Objective:To establish a rapid and accurate method for the detection of Klebsiella pneumoniae carbapenemase (KPC) carbapenemase gene based on recombinase aided amplification (RAA)-CRISPR-Cas13a (CRISPR-Cas13a) technology. Methods:Twenty-five clinical isolates of carbapenem-resistant Klebsiella pneumoniae (CRKP) and five carbapenem-sensitive Klebsiella pneumoniae (CSKP) strains preserved in 2020-2021 in Beijing Chuiyangliu Hospital were randomly collected, and the total DNA samples of the strains was extracted. RAA primers specific for KPC DNA and CRISPR RNA (crRNA) were designed to establish a rapid and accurate method for the detection of KPC carbapenemase gene based on RAA-CRISPR-Cas13a technology. The method was evaluated by plasmids and clinical sample strains, and the detection was also performed by Quantitative real-time PCR (qPCR) method to compare the detection rate and consistency of the two methods. Results:The RAA-CRISPR-Cas13a method can detect KPC plasmids and samples with a sensitivity of 1 copy/μl, which is higher than that of qPCR (10 1 copies/μl). Among the 30 clinical strains (including 25 CRKP strains and 5 CSKP strains), 23 strains were detected to carry KPC gene by both RAA-CRISPR-Cas13a method and qPCR method, and 7 strains were not detected with KPC gene. The detection rate of KPC gene in the 25 CRKP strains was 92% (23/25). The positive coincidence rate of the two methods was 100% (23/23). Conclusions:This study combined RAA amplification technology with CRISPR-Cas13a technology to establish a rapid and accurate method for detecting KPC carbapenemase gene. The method is useful for accurate screening of KPC carbapenemase-producing strains. It has a wide application prospect in drug resistance monitoring and infection control.

Objective:To evaluate the efficacy and safety of intravitreal ranibizumab combined with laser (IVR+ Laser) and the intravitreal ranibizumab (IVR) monotherapy for the treatment of diabetic macular edema (DME).Methods:A meta-analysis was conducted on randomized controlled trial (RCT) literature related to IVR+ Laser therapy and IVR alone for DME.Databases including Cochrane Library, PubMed, EMbase, Web of Science, SinoMed, CNKI, VIP and WanFang Data were searched from their inception to April 2022.Literature screening, data extraction, quality evaluation and cross-checking were conducted independently by two researchers according to inclusion and exclusion criteria.Then a meta-analysis was conducted using RevMan 5.4.1 software.The two therapies were compared in terms of best corrected visual acuity (BCVA), central macular thickness (CMT), mean number of injections and adverse events.Results:Twelve RCTs involving 1 695 eyes were included in the study.Meta-analysis showed that at the end of follow-up, IVR+ Laser demonstrated better improvement in BCVA and CMT than IVR alone, and there were significant differences in the changes in BCVA and CMT between the two groups (weighted mean difference[WMD]=-0.66, 95% confidence interval[ CI]: -1.11--0.21, P<0.01; WMD=-5.05, 95% CI: -9.21--0.89, P=0.02).IVR+ Laser required significantly fewer injections than IVR alone (WMD=-1.16, 95% CI: -2.07--0.25, P=0.01).There were no significant differences in the adverse events incidence between the two therapies (all at P>0.05). Conclusions:The safety of IVR+ Laser is comparable to IVR alone, and it requires fewer injections for the treatment of DME.

Objective:To investigate the expression and role of asparte-specific cysteine protease (Caspase)-1, inflammasomes key molecule, in hepatitis B virus (HBV)-related diseases.Methods:HBV-related liver disease patients' serum (438 cases) and liver tissue (82 cases) samples were collected from Beijing You'an Hospital affiliated with Capital Medical University. The mRNA expression level of caspase-1 in liver tissue was detected by real-time fluorescence quantitative PCR (qRT-PCR). The protein expression level of Caspase-1 in liver tissue was detected by the immunofluorescence method. The activity of Caspase-1 was detected using the Caspase-1 colorimetric assay kit. The level of Caspase-1 in the serum was detected by an ELISA kit.Results:The results of qRT-PCR showed that the mRNA level of Caspase-1 was downregulated in patients with chronic hepatitis B (CHB), cirrhosis (LC), and hepatocellular carcinoma (HCC), while up-regulated in patients with acute-on-chronic liver failure (ACLF) ( P＜0.01) compared with normal subjects. Immunofluorescence assays showed that Caspase-1 protein levels were elevated in ACLF patients, decreased in HCC and LC patients, and slightly elevated in CHB patients. The activity of Caspase-1 was slightly higher in liver tissue from CHB, LC, and HCC patients than in the normal control group, and there was no statistically significant difference between the groups. Additionally, compared with the control group, Caspase-1 activity was significantly reduced in the ACLF group ( P＜0.01). Serum Caspase-1 levels were significantly lower in patients with CHB, ACLF, LC, and HCC than in normal subjects, and serum Caspase-1 levels were lowest in patients with ACLF ( P＜0.001). Conclusion:Caspase-1, a key molecule of inflammasomes, plays an important role in HBV-related diseases and has significant differences, showing distinct features for ACLF than other HBV-related diseases.

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.

Objective:To investigate the mechanism of action of peroxisome proliferator-activated receptor α (PPARα)-mediated CCAAT/enhancer binding protein homologous protein (CHOP) signaling molecule with response to inflammation in mice with acute liver failure.Methods:C57BL/6 mice were used as the research subjects, and D-galactose (D-GalN) combined with lipopolysaccharide (LPS) was injected intraperitoneally to establish a mouse model of acute liver failure. PPARα was activated by Wy-14643. CHOP expression was promoted by plasmids. Liver pathological changes and serum transaminases (ALT and AST) were detected in mice to evaluate liver function. The mRNA expression level of inflammatory factors in liver tissue was detected by real-time fluorescence quantitative PCR. LPS-stimulated macrophage was used to establish an inflammation model. PPARα and CHOP expression was inhibited by siRNA. The mRNA expression level of inflammatory factors in the cells was detected by real-time fluorescence quantitative PCR.Results:Promoted PPARα activation had inhibited liver hemorrhage and inflammation in mice with acute liver failure induced by D-GalN/LPS. In addition, the serum level of transaminases and genetic level of inflammatory factors in liver tissues were reduced ( P < 0.01). CHOP accelerated expression had reversed the hepatoprotective effect of PPARα activation, aggravated liver injury, and increased inflammatory factors expression ( P < 0.01). At the cellular level, the inhibition of PPARα activation had accelerated the increase of inflammatory factors ( P < 0.01), while the inhibition of CHOP activation had all over again decreased the inflammatory factors ( P < 0.01). Conclusion:PPARα and CHOP are important signaling molecules to regulate the inflammatory response in acute liver failure and liver injury. PPARα acceleration can down-regulate CHOP to inhibit inflammatory factors, which might play a protective role in mice with acute liver failure.

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.

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.

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.

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.

OBJECTIVETo investigate the protective mechanism of endoplasmic reticulum stress (ERS) inhibition against inflammation-induced acute liver injury using a mouse model.

METHODSMarrow-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.

RESULTSExpression 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.

CONCLUSIONERS 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.

Animals ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; Inflammation ; Interleukin-6 ; Lipopolysaccharides ; Liver ; Macrophages ; Mice ; Toll-Like Receptor 4 ; Up-Regulation