Establishment of a droplet digital PCR method for the detection of hepatitis B virus covalently closed circular DNA / 临床肝胆病杂志

ObjectiveTo establish a droplet digital PCR (ddPCR) method for detecting hepatitis B virus (HBV) covalently closed circular DNA (cccDNA). MethodsHBV cccDNA standard substance was constructed, and HBV cccDNA primers and probes were designed based on the structural differences between HBV cccDNA and relaxed circular DNA (rcDNA). HBV plasmid was amplified to obtain HBV cccDNA standard substance, and a ddPCR detection method was established with the standard substance after gradient dilution as the template for HBV cccDNA detection; the limit of detection and repeatability of this method were analyzed. Liver tissue samples were collected from 20 patients who attended Beijing YouAn Hospital, Capital Medical University, from June 2017 to October 2020, all of whom were diagnosed with HBV infection, and DNA of the samples was extracted and digested with plasmid-safe ATP-dependent DNA enzyme to obtain HBV cccDNA template; the ddPCR detection method was evaluated in clinical samples and was compared with the quantitative real-time PCR (qPCR) detection method. The chi-square test was used for comparison of categorical data between the two groups. ResultsThe HBV cccDNA detection method based on ddPCR was established, which accurately detected HBV cccDNA in standard substance after gradient dilution, with a limit of detection of 1 copy/μl, and the coefficients of variation of 1×103, 1×102, and 1×101 copies/μl standard substances were 441%, 3.98%, and 5.09%, respectively. HBV cccDNA was detected in the samples of 20 patients with HBV infection; the ddPCR detection method detected HBV cccDNA in 17 patients, with a positive rate of 85%, while the qPCR detection method detected HBV cccDNA in 11 patients, with a positive rate of 55%, and there was a significant difference between the two methods (χ2=4.286, P=0038). ConclusionThe established ddPCR method for detecting HBV cccDNA has a low limit of detection and good repeatability, which provides an effective tool for further clinical detection.

Study of endoplasmic reticulum stress role in hepatic failure induced by severe hepatitis B / 中华肝脏病杂志

Objective@#To investigate the endoplasmic reticulum stress (ERS) role in the course of liver failure induced by severe hepatitis B virus (HBV) infection and its related mechanism.@*Methods@#Liver tissue samples and clinical data [chronic hepatitis B patients (12 cases, chronic hepatitis B group), hepatic failure induced by severe hepatitis B virus (12 cases, severe hepatitis B virus liver failure group), and normal subjects (8 cases, control group)] were collected from the Beijing You'an Hospital affiliated to Capital Medical University between 2009 to 2011. Statistical analysis was performed on the clinical indicators of each group. The structure of endoplasmic reticulum in liver tissue was observed by transmission electron microscopy. Western blot and qRT-PCR were used to detect the expression of endoplasmic reticulum stress and apoptosis-related factors, including glucose-regulated protein (Grp), and C/EBP homologous protein (CHOP). Frozen sections of liver tissues were prepared for immunofluorescence test. All data were expressed as mean ± standard deviation. LSD-t test was used to compare the results between groups. A p value < 0.05 was considered as statistically significant.@*Results@#Transmission electron microscopy showed that the morphological structure of the endoplasmic reticulum was damaged in both groups (chronic hepatitis B and liver failure induced by severe hepatitis B virus), and liver failure induced by severe hepatitis B virus group was more critical. Western blot and qRT-PCR showed that Grp78, Grp94 and Caspase-4 were highly expressed in normal group and chronic hepatitis B group, and the relative protein expressions were 1.20 ± 0.13 and 0.78 ± 0.11, 0.90 ± 0.06 and 0.11 ± 0.01, 0.15 ± 0.02 and 0.22 ± 0.04, respectively. The expression of protein was weakened in liver failure induced by severe hepatitis B virus group (relative protein expression was 0.01 ± 0, 0.01 ± 0, and 0.11 ± 0.02, respectively).There was a statistically significant difference between the two groups (P < 0.05). The expression of CHOP was consistent with the results of immunofluorescence, and increased with the stressing of injury.@*Conclusion@#During the course of severe hepatitis B infection, dysregulated endoplasmic reticulum stress activated mild stress in chronic hepatitis B group, while severe stress in hepatic failure induced by severe hepatitis B virus group. Therefore, endoplasmic reticulum stress plays an important and complex role in the pathogenesis of hepatic failure induced by severe hepatitis B virus.

Protective effect of glycogen synthase kinase 3β inhibition via peroxisome proliferator-activated receptor alpha activation in mice with acute liver failure / 中华肝脏病杂志

Objective@#To investigate the role of the glycogen synthase kinase 3β (GSK3β) and the peroxisome proliferator-activated receptor alpha (PPARα) signaling pathway in acute liver failure and related mechanisms in a mouse model of acute liver failure induced by D-galactosamine/lipopolysaccharide (D-GalN/LPS).@*Methods@#C57BL/6 mice were given intraperitoneal injection of D-GalN/LPS to establish a mouse model of acute liver failure. SB216763 was used to inhibit the activity of GSK3β and PPARα siRNA was used to inhibit the expression of PPARα. Western blotting was used to measure the expression of PPARα protein. The changes in liver pathology were observed to evaluate liver injury, and the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured to assess liver function. Quantitative real-time PCR was used to measure the mRNA expression of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-12p40 (IL-12p40), and PPARα. A one-way analysis of variance was used for comparison of means between multiple groups; the least significant difference test was used for data with homogeneity of variance, and the Games-Howell method was used for data with heterogeneity of variance.@*Results@#In the mice with liver failure induced by D-GalN/LPS, GSK3β inhibition promoted the mRNA and protein expression of PPARα (F = 13.18 and 301.36, P = 0.00 and 0.00). In the mice with acute liver failure induced by D-GalN/LPS, GSK3β inhibition alleviated liver bleeding, inflammation, and necrosis and reduced the serum levels of ALT (F = 25.16, P = 0.000) and AST (F = 12.96, P = 0.001), as well as the mRNA expression of TNF-α (F = 32.17, P = 0.00), IL-1β (F = 11.57, P = 0.005), and IL-12p40 (F = 14.17, P = 0.015) in liver tissue. The inhibition of PPARα expression reversed the liver-protecting effect of GSK3β inhibition, which manifested as aggravation in liver bleeding, inflammation, and necrosis, increases in the serum levels of ALT (F = 25.16, P = 0.001) and AST (F = 12.96, P = 0.000), and an increase in the mRNA expression of TNF-α (F = 32.17, P = 0.00), IL-1β (F = 11.57, P = 0.024), and IL-12p40 (F = 14.17, P = 0.001) in liver tissue.@*Conclusion@#In mice with acute liver failure induced by D-GalN/LPS, the GSK3β-PPARα-inflammatory factor signaling pathway may play an important role. GSK3β inhibition has a protective effect in mice with acute liver failure possibly by activating the inhibitory inflammatory factor of PPARα.

Application of enhanced recovery program in laparoscopic distal pancreatectomy / 浙江大学学报·医学版

Objective: To evaluate the feasibility and safety of applying enhanced recovery after surgery (ERAS) protocol in patients undergoing laparoscopic distal pancreatectomy. Methods: Data of 36 patients undergoing laparoscopic distal pancreatectomy from May 2016 to May 2017 in the First Affiliated Hospital, Zhejiang University School of Medicine were reviewed. The patients were divided into ERAS group (n=12) and control group (n=24). The patients in ERAS group received a series of enhanced recovery procedures, including multimodal analgesia, early off-bed activity and early oral food-taking, etc. Operation time, intraoperative blood loss, time to first flatus, postoperative complications, and length of postoperative hospital stay were evaluated. Results: There were no statistically significant differences in operation time and intraoperative blood loss between ERAS group and control group (all P0.05). The time to first flatus and length of postoperative hospital stay were significantly shortened in ERAS group (all PP0.05). Conclusion: The ERAS protocol for laparoscopic distal pancreatectomy can significantly promote gastrointestinal function recovery and shorten postoperative hospital stay, and may reduce the incidence of postoperative complications.

Caspase-1 aggravates the D-galactosamine/lipopolysaccharide induced acute liver failure in mice through activating glycogen synthase kinase-3β / 中华微生物学和免疫学杂志

Objective To analyze the role of cysteinyl aspartate specific proteinase-1 (caspase-1) in a mouse model of D-galactosamine (D-GalN) and lipopolysaccharide (LPS) induced acute liver failure (ALF) and to study the possible mechanism. Methods C57BL/ 6 mice were randomly divided into four groups including control group, Z-WEHD-FMK (caspase-1 inhibitor) treatment group, ALF model group and Z-WEHD-FMK-treated ALF group. The mouse model of ALF was established by intraperitoneally injec-ting the mice with D-GalN (450 mg/ kg) and LPS (10 μg/ kg). The damages in liver tissues were evaluated based on the histopathological examination and the levels of alanine transaminase (ALT) and aspartate trans-aminase (AST) in serum samples. Western blot assay was performed to analyze the expression of caspase-1 and the phosphorylation of glycogen synthase kinase 3β (GSK-3β). The qRT-PCR was used to measure the expression of inflammatory cytokines at transcriptional level. Results The expression of caspase-1 at both mRNA and protein levels were gradually increased during the development of ALF. Compared with the mice with ALF, those in the Z-WEHD-FMK-treated ALF group showed less severe liver damages on histopatholog-ical examination and decreased levels of ALT and AST in serum samples [ALT: (479. 2±39. 5) U/ L vs (998. 5±60. 4 ) U/ L, P<0. 05; AST: ( 478. 5±28. 6) U/ L vs ( 1 180. 7±91. 4) U/ L, P<0. 05]. The expression of TNF-α, IL-1β, IL-18 and IL-33 at transcriptional level were significantly suppressed in mice with ALF upon the Z-WEHD-FMK intervention. Results of the Western blot assay indicated that Z-WEHD-FMK suppressed the activities of GSK-3β by enhancing its phosphorylation. Conclusion This study demon-strated that caspase-1 could promote the activation of GSK-3β resulting in the development of inflammation responses and liver damages during the development of ALF in mice.

Activated peroxisome proliferator-activated receptor αinhibiting the lipopolysaccharide-induced macrophage-mediated inflammatory responses by promoting the autophagy / 中华微生物学和免疫学杂志

Objective To investigate the effects of peroxisome proliferator-activated receptor α( PPARα) on macrophage-mediated inflammatory responses with the interference of lipopolysaccharide and the possible mechanism.Methods The bone marrow stem cells were isolated from the femora of mice.The granulocyte-macrophage colony stimulating factor ( GM-CSF) was used to stimulate the in vitro differentiation from bone marrow stem cells into primary macrophages.An in vitro model with cultured cells expressing in-flammatory cytokines was established by treating the primary macrophages with lipopolysaccharide ( LPS) .A specific chemical agonist, Wy-14643, was used to activate PPARα. Autophagy inhibitors including 3-methyladenine (3-MA) and small interfering RNA against Atg7 ( Atg7 siRNA) were used to inhibit the autophagy.Western blot assay was performed to detect the expression of autophagy-related proteins ( Atg5, Atg7, Beclin-1 and LC3).The transcriptional levels of TNF-α, IL-1β, IL-6, Atg5, Atg7 and Beclin-1 were analyzed by qRT-PCR.Results Compared with the macrophages treated with LPS alone, those pretreated with various concentrations of Wy-14643 (10 μmol/L, 25 μmol/L and 50 μmol/L) showed inhibited ex-pression of proinflammatory cytokines ( TNF-α,IL-1βand IL-6) and enhanced expression of autophagy-relat-ed proteins (Atg5, Atg7 and Beclin-1) at mRNA level in a dose-dependent manner.The expression of auto-phagy-related proteins (Atg5, Atg7, Beclin-1 and LC3) by macrophages was promoted with the pretreatment of Wy-14643 as indicated by Western blot assay.The transcriptional levels of TNF-α, IL-1βand IL-6 were increased in Wy-14643 pretreated-macrophages after stimulation with 3-MA or Atg7 siRNA .Conclusion PPARαsuppressed the macrophage-mediated inflammatory responses by promoting autophagy, suggesting that the PPARα-autophagy pathway might be one of the signaling pathways regulating LPS induced-inflamma-tory responses.

Endoplasmic reticulum stress collaborates with lipopolysaccharide to promote the inflammatory response in macrophages / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the protective mechanism of endoplasmic reticulum stress (ERS) inhibition against inflammation-induced acute liver injury using a mouse model.</p><p><b>METHODS</b>Marrow-derived stem cells were isolated from mouse femur and used to derive macrophages for analysis in experimental inflammation conditions, established by exposure to LPS and consequent activation of TLR4. Tunicamycin, an ERS chemical inducer, was applied to interfere the inflammation model condition.Affect on the inflammation-related factor MAPK was detected by western blot, and affects on gene expression of inflammatory factors were measured by real-time quantitative PCR. Affect on TNFa was also measured by ELISA.</p><p><b>RESULTS</b>Expression of TNFa, IL-6 and IL-1b was induced upon exposure to LPS, with the peak levels being reached at 4 hours of exposure (TNFa, 0.82+/-0.24; IL-1 b, 2.20+/-0.69; IL-6, 0.330+/-0.150). Tunicamycin significantly enhanced the LPS-induced up-regulation of TNFa, IL-6 and IL-1b expression (TNFa, 1.44+/-0.38, t=2.8, P<0.05; IL-1b, 16.063.40, t =7.93, P<0.05; IL-6, 31.1610.60, t=5.08, P<0.05). The tuniamycin treatment also enhanced the LPS-induced up-regulation of the protein expression of phospo-p38, phospho-JNK and phoshpo-ERK.</p><p><b>CONCLUSION</b>ERS collaborates with LPS to promote the TLR4-mediated inflammatory response of macrophages, and this collaboration may be a pathogenic mechanism underlying progressive development of acute liver injury.</p>

Role of endoplasmic reticulum stress in D-GalN/LPS-induced acute liver failure / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the role of endoplasmic reticulum stress (ERS) in acute liver failure (ALF) using a mouse model of D-Galactosamine/lipopolysaccharide (D-GalN/LPS)-induced ALF.</p><p><b>METHODS</b>The ALF model was established by administering intraperitoneal (i.p.) injections of D-Ga1N (700 mg/kg) and LPS (10 mug/kg) to six C57BL/6 mice. Three of the modeled mice were also administered 4-phenylbutyrate (4-PBA; 100 mg/kg i.p.) at 6 hours before the onset of ALF and served as the intervention group. Non-modeled mice served as controls. All mice were analyzed by western blotting and qRT-PCR to determine the expression levels of ERS-related proteins in liver tissue. Liver function was assessed by measuring levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. Extent of injury to the liver tissue was assessed by hematoxylin-eosin staining and histological analysis. qRT-PCR was also used to detect differences in expression of inflammation-related genes, and western blotting was also used to detect differences in expression of the apoptosis related protein Caspase-3.The extent of apoptosis in liver tissue was assessed by TUNEL assay.</p><p><b>RESULTS</b>The ERS markers GRP78 and GRP94 showed increased expression at both the gene and protein levels which followed progression of ALF. The ERS effector proteins XBP-1, ATF-6 and IRE 1 a involved in the unfolded protein response were activated in the early stages of ALF, and the ERS-induced apoptosis regulators Caspase-12 and CHOP were activated in the late stage of ALF. Inhibition of ERS by 4-PBA intervention protected against injury to liver tissue and function, as evidenced by significantly lower levels of serum ALT and AST and a remarkably decreased extent of histological alterations. Furthermore, the inhibition of ERS suppressed expression of the proinflammatory cytokines TNFa, IL-6 and IL-1 β, and reduced the extent of hepatocyte apoptosis.</p><p><b>CONCLUSION</b>ERS is activated in the mouse model of D-GalN/LPS-induced ALF. Inhibition of ERS may be protective against liver injury and the mechanism of action may involve reductions in inflammatory and apoptotic factors and/or signaling. Therefore, inhibiting ERS may represent a novel therapeutic approach for treating ALF.</p>

Construction of gold film solid-phase carrier of myoglobin monoclonal antibody based on thiol self-assembly / 生物工程学报

In order to immobilize myoglobin (Mb) monoclonal antibody on gold film solid-phase carrier, we grew a mixed self-assembled monolayers (SAMs) of acid thiol and mercapto ethanol on gold film. Then we analyzed the property of the sample by atomic force microscopy and X-ray photoelectron spectroscopy. Then, we used 1-(3-dimethyl aminopropyl)-3-ethyl carbodiimide hydrochloride as catalyst to couple SAMs with amino of antibody so that we immobilized antibody on surface of gold film, followed by detecting myoglobin antigen. Results showed that, by optimizing experimental conditions, when we treated gold film by a mixture of mercapto hexadecanoic acid and mercapto undecanol ethanol solution of concentration of 50 mmol/L at temperature of 60 degrees C for 3 hours, and Mb monoclonal antibody of concentration of 40 mg/L for 3 hours, respectively, antibody had high immobilization efficiency and the MbAg was detected to 30 microg/L. The method provided a theoretical and practical basis for using magnetoresistence biosensors to diagnosis myocardial infarction.