ObjectiveTo observe the therapeutic effect of Qiwei Baizhusan（QWBZS） on diabetic encephalopathy（DE） rat model， and to explore the possible mechanism of QWBZS in the treatment of DE based on phosphatidylinositol 3-kinase（PI3K）/protein kinase B（Akt）/glycogen synthase kinase-3β（GSK-3β） signaling pathway. MethodForty-eight SPF male Wistar rats were randomly divided into blank group（8 rats） and high-fat diet group（40 rats）. After 12 weeks of feeding， rats in the high-fat diet group were intraperitoneally injected with 35 mg·kg^-1 of 1% streptozotocin（STZ） for 2 consecutive days to construct a DE model， and rats in the blank group were injected with the same amount of sodium citrate buffer. After successful modeling， according to blood glucose and body weight， model rats were randomly divided into model group， low， medium and high dose groups of QWBZS（3.15， 6.3， 12.6 g·kg^-1）， combined western medicine group（metformin+rosiglitazone， 0.21 g·kg^-1）， with 6 rats in each group. The administration group was given the corresponding dose of drug by gavage， and the blank group and the model group were given an equal volume of 0.9% sodium chloride solution by gavage， 1 time/day for 6 weeks. Morris water maze was used to detect the spatial memory ability of DE rats. Fasting insulin （FINS） level was detected by enzyme-linked immunosorbent assay（ELISA） and insulin resistance index（HOMA-IR） was calculated. Hematoxylin-eosin（HE） staining was used to observe the morphological changes of hippocampus in rats， ELISA was used to detect the indexes of oxidative stress in hippocampal tissues， real-time fluorescence quantitative polymerase chain reaction（Real-time PCR） was used to detect mRNA expression levels of PI3K， Akt， nuclear transcription factor-κB（NF-κB）， tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β） in hippocampus， and Western blot was used to detect the protein expression of PI3K， Akt， phosphorylated（p）-Akt， GSK-3β and p-GSK-3β in hippocampus of rats. ResultCompared with the blank group， FINS and HOMA-IR values of the model group were significantly increased（P<0.01）， the path of finding the original position of the platform was significantly increased， and the escape latency was significantly prolonged（P<0.01）， the morphology of neuronal cells in hippocampal tissues was disrupted， the levels of reactive oxygen species（ROS） and malondialdehyde（MDA） in hippocampus of rats were increased， and the activity of superoxide dismutase（SOD） was decreased（P<0.05， P<0.01）， mRNA expression levels of PI3K and Akt were decreased（P<0.01）， mRNA expression levels of NF-κB， TNF-α and IL-1β were increased（P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly decreased， and the protein expression of GSK-3β was significantly increased（P<0.01）. Compared with the model group， the FINS and HOMA-IR values of the medium dose group of QWBZS and the combined western medicine group were significantly decreased（P<0.01）， the path of finding the original position of the platform and the escape latency were significantly shortened（P<0.01）， the hippocampal tissue structure of rats was gradually recovered， and the morphological damage of nerve cells was significantly improved， the contents of ROS and MDA in hippocampus of rats decreased and the level of SOD increased（P<0.01）， the mRNA expression levels of PI3K and Akt were increased（P<0.01）， and the mRNA expression levels of NF-κB， TNF-α and IL-1β were decreased （P<0.05， P<0.01）， the protein expression levels of PI3K， p-Akt and p-GSK-3β were significantly increased（P<0.01）， and the expression of GSK-3β was significantly decreased（P<0.01）. ConclusionQWBZS can alleviate insulin resistance in DE rats， it may repair hippocampal neuronal damage and improve learning and cognitive ability of DE rats by activating PI3K/Akt/GSK-3β signaling pathway.

Hepatitis C virus infection is a global public health issue， and the emergence of direct-acting antiviral agents has brought revolutionary breakthroughs in the treatment of hepatitis C patients. Although direct-acting antiviral agents have a marked therapeutic effect in adult patients， there are still many challenges in the treatment of special populations such as pregnant women， infants， young children， and adolescents. This article reviews the current status of antiviral therapy for these special populations with hepatitis C and the problems that need to be solved， in order to provide reference and guidance for clinical workers.

OBJECTIVE To develop a high-throughput screening assay for the discovery of Omicron variant main protease(OM-Mpro) inhibitors based on the principle of fluorescence resonance energy transfer(FRET). METHODS The recombinant OM-Mpro enzyme was expressed in Escherichia coli Rosetta(DE3) cells, and further purified by a HisTrapTM chelating column. Subsequently, the enzymatic activity of OM-Mpro and wild type main protease(WT-Mpro) enzymes and inhibition of nirmatrelvir against both proteases were measured using FERT assay. With the FRET assay, OM-Mpro inhibitors were identified via high-throughput screening of an approved drug library. RESULTS The active OM-Mpro enzyme was successfully prepared from E. coli cells. OM-Mpro and WT-Mpro enzymes possessed the same enzymatic activity, and OM-Mpro remained susceptible to nirmatrelvir in vitro. Through high-throughput screening of the marketed drug library, it was found that cetylpyridinium chloride(CPC) is a mixed-type OM-Mpro inhibitor in vitro with an IC50 value of 8.76 μmol·L−1. CONCLUSION A robust FRET assay has been successfully developed based on the production of active OM-Mpro enzyme for screening of its inhibitors, and CPC is identified as a potential lead compound against OM-Mpro in vitro. This study provides a promising avenue for rapid discovery of broad-spectrum antivirals against coronavirus protease.

Objective: The current investigation aimed to determine the appropriate dosage form by comparing solid dispersion and liposome to achieve the purpose of improving the solubility and bioavailability of linarin. Methods: Linarin solid dispersion (LSD) and linarin liposome (LL) were developed via the solvent method and the thin film hydration method respectively. The Transwell chamber model of Caco-2 cells was established to evaluate the absorption of drug. The pharmacokinetics of linarin, LSD and LL in rats after ig administration were carried out by high performance liquid chromatography (HPLC) method. Results: The solubility of LSD and LL was severally 3.29 times and 3.09 times than that of linarin. The permeation coefficients of LSD and LL were greater than 10

Objective:To analyze the clinical characteristics and prognosis of pregnant women with hemorrhagic fever with renal syndrome (HFRS).Methods:A total of 11 pregnant women with HFRS admitted to The Second Affiliated Hospital of Xi′an Jiaotong University (four cases), The Second Affiliated Hospital of Air Force Medical University (four cases), The First Affiliated Hospital of Xi′an Jiaotong University (one case) and Central Hospital of Xianyang City (two cases) between November 2009 and February 2019 were included as the study group, and 24 age-matched non-pregnant women with HFRS were selected as the control group. The age, complications, clinical classification and laboratory indexes of the two groups were analyzed retrospectively, and the clinical outcomes of pregnant women and their fetuses in the study group were followed up. The data between two groups were compared using Mann-Whitney U test or chi-square test. Results:Patients in the study and control groups were 29 (22, 33) and 32 (24, 37) years old, respectively. Seven of 11 patients in study group were severe and critical cases, which was significantly higher than that in the control group (16.7%(4/24), χ2=7.722, P=0.015). In the study group, 10 patients had hypervolemic syndrome, 10 patients had pulmonary edema and six patients had overlapping hypotension shock phase and oliguria phase, which were all higher than those in the control group ((2/24, 8.3%), (2/24, 8.3%) and (2/24, 8.3%), respectively; χ2=22.828, 22.828 and 9.135, respectively, all P<0.01). Compared with the control group, the pregnant patients in study group had a higher urea nitrogen maximum and serum creatinine maximum, and the differences were both statistically significant ( Z=-2.453 and -2.336, respectively, both P<0.05), while they had a lower serum albumin minimum, hemoglobin maximum and hemoglobin minimum, and the differences were all statistically significant ( Z=-3.742, -3.350 and -4.034, respectively, all P<0.01). All pregnant women with HFRS recovered. Nine pregnant women gave birth to nine healthy infants. All of them received breastfeeding and the feeding duration were more than six months. No abnormal growth and development were found during an average follow-up of three years. Conclusions:Pregnancy can aggravate the severity of HFRS, and pregnant women have higher risk of the multiple stages overlap and the complications such as hypervolemic syndrome and acute pulmonary edema. After recovery from HFRS, mother may carry to full-term pregnancy.

Objective:To study the real-world outcome of China FDA-approved Sofosbuvir (SOF)/Velpatasvir (VEL) in Northwest China.Methods:In this multicenter, prospective, real-world cohort study, we recruited patients from 10 sites from Northwest China, who were chronically infected with HCV GTs 1-6 from 06/2018 to 09/2019. Patients received SOF (400mg)/VEL (100mg) for 12 weeks, and with ribavirin 900-1200 mg for GT3 cirrhosis and for any genotype decompensated cirrhosis. The primary endpoint was sustained virological response at 12-weeks post-treatment (SVR12) and safety. The secondary endpoint was the change of liver function after the achievement of SVR12.Results:Totally, 143 patients were enrolled in the study, four patients were lost to follow-up and one died during the follow-up, 138 patients were included in per-protocol analysis. Of the 138 patients, the mean age 53 years, 53.6% male, 94.2% Han nationality, 53.6% liver cirrhosis, 10.1% HBsAg +, 6.5% renal dysfunction, 5.1% treatment-experienced, and 16.7% patients received ribavirin treatment. The genotype distribution was as follows: 35.5% GT1, 42.8% GT2, 15.9% GT3, and 5.8% un-typed. The SVR12 rate was 96.5% (138/143, 95% CI: 93.5%-99.6%) for intention-to-treat analysis, and in per-protocol analysis, all 138 patients obtained SVR12 (100%). Compared with baseline, the serum total bilirubin, ALT and AFP levels decreased (all P < 0.05), as well as increased ALB and platelet count (all P < 0.001) at post-treatment 12-weeks. Overall adverse events (AEs) rate is 29.0%, and the most common AEs were anemia (14.5%) and fatigue (8.0%). Severe side effects (edema and fatigue) occurred in 2 patients, one of whom needed a short-term interruption of treatment due to fatigue. Conclusion:In this real-world cohort study, 12-week SOF/VEL regimen with or without ribavirin achieved high SVR12 rates (96.5%-100% overall) with excellent safety profile among patients with HCV GT1/2/3 infection including patients with GT3 and cirrhosis, and led to improvement of liver function.

Objective: To explore the application value of serum cystatin C (Cys-C) and β2-microglobulin (β2-MG) in the diagnosis and short-term efficacy evaluation of multiple myeloma (MM). Methods: A total of 450 patients with MM admitted to Anqing Hospital of Chinese People′s Liberation Army Navy and Jinhua Central Hospital of Zhejiang Province from October 2016 to October 2018 were selected as subjects (MM group), according to the Durie-Salmon staging criteria, including 150 patients in stage Ⅰ, Ⅱ and Ⅲ. A total of 150 healthy subjects were selected as the control group. The levels of Cys-C and β2-MG in the serum of the subjects were determined. The differences of Cys-C and β2-MG levels between the two groups and the MM patients with different Durie-Salmon stages were compared. The differences of Cys-C and β2-MG levels between the patients with different short-term efficacy were compared. The receiver operating characteristic (ROC) curve was used to analyze the value of the two indicators in the evaluation of MM efficacy, and the correlation between Cys-C and β2-GM was analyzed. Logistic regression analysis was used to analyze the multiple factors affecting the clinical efficacy of MM patients. Results: The levels of Cys-C and β2-MG in the serum of the patients with MM were (2.11±0.78) mg/L and (6.07±3.08) g/L respectively, and those in the control group were (0.75±0.20) mg/L and (1.78±0.59) g/L, with significant differences (t=33.848, P<0.001; t=28.084, P<0.001). The Cys-C levels of Durie-Salmon stage Ⅰ, Ⅱ and Ⅲ patients were (0.99±0.21) mg/L, (1.36±0.17) mg/L and (3.07±1.02) mg/L respectively, and the difference was statistically significant (F=44.157, P<0.001). The β2-MG levels in the serum of patients with stage Ⅰ, Ⅱ and Ⅲ were (2.57±0.75) g/L, (4.66±1.43) g/L, (8.63±2.26) g/L respectively, and the difference was statistically significant (F=57.285, P<0.001). In all the patients, 338 patients were effective, accoun-ting for 75.11%, and 112 patients were ineffective, accounting for 24.89%. The levels of Cys-C and β2-MG in the serum of the effective MM patients were (1.28±0.23) mg/L and (2.82±0.78) g/L, and those of ineffective patients were (2.97±0.77) mg/L and (6.22±1.92) g/L, with statistically significant differences (t=35.874, P<0.001; t=26.633, P<0.001). The sensitivity of serum Cys-C for predicting short-term efficacy was 83.0%, the specificity was 76.6%, and those of serum β2-MG were 89.3% and 73.6%. The area under curve (AUC) of the serum Cys-C was 0.813 (95%CI: 0.764-0.862), and the AUC of serum β2-MG was 0.865 (95%CI: 0.825-0.906), with a statistically significant difference (Z=2.490, P=0.011). Spearman correlation analysis showed a positive correlation between serum Cys-C and β2-MG (r=0.539, P=0.041). Logistic regression analysis showed that both β2-MG (95%CI: 2.386-5.144, P<0.001) and Cys-C (95%CI: 2.367-9.702, P<0.001) were independent factors affecting the short-term efficacy of MM. Multivariate analysis showed that β2-MG (95%CI: 3.549-13.739, P=0.001) was an independent factor affecting the efficacy of MM. Conclusion The levels of serum Cys-C and β2-M in MM patients are significantly higher than those in healthy people, and they show an increasing trend with the progression of MM disease, which can be used as markers for the pathological staging diagnosis of MM patients. The short-term efficacy of the patients can be evaluated by using the two indicators, and the clinical significance in efficacy evaluation of β2-MG is slightly better than that of Cys-C.

Objective To construct a lentivirus vector over-expressing Tumor necrosis factor alpha induced protein 1 (TNFAIP1) gene and detect its expression level in vitro.Methods The full length of TNFAIP1 gene fragments were amplified by polymerase chain reaction (PCR).The pEZ-Lv105 vectors were digested by restriction endonuclease which was then linked to the full length of TNFAIP1 gene fragments by using T4DNA ligase.The plasmids were transfected into E.coli stbl3 and then we obtained the highly expressing positive clones by screening and identifying.The lentivirus vectors containing TNFAIP1 gene were transfected into 293T cells for package according to the packing kit manual.Results TNFAIP1 gene was amplified and successfully bound to the pEZ-Lv105 lentivirus vectors.The sequences of the recombinant plasmids were confirmed correctly by PCR and DNA sequence.The enhanced green fluorescent protein (eGFP) could be observed after recombinant lentiviruses were cotransfected into 293T cells.Conclusions The TNFAIP1 overexpressed lentivirus vector is successfully constructed,which provides a molecular tool for further study of TNFAIP1 gene in optic nerve glioma.

To screen the specific anti-human intercellular adhesion molecule-1 (ICAM-1) single chain fragment variable (scFv) using phage display library technology and to identify its biological activity. P1 peptide was used as antigen, and the phage antibodies against human ICAM-1 antigen were panned by four binding-eluting-amplifying cycles using Tomlinson I+J phage display library. After four rounds of selective enrichment screening, the positive clones were determined by PCR, enzyme linked immunosorbent assay (ELISA)-based antigenic cross reaction and Dot blotting. Then the binding specificity and biological activity of purified scFv were identified by Western blotting, competitive ELISA and cell adhesion inhibition assay respectively. Furthermore, four positive clones were first panned through P1 peptide coated-ELISA assay, and then J-A1 was obtained and identified by PCR, ELISA-based antigenic cross reaction and Dot blotting, which could show a specific binding between P1 peptide and human ICAM-1 protein antigen. Subsequently, the purified scFv showed a satisfactory specificity and anti-adhesive activity in competitive ELISA and the cell adhesion inhibition assay. The specific anti-human ICAM-1 scFv was prepared successfully from Tomlinson I+J phage display library, which pave the way for further application of anti-human ICAM-1 scFv for inflammation diseases therapeutics.
Antibodies ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin Variable Region ; Intercellular Adhesion Molecule-1 ; immunology ; Peptide Library ; Single-Chain Antibodies

Humans ; Models, Molecular ; Repressor Proteins ; chemistry ; metabolism ; Retinoblastoma-Binding Protein 4 ; chemistry ; metabolism