Study on early markers of death in patients with COVID-19.

OBJECTIVE: Coronavirus disease 2019 (COVID-19) was first discovered in December 2019, and since then rapidly spread worldwide. Our study aimed to investigate the early indicators of death in patients suffering from severe and critical COVID-19. PATIENTS AND METHODS: A retrospective cohort study was conducted on patients with severe and critical COVID-19, admitted to the Seventh Hospital of Wuhan. Clinical information was collected from electronic medical records according to standardized data collection tables. Patients were divided into non-survival and survival groups based on the disease outcome. Using univariate and multivariate logistic regression analysis, and calculating odds ratios (OR) and 95% confidence intervals (CI), independent risk factors for death in severe and critically ill COVID-19 patients were identified. RESULTS: The median age of 162 patients (57.4% males) was 67.5 years old. Patients in the non-survival group had significantly higher white blood cell count, decreased lymphocyte count, anemia and thrombocytopenia compared to patients in the survival group (p < 0.05). A 28-day mortality rate of the study cohort was 31.5%. Multivariate logistic regression analysis showed that underlying heart disease, lymphocyte count < 1.0 × 109/L, glomerular filtration rate < 66, lactate > 2.2 mmol/L, higher Sequential Organ Failure Assessment (SOFA) score, lower oxygenation index (OR 1.748; 95% CI 1.024-2.984; p=0.041) and higher "multi-lobar infiltration, hypo lymphocytosis, bacterial co-infection, smoking history, hypertension and age" (MuLBSTA) score (OR 1.601; 95% CI 1.062-2.415; p=0.025) were risk factors associated with death in patients with severe and critical COVID-19. CONCLUSIONS: Underlying heart disease, lymphocyte count, glomerular filtration rate, lactate, oxygenation index, SOFA score, and MuLBSTA score were associated with the risk of death in severe and critical COVID-19 patients.

[Cytotoxicity and underlying mechanism of evodiamine in HepG2 cells].

OBJECTIVE: To investigate evodiamine (EVO)-induced hepatotoxicity and the underlying mechanism. METHODS: HepG2 cells were treated with EVO (0.04-25 µmol/L) for different time intervals, and the cell survival rate was examined by cell counting kit-8 (CCK-8) method. After HepG2 cells were treated with EVO (0.2, 1 and 5 µmol/L) for 48 h, the alanine transaminase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), alkaline phosphatase (ALP) activities and total bilirubin (TBIL) content of supernatant were detected. A multifunctional microplate reader was used to detect the intracellular superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in HepG2 cells to evaluate the level of cell lipid peroxidation damage. The interactions between EVO and apoptosis, autophagy or ferroptosis-associated proteins were simulated by molecular docking. The HepG2 cells were stained by mitochondrial membrane potential (MMP) fluorescent probe (JC-10) and annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI), and MMP and apoptosis in HepG2 cells were detected by flow cytometry. The protein expression levels of caspase-9, caspase-3, bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2) were detected by Western blot. RESULTS: The cell survival rate was significantly reduced after the HepG2 cells were exposed to EVO (0.04-25 µmol/L) in a time- and dose-dependent manner. The half maximal inhibitory concentration (IC50) of the HepG2 cells treated with EVO for 24, 48 and 72 h were 85.3, 6.6 and 4.7 µmol/L, respectively. After exposure to EVO (0.2, 1 and 5 µmol/L) for 48 h, the ALT, AST, LDH, ALP activities and TBIL content in the HepG2 cell culture supernatant, and the MDA content in the cells were increased, and SOD enzyme activity was decreased. Molecular docking results showed that EVO interacted with apoptosis-associated proteins (caspase-9 and caspase-3) better. JC-10 and Annexin V-FITC/PI staining assays demonstrated that EVO could decrease MMP and promote apoptosis in the HepG2 cells. Western blot results indicated that the protein expressions of cleaved caspase-9 and cleaved caspase-3 were upregulated in the HepG2 cell treated with EVO for 48 h. In contrast, the protein expressions of pro-caspase-3, BSEP and MRP2 were downregulated. CONCLUSION: These results suggested that 0.2, 1 and 5 µmol/L EVO had the potential hepatotoxicity, and the possible mechanism involved lipid peroxidation damage, cell apoptosis, and cholestasis.

[Analysis of incidence and risk factors for postoperative venous thromboembolism in patients with stageâ a non-small-cell lung cancer].

Objective: To analyze the incidence and risk factors for postoperative venous thromboembolism(VTE) in patients with stage Ⅰa non-small-cell lung cancer(NSCLC), so as to find evidence for further research of prophylactic anticoagulation. Methods: A total of 132 patients with stage Ⅰa NSCLC, 42 males and 90 females aged from 26 to 79 years with an average of (57±10) years, were retrospectively included in this study. All of them underwent surgical treatment at the Department of Thoracic Surgery of Beijing Chaoyang Hospital Affiliated with Capital Medical University from January 2017 to October 2020. A lower extremity venous ultrasound was performed before and after the operation. Participants were divided into VTE group (n=11) or non-VTE group (n=121) according to whether or not VTE occurred after operation. The surgical conditions, test indicators, imaging information, pathology information were compared between the two groups. Logistic regression analysis was performed to test the associations of VET with putative risks factors in which significant differences were observed. The independent risk factors of VET were determined by this way. Results: Postoperative VTE occurred in 11 cases (8.3%), including 10 cases (90.9%) of deep vein thrombosis (DVT) of lower limbs and 1 case (9.1%) of DVT complicated with pulmonary embolism (PE). The mean age of Patients in the VTE group was older than that in non-VTE Group ((65±9) years vs (57±10) years, P=0.009). On the fifth day after operation, patients in both groups had significantly higher D-dimer level compared with that before operation (3.18(1.55, 5.15) vs 1.54(1.09, 2.57); 2.66(1.17, 4.65) vs 1.34(0.78, 2.04))(both P<0.05). The value of neuron-specific enolase (NSE) and the number of lymph nodes removed during the operation in the VTE group were significantly higher than those in the non-VTE group ((21.54±12.37) vs (14.72±5.75); (19.7±8.2) vs (13.0±7.9)) (both P<0.05). There was no statistically significant difference in the approach of surgery, imaging features (tumor location, vascular cluster signs, etc.), and pathological information (pathological types, etc.) (all P>0.05). The logistic regression analysis showed that the number of lymph nodes removed during the operation was an independent risk factor related to the occurrence of VTE (OR=1.306, 95%CI:1.000-1.600,P<0.05). Conclusions: The incidence of postoperative VTE in patients with stage Ⅰa NSCLC is approximately 8.3%. The number of lymph nodes removed during the operation may be an independent risk factor for postoperative VTE in patients with stage Ⅰa NSCLC.

[Study of effects of micheliolide on ovarian cancer cell lines and its mechanism].

Objective: To study the roles of micheliolide on ovarian cancer cells. Methods: Firstly, human ovarian cancer cell lines HeyA8, SKOV3 and A2780/DDP were treated with different concentration of micheliolide(0.25, 0.5, 1, 2.5, 5, 10, 20, 50 µmol/L)for 72 hours, then methyl thiazolyl tetrazolium(MTT)assay was used wo detect the growth of the human ovarian cancer cell lines and the stongest inhibited cell line were selected for the following test. Secondly, after HeyA8 cell line was treated with different concentration(5, 10, 20 µmol/L)of micheliolide for 24 hours, the HeyA8 cell apoptosis was measured byflow cytometry. Thirdly, the expression of RelA mRNA in HeyA8 cell was detected through real-time PCR, the expressions of nuclear factor κB(NF-κB)signal pathway related protein RelA and the activited cysteinyl aspartate specific proteinase(caspase-9)were detected by western blot analysis. Results: (1)The growth of HeyA8, SKOV3 and A2780/DDP cells were all significantly inhibited after being treated with different concentration of micheliolide for 72 hours and the roles of inhibition were all concentration dependant(P<0.05). The half maximal inhibitory concentration(IC50)of HeyA8, SKOV3 and A2780/DDP were(9.8±2.2),(12.0±2.1)and(12.8±1.8)µmol/L, respectively. We chose HeyA8 cell to do the following expreriments because of its best inhibited effect.(2)After HeyA8 cell was treated with micheliolide of different concentrations, as the concentration increased(20 and 0 µmol/L, for example), the apoptosis rate of HeyA8 cell raised from(7.2±1.0)% to(17.4±1.1)%, the percentage of survived cells reduce from(92.8 ± 1.3)% to(82.6 ± 1.4)%, and the relative mRNA level of RelA decreased from 1.00 ± 0.13 to 0.18 ± 0.00(P<0.01); furthermore, the expression of RelA protein was weaken and the activited caspase-9 protein expression was increased gradually. Conclusions: Micheliolide plays a significantly inhibited role in HeyA8, SKOV3 and A2780/DDP cells. The inhibited role of micheliolide inovarian cancer cells might through inhibiting nuclear factor-kappa B(NF-кB)signaling pathway, and inducing the expression of activited caspase-9 protein to promoting apoptosis of HeyA8 cell.

[Study on the application effect of Once Vex(GM)(-)(CSF) and adriamycin in the interventional therapy of malignant tumor in clinical curative effect].

OBJECTIVE: To investigate the application effect of oncolytic herpes simplex virus Once Vex(GM-CSF) and adriamycin in malignant tumor. METHODS: A total of 102 cases of malignant tumor were analyzed retrospectively from April 2013 to April 2015 of Cancer Treatment Center of Affiliated Hospital of Beihua University, and these cases were randomly divided into trial group (n=51) and control group (n=51). The control group was treated with adriamycin and the trial group patients received conditionally replicating adenoviruses on the basis of the control group.The adenovirus proliferation was analyzed via quantitative PCR to investigate the malignant tumor cell inhibitory action in two groups. RESULTS: Treatment group total effective rate (96.1%, 49/51) was significantly higher than the control group (78.4%, 40/51) (χ(2)=7.845, P=0.014). There were statistically significant difference of the killing capability between trial group (86.3%, 44/51) and control group (23.5%, 12/51) (χ(2)=7.859, P=0.013). Tumor shrinkage rate of trial group(76.5%, 39/51) was superior to that of the control group(19.6%, 10/51), the difference was statistically significant (χ(2)=7.654, P=0.019). There was statistically significant difference of the decrease of tumor marker CA125 between trial group (74.5%, 38/51) and control group (23.5%, 12/51) (χ(2)=7.342, P=0.023). CONCLUSION: Once Vex(GM-CSF) can effectively kill the tumor cells in the treatment of malignant tumor, and shrink the tumor significantly.

1H NMR structural analysis of human ghrelin and its six truncated analogs.

Human ghrelin, the first recognized natural ligand of growth hormone secretagogue growth hormone secretagogue receptors (GHS-Rs) (M. Kojima, H. Hosada, Y. Date, M. Nakazato, H. Matsuo, and K. Kangawa, Nature, 1999, Vol. 402, pp. 656-660), consists of 28 amino acids of which Ser3 is modified by n-octanoylation. This new peptide hormone has been implicated not only in regulation of the GH secretion but also in regulation of food intake. The discovery of ghrelin opens up more opportunities to study the relationship of ghrelin with metabolic diseases. Until now, only mass spectometry analysis has been reported on the structure of ghrelin. NMR analysis is a suitable way to study if any tertiary structure of unbound ghrelin is present in solution. NMR studies were carried out on human ghrelin and its five truncated analogs. The full-length ghrelin and its fragments exhibited random coil behavior in aqueous solution. Additional studies were carried out on the shortest active segment of human ghrelin, which consists of the first five amino acids of the ghrelin sequence (M. A. Bednarek, S. D. Feighner, S.-S. Pong, K. K. McKee, D. L. Hreniuk, M. V. Silva, V. A. Warrem, A. D. Howard, L. H. Y. Van der Ploeg, and J. V. Heck, Journal of Medical Chemistry, 2000, Vol. 43, pp. 4370-4376), to compare the spectral features with their counterparts in the full-length ghrelin. The NMR data showed behavior similar to ghrelin except for two additional nuclear Overhauser effects (NOEs) between the Phe4 NH and the protons of the beta-methylene of Ser3. CD on human ghrelin and its short active analog in water were indicative of random coil peptides. Molecular modeling based on NMR data was carried out to probe which structural features were similar to growth hormone-releasing peptide-6 (GHRP-6), a hexapeptide that binds to GHS-R releasing GH and stimulating food intake. Modeling suggested some similarities, but they were not of a nature to account for binding properties of these compounds.

Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.