Unraveling the pharmacodynamic substances and possible mechanism of Trichosanthis Pericarpium in the treatment of coronary heart disease based on plasma pharmacochemistry, network pharmacology and experimental validation.

ETHNOPHARMACOLOGICAL RELEVANCE: Coronary heart disease (CHD) is a chronic disease that seriously threatens people's health and even their lives. Currently, there is no ideal drug without side effects for the treatment of CHD. Trichosanthis Pericarpium (TP) has been used for several years in the treatment of diseases associated with CHD. However, there is still a need for systematic research to unravel the pharmacodynamic substances and possible mechanism of TP in the treatment of coronary heart. AIM OF THE STUDY: The purpose of current study was to explore the pharmacodynamic substances and potential mechanisms of TP in the treatment of CHD via integrating network pharmacology with plasma pharmacochemistry and experimental validation. MATERIALS AND METHODS: The effect of TP intervention in CHD was firstly assessed on high-fat diet combined with isoprenaline-induced CHD rats and H2O2-induced H9c2 cells, respectively. Then, the LC-MS was utilized to identify the absorbed components of TP in the plasma of CHD rats, and this was used to develop a network pharmacology prediction to obtain the possible active components and mechanisms of action. Molecular docking and immunohistochemistry were used to explore the interaction between TP and key targets. Subsequently, the efficacy of the active ingredients was investigated by in vitro cellular experiments, and their metabolic pathways in CHD rats were further analyzed. RESULTS: The effects of TP on amelioration of CHD were verified by in vivo and in vitro experiments. Plasma pharmacochemistry and network pharmacology screened six active components in plasma including apigenin, phenylalanine, quercetin, linoleic acid, luteolin, and tangeretin. The interaction of these compounds with potential key targets AKT1, IL-1ß, IL-6, TNF-α and VEGFA were preliminarily verified by molecular docking. And immunohistochemical results showed that TP reduced the expression of AKT1, IL-1ß, IL-6, TNF-α and VEGFA in CHD rat hearts. Then cellular experiments confirmed that apigenin, phenylalanine, quercetin, linoleic acid, luteolin, and tangeretin were able to reduce the ROS level in H2O2-induced HUVEC cells and promote the migration and tubule formation of HUVEC cells, indicating the pharmacodynamic effects of the active components. Meanwhile, the metabolites of TP in CHD rats suggested that the pharmacological effects of TP might be the result of the combined effects of the active ingredients and their metabolites. CONCLUSION: Our study found that TP intervention in CHD is characterized by multi-component and multi-target regulation. Apigenin, phenylalanine, linoleic acid, quercetin, luteolin, and tangeretin are the main active components of TP. TP could reduce inflammatory response and endothelial damage by regulating AKT1, IL-1ß, IL-6, TNF-α and VEGFA, reduce ROS level to alleviate the oxidative stress situation and improve heart disease by promoting angiogenesis to regulate endothelial function. This study also provides an experimental and scientific basis for the clinical application and rational development of TP.

Application of Extracorporeal Membrane Oxygenation in Patients With Severe Acute Respiratory Distress Syndrome Caused by Pneumocystis jirovecii Pneumonia Following Kidney Transplantation: A Case Series.

Objective: To investigate the application effect of extracorporeal membrane oxygenation (ECMO) in patients with severe acute respiratory distress syndrome (ARDS) caused by Pneumocystis jirovecii pneumonia (PJP) after kidney transplantation. Methods: This is a case series on 10 kidney transplant recipients with severe ARDS caused by PJP at the People's Hospital of Zhengzhou, who were enrolled as the case group. A total of 17 cases of PJP diagnosed with severe ARDS without ECMO were selected as the control group. The timing and mode of ECMO support and treatment complications were summarized. The primary aim of this study was mortality and secondary was imaging and complications. Results: The enrolled patients' oxygenation index before the start of ECMO ranged from 25 to 92, and the time from admission to the start of ECMO was 1-17 days, with an average of 5.56 days. In the case group, one patient died of hemorrhagic shock due to abdominal hemorrhage, but the other nine patients were successfully weaned from ECMO. Of these patients, one died due to sepsis following weaning. The survival rate in the case group was 80.0% (8/10), and the survival rate in the control group was 35.29% (6/17). The vein-vein ECMO support time in the nine successfully weaned patients in the case group ranged from 131 to 288 h, with an average of 215.5 h. Of the eight patients who survived, deterioration of renal function after transplantation occurred in two patients, but no fatal complications occurred. Conclusion: Overall, Patients with severe ARDS caused by postoperative PJP infection following kidney transplantation have a poor prognosis. The mortality was lower in patients who were treated with ECMO compared to standard care.

[Development of preparation process of icaritin-coix seed oil microemulsion based on quality by design concept].

To optimize the formulation and preparation process of icaritin-coix seed oil microemulsion(IC-MEs) based on quality by design(QbD) concept. IC-MEs were prepared by water titration. Firstly, the risk factors that may affect the quality of IC-MEs were evaluated. Then Plackett-Burman design was used to screen out prescription factors and process parameters that had a significant effect on the indicators. Finally, Box-Behnken design was used to optimize the prescription ratio of IC-MEs. Through the risk assessment and Plackett-Burman design, three formulation factors [drug loading efficiency, the ratio of mixed-oil(coix seed oil-Glycerol tributyrate) to mixed-surfactant(HS15-RH40) and water addition] were determined as the key factors affecting IC-MEs. The regression model established by Box-Behnken design had a good predictability. The optimal formula was as following: the drug loading efficiency of 0.92%, the ratio of mixed-oil(coix seed oil-glycerol tributyrate) to mixed-surfactant(HS15-RH40) of 4∶6, and the water addition of 5.7 mL. According to this prescription, IC-MEs were prepared, and its encapsulation efficiency after 1 week was 92.45%±1.00%. Therefore, the stability of IC-MEs could be improved by optimizing prescription and process parameters of IC-MEs based on the QbD concept, which can provide certain reference value for the future development of IC-MEs.

[Sensitivity of AML Cells with FLT3- to PKC412 In Vitro].

OBJECTIVE: To explore the clinical value of PKC412 (midostaurin) in treatment of AML patients with FLT3-. METHODS: The bone marrow or peripheral blood were collected and heparinized from 21 newly diagnosed FLT3- AML patients, then the mononuclear cells from bone marrow or peripheral blood were isolated by density-gradient method. The sensitivity of leukemia cells to PKC412 of 8 concentration in vitro was detected by ATP-bioluminescence-tumor chemosensitivity assay (ATP-TCA), and the relationship among sensitivity results in vitro, risk stratification and therapeutic efficacy was analyzed. RESULTS: The leukemia cells of 21 patients with AML displayed different sensitivities to PKC412 in vitro. The rate of sensitivity in vitro was 42.9%, and sensitive concentration in vitro were between 1 µmol/L and 5 µmol/L. There was no significant relationship between risk stratification and sensitivity results of PKC412 in vitro. There was also no significant relationship between clinical efficacy and sensitivity results of PKC412 in vitro. The survival of patients in low-risk and intermediate-risk groups was better than that of patients in high-risk groups (P=0.015). CONCLUSION: PKC412 can be one of the effective therapeutic method for AML patients without FLT3 mutation. The sensitivity of leukemia cells to PKC412 may become a prognostic marker for evaluating clinical efficacy of PKC412, which is independent of other factors.

Preparation of novel butyryl galactose ester-modified coix component microemulsions and evaluation on hepatoma-targeting in vitro and in vivo.

The butyryl galactose ester-modified coix component microemulsions (But-Gal-CMEs) was developed for enhanced liver tumor-specific targeting. The study was aimed to evaluate the hepatoma-targeting potential of But-Gal-CMEs in vitro and in vivo. But-Gal-CMEs with a uniform spherical shape exhibited a small particle size (56.68 ± 0.07 nm), a narrow polydispersity (PDI, 0.144 ± 0.005) and slightly negative surface charge (-0.102 ± 0.008 mV). In the cell uptake studies, But-Gal-CMEs showed a significant enhancement on the intracellular fluorescent intensity on HepG2 cells model, which was 1.93-fold higher relative to coix component microemulsions (CMEs). The IC50 of But-Gal-CMEs against HepG2 cells was 64.250 µg/mL, which was notably stronger than that of CMEs. In the cell apoptosis studies, compared with CMEs, But-Gal-CMEs (50 µg/mL) treatment resulted in a 1.34-fold rise in total apoptosis cells of HepG2. In the biodistribution studies in vivo, the intratumorous fluorescence of Cy5-loaded But-Gal-CMEs was 1.43-fold higher relative to that of Cy5-loaded CMEs, suggesting an obviously enhanced accumulation in the tumor sites. Taken as together, But-Gal could be incorporated into the coix component microemulsions as a novel ligand for realizing hepatoma-targeting drugs delivery.

[Analysis of Differentially Expressed Signaling Pathway Proteins in Myelodysplastic/Myeloproliferative Neoplasms-Unclassifiable].

OBJECTIVE: To screen signaling pathway proteins in myelodysplastic/myeloproliferative neoplasms-unclassifiable (MDS/MPN-U), and to explore the possible role of the differentially expressed signaling pathway proteins in pathogenesis of MDS/MPN-U. METHODS: Protein Pathway Array (PPA) was applied to analyze the differential expression levels of signaling pathway proteins in 10 patients with MDS/MPN-U and normal controls, and furthermore to identify the signaling pathways and network in which these proteins were analyzed by Ingenuity pathway analysis program. RESULTS: The expressions of 25 signaling proteins in MDS/MPN-U were significantly different, compared with the control group. Among them 15 proteins were upregulated in MDS/MPN-U patients, while 10 proteins were downregulated. These dysregulated proteins were involved in 10 major signaling pathways related with cell proliferation and immunity. The complicated interactive network was established by these proteins and pathways. CONCLUSION: The differentially expressed signaling proteins screened from the MDS/MPN-U patients by PPA might be helpful to reveal the pathogenesis of MDS/MPN-U and to discover the therapeutic targets.

[Transformation of icariin by immobilized ß-glucosidase and snailase].

This study was performed to prepare immobilized ß-glucosidase and snailase, then optimize and compare the process conditions for conversion of icariin. Immobilized ß-glucosidase and snailase were prepared using crosslink-embedding method. The best conditions of the preparation process were optimized by single factor analysis and the properties of immobilized ß-glucosidase and snailase were investigated. The reaction conditions including temperature, pH, substrate ratio, substrate concentration, reaction time and reusing times of the conversion of icariin using immobilized ß-glucosidase or snailase were optimized. Immobilized ß-glucosidase and snailase exhibited better heat stabilities and could remain about 60% activity after storage at 4 degrees C for 4 weeks. The optimized conditions for the conversion of icariin were as follows, the temperature of 50 degrees C, pH of 5.0, enzyme and substrate ratio of 1 : 1, substrate concentration of 0.1 mg x mL(-1), reaction time of 6 h for ß-glucosidase and 2 h for snailase, respectively. In 5 experiments, the average conversion ratio of immobilized ß-glucosidase and snailase was 70.76% and 74.97%. The results suggest an effect of promoted stabilities, prolonged lifetimes in both ß-glucosidase and snailase after immobilization. The immobilized ß-glucosidase and snailase exhibited a higher conversion rate and reusability compared to the free ß-glucosidase and snailase. Moreover, the conversion rate of immobilized snailase was higher than that of immobilized ß-glucosidase. The process of icariin conversion using immobilized ß-glucosidase and snailase was moderate and feasible, which suggests that immobilized enzymes may hold a promise for industrial usage.

[Genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4].

This study was aimed to investigate the genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4, and evaluate its application in measuring telomere length by Flow-FISH. Molt-4 cell line was cultured in suspension and subcultured regularly. Eight different passages of Molt-4 cells in exponential stage were selected.The growth curves were drawn by cell counting method, meanwhile calculating the population doubling times of cells,DNA ploidies were determined by flow cytometry,karyotypes were analyzed by G-banding and telomere lengths were measured by Southern blot. The results showed that the population doubling time of Molt-4 cell line was (1.315 ± 0.062) d, DNA ploidy index was (2.085 ± 0.0093) , and the telomere length was (32.05 ± 5.27) kb. There were no significant difference among different passages (P = 0.931,0.888 and 0.935 separately). The karyotypes showed that the chromosome numbers of Molt-4 cell line were from 91 to 99 in different metaphases, and the majority of them were hypertetraploid, and stable and recurrent structural abnormalities of chromosomes could be kept. It is concluded that the stable genetic characteristics and the longer telomere length of Molt-4 cell line makes it be a feasible control cells in measurement of telomere length by Flow-FISH.

[Application of flow cytometry-fluorescence in situ hybridization in the measurement of relative telomere length of bone marrow CD34(+) cells in patients with myelodysplastic syndrome].

This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was counterstained and the relative telomere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further.

Clinical outcome of autologous peripheral blood stem cell transplantation in opticospinal and conventional forms of secondary progressive multiple sclerosis in a Chinese population.

To evaluate clinical outcomes of autologous peripheral blood stem cell transplantation (APBCST) between opticospinal multiple sclerosis (OSMS) and conventional multiple sclerosis (CMS) during disease progressive stage in a Chinese population. Thirty-six secondary progressive MS patients, among whom 21 were with OSMS and 15 with CMS, underwent APBSCT and were followed up for an average of 48.92 months (range, 10-91 months). Peripheral blood stem cells were obtained by leukapheresis after mobilization with granulocyte colony-stimulating factor. Modified BEAM conditioning regimen (Tiniposide, melphalan, carmustin, and cytosine arabinoside) were administered. Outcomes were evaluated using the expanded disability status scale (EDSS). No maintenance treatment was administered if there was no disease progression. No treatment-related mortality occurred. Among the 36 patients, one OSMS patient dropped during the follow-up. Among the 22 relapse-free patients, 20 were with continuous neurological improvement without any relapse events, and two remained in neurologically stable states. Among the 13 relapse patients, seven had experienced of neurological relapse, but with no progression during the follow-up period; and six experienced neurological deterioration after transplantation and needed further immunosuppressant treatment. The confirmed relapse-free survival rate was 62.9% and progression-free survival rate was 83.3% after 91 months according to Kaplan and Meier survival curves. Eleven of the 20 OSMS patients (55%) and two of the 15 CMS patients (13.3%) stayed in disease active group (P = 0.014). For the 20 OSMS patients, the overall EDSS score decreased significantly after transplantation (P = 0.016), while visual functions had no significant improvement (P = 0.716). Progressive OSMS has a higher relapse rate than CMS following APBSCT.

[Multiparameter flow cytometric evaluation of bone marrow involvement in B cell non-Hodgkin's lymphoma].

The objective of study was to evaluate the clinical values of multiparameter flow cytometry (MPFC) and cytomorphology of bone marrow aspiration(BMA) in detecting bone marrow involvement in patients with B cell Non-Hodgkin's lymphoma (B-NHL). 96 bone marrow samples from the patients with B-NHL were measured by MPFC using CD45/SSC and CD20/SSC gating strategy combined with anti-kappa and anti-lamda monoclonal antibodies, and then compared with results acquired by cytomorphologic analysis of BMA. The results showed that the bone marrow involvement was confirmed by MPFC in 38 cases (39.6%), while it was detected by cytomorphologic analysis of BMA only in 12 cases (12.5%). There was a significant difference between the two methods (p<0.05). 12 positive cases detected by cytomorphologic analysis of BMA were also positive by MPFC. There was no difference of 3-year overall survival rate between negative and positive cases detected by MPFC, but their 4-year overall survival rate was 73.18+/-6.65% and 44.13%+/-19.55% respectively (p<0.05). It is concluded that the MPFC is a more sensitive method for detecting bone marrow involvement in patients with B-NHL than cytomorphologic analysis of BMA. The 4-year overall survival rate of the patients without bone marrow involvement was significant higher than those of patients with bone marrow involvement. Bone marrow involvement in B-NHL detected by MPFC can be useful for clinical evaluation and prognosis prediction.

[Application of interphase FISH on cell smears in detection of hematological diseases].

The study was aimed to investigate the application value of interphase fluorescence in situ hybridization (FISH) on cell smears in hematological diseases. Both interphase FISH on peripheral blood smears and bone marrow smears treated by methanol/acetic acid, and routine interphase FISH of bone marrow cells dropped on slides were done at the same time, in order to detect Ph chromosome by BCR/ABL dual color, dual fusion probe in 20 patients with chronic myelogenous leukemia or acute lymphoblastic leukemia which had been proven to display Ph chromosome positive. The results indicated that as compared with routine interphase FISH, the interphase FISH on cell smears could also offer reliable result. It is concluded that interphase FISH on cell smears is a kind of reliable and time-saving technique, which is also suitable for retrospective research and worthy to further apply in clinic.

[Megakaryocytic dysplasia and leukemia associated phenotype in elderly patients with acute myeloid leukemia].

This study was purposed to investigate the megakaryocytic dysplasia and leukemia-associated phenotypes (LAP) of acute myeloid leukemia (AML) in the elderly. The megakaryocytic dysplasia, lineage infidelity, asynchronous antigen expression, total WBC count, and karyotypes were observed in the 147 none M(3)-AML patients. Logistic regression were used to analyzed the difference between the elderly (age > or = 60) and the control. The results showed that out of the total 147 patients (66 elderly patients, and 81 younger patients) 124 patients accepted induction chemotherapy, in which 70 cases achieved complete remission (elderly 18, younger 52, p = 0.008); megakaryocytic dysplasia was found in 32 patients (21.8%); CD33 and CD19/CD7 (lineage infidelity) was co-expressed in 55 patients (37.4%), CD34 and CD11b (asynchronous antigen expression) was co-expressed in 65 patients (44.2%); white blood cell count > 25 x 10(9)/L was found in 52 patients (35.4%). By the Logistic regression, compared with the control, in the elderly patients there was difference in the megakaryocytic dysplasia, and the co-expression of CD33/CD19/CD7 and CD34/CD11b (OR = 4.315, 2.761, 0.397; p = 0.001, 0.006, 0.020), but there was no difference in the total WBC count and karyotypes (OR = 0.802, 1.096; p = 0.646, 0.813). It is concluded that the incidence of megakaryocytic dysplasia, such as lineage infidelity, and asynchronous antigen expression, in elderly patients is higher than that in younger patients.

Clinical outcomes after autologous haematopoietic stem cell transplantation in patients with progressive multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a continuously disabling disease and it is unresponsive to high dose steroid and immunomodulation with disease progression. The autologous haematopoietic stem cell transplantation (ASCT) has been introduced in the treatment of refractory forms of multiple sclerosis. In this study, the clinical outcomes followed by ASCT were evaluated for patients with progressive MS. METHODS: Twenty-two patients with secondary progressive MS were treated with ASCT. Peripheral blood stem cells were obtained by leukapheresis after mobilization with granulocyte colony stimulating factor. Etoposide, melphalan, carmustin and cytosine arabinoside were administered as conditioning regimen. Outcomes were evaluated by the expanded disability status scale and progression free survival. No maintenance treatment was administered during a median follow-up of 39 months (range, 6 to 59 months). RESULTS: No death occurred following the treatment. The overall confirmed progression free survival rate was 77% up to 59 months after transplantation which was significantly higher compared with pre-transplantation (P = 0.000). Thirteen patients (59%) had remarkable improvement in neurological manifestations, four (18%) stabilized their disability status and five (23%) showed clinical recurrence of active symptoms. CONCLUSIONS: ASCT as a therapy is safe and available. It can improve or stabilize neurological manifestations in most patients with progressive MS following failure of conventional therapy.

[Expansion ex vivo of human bone marrow mesenchymal stem cells and cord blood CD34+ cells].

The aim was to study the roles that the bone marrow mesenchymal stem cells (MSC) and cytokines play in cord blood CD34(+) cell expansion ex vivo and the influence of culture ex vivo on expression of the adhesive molecule of CD44. CD34(+) cells sorted from cord blood cells had been cultured in each well of 24 well culture plates containing culture medium supplemented with mesenchymal stem cells layer or/and cytokines for a week, and then all kinds of indexes of different groups were compared. The results showed that as for cord blood cell expansion, there was no significant difference between the groups with cytokines SDF-1alpha + SCF + TPO + FL and SCF + TPO + FL no matter if MSC layer existed or not. The groups with MSC layer and cytokines were superior to the corresponding groups without MSC layer. In addition, the expression of the adhesion molecule CD44 had no distinct change after culture. It is concluded that SDF-1alpha has no distinct influence on the effect of cytokines SCF + TPO + FL on cord blood cell expansion ex vivo. MSC enhance the effect of cytokines on cord blood cell expansion ex vivo. Such expansion ex vivo may not influence the expression of the adhesive molecule CD44 on cord blood cells.