The antimicrobial peptide YD attenuates inflammation via miR-155 targeting CASP12 during liver fibrosis

@#The antimicrobial peptide APKGVQGPNG (named YD), a natural peptide originating from Bacillus amyloliquefaciens CBSYD1, exhibited excellent antibacterial and antioxidant properties in vitro. These characteristics are closely related to inflammatory responses which is the central trigger for liver fibrosis. However, the therapeutic effects of YD against hepatic fibrosis and the underlying mechanisms are rarely studied. In this study, we show that YD improved liver function and inhibited the progression of liver fibrosis by measuring the serum transaminase activity and the expression of α-smooth muscle actin and collagen I in carbon tetrachloride-induced mice. Then we found that YD inhibited the level of miR-155, which plays an important role in inflammation and liver fibrosis. Bioinformatics analysis and luciferase reporter assay indicate that Casp12 is a new target of miR-155. We demonstrate that YD significantly decreases the contents of inflammatory cytokines and suppresses the NF-κB signaling pathway. Further studies show that transfection of the miR-155 mimic in RAW264.7 cells partially reversed the YD-mediated CASP12 upregulation, the downregulated levels of inflammatory cytokines, and the inactivation of the NF-κB pathways. Collectively, our study indicates that YD reduces inflammation through the miR-155–Casp12–NF-κB axis during liver fibrosis and provides a promising therapeutic candidate for hepatic fibrosis.

Preparation and drug release effect evaluation of drug-loaded cross-linked decellularized corneal stromal lenticules in vitro / 中华实验眼科杂志

Objective:To prepare a drug release system of drug-loaded cross-linked decellularized corneal stromal lenticules and evaluate its drug release characteristics in vitro. Methods:Lenticules were obtained during femtosecond laser-assisted small incision lenticule extraction (SMILE) surgery in Chongqing Aier Ophthalmology Hospital.Decellularized corneal stromal lenticules were prepared using high concentration sodium chloride (NaCl) combining nuclease.The decellularized corneal stromal lenticules were randomly divided into normal group, 0.5% levofloxacin group, 3% levofloxacin group and 5% levofloxacin group, with 4 lenticules in each group.The lenticules did not receive any treatment in the normal group, and drug-loading those were soaked in different doses of levofloxacin solution for three hours according to grouping.In the crosslinking test, 12 decellularized corneal stromal lenticules were randomly divided into non-crosslinking group, 0.01 mmol 1-(3-dimethylamino) propylimine (EDC) group, 0.05 mmol EDC group and 0.25 mmol EDC group.The lenticules for cross-linking were soaked in different contents of mixed solution of EDC with N-hydroxysuccinyl (NHS) for four hours respectively according to grouping, and then in 3% levofloxacin solution for three hours.Only 3% levofloxacin solution soaking was carried in the non-crosslinking group.High performance liquid chromatography (HPLC) was employed to detect the drug release concentration of the lenticules, and spectral scanning method was performed to measure light transittance of the lenticules.The surface ultrastructure of the decellularized lenticules among different cross-linking groups was examined and compared with scanning electron microscope.The use of the human corneal lenticules was approved by an Ethics Committee of Chongqing Aier Ophthalmology Hospital (No.2019012). Written informed consent was obtained from each patient before surgery.Results:The release concentrations of decellularized corneal stroma lenticules were significantly different at 1 day, 7, 14, and 21 days among 0.5%, 3%, and 5% levofloxacin group ( P<0.05) or also among the 0.01 mmol EDC, 0.05 mmol EDC, and 0.25 mmol EDC cross-linked groups ( P<0.01). The drug release concentrations in 0.05 mmol EDC group were the highest at various time points, and the release time of the three cross-linked groups lasted until 21 days after release concentrations of decellularized corneal stroma lenticules.The drug release concentrations in cross-linked groups and non-crosslinking group were gradually declined with the prolong of drug-loading time, showing a significant difference at different time points ( P<0.05). The transmittance of the lenticules was (88.68±1.19)% and (91.55±1.16)% in the non-crosslinking group and normal group, respectively, with no significant difference ( P>0.05). The average transmittance of the lenticules was significantly reduced in the drug-loaded groups compared with the normal group ( P<0.05). The smaller collagen fiber voids and closely arranged collagen fibers were displayed in the cross-linking groups under the scanning electron microscope with the best effect in the 0.25 mmol EDC group. Conclusions:EDC/NHS cross-linking can improve the drug-loading effect of decellularized corneal stromal lenticules probably by lessening collagen fiber voids.The drug-loaded cross-linked decellularized corneal stromal lenticules have a good drug release effect in vitro.

In Vitro Metabolism of Two Novel Tacrine Derivates ST09 and ST10 / 中国药学杂志

OBJECTIVE: To determin the in vitro metabolic stability of ST09 and ST10 in human liver microsomes (HLMs), and to evaluate their potential inhibitions on five HLM cytochrome P450 isoforms. METHODS: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess remaining concentration of ST09 and ST10 at designed time points during the HLM incubation. Six major metabolites of cytochrome P450 were simultaneously measured with LC-MS/MS, and the inhibitory effects of ST09 and ST10 were respectively evaluated with IC50 values. RESULTS: ST09 was extremely unstable in vitro, and t1/2 was less than 1 min. However, ST10, the major metabolite of ST09, was NADPH-independent metabolized in HLMs, while its t1/2 and microsomal intrinsic clearance (CLint) were 32 min and 0.043 mL·min-1·mg(protein)-1, respectively. IC50 values of ST09 and ST10 on CYP3A4 (midazolam as substrate), CYP3A4 (testosterone as substrate), CYP1A2, CYP2C9, CYP2C19 and CYP2D6 were 0.42/0.25, 1.27/0.81, 24.92/18.21, 36.53/54.34, 67.64/144.90, 6.43/5.30 μmol·L-1, respectively. CONCLUSION: ST09 and ST10 are extensively metabolized in vitro and both compounds had significant inhibition on CYP3A4 and CYP2D6.

Application value of urinary calprotectin in differential diagnosis between prerenal and intrinsic pediatric acute renal injury / 中华实用儿科临床杂志

Objective To investigate the value of urinary calprotectin in differential diagnosis between prerenal and intrinsic pediatric acute renal injury(AKI).Methods A total of 68 cases with AKI were enrolled in this study,and they were divided into prerenal AKI group(25 cases) and intrinsic AKI group(43 cases) according to their tissue perfusion.The general data was collected,and the blood urea nitrogen(BUN),serum creatinie(Scr),BUN/Scr,potassium,fractional excretion of filtrated sodium(FENa),urine osmotic pressure,B-type natriuretic peptide (BNP),urinary calprotectin,neutrophil gelatinase-associated lipocalin (NGAL),kidney injury molecule 1 (Kim-1) were recorded and compared between the 2 groups.Results There were significant differences between prerenal AKI group and intrinsic AKI group in Scr[(439.0 ± 278.0) μmol/L vs.(603.0 ± 286.0) μmol/L,t =2.30,P ＜ 0.05],BUN/Scr (20.58 ±5.62 vs.14.93 ±4.32,t =4.65,P＜0.05),FENa[(1.5 ±0.6)％ vs.(8.1 ±2.6)％,t =12.46,P＜ 0.05],BNP[95.6(54.0,109.4) ng/L vs.512.3(320.1,520.3) ng/L,Z =2.21,P ＜0.05],urinary calprotectin [20.7(4.3,42.4) μg/L vs.402.4(60.1,498.7) μg/L,Z=3.13,P＜0.05] and NGAL[74.9(14.5,365.5) μg/L vs.684.2(56.2,1 502.5) μg/L,Z =2.35,P ＜0.05].Receiver operating characteristic curve analysis showed that urinary calprotectin[area under the curve(AUC) =0.940] and BNP(AUC =0.909) both had higher value in differential diagnosis.When the cut off value of calprotectin was 240.6 μg/L,its specificity was 96.0％ and the sensitivity was 86.0％.When the cut off value of BNP was 120.6 ng/L,its specificity was 92.0％ and the sensitivity was 90.7％.The diagnostic accuracy was low in Scr,but moderate in BUN,Scr,FENa and NGAL.BUN,potassium,urine osmotic pressure and Kim-1 had no diagnostic value.Conclusions Urinary calprotectin may have higher diagnostic value in the differential diagnosis between prerenal and intrinsic pediatric AKT.It can be used in the clinical diagnosis as a reference index.

Analysis of non-bacterial respiratory pathogen infection in children with asthmatic diseases / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the association of non-bacterial respiratory pathogens with asthmatic diseases in children, and the clinical significance of total serum IgE levels and peripheral eosinophil count in infection with non-bacterial respiratory pathogens.</p><p><b>METHODS</b>Indirect immunofluorescence assay was used to detect IgM antibodies against nine types of non-bacterial respiratory pathogens in the sera of 490 children with asthmatic diseases between September 2010 and September 2011. Pathogens were analyzed and total serum IgE levels and peripheral eosinophil count were measured in IgM-positive cases.</p><p><b>RESULTS</b>Of the 490 children with asthmatic diseases, 47.6% (233 cases) were positive with IgM antibodies against non-bacterial respiratory pathogens, the most common being Mycoplasma pneumoniae (MP) (25.3%), followed by adenovirus (ADV) (8.9%) and influenza B virus (Flu B) (8.8%). Thirty-six cases suffered from co-infection of two or more non-bacterial pathogens, mainly comprising MP and other pathogens (94%). There were significant differences in the total detection rate of IgM antibodies among all age groups (0-30 days: 50.0%; 1-6 months: 67.3%; 0.5-1 year: 33.1%; 1-3 years: 57.3%; 3-8.9 years: 61.7%). The positive rate of IgM antibodies against respiratory pathogens was highest in children with bronchial asthma, followed by children with asthmatic bronchitis, and it was lowest in children with bronchiolitis. IgM-positive children had significantly decreased blood eosinophils and significantly increased total serum IgE levels.</p><p><b>CONCLUSIONS</b>The main non-bacterial respiratory pathogens include MP, ADV and Flu B in children with asthmatic diseases, and co-infection of MP and other non-bacterial pathogens is common. Infants aged 1 to 6 months have a higher infection rate than other age groups. Monitoring the changes in total serum IgE levels and peripheral eosinophil count has great significance for the clinical diagnosis and treatment of asthmatic diseases in children.</p>

PPM1D silencing by lentiviral-mediated RNA interference inhibits proliferation and invasion of human glioma cells / 华中科技大学学报（医学）（英德文版）

To construct a lentiviral shRNA vector targeting human protein phosphatase 1D magnesium-dependent (PPM1D) gene and detect its effectiveness of gene silencing in human gliomas, specific siRNA targets with short hairpin frame were designed and synthesized. DNA oligo was cloned into the pFU-GW-iRNA lentiviral expression vector, and then PCR and sequencing analyses were conducted to verify the constructs. After the verified plasmids were transfected into 293T cells, the lentivirus was produced and the titer of virus was determined. Real-time quantitative PCR and Western blot were performed to detect the PPM1D expression level in the infected glioma cells. PCR and Western blot analyses revealed the optimal interfering target, and the virus with a titer of 6×10(8) TU/mL was successfully packaged. The PPM1D expression in human glioma cells was knocked down at both mRNA and protein levels by virus infection. The expression of PPM1D mRNA and protein was decreased by 76.3% and 87.0% respectively as compared with control group. The multiple functions of human glioma cells after PPM1D RNA interference were detected by flow cytometry and cell counting kit-8 (CCK-8). Efficient down-regulation of PPM1D resulted in significantly increased cell apoptosis and reduced cell proliferation and invasion potential in U87-MG cells. We have successfully constructed the lentiviral shRNA expression vector capable of stable PPM1D gene silencing at both mRNA and protein levels in glioma cells. And our data gave evidence that the reduced cell growth observed after PPM1D silencing in glioma cells was at least partly due to increased apoptotic cell death.

Expression of PirB in normal and injured spinal cord of rats / 华中科技大学学报（医学）（英德文版）

The expression of paired immunoglobulin-like receptor B (PirB) in normal and injured spinal cord of rats was investigated. The SD rat hemi-sectioned spinal cord injury (SCI) model was established. Before and 1, 3, 7, 10 days after SCI, the spinal cord tissues were harvested, and Western blot and immunohistochemistry were used to examine the expression and location of PirB. The results showed that the expression level of PirB in the normal spinal cord of SD rats was low. At the first day after SCI, the expression of PirB was obviously increased, and that in the injured spinal cord from the first day to the 10th day was significantly higher than in the normal spinal cord. The positive expression of PirB in neurons from different regions of gray matter of the injured spinal cord was seen. It was concluded that the expression of PirB in the normal spinal cord of rats was low. The expression of PirB in SCI was significantly increased till at least the 10th day.

The application of proton magnetic resonance spectroscopy and computerized tomography in evaluating nonalcoholic fatty liver disease / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To investigate the application of proton magnetic resonance spectroscopy (1HMRS) and computerized tomography (CT) in evaluating nonalcoholic fatty liver disease (NAFLD).</p><p><b>METHODS</b>Twenty-two NAFLD patients were selected, according to the Chinese Medical Association standard, and compared with 20 healthy persons (as the control group). Their body mass index (BMI), waist to hip ratio (WHR), and blood pressure (BP) were examined. The serum ALT, the concentration of fasting blood glucose (FBG), total cholesterol (TC), triglyceride (TG), and uric acid (UA) were tested simultaneously. The severity of hepatosteatosis was evaluated by 1HMRS and CT scans of their livers. The intrahepatic content of lipid (IHCL) and CT liver and spleen ratios were measured.</p><p><b>RESULTS</b>The BMI, WHR, serum ALT, FBG, TG, and UA were all elevated significantly in the NAFLD group and were (28.4+/-2.4) kg/m2, 0.91+/-0.04, (71.5+/-24.8) U/L, (5.67+/-0.61) mmol/L, (2.48+/-1.46) mmol/L, (420.7+/-57.5)micromol/L, respectively, P less than 0.01 or 0.05. Meanwhile, in the NAFLD group, the IHCL calculated by 1HMRS were increased and CT value ratios were decreased significantly compared with those of the control group (27.49%+/-12.27% vs 1.34%+/-0.79%, P less than 0.01). However, there was no correlation between the clinical features and the IHCL and between the clinical features and CT value ratios, but a negative correlation existed in the CT value ratio and IHCL.</p><p><b>CONCLUSIONS</b>The intrahepatic content of lipids can be measured precisely by 1HMRS, and 1HMRS is better than CT in quantitative evaluations of NAFLD.</p>