PURPOSE AND METHODS In order to investigate the effects of Zhaolian on anti tumor promotion,we studied its influence on liver precancerous lesion in rats induced by DEN.RESULTS (1)Liver function of rats was protected ( P

OBJECTIVE: To establish a model of alcoholic liver fibrosis (ALF) in rats induced by complex factors. METHODS: Forty-seven healthy male rats were divided into three groups: normal control group (n=12), minor CCl4 group (n=12) and complex factors group (n=27). The rats in the complex factors group were fed a complex diet including alcohol, corn oil and pyrazole, and administered with intraperitoneal injection of minor CCl4 to induce ALF. During induction process, the histopathological changes of liver tissue and the values of liver-to-body weight ratio were both observed regularly. The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyltransferase (gamma-GT) in these three groups were all examined at the 12th week of the induction process. RESULTS: At the 12th week of the induction process, the model of ALF induced by complex factors was successfully established in rats, and the histopathological presentations showed alcoholic fatty liver, hepatitis and liver fibrosis in a sequence along with the induction process. The value of liver-to-body weight ratio and the serum levels of ALT, AST and gamma-GT of rats in the complex factors group were all significantly different from those in the other two groups. CONCLUSION: It is a steady and effective way to induce ALF in rats with complex diet and minor CCI4 injection.

BACKGROUND:Hydroxyapatite (HA) is a good scaffold material, and recombinant human bone morphogenetic protein-2 (rhBMP-2) possesses a strong osteogenic ability, therefore, by which preparing a novel composite material wil be helpful for bone repair. OBJECTIVE:To explore the effects of the hol ow HA/rhBMP-2 microspheres on the osteogenesis and biomechanics of rabbit bone defects. METHODS:Forty-eight male healthy adult New Zealand white rabbits were randomly divided into three groups (n=16 per group), including composite, single and control groups. Radical defect models were prepared, and the hol ow HA/rhBMP-2 and hol ow HA scaffolds were implanted into the composite and single groups, respectively. The control group received no treatment. At the 1st day of 4, 8, 12, and 16 weeks after implantation, the level of serum alkaline phosphatase was detected, and the bone healing was assessed through X-ray, three-dimensional CT, radionuclide bone scan and biomechanics testing, respectively. RESULTS AND CONCLUSION:The level of serum alkaline phosphatase, X-ray scale scores, osteogetic effect, region of interest volume, three-dimensional CT and biomechanical strength in the composite group were superior to those in the single group. In the meanwhile, the bone healing was unsatisfactory in the control group. Our findings indicate that the hol ow HA/rhBMP-2 artificial bone exhibits a good osteogenic ability and mechanical strength, contributing to bone healing.

The recombinant chimeric enzyme of AnsB-TTP-P277 comprising L-asparaginase, a tetanus toxin peptide spacer and P277 was expressed as a soluble protein in Escherichia coli. The purified chimeric enzyme exhibited primary activity of the native asparaginase. Prediabetic NOD mice immunized with the chimeric enzyme could induce specific antibodies against P277 and the specificity of anti-P277 antibodies was verified by Western blot assay. The study showed that displaying the P277 epitope on the surface of asparaginase could effectively overcome the weak antigenicity of the P277 epitope and evoke a strong P277-specific immune response in mice. Moreover, the concentration of blood glucose was measured by an automated analyzer.Histochemical analysis of mice pancreas tissues showed that the administration of the chimeric enzyme to NOD mice could prevent the development of diabetes more efficiently than the peptide P277 itself.

Objective:To study the the mechanism of action of Huanglong Mixture in the treatment of cough variant asthma (CVA) in children based on the IL-4/signal transduction and activator of transcription 6 (STAT6) signaling pathway using network pharmacology methods, molecular docking techniques, and in vitro cell experiments.Methods:The components and targets of various TCM components in Huanglong Mixture were searched in TCMSP database, HERB database and literature, and the disease targets of CVA were found in Gene Cards database, OMIM database, DrugBank database and PharmGkb database. The STRING database was used to construct the protein-protein interaction network, and Cytoscape 3.9.1 was used for topology analysis to screen out the core targets. The disease-drug-component-target network was constructed to screen out the core components. The KEGG enrichment analysis and GO enrichment analysis of the intersection targets were performed using Metascape software. PDB protein database, PubChem, Autodock and R language were used for molecular docking verification of core targets and core drug components. Finally, rat primary airway smooth muscle cells were cultured, modeled with interleukin-4 (IL-4), and p-STAT6 expression in the cytoplasm and nucleus was detected by Western blot.Results:A total of 122 effective components were obtained, including quercetin, kaempferol, luteolin and so on. The core targets included JUN, ESR1, TP53, MYC, HIF1, etc. GO enrichment analysis involved biological processes such as response to external stimuli, response to oxygen levels, positive regulation of protein phosphorylation, and regulation of cellular stress response. KEGG enrichment analysis showed that the main pathways of Huanglong Mixture in treating CVA included advanced glycation end product-glycation end product receptor (AGE-RAGE) signaling pathway, phosphatidylinositol-3-kinase-protein kinase B (PI3K-Akt) signaling pathway, tumor necrosis factor (TNF) signaling pathway, Janus kinase/signal transduction activation factor (JAK-STAT) signaling pathway. Molecular docking found that the core targets and core drug components had good combination. Cell experiments also confirmed that Huanglong Mixture could inhibit p-STAT6 entering the nucleus.Conclusions:The effective components and targets of Huanglong Mixture in the treatment of CVA are successfully predicted. The mechanism of Huanglong Mixture in the treatment of children with CVA may be related to the inhibition of IL-4/STAT6 signaling pathway.

Objective:To investigate the clinical efficacy of percutaneous transhepatic cholangioscopic lithotripsy with one-step biliary fistulation (PTCSL-OBF) for the treatment of hepatolithiasis.Methods:The retrospective and descriptive study was conducted. The clinicopathological data of 59 patients with hepatolithiasis who were admitted to the First Affiliated Hospital of Nanjing Medical University from March 2017 to December 2019 were collected. There were 16 males and 43 females, aged from 16 to 84 years, with a median age of 60 years. All patients underwent PTCSL-OBF. Observation indicators: (1) surgical and postoperative situations; (2) follow-up; (3) introduction of special cases. Follow-up using outpatient examination and telephone interview was performed to detect the hepotolithiasis-related symptoms, recurrence of hepatolithiasis , and survival of patients up to December 2019. Measurement data with skewed distribution were represented as M (range). Count data were represented as absolute numbers or percentages. Results:(1) Surgical and postoperative situations: 59 patients underwent PTCSL-OBF successfully. Of the 59 patients, 48 cases had 1 biliary sinus tract, 8 cases had 2 biliary sinus tracts, 2 cases had 3 biliary sinus tracts and 1 case had 4 biliary sinus tracts. A total of 106 times of cholangioscopic lithotripsy procedure were performed in the 59 patients, including 20 cases with once of cholangioscopic lithotripsy procedure, 31 cases with twice of cholangioscopic lithotripsy procedure and 8 cases with 3 times of cholangioscopic lithotripsy procedure. Among the 59 patients, 14 cases were diagnosed with residual stones after operation, 45 cases had stones removed completely with the stones removal rate of 76.3%(45/59). The duration of hospital stay of the 59 patients were 24 days(range, 3-88 days). Of the 59 patients, 11 cases had 14 times of postoperative complications. The total incidence of complications was 18.6%(11/59). Among the 14 times of complications, there were 4 times of pleural effusion combined with infection, 2 times of liver abscess, 2 times of ascites, 2 times of intraoperative hypotension septic shock, 2 times of bleeding, 1 time of colon perforation and 1 time of bile leakage. Patients with complications were cured with symptomatic support treatment including puncture drainage, anti-infection and hemostasis. (2) Follow-up: 59 patients were followed up for 1-34 months, with a median follow-up time of 18 months. Of the 59 patients, 9 had recurrence of hepatolithiasis, with a recurrence rate of 15.3%(9/59); 17 had hepatolithiasis related symptoms, with an incidence of 28.8%(17/59). Of the 45 patients who had stones removed completely, 6 had recurrence of hepatolithiasis, with a recurrence rate of 13.3%(6/45); 14 had hepatolithiasis related symptoms, with an incidence rate of 31.1%(14/45). Of the 14 patients who had residual stones postoperatively, 3 had recurrence of hepatolithiasis, with an incidence rate of 21.4%(3/14); 3 had hepatolithiasis related symptoms, with an incidence rate of 21.4%(3/14). During the follow-up, 7 of 59 patients died, 2 of whom died of biliary neoplasms and 5 died of decompensated cirrhosis. (3) Introduction of special cases: among the 5 patients with biliary cirrhosis, 3 cases had stones removed completely, but the original symptoms of the 2 cases were not improved postoperatively and cholangitis occurred repeatedly. The other 2 cases underwent only once of cholangioscopic lithotripsy procedure because of severe cirrhosis combine with massive ascites.Conclusion:PTCSL-OBF can be used in the treatment of hepatolithiasis with appropriate indications, careful operation and strengthened perioperative management to reduce complications.

Objective:To investigate the clinical characteristics of elderly patients with coronavirus disease 2019(COVID-19), in order to provide scientific evidence for the diagnosis and treatment of COVID-19 in elderly patients.Methods:Clinical data of 102 patients with COVID-19 admitted to the B11 East Ward of the Zhongfaxincheng campus and the E1-3 ward of the Guanggu Campus of Tongji Hospital affiliated to Huazhong University of Science and Technology in Wuhan from 1 February 2020 to 28 February 2020 were retrospectively collected and analyzed.Patients were categorized into 2 groups: the elderly group(≥60 years old)and the young and middle-aged group(<60 years old). Differences in epidemiological features, demographics, clinical symptoms, laboratory results and imaging findings between the two groups were retrospectively analyzed.Results:Among 102 patients with COVID-19, 58 were in the elderly group(≥60 years old), with a median age of 67.0(63.8, 71.0)years old, and 44 in the young and middle-aged group(<60 years old), with a median age of 47.5(38.0, 51.8)years old.There was no significant difference in gender ratio between the two groups( χ2=0.033, P=0.855). Of 102 patients, 42.0%(21/50)had close contact with an infected person, 14.0%(7/50)were from infection clusters, and 18.0%(9/50)had suspected hospital-acquired infections.Fever and cough remained the most common symptoms, but gastrointestinal symptoms such as nausea, poor appetite, diarrhea and muscle cramps were also warning signs.Fatigue and cough were the most common presenting symptoms in elderly male patients.Bilateral patchy infiltrates(57.9%, 22/38)and ground-glass opacities(42.1%, 16/38)were the main imaging features and 42.1%(16/38)patients had multiple areas of the lungs involved.Over 50% patients had increased levels of blood glucose, D-dimer, fibrinogen, C-reactive protein, procalcitonin, multiple cytokines and neutrophil-to-lymphocyte ratio, as well as decreased levels of albumin, hemoglobin, hematocrit, lymphocytes and serum calcium.Compared with the young and middle-aged group, the elderly group had higher rates of abnormality in levels of D-dimer and serum calcium( χ2=7.067 and 4.166, P=0.008 and 0.041). Conclusions:Fever and cough are the most common symptoms in elderly patients with COVID-19.Elderly patients with COVID-19 have multiple abnormalities in clinical laboratory test results, which show a certain level of specificity compared with young and middle-aged patients.

Objective:To identify a two-component signaling system (TCSS) composed of β-lactam antibiotic resistance-associated intracellular CiaR and transmembrane serine/threonine kinase StkP of Streptococcus pneumoniae ( S. pneumoniae). Methods:The intracellular segment of stkP gene (IC- stkP) was amplified by PCR and the PCR product was sequenced after T-A cloning. A prokaryotic expression system for IC- stkP segment was established. SDS-PAGE was used to detect the expression of the target recombinant proteins (rIC-StkP and rCiaR) by the established prokaryotic expression system and a previously established prokaryotic expression system for ciaR gene. Ni-NTA affinity chromatography was used to purify the recombinant proteins. The rIC-StkP-captured target proteins of S. pneumoniae were identified by LC-MS/MS after Co-IP. The ability of rCiaR to bind to rIC-StkP was detected by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). Results:The established prokaryotic expression system for IC- stkP segment could effectively express rIC-StkP. Both rIC-StkP and rCiaR after purification showed a single protein band on SDS-PAGE. CiaR could be specifically co-precipitated with rIC-StkP. Three extracted cleaved peptides were found in CiaR molecule with exactly matched sequences. SPR and ITC showed that rCiaR could strongly bind to rIC-StkP with high affinity and the KD values were 1.526×10 -9 mol/L and 1.980×10 -6 mol/L, respectively. Conclusions:S. pneumoniae CiaR could act as the downstream response regulatory protein of StkP kinase to compose StkP/CiaR TCSS.

BACKGROUND:Nano-zirconium dioxide has good application potential in the field of bone tissue repair.Studying the effect of nano-zirconium dioxide on osteogenic differentiation will help to promote the clinical application of nano-zirconium dioxide in the treatment of bone defects. OBJECTIVE:To explore the effect of nano-zirconium dioxide on the osteogenic differentiation of ectomesenchymal stem cells in the nasal mucosa. METHODS:Ectomesenchymal stem cells derived from rat nasal mucosa were isolated and cultured,and the biotoxicity of nano-zirconium dioxide to the cells was detected by CCK-8 assay.The biosafety concentration was selected according to the cytotoxicity,and the cells were randomly divided into a control group,a nano-zirconium dioxide group,and a nano-hydroxyapatite group.Osteogenic differentiation of cells was directionally induced in each group.On day 7 of induced differentiation,alkaline phosphatase staining was performed.qRT-PCR and western blot assay were used to detect the expression of early osteogenic markers(Runx2 and Osx).On day 21 of induced differentiation,alizarin red staining was conducted.qRT-PCR and western blot assay were utilized to determine the expression levels of late osteogenic markers(OPN and OCN). RESULTS AND CONCLUSION:(1)The median lethal concentration of nano-zirconium dioxide on ectomesenchymal stem cells in nasal mucosa was 0.6 mg/mL.In the experiment,the mass concentration of 200 μg/mL was selected for intervention.Zirconium dioxide had no significant effect on the proliferation of the cells.(2)Compared with the control group,the alkaline phosphatase staining of the cells in the nano-zirconium dioxide group was more obvious and the level of cell mineralization was higher,but there was no significant difference compared with the nano-hydroxyapatite.(3)Compared with the control group,the expression of bone-related genes and proteins increased significantly,but there was no significant difference compared with nano-hydroxyapatite.(4)The results show that nano-zirconium dioxide has good biological safety and can promote the osteogenic differentiation of ectomesenchymal stem cells in the nasal mucosa.This promoting effect is equivalent to that of nano-hydroxyapatite.

Objective:To study the clinical application of a new classification on location of hepatolithiasis in guiding treatment using percutaneous transhepatic choledochoscopic lithotomy (PTCSL).Methods:The clinical data of 85 consecutive patients with preoperatively diagnosed hepatolithiasis who underwent PTCSL at the First Affiliated Hospital of Nanjing Medical University from January 2017 to July 2021 were prospectively collected. There were 27 males and 58 females, aged from 15 to 86(62±14) years. Hepatolithiasis was classified into five types of stone location based on preoperative imagings: type Ⅰ ( n=12) , stones located in central bile duct, including hilar bile duct and common hepatic duct; type Ⅱ ( n=17) in unilateral hepatic duct with multiple branches; type Ⅲ ( n=24) in unilateral hepatic duct with multiple branches plus central bile duct; type Ⅳ ( n=31) in bilateral hepatic ducts with multiple branches; and type Ⅴ ( n=1) in unilateral hepatic duct with a single branch. Fistulation path, number of procedures, number of bile duct fistula, and complications were recorded. The residual stone rate and stone recurrence rate were compared among the five types. The follow-up was performed to analyse prognosis. Results:A total of 99 biliary fistulae were performed, with one single tract created in 74 patients, two tracts in 9 patients, three tracts in 1 patient, and four tracts in 1 patient. The fistulation path was B2 in 12 patients, B3 in 18 patients, B4 in 1 patient, B5 in 4 patients, B6 in 10 patients, B7 in 4 patients, and B8 in 50 patients. Altogether, 151 choledochoscopic lithotomy procedures were performed (1-3 times per patient, mean 1.78 times). For the 9 patients with residual stones (10.6%, 9/85), there were 3 patients with type Ⅱ and 6 patients with type Ⅳ. There were significant differences in the residual stone rates among the 5 types (χ 2=11.13, P=0.025). Stone recurrence developed in 33 (38.8%) patients, including 2 patients with type Ⅰ, 7 patients with type Ⅱ, 10 patients with type Ⅲ and 14 patients with type Ⅳ (χ 2=9.07, P=0.046). The total intraoperative and postoperative complications rates was 28.2% (24/85). The follow-up period was 4-58 months with the median follow-up time of 30 months. Twelve patients died during the follow-up period, including 1 patient who died from postoperative bleeding, 3 cholangiocarcinoma, 7 biliary cirrhosis-related liver failure, and 1 stone-unrelated disease. Conclusion:Type Ⅳ in the location classification of hepatolithiasis based on PTCSL had significantly higher rates of residual stones and stone recurrence. This new classification is helpful for clinicaians to determine the optimal path using a smaller number of fistulation tracts to clear stones. It improved the efficacy of PTCSL in treating hepatolithiasis.