induced by injection of more than 200 mg/kg D-GalN. Hepatic mitochondria smac was reduced with dosage of D-GalN and, on the contrary, the activity of caspase3 was increased. D-GalN at 200 mg/kg increased Caspase9 while D-GalN at 300 mg/kg decreased caspase9. Mitochondria signal channel plays an important role in the endotoxin-induced apoptosis of hepatic cells by promoting the release of smac from mitochondria to cytoplasm and activating caspase9 and caspase3 in its low-level channel.
Apoptosis/*drug effects ; Carrier Proteins/*metabolism ; Caspase 3/metabolism ; Caspase 9/metabolism ; Cell Line ; Cells, Cultured ; Endotoxins/*pharmacology ; Liver/cytology ; Liver/*metabolism ; Liver/pathology ; Liver Failure/chemically induced ; Liver Failure/pathology ; Mitochondrial Proteins/*metabolism ; Rats, Wistar

Objective To study the effect of a new mucA gene mutation on the biofilm formation process and the morphology of matured biofilm of Pseudomonas aeruginosa. Methods The mucA gene of PAO1 was cloned into the Pseudomonas aeruginosa expression plasmid pUCP20. The recombinant plasmid was transformed to mucoid PA17 which contained a new mucA mutation. Positive clones of the plasmids were identified by enzyme digestion and sequencing. The expression levels of algD in the positive clones were assessed by semi-quantitative RT-PCR. The modified plate culture method was used to establish the biofilm models of PA17, PA17 with recombinant plasmid and PAO1 in vitro. Results Transformation was identified by the decreased expression of algD in positive clones. The rate of biofilm formation of the positive clones was between those of PAO1 and PA17. The irreversible adhesion occurred after 8 h and the matured biofilm was observed on day 6. The morphologies of PA17, PAO1 and PA17 with recombinant plasmid were the same. Conclusion The mucA gene mutation of PA17 delays the formation of irreversible adhesion of PA17 biofilm, but it has no effect on the morphology of matured biofilm.

To examine the effect of Gankang Suppository on duck hepatitis B virus (DHBV), the serum biochemistry and hepatic histology in an animal model of DHBV infection, a model of DHBV infection was established by infecting 1-day-old Yingtaogu ducklings with DHBV-positive serum.The successful model was confirmed by PCR assay and 48 ducklings infected with DHBV were randomly divided into 3 groups: a Gankang Suppository treatment group, an acyclovir (ACV) group and a DHBV model group (control), with each group having 16 animals. All the animals were .given the medicines for 4 weeks in a row. The serum of the animals was taken 14 and 28 days after the medication and 7 days after drug discontinuation. Real-time PCR was performed to detect the copy numbers of DHBV DNA in the serum. ALT and AST were dynamically monitored. The ducklings were sacrificed on the 7th day after the discontinuation of the treatment and livers were harvested and examined for inflammation and degeneration of liver cells by using HE staining. The results showed that on day14, 28 after the treatment and day 7 after the withdrawal, the logarithmic values (log) of DHBV DNA copy numbers in ducklings of Gankang Suppository treatment group were significantly lower than that before the treatment (P=0.0092, P=0.0070, P=0.0080, respectively). Compared with DHBV model control group, the ALT level was significantly decreased (P=0.0020, P=0.0019, respectively)on day 28 after the treatment and on day 7 after the withdrawal. The AST level was also reduced on day 14 after the treatment (P=0.0298). Compared with the ACV control group, the level of ALT was lower on day 7 after the withdrawal (P=0.0016). Histologically, the hepatocyte swelling, vacuolous degeneration and acidophilic degeneration in Gankang Suppository treatment group were alleviated 7days after the withdrawal as compared with model control group(P=0.0282, P=0.0084, P=0.0195,respectively). It is concluded that Gankang Suppository can effectively suppress DHBV replication,reduce the levels of serum ALT and AST and improve hepatic histology.

To study the effect of endotoxin on liver apoptosis, LO2 liver ceils were cultured and passaged in vitro, and then stimulated by endotoxin at 10 mg/mL for 4, 8, 16 and 24 h respectively. Liver apoptosis was flow cytometrically and fluorescently detected. Immunohistochemistry was used to detect the delivery of smac and caspase9. The delivery of liver cell smac and the activity of caspase3 were measured by caspase3 assay kit. The hepatic failure models of rats were established by using D-galactosamine. The blood serum and liver tissues were collected for the detection of the liver function, the level of endotoxin and the activity of caspase3 by using chromogenic substrate limulus amebocyte lysate method (LAL) and caspase3 active assay kit. The expression of smac and caspase9 in liver ceils was detected by Western blotting. With in vitro study, the LO2 cells stimulated by LPS condensed into conglobation and formed apoptotic bodies. After those cells were stained.by hoechst,the apoptotic cells displayed blue color under the fluorescent microscope. The apoptosis rate was increased over time and the apoptosis was mainly of advanced stage. Meanwhile, the rate of smac delivery and activity of caspase9 and caspase3 were increased on LO2 cell membrane. In vivo, hepatic failure and obvious endotoxemia were induced by injection of more than 200 mg/kg D-GaIN. Hepatic mitochondria smac was reduced with dosage of D-GaIN and, on the contrary, the activity of caspase3 was increased. D-GaIN at 200 mg/kg increased Caspase9 while D-GaIN at 300 mg/kg decreased caspase9. Mitochondria signal channel plays an important role in the endotoxin-induced apoptosis of hepatic cells by promoting the release of smac from mitochondria to cytoplasm and activating caspase9 and caspase3 in its low-level channel.

OBJECTIVE: To explore the molecular pathogenesis of a Chinese pedigree affected with inherited protein C (PC) deficiency. METHODS: The protein C activity (PC:A) and protein C antigen (PC:Ag) of the proband and his family members were determined by a chromogenic substrate method and enzyme-linked immunosorbent assay, respectively. The proband was subjected to whole exome sequencing. Candidate variants were verified by Sanger sequencing of other members of the pedigree. RESULTS: The PC:A and PC:Ag of proband were reduced to 15% and 11%, respectively. The above parameters of his parents and elder sister were also decreased to approximately 50% of reference values. Next generation sequencing has revealed that the proband has harbored a heterozygous c.572_574delAGA (p.Glu191_Lys192delinsGlu) variant in exon 7 and a missense c.752C>T (p.Ala251Val) variant in exon 8 of the PROC gene. His father was heterozygous for the c.572_574delAGA variant, while his mother and elder sister were heterozygous for the c.752C>T variant. According to the American College of Medical Genetics and Genomics Standards and Guidelines, the c.572_574delAGA (p.Glu191_Lys192 delinsGlu) variant was predicted to be likely pathogenic (PS1+PM4+PP3). c.752 C>T (p.Ala251Val) variant was also likely pathogenic (PS1+PM1+PP3). CONCLUSION The deletional variant of c.572_574delAGA (p.Glu191_Lys192delinsGlu) in exon 7 and missense variant c.752C>T (p.Ala251Val) in exon 8 of the PROC gene probably underlay the inherited protein C (PC) deficiency in this pedigree. Above finding has enriched the spectrum of PROC gene variants and provided a basis for genetic counseling for this pedigree.
Humans ; China ; Mutation ; Pedigree ; Protein C/genetics* ; Protein C Deficiency/genetics* ; Male ; Female