OBJECTIVEEffects of ginsenoside Rb1, Rg1 and Re on neurotrophic factor signal transduction pathway using liposome-mediated transfection of eukaryotic cells approach.

METHODThe injury model was established by treating SH-SY5Y cells with 0.6 mmol x L(-1) of corticosterone (CORT) by 24 h. SH-SY5Y cell were pretreated with CORT for 30 min followed by co-treated with 120,60 and 20 micromol x L(-1) of Rb1, 120, 80 and 40 micromol x L(-1) of Rg1 and 120, 80 and 40 micromol x L(-1) of Re for 24 h. Cells viability was determined by Cell Counting Kit (CCK) assay. CREB expressing Luciferase reporter gene was constructed and transfected with plasmid containing hRaf, hcAMP, hAkt, hCaMK gene into human embryonic kidney (HEK293) cells using liposornal transfection reagent lipofection 2000. The expression of CREB before and after it addion of Rb1, Rg1 and Re was examined by Luc assay system and Western blotting.

RESULTCompared with normal control group, CORT significantly decreased the viability of SH-SY5Y cells to 67.21% (P < 0.01). CCK results show that Rb1 (60 micromol x L(-1)), Rg1 (80 micromol x L(-1)) and Re (80 micromol x L(-1)) on SH-SY5Y cells have significant protective effect (P < 0.01). Lucassay and Western blotting results show that the gene and protein levels of CREB increased significantly through the pathway of Raf and Akt with Rb1 and Rg1 (P < 0.01), Re can increase significantly the gene and protein levels of CREB through the pathway of Raf and CaMK II.

CONCLUSIONRb1, Rg1 and Re protects SH-SY5Y cells from CORT-induced damage and the neuroprotective mechanism may be associated with the Raf-CREB, Akt-CREB and CaMK II -CREB pathways.

Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Cell Line ; Cell Survival ; drug effects ; Cyclic AMP Response Element-Binding Protein ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Genes, Reporter ; Ginsenosides ; pharmacology ; Humans ; Panax ; chemistry ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Signal Transduction ; drug effects ; raf Kinases ; genetics ; metabolism

Arteriovenous Fistula ; complications ; etiology ; Cardiac Output, High ; Femoral Artery ; Femoral Vein ; Heart Failure ; etiology ; Humans ; Intra-Aortic Balloon Pumping ; adverse effects ; Male ; Middle Aged

Background Heparanase degrade heparan sulfate side chains of heparan sulfate proteoglycans in the extracellular matrix.Heparanase induces angiogenesis and likely promotes the vascularization of tumor.ObjectiveThe present study is to investigate the expression of heparanase and perlecan in retinas with oxygen-induced retinopathy.Methods Sixty-five clean neonatal C57BL/6J mice were raised in a hyperbaric oxygen box with a volume percentage of 75%±2% for 5 days and then returned to the normal air room.Another 65 matched mice were raised in the normal environment as controls.Evans blue was infused by the superior vena cava in all the mice on postnatal days 12,13,17,21 and 30,afterwards fluorescein angiography was performed and then the mice were sacrificed.The retinas of mice were isolated and prepared and the retinal vessels were examined under a fluorescent microscope and optical microscope.Heranase and perlecan mRNA was detected using reverse transcription PCR (RT-PCR).Heranase and perlecan proteins were detected by Western blot.The analysis of variance was used to compare the mRNA and the protein levels of heranase and perlecan between the experimental and control groups.Results The expression of heparanase mRNA in the retinas of different ages of mice and the different groups showed significant differences (F_(group)=16.303,P=0.000;F_(time)=18.614,P=0.000;F_(interaction)=11.299,P=0.000),and the expression of heparanase mRNA was significantly enhanced in mice from postnatal days 12,13,17 and 21 compared with normal control mice (P=0.001,0.000,0.000,0.001,respectively).The expression of heparanase protein in the retinas of different ages of mice and the different groups followed the same tendency(F_(group)=458.134,P=0.000;F_(time)=78.466,P=0.000;F_(interaction)=71.398,P=0.000).The expression of perlecan mRNA in the retinas of different ages of mice and the different groups showed significant differences (F_(group)=7.351,P=0.013;F_(time)=9.098,P=0.000;F_(interaction)=3.349,P=0.000),and increase in differences also were clearly seen in mice from postnatal days 13,17 and 21 compared with normal control mice (P=0.048,0.000,0.003,respectively).Conclusion The expression of heparanase and perlecan is associated with the development and progression of retinal neovascularization,and perlecan and heparanase together produce a synergistic effect.Heparanase and perlecan may participate in the angiogenesis of oxygen-induced retinopathy.

OBJECTIVETo study the molecular mechanism of Yangjing Zhongyu Decoction (YZD) n-butanol extracts (ZDC) and ethyl acetate extracts (YSYZ) in reducing androgen in porcine granulose cells by mitogen-activated protein kinase (MAPK) pathway.

METHODSPorcine granulose cells were isolated and cultured. They were inoculated by MAPK inhibitor PD98059 at different concentrations, and then they were divided into the blank control group (0), 1, 3, 10, and 25 micromol/L groups. After 24-h culture the cytochrome P450c17a (CYP17) mRNA expression level was detected using Real-time fluorescent quantitative PCR. Contents of androgen (testosterone) in the supernate were detected using RIA and optimal PD98059 concentration screened. After intervened by 10 micromol/L PD98059 for 24 h, the culture solution was intervened by effective ingredients of with or without YZD or YSYZ at various concentrations (0, 1 , 5, 25, 50 mg/mL) at various time points (3, 6, 18, 24 h). Expression levels of p-ERK1/2, c-Fos and CYP17 were detected by Western blot. Testosterone content in the supernate was determined by radioimmunoassay (RIA).

RESULTSTen pLmol/L PD98059 could obviously decrease p-ERK1/2 protein expression and increase CYP17 mRMA expression, and elevate testosterone content in the supernate (P < 0.05). ZDC and YSYZ at 25 ng/mL could increase p-ERK1/2 protein expression and c-Fos levels, and reduce CYP17 protein expression, and lower testosterone content in the supernate after 6-h intervention (P < 0.01).

CONCLUSIONEffective ingredients of YZD could reduce androgen production in porcine granulose cells through increasing activities of MAPK.

Androgens ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Flavonoids ; Granulosa Cells ; metabolism ; Mitogen-Activated Protein Kinase 3 ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; RNA, Messenger ; Swine

BACKGROUND: Strontium-doped calcium polyphosphate (SCPP) is a new type of bone repair materials with good biocompatibility and controlled degradation. The preliminary studies of our group indicate their role in promoting angiogenesis,but its mechanism is unclear.OBJECTIVE: By co-culturing osteoblasts ROS17/2.8 with SCPP in vitro to observe cell proliferation and the secretion of vascular endothelial growth factor (VEGF).DESIGN, TIME AND SETTING: A contrast study was performed at the Laboratory of Tissue Engineering of Sichuan University from October 2008 to June 2009.MATERIALS: A series of calcium polyphosphate (CPP) respectively containing 0%, 1 %, 2%, 5%, 8%, and 10% Sr~(2+) were prepared. ROS17/2.8 osteoblastic cell strain was provided by Laboratory of Transplantation Immunity and Transplantation Engineering, West China Hospital, Sichuan University.METHODS: ①Preparation of cell scaffold complexes: The materials were placed in 24-well plates, then 300 μL cell suspension with a concentration of 2×10~7 cells/Lwas inoculated into each hole. These complexes were cultured for 14 days and the liquid was changed every two days. ②These complexes were measured by MTT assay to observe the proliferation of osteoblasts on the 1~(st), 3~(rd), 5~(th), 7~(th), 10~(th) and 14~(th) days, respectively. ③ The centrifugal supernatant of the complex cultured for seven days was measured by ELISA assay to check the secretion of VEGF.MAIN OUTCOME MEASURES: The proliferation of osteoblastic cells on SCPP and CPP was observed. The amount of VEGF protein secreting from osteoblastic cells was detected.RESULTS: The results of MTT showed that, compared with the CPP group, SCPP groups could promote the proliferation of osteoblasts, and 8% SCPP group was the best; ELISA results showed that, compared with the CPP group, SCPP groups could increase the amount of VEGF protein secretion, of which the promoting role of 8% SCPP was the most obvious (P < 0.05).CONCLUSION: When cultured with osteoblasts, SCPP can promote cell proliferation, and can significantly increase the secretion of VEGF; moreover, 8% SCPP is the best, which reveals a certain mechanism of its promoting angiogenesis.

OBJECTIVETo observe the anti-inflammatory effect of total saponins of Panax japonicus on LPS-induced RAW264. 7 macrophages.

METHODThe effect of total saponins of P. japonicus of different concentrations on RAW264. 7 cell viability was determined with the MTT method. The NO kit assay was adopted to detect the NO release of total saponins of P. japonicus to LPS-induced RAW264. 7 cells. The enzyme linked immunosorbent assay (ELISA) was used to detect the secretion of tumor necrosis factor-alpha (TNF-alpha) and interleukin 1-beta (IL-1beta). The reverse transeriptase-polymerase chain reaction (RT-PCR) was used to determine the expression of inducible nitric oxide synthase (iNOS) ,TNF-alpha,IL-1beta. The protein expression of nuclear transcription factor-kappaB p65 (NF-kappaB p65) was tested by Western blot.

RESULTThe safe medication range of total saponins of P. japonicus was less than 80 mg x L(-1). Compared with the LPS model group, total saponins of P. japonicus high, middle and low dose groups (0.1, 1, 10, 40 mg x L(-1)) could significantly reduce the secretion of NO, TNF-alpha, IL-1beta of LPS-induced RAW264. 7 cells, and inhibit the expressions of iNOS, TNF-alpha and IL-1beta mRNA and the protein expression of NF-kappaB p65.

CONCLUSIONThis study preliminarily proves the protective effect of total saponins of P. japonicus on LPS-induced RAW264.7 macrophages. Its action mechanism may be related to NF-kappaB signal pathway.

Animals ; Anti-Inflammatory Agents ; pharmacology ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Inflammation ; drug therapy ; genetics ; immunology ; Interleukin-1beta ; genetics ; immunology ; Lipopolysaccharides ; adverse effects ; Macrophages ; drug effects ; immunology ; Mice ; NF-kappa B ; genetics ; immunology ; Nitric Oxide ; immunology ; Nitric Oxide Synthase Type II ; genetics ; immunology ; Panax ; chemistry ; Protective Agents ; pharmacology ; Saponins ; pharmacology

Objective To research the protective effects of totalPanax Japonicus extract on learning memory, antioxidation, and anti-apoptosis of aging mice,and explore the mechanism. Methods Male mice were randomly divided into normal group, model group, Vitamin E (VE) group, Panax Japonicus extract low and high dose group. Except for the normal group, the other groups were injected withD-gal on the back of the neck subcutaneously to establish aging model. Normal group and model group were given a gavage with saline and each treatment group was given a gavage with totalPanax Japonicus extract and VE once a day for 7 weeks after the aging model established. All mice were be measured their learning and memory ability in the eighth week. After the test, the morphological changes of CA1 neurons were observed by HE stain. SOD, GSH-Px, MDA levels in brain tissue were measured by biochemical method, the expressions of Bcl-2 and Bax mRNA were evaluated by RT-PCR.Results Mice inPanax Japonicus extract low and high dose group could spend less time in searching for the platform, improve the learning and memory ability. TotalPanax Japonicus extract increased the activity of SOD and GSH-Px, while decreased the content of MDA. In addition, it could increase the expression level of Bcl-2 mRNA and reduce the expression level of Bax mRNA as well.Conclusion TotalPanax Japonicus extract has anti-aging effect.

Objective To evaluate the feasibility and outcomes of different surgical approaches on the basis of sentinel lymph node biopsy (SLNB) in treating early-stage vulvar cancer, and discuss the proper strategy for individualized treatment. Methods The medical charts of patients with early-stage vulvar cancer treated in Sun Yat-sen University between January 2004 and December 2013 were retrospectively collected. A total of 74 patients who received sentinel lymph node(SLN)detection in primary surgery were enrolled (average age 55). The surgical approaches contained SLNB, inguinal lymphadenectomy (IL), and extensive vulvectomy. The SLN were examed on intraoperative frozen sections. The treatment protocols, lymphatic metastasis, postoperative recovery condition, recurrence and survival data were collected and analyzed. Results At least one SLN was successfully detected in 68 (92%,68/74) patients. SLN were positive in 21 patients, of whom 12 (group A) underwent bilateral IL, and 9 (group B) received radiotherapy without performed IL. SLN were negative in 47 patients, of whom 26 (group C) underwent bilateral IL and one of them had a non-SLN metastasis, and 21 (group D) were advised to follow-up. The coincidence of pathological results between frozen and paraffin sections was 100%. The sensitivity and specificity of SLNB for diagnosis of lymph node metastasis were 95% and 100%, respectively. A total of 44 complications happened in patients underwent SLNB and IL (group A and C), including 16 poor wound healing, 14 lymphedema, 8 lymphatic fistulas, 3 phlebothrombosis and 3 infections. There were no complications happened in patients underwent SLNB alone (group B and D), among whom the operation time, bleeding amount, and hospital stay were also significantly less than those in patients underwent SLNB and IL. The median follow-up time was 41 months and the 3-year overall survival rate was 85% in the whole series. Recurrences were observed in 11 patients and 9 of them died of the tumor with the median survival time of 15 months. In patients with positive SLN (group A and B), the 3-year overall survival rate was 58% with 8 patients died of the disease, including 4 in group A and 4 in group B. In patients with negative SLN (group C and D), the 3-years overall survival rate was 97% with one patient in group D died of the tumor, and significantly higher than that of patients with positive SLN (P=0.003). The 3-year overall survival rate was significantly difference. In univariate analysis by log-rank test showed that, neither in patients with nor without SLN metastasis the prognosis differed with respect to surgical approaches (group A vs B, P=0.709;group C vs D, P=0.253). Univariate analysis by log-rank test showed that, lymph node metastasis, pathological grade, depth of invasion, and tumor location could significantly affected survival (P<0.05), whereas age, tumor diameter, and surgical approach didn′t (P>0.05). Multivariate analysis showed that lymph node metastasis (RR=21.57, 95%CI:2.68-173.10, P=0.002) and tumor location (RR=7.85, 95%CI:1.79-34.50, P=0.024) were the independent factors for overall survival. Conclusions Lymph node metastasis is an independent prognosis factor for patients with early-stage vulvar cancer. SLNB could accurately diagnose the status of lymph nodes and help to decide subsequent treatment. The omissions of IL in patients with negative SLN avoid surgical morbidity and shorten postoperative recovery period without an increased risk of recurrence.

Objective:To explore the efficacy and prognosis factors of acute myeloid leukemia with a combination therapy of venetoclax.Methods:A retrospective analysis was performed on the clinical data of AML patients treated with a combination therapy of venetoclax from March 2020 to April 2023 in the First Hospital of Lanzhou University.The efficacy,adverse reactions and survival were observed,and the influencing factors were analyzed.Results:A total of 74 AML patients were included in this study,including 43 initially treated AML and 31 relapsed or refractory AML(R/R AML).The median age of 43 initially treated AML patients was 65 years old,the composite complete remission(cCR)rate was 67.4％(29/43),the objective response rate(ORR)was 72.1％(31/43),and the median overall survival(OS)was 17.3 months.The median age of 31 R/R AML patients was 51 years old,with a cCR rate of 38.7％(12/31),an ORR of 58.1％(18/31),and a median OS of 7.1 months.Sex,the blood cell count before VEN,gene mutation and prognosis stratification were related to whether to obtain cCR.Failure to obtain cCR was an independent risk factor for adverse outcomes.Conclusion:A combination therapy of venetoclax is safe and efficacious for AML.Its efficacy and survival are affected by molecular biology,cytogenetics and other factors.

Objective:To explore the correlation between gene mutations and clinical characteristics,prognosis of myelodysplastic syndromes(MDS).Methods:Clinical data of 131 patients with MDS were collected from the First Hospital of Lanzhou University from June 2015 to February 2023,which 19 of them developed into secondary acute myeloid leukemia(sAML)during follow-up time.Second generation sequencing technology was used to detect the mutation types of MDS disease-related genes,drawn gene maps,and analyzed their correlation and prognosis based on the clinical data of patients.Results:The median age of 131 MDS patients was 58(17-86)years old.The ratio of male to female was 1.3:1.A total of 148 gene mutations and 25 types were found in the center.U2AF1 and ASXL1 were often co-mutations with other genes,which were accompanied by 20q-and normal karyotype(NK)respectively.SETBP1 and SRSF2 were more common in patients over 60 years old,while NPM1 and WT1 under 60 years.Older patients had a higher the number of genetic mutations than younger patients.The incidence of SF3B1 and RUNX1 in males was higher than females and DNMT3A in females was higher than males.The number of gene mutations in sAML was higher than MDS(1.8 vs 1.0,P=0.006).The univariate and multivariate analysis showed that IPSS-R prognostic score≥ 3.5,TP53 were adverse factors for poor prognosis in MDS patients.Patients with monoallelic mutation(ma-TP53)and wild-type(wt-TP53)TP53 had OS better than biallelic mutation(bi-TP53)(P=0.003).The OS of MDS patients was better than sAML(P=0.01)and transplant patients was significantly better than non-transplant patients(P=0.036).Conclusion:Gene mutation is closely related to cytogenetic indexes and clinical features(peripheral blood cell count,sex,age).IPSS-R prognostic score and TP53 were risk factors affecting OS in MDS patients.