Considerations on investigation on quality standard of Chinese patent medicine / 药学学报

Chinese patent medicine (CPM) is an important part of traditional and Chinese medicine (TCM). Its quality has direct impact on the safety and effectiveness of clinical use. The quality standard is the pivotal approach to guarantee the quality of CPM. Due to the complex material basis, multitudinous quality influencing factors and unveiled active ingredients, dose-effect relationship and action mechanism, the investigation on quality standard faces many difficulties. This paper surveys the current quality status of CPM and the general situation of CPM standards. At present, the dosing problem has the crucial impact on the quality of CPM. The current quality standard system of CPM is confirmed and the limitations are indicated. Based on the above analysis, the principles and considerations on investigation of quality standard are proposed as follows: ① Adhere to safety as the bottom line, strengthen the risk-control ability of the standard of CPM; ② Adhere to theory of TCM and comprehensive quality, improve the integrative control level of the CPM standard; ③ Emphasize technological development and innovation, promote the quality control competence of CPM standard; ④ Facilitate planning and coordination, optimize the management of the CPM standard system; ⑤ Reinforce investigation on evaluation method, develop grade evaluation standard, accelerate high-quality development of CPM. Finally, the future perspective on investigation of CPM quality standard is prospected.

Value of (11)C-PiB PET/MRI in the evaluation of organ involvement in primary systemic light chain amyloidosis / 中华血液学杂志

Objective: To analyze the value of (11)C-PiB PET/MRI for evaluating organ involvement in patients with primary light chain amyloidosis (pAL) . Methods: The clinical data of 20 patients with pAL and 3 healthy volunteers from January 2019 to October 2021 were retrospectively analyzed. The correlation between the organ involvement evaluated by clinical standards and PET/MRI was compared. The relationship between cardiac-related biological indicators, disease stage, and the maximum standardized uptake value (SUVmax) were analyzed. The relationship between 24-hour urinary protein quantification and kidney SUVmax was analyzed. Results: ①In 20 patients (18 newly diagnosed patients and 2 non-newly diagnosed patients) ,(11)C-PiB positive uptake was observed in the heart (15 patients, 75%) , lung (8 patients, 40%) , bone marrow (10 patients, 50%) , muscle (10 patients, 50%) , tongue muscle (7 patients, 35%) , thyroid (6 patients, 30%) , salivary gland (4 patients, 20%) , spleen (2 patients, 10%) , and stomach wall (1 patient, 5%) . ②Organ involvement on (11)C-PiB PET/MRI showed good correlations with the clinical evaluation criteria for the heart and bone marrow. The positive rate of PET/MRI evaluation in the lung, spleen, gland, muscle, and tongue muscle was significantly higher than the clinical criteria. However, (11)C-PiB PET/MRI has limitations in the evaluation of the nervous system and fat tissue. ③To analyze the relationship between cardiac-related biological indexes and the SUVmax of the heart in 13 newly diagnosed patients. Patients with left ventricular ejection fraction (LVEF) <50% and interventricular septal thickness (ISV) ≥1.2 cm showed a higher SUVmax than patients with LVEF ≥50% and ISV<1.2 cm (P<0.05) .There are significant differences in the SUVmax of the heart between the Mayo2004 stage and the Mayo2012 stage. The later the disease stage, the higher the SUVmax (P<0.05) . The SUVmax of the heart was positively correlated with cardiac troponin I (cTnI) and N-terminal pro-brain natriuretic peptide (NT-proBNP) (P<0.01) .There was no significant correlation between renal SUVmax and 24-hour urine protein (P>0.05) . Conclusion: Whole body (11)C-PiB PET/MRI, as a visualization system of amyloid protein, is used to qualitatively evaluate organ involvement, which can improve the level of early non-invasive diagnosis. Whole body (11)C-PiB PET/MRI can be used to perform quantitative evaluation of organ levels, especially the heart, which is expected to evaluate organ function and predict disease prognosis more accurately.

Study on potential hepatotoxicity of rhein in Rhei Radix et Rhizoma based on liver metabolism / 中国中药杂志

The bilirubin metabolism mediated by the phase Ⅱ metabolizing enzyme UGT1A1 in the liver was evaluated to study the potential hepatotoxicity risk based on investigation on the inhibitory effect of rhein and its metabolites on the UGT1A1 enzyme in Rhei Radix et Rhizoma. Firstly, in vitro liver microsomes incubation was used to initiate the phase Ⅱ metabolic reaction to investigate the inhibitory effect of rheinon UGT1A1 enzyme. Secondly, the phase Ⅰ and phase Ⅱ metabolic reactions were initiated to investigate the hepatotoxicity risk of rhein metabolites. It was found that the rhein and its phase Ⅱ metabolites had no significant inhibitory effect on UGT1A1 enzyme, but its phase Ⅰ metabolites significantly reduced UGT1A1 enzyme activity. Based on the metabolites analysis, it is speculated that the rhein phase Ⅰ metabolite rheinhydroxylate and its tautomers have certain hepatotoxicity risks, while the toxicity risk induced by the prototype and phase Ⅱ metabolites of rheinglucoside, rheinglucuronic acid and rhein sulfate is small.

Research progress on chemical constituents and quality control of Tripterygium wilfordii preparations / 中国中药杂志

Tripterygium wilfordii preparations,with various biological activities such as immunosuppressive,anti-inflammatory and anti-cancer effects,are widely used in the treatment of autoimmune diseases such as rheumatoid arthritis,lupus erythematosus,and nephrotic syndrome. They have definite therapeutic effect,but often cause serious adverse reactions and result in damages to liver,kidney,blood,reproduction,and other systems due to their complex compositions,great toxicity,and narrow margin between the toxic and therapeutic dosages. At present,T. wilfordii preparations produced by different manufacturers exhibit large variations in clinical efficacy and side effects in account of their different chemical compositions and quality fluctuation due to differences in raw materials and production process. However,the existing quality standards are controversial in terms of index components and content limit,which cannot be effectively used for the overall quality control of the preparations. In this paper,the research progress on chemical constituents,quality standard and quality control methods of four T. wilfordii preparations including Tripterygium Tablets,Tripterygium Zongtie Tablets,Tripterygium Shuangceng Tablets and Tripterygium Glycosides Tablets was reviewed,in order to provide ideas and reference for the quality improvement of this type of preparations.

Study on hepatotoxicity of physcion based on liver metabolism in vitro / 中国中药杂志

To evaluate the hepatotoxicity risks of physcion on the basis of the bilirubin metabolism mediated by glucuronidation of UDP-glucuronosyltransferases 1A1(UGT1A1 enzyme). The monomers were added into the rat liver microsomes to test the hepatotoxicity by using bilirubin as UGT1A1 enzyme substrate, with apparent inhibition constant K_i as the evaluation index. Liver microsome incubation in vitro was adopted to initiate phase Ⅱ metabolic reaction and investigate the inhibitory effect of physcion. Then the phase Ⅰ and Ⅱ metabolic reactions were initiated to investigate the comprehensive inhibition of metabolites and prototype components. The results showed that when only the phase Ⅱ reaction was initiated, physcion directly acted on the UGT1A1 enzyme in a prototype form, exhibited weak inhibition and the inhibition type was mixed inhibition; When the phase Ⅰ and Ⅱ reactions were initiated simultaneously, the inhibitory effects of physcion on UGT1A1 enzyme became strong and the inhibition type was mixed inhibition, suggesting that physcion had phase Ⅰ and Ⅱ metabolic processes, and the metabolites had strong inhibitory effect on UGT1A1 enzyme. This experiment preliminarily proved that the metabolites of physcion may be the main components to induce hepatotoxicity.

Prediction of potential drug interactions of apigenin based on molecular docking and in vitro inhibition experiments / 中国中药杂志

The purpose of this study was to investigate the effect of apigenin on UGT1 A1 enzyme activity and to predict the potential drug-drug interaction of apigenin in clinical use. First,on the basis of previous experiments,the binding targets and binding strength of apigenin to UGT1 A1 enzyme were predicted by computer molecular docking method. Then the inhibitory effect of apigenin on UGT1 A1 enzyme was evaluated by in vitro human liver microsomal incubation system. Molecular docking results showed that apigenin was docked into the active region of UGT1 A1 enzyme protein F,consistent with the active region of bilirubin docking,with moderate affinity. Apigenin flavone mother nucleus mainly interacted with amino acid residues ILE343 and VAL345 to form hydrophobic binding Pi-Alkyl. At the same time,the hydroxyl group on the mother nucleus and the amino acid residue LYS346 formed an additional hydrogen bond,which increased the binding of the molecule to the protein. These results suggested that the flavonoid mother nucleus structure had a special structure binding to the enzyme protein UGT1 A1,and the introduction of hydroxyl groups into the mother nucleus can increase the binding ability. In vitro inhibition experiments showed that apigenin had a moderate inhibitory effect on UGT1 A1 enzyme in a way of competitive inhibition,which was consistent with the results of molecular docking. The results of two experiments showed that apigenin was the substrate of UGT1 A1 enzyme,which could inhibit the activity of UGT1 A1 enzyme competitively,and there was a risk of drug interaction between apigenin and UGT1 A1 enzyme substrate in clinical use.

Quality control of Guci tablets using UPLC-ELSD fingerprint analysis coupled with chemometrics / 中国中药杂志

Ultra-performance liquid chromatography-evaporative light scattering detection (UPLC-ELSD) fingerprint analysis method was established for quality control of Guci tablets. Chromatographic separation was performed on Waters Acquity UPLC BEH C₁₈ column (2.1 mm×100 mm, 1.7 μm) at 30 °C of column temperature. Acetonitrile-0.1% formic acid solution was adopted as mobile phase for gradient elution. The flow rate was set at 0.3 mL·min⁻¹, and the injection volume was 3 μL. Detection was carried out on an ELSD with a nitrogen pressure of 0.28 MPa, drift tube temperature of 60 °C, and gain of 400. A total of 39 batches of samples produced by six manufacturers were measured by using the above method and the data were analyzed by ChemPattern software. The peak present in more than 75% of the samples was defined as a common peak, and 30 common peaks were determined. Among them, 19 peaks were identified by rapid resolution liquid chromatography/tandem mass spectrometry (RRLC-MS/MS) method, 16 of which were confirmed by reference substances. The similarity of the tested samples was 0.47-0.98, suggesting that the quality of the samples from different manufacturers varied greatly. Furthermore, principal component analysis (PCA) and hierarchical analysis (HCA) were performed to clarify the main different components in samples. The results indicated that there might be some feeding problems about Paeoniae Radix Alba, Notoginseng Radix et Rhizoma, and Clematidis Radix et Rhizoma in a few manufacturers. This study provided some evidences for the overall quality control of Guci tablets, as well as its quality standard improvements.

Analysis on clinicopathological features of 617 patients with colorectal cancer / 中国内镜杂志

Objective To summarize the clinicopathological features with 617 cases colorectal cancer and explore reliable clues for early diagnosis. Methods Retrospective analysis of clinical, endoscopic, pathological features and DNA mismatch repair of 617 cases of colorectal cancer was made from January 2008 to March 2017. Results The overall diagnostic yield of colorectal cancer was 2.35% (596/25 308). 18 patients were diagnosed as simultaneous multiple colorectal cancer (3.02%, 18/596). Males and females ratio is 1.34 : 1.00. The average age diagnosed was 66.8 years old. The proportion of colon cancer was 76.68% (457/596), while cancer located in right side of the colon was 39.17%. Occurrence rate of right colonic cancer were higher in female group (47.34%) than that in male group (33.46%) (P = 0.003). Well and moderately differentiated adenocarcinoma was observed in 84.60% (522/617) of the patients. The ratio of mucinous adenocarcinoma was 6.81% (42/617). Totally 230 patients received the DNA mismatch repair, and 57 patients were diagnosed as defective DNA mismatch repair (24.78%). Defective DNA mismatch repair (dMMR) was associated with right colonic cancer, poorly differentiated adenocarcinoma, signet-ring carcinoma and mucinous adenocarcinoma (P < 0.05). Conclusions Colonoscopy screening in the elderly patients deserves great attention. Raise awareness of simultaneous multiple colorectal cancer. Pay attention to the screening of right colon cancer in female. The DNA mismatch repair should be detected in right colonic cancer, poorly differentiated adenocarcinoma, signet-ring carcinoma and mucinous adenocarcinoma.

Identification of commercial Bupleuri Radix and its adulterants based on ITS2 barcode / 中草药

Objective DNA barcoding technology, a molecular identification method, is applied to distinguishing Bupleuri Radix from its adulterants in order to ensure the quality and clinical curative effect. Methods In this study, the internal transcribed spacer 2 (ITS2) regions of 85 samples were amplified by PCR and sequenced bi-directionally. Obtained sequences were assembled using CodonCode Aligner. The genetic distances were computed by MEGA 6.0 in accordance with the kimura 2-parameter (K2P) model and the phylogenetic tree was constructed by Neighbor-joining (NJ) method. Moreover, the secondary structure of ITS2 was predicted using ITS2 database websites. Results The intra-specific genetic distances were smaller than inter-specific ones in ITS2 regions of Bupleuri Radix. NJ tree and secondary structure results could distinctly differentiate quality product and adulterants. Only 64.7% of the 85 samples were in accordance with the requirements of Chinese Pharmacopoeia. Conclusion ITS2 sequence can accurately and reliably identify the authenticity of Bupleuri Radix and could provide a new technique to ensure clinical safety of this traditional Chinese medicine.

Simultaneous determination of ten bioactive constituents in Lonicerae Flos by HPLC-MS-MS / 中国中药杂志

In this study, an HPLC-MS/MS method was developed and validated for simultaneous determination of six iridoids and four flavonoids in batches of Lonicerae Flos samples. Chromatographic separation was performed on a Shiseido Capcell Pak-C₁₈ column (4.6 mm×250 mm, 5 μm). 0.1% Aqueous formic and acid (A) and acetonitrile (B) were adopted as mobile phase. Detection was carried out on a triple quadrupole mass spectrometer in the negative ion mode using an electrospray source. Multiple reaction monitoring (MRM) mode was employed. The developed method showed good linearity (R² ≥0.999 0) for all the analytes within the test ranges and the limits of quantification (LOQs) ranged from 7.4 to 31.0 μg•L⁻¹. The recoveries varied between 94.16% and 105.3%. The quantitative data indicated that the total content of iridoids (0.338%-1.440%) was much higher than that of flavonoids (0.015 4%-0.057 5%) in all samples. Moreover, it was found that there were significant differences in the content of six compounds among the samples from three different original plants, which might provide scientific evidences for the origin identification and quality control of Lonicerae Flos.

Chemical constituents from roots of Illicium majus / 中国中药杂志

Ten compounds, including seven sesquiterpenes, two phenols and one phenylpropanoid, were isolated from the roots of Illicium majus by means of silica gel, ODS, Sephadex LH-20, and preparative HPLC. On analysis of MS and NMR spectroscopic data , their structures were established as cycloparviflorolide (1), cycloparvifloralone (2), tashironin (3), tashironin A (4), anislactone A(5), anislactone B (6), pseudomajucin (7), syringaldehyde (8), methyl-4-hydroxy-3, 5-dimethoxybenzoate (9), and (E)-3-methoxy-4,5-methylenedioxycinnamic alchol (10), respectively. Compounds 1-4 and 8-10 were first isolated from this plant. In the in vitro assays, at a concentration of 1.0 x 10(-5) mol x L(-1), compounds 5 and 6 were active against LPS induced NO production in microglia with a inhibition rate of 75.31% and 53.7%, respectively.

Study on the diterpenoids from radix illicii Maji and their antiviral activity against coxsackie B virus / 国际药学研究杂志

Objective To investigate he diterpenoids from he roots of Illicium majus(Radix Illcii Maji) and their antiviral activity against the Coxsackie B virus. Methods The compounds were isolated by column chromatography over silica gel, octadecylsi-ane chemically bonded silica gel(ODS), and Sephadex HL-20 coupled with preparative HPLC. Their stuctures were elucidated by spectroscopic analysis and the in situ dimolybdenum circular dichroism(CD) method, and their antiviral activities against the Coxsackie B3 virus were evaluated by cytopathic effect(CPE) method. Results Twelve diterpenoids were isolated from the roots of Illicium ma-jus, which were identified as 4-epi-dehydroabietic acid(l), 8,11,13,15-abietatraen-19-oic acid(2), jiadifenoic acids B(3), C(4), G(5) and 1(6), majusanic acids B(7) and D(8), lambertic acid(9), angustanoic acids F(10) and G(ll), and 13-hydroxy-8,11, 13-podocarpatrien-19-oic acid(12). These diterpenoids displayed antiviral activity against the Coxsackie B3 virus, with IC50 values of 3. 3-66. 7 μmol/ml. Conclusion The antiviral activity and cytotoxicity of the diterpenoids relate o he substituent species and position. Compounds 3-6 and 9 were obtained from his plant for the first time.

Hepatitis B virus reactivation in a chronic myeloid leukemia patient treated with imatinib mesylate / 中华医学杂志(英文版)

Imatinib mesylate is a molecular targeted agent for treating chronic myeloid leukemia (CML) and gastrointestinal stromal tumor. Although imatinib mesylate is not regarded as an immunosuppressive agent, few studies have also shown that it may impair immune response. In this report, we present a case of transient hepatitis B virus (HBV) reactivation during imatinib mesylate treatment for CML.

Reactivation of chronic hepatitis B infection related to imatinib mesylate therapy in patients with chronic myeloid leukemia: two cases report and literatures review / 中华血液学杂志

<p><b>OBJECTIVE</b>To probe the cause for triggering HBV reactivation and possible management of the chronic hepatitis B individuals received imatinib.</p><p><b>METHODS</b>This study presented two cases of transient hepatitis B virus (HBV) reactivation and hepatic dysfunction during oral imatinib for chronic myeloid leukemia (CML) and made a literatures review about the pathogenesis, possible prophylactic and therapeutic management of such chronic hepatitis B individuals receiving imatinib.</p><p><b>RESULTS</b>Two CML patients, without prior liver dysfunction but with chronic HBV infection, suffered from transient HBV reactivation occurred during oral imatinib. Both of them finally obtained good outcome following the additional oral nucleotide antiviral therapy.</p><p><b>CONCLUSION</b>It remained unclear whether imatinib induced the reactivation of HBV in patients with a latent HBV infection. From our study, all candidates receiving oral imatinib should be screened for HBsAg and anti-HBc antibodies prior to initiation of imatinib. Prophylactic antiviral therapy should be offered to HBV-infected individuals along with a close monitoring for signs of reactivation.</p>

Effect of RNA interference targeting brain-derived neurotrophic factor gene on HeLa cell proliferation and apoptosis / 中华病理学杂志

<p><b>OBJECTIVE</b>To screen effective sequences of short hairpin RNA on brain-derived neurotrophic factor (BDNF) gene and the effect of RNA interference on the proliferation and apoptosis of HeLa cells, a cervix carcinoma cell line with high expression of BDNF.</p><p><b>METHODS</b>Two recombinant eukaryotic human-BDNF siRNA expression vectors were designed and constructed. Sequences were confirmed by restrictive endonuclease digestion and DNA sequencing. The empty vector pGenesil-1 and two recombinant plasmids, pGenesil-shRNA-BDNF1 and pGenesil-shRNA-BDNF2, were transfected into HeLa cells using Lipofectamine 2000 (groups: P(0), P(1) and P(2), respectively). The mRNA and protein levels of BDNF in HeLa cells were detected by RT-PCR and Western blot, respectively. The cellular proliferation rates were determined by MTT assay and the apoptotic rates were measured by flow cytometry and Hoechest 33258.</p><p><b>RESULTS</b>The recombinant eukaryotic BDNF siRNA expression vectors were successfully constructed. The expression of mRNA and protein of BDNF in P(1) group were significantly decreased, comparing with non-transfected group, P(0) and P(2) groups (F = 48.19, P < 0.01). P(2) group failed to meet the expected results (P > 0.05). In addition, the proliferation activity was reduced in P(1) group and the peak point of proliferation curve was prolonged. Moreover, the early cell apoptotic rates were statistically increased in P(1)[(53.4 +/- 4.2)%] VS. non-transfected [(0.8 +/- 0.4)%], P(0) [(5.1 +/- 1.8)%] and P(2)[(7.9 +/- 2.4)%] groups (F = 269.77, P < 0.01).</p><p><b>CONCLUSION</b>HeLa cells express a high level of BDNF. BDNF gene silencing by RNA interference increases the apoptosis of HeLa cells and inhibits cell proliferation, offering a possible target for efficient tumor therapy.</p>

Antitumor effect of anti-brain derived neurotrophic factor monoclonal antibody in human multiple myeloma xenograft animal model / 中国实验血液学杂志

This study was aimed to further explore whether brain derived neurotrophic factor (BDNF) pathway is a potential therapeutic target in multiple myeloma (MM) and whether anti-BDNF monoclonal antibody can prevent the development of this disease. The in vivo antitumor effect of anti-BDNF monoclonal antibody (McAb) on a human myeloma xenograft animal model was evaluated. The model of xenograft tumors was established in the nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice by subcutaneous injection of human myeloma cell line RPMI8226. The antibodies were injected intraperitoneally at a dose of 20 microg/mouse at day 1, 2, 3 after inoculation or at a dose of 100 microg/mouse once a week after tumors were detected. The microvascular densities in tumors were analyzed by immunohistochemistry study. The effect of anti-BDNF McAb on the proliferation of RPMI8226 cells in vitro and on endothelial cells network formation in the co-culture system were determined by using a (3)H-thymidine incorporation assay and a Matrigel network formation assay, respectively. The results showed that multiple injections of anti-BDNF McAb reduced the tumor size, decreased the microvascular density and significantly prolonged tumor-free time and survival time. Moreover, the proliferation of RPMI8226 cells was inhibited in vitro by anti-BDNF McAb, but not by the control IgG. Anti-BDNF McAb also inhibited RPMI8226-induced network formation in endothelial cells in vitro. It is concluded that anti-BDNF monoclonal antibody can inhibit cell growth and angiogenesis in subcutaneous plasmacytoma.

Role of multiple myeloma cells on normal endothelial cells in co-culture system / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of multiple myeloma (MM) cells on normal endothelial cells in co-culture system in vitro.</p><p><b>METHODS</b>A co-culture system of human MM cell line RPMI8226 with human umbilical vein endothelial cells (HUVECs) was established in vitro. Mono-cultured normal endothelial cells were used as control. Light microscopy and transmission electron microscopy were used to observe the morphology of the endothelial cells. The effects of HUVECs co-cultured with RPMI8226 on HUVECs angiogenesis were studied by modified transwell migration assay and net-like formation assay. The protein expression of brain derived neurotrophic factor (BDNF), TrkB, Endoglin, Tie-2, beta 3 integrin and vascular cell adhesion molecule-1 (VCAM-1) in HUVECs were determined by FACS and Western blot analysis, respectively.</p><p><b>RESULTS</b>The morphology of HUVECs co-cultured with RPMI8226 cells became a narrower apart of extended shape as they began to align themselves. The sizes of nucleus and nucleolus were enlarged with an increased ratio of nuclear to nucleoplasm. The endoplasm was lose and distorted and the number of surface microvilli decreased. The RPMI8226 cell stimulated the migration and net-like formation of HUVEC, the number of net-like structure and migration cell being increased by 112% and 136%, respectively, compared with that of mono-cultured HUVECs. The expressions of BDNF, TrkB, Endoglin, Tie-2, beta 3 integrin and VCAM-1 in the ECs co-cultured with RPMI8226 were all up-regulated in comparison with those in the controls.</p><p><b>CONCLUSION</b>The MM cells promote formation of new vessels in co-cultured endothelial cells and the endothelial cells in MM are different from the normal ECs in character, and behavior.</p>

Brain derived neurotrophic factor induces endothelial cells angiogenesis through AKT and ERK1/2 signal pathway / 中国实验血液学杂志

Our previous studies have demonstrated the effects of brain derived neurotrophic factor (BDNF) on promoting proliferation of multiple myeloma (MM) cells and inducing angiogenesis in MM in vitro. To further investigate whether the PI3K/Akt and MEK1/ERK pathway play a role in the BDNF-induced angiogenesis in vitro and to explore the further molecular mechanisms, two ways to establish human myeloma xenograft animal model were developed, their advantages and disadvantages were elucidated. The phosphorylation of AKT and ERK1/2 were detected in human umbilical vein endothelial cells (HUVECs) by Western blot. The angiogenic activity in vitro was evaluated by transwell migration assay and tubule formation assay. Cell proliferation was determined by crystal violet staining. Cell apoptosis was detected by FITC-Annexin-V/PI double staining and flow cytometry. The results showed that the BDNF activated the PI3K/Akt and MEK1/ERK pathway in the time-dependent manner. Ly294002 and PD98059 blocked the activation of Akt and ERK1/2 respond to BDNF. 100 ng/ml BDNF significantly increased HUVEC tube formation, migration and proliferation in vitro at a similar degree of 25 ng/ml VEGF. Furthermore, tube formation of HUVECs toward BDNF was significantly inhibited by 57% and 40% with 20 micromol/L Ly294002 and 20 micromol/L PD98059 treatment, respectively. At the same time, Ly294002 and PD98059 reduced the BDNF-induced migration of HUVECs by 74% and 36%, respectively. While BDNF-induced survival was only blocked by Ly294002 and BDNF-induced proliferation was only inhibited by PD98059. It is concluded that BDNF promotes angiogenesis of HUVECs in vitro. ERK and Akt are two crucial events in BDNF-mediated signal transduction leading to HUVECs angiogenesis by different mechanisms. Moreover, the latter is more important.

Monoclonal antibody against brain derived neurotrophic factor inhibits myeloma growth and angiogenesis in the xenograft NOD/SCID animal model / 中华血液学杂志

<p><b>OBJECTIVE</b>To evaluate the in vivo antitumor effect of anti-brain derived neurotrophic factor (BDNF) monoclonal antibody (MoAb) on a human myeloma xenograft animal model.</p><p><b>METHODS</b>The xenograft tumor model was established in the nonobese diabetic/severe combined immunodeficiency (NOD/ SCID) mice by subcutaneous injection of human myeloma cell line RPMI 8226. The antibodies were injected intraperitoneally at a dose of 20 microg/mouse at day 1, day 2, day 3 after tumor cell inoculation or at a dose of 100 microg/mouse once a week after tumors were developed. The histologic and cytologic examination were performed to confirm the development of plasmacytomas. The microvascular densities (MVD) in tumors were analyzed by immunohistochemistry. The effect of anti-BDNF MoAb on the proliferation of RPMI 8226 cells in vitro and on endothelial cell network formation in the co-culture system were determined by 3H-thymidine incorporation assay and Matrigel network formation assay, respectively.</p><p><b>RESULTS</b>The xenograft NOD/SCID animal model had high capacity for growth of RPMI 8226 subcutaneous tumors and presented pathologic features of plasmacytomas. After subcutaneous injection of RPMI 8226 cells, all mice developed localized tumors in (20 +/- 2) d. On 20 microg anti-BDNF MoAb 3 consecutive treatment, the mean tumor-free time was extended to (30 +/- 6) d and survival was significantly prolonged compared with IgG-treated group [(57 +/- 7) d vs (48 +/- 4) d, P < 0.05]. When mice died naturally, the tumors size in anti-BDNF MoAb treated ones was also reduced compared with control group [(157.9 +/- 21.6) mm3 vs (405.5 +/- 35.2) mm3, P < 0.05]. When the antibody treatment (100 microg/mouse) underwent from 27 th to 60 day once a week after tumor inoculation, the local tumor growth was inhibited partially and necrosis and infiltration were observed in the tumors. The median MVD in the antibody-treated mice (100 microg/mouse) was 11 vessels/0.216 mm2. The IgG treated mice had no decrease in MVD of subcutaneous tumors compared with untreated mice. In vitro, anti-BDNF MoAb (1.5 microg/ml) significantly but partially inhibited HUVEC network formation induced by RPMI 8226 (68.2% reduction) and significantly inhibited RPMI 8226 proliferation, too. The IgG (1.5 microg/ml) treated mice had no significant effect on both of two assays.</p><p><b>CONCLUSIONS</b>Anti-BDNF monoclonal antibody could inhibit growth and angiogenesis in subcutaneous myeloma tumors. BDNF is a potential therapeutic target in MM.</p>

Human multiple myeloma cell line RPMI8226 activates brain derived neurotrophic factor autocrine loop of co-cultured endothelial cells / 中华血液学杂志

<p><b>OBJECTIVE</b>To study the influence of multiple myeloma cells on normal endothelial cells in co-culture system.</p><p><b>METHODS</b>Human multiple myeloma cell line RPMI8226 was co-cultured with human umbilical vein endothelial cells (HUVECs). HUVECs cultured alone were used as control. The expression of brain derived neurotrophic factor (BDNF) and its specific acceptor TrkB mRNA and protein in HUVECs were determined by RT-PCR and Western blot, respectively, BDNF levels in culture supernatant by enzyme-linked immunosorbent assay (ELISA). After transferring the co-culture, the effects RPMI8226 on HUVECs angiogenesis were studied by modified transwell migration assay and net-like formation assay.</p><p><b>RESULTS</b>The median BDNF concentration in culture supernatant was increased in co-cultured HUVECs compared with that in HUVECs cultured alone [(31.6 +/- 7.2) ng/ml vs (12.4 +/- 5.1) ng/ml, P < 0.05]. The expression of BDNF transcript demonstrated by RT-PCR did the same in the two culture systems (1.7 fold increase, P < 0.05). TrkB mRNA was hardly detected in culture of HUVECs alone but was increased in co-cultured HUVECs (4.4- fold increase, P < 0.05). The BDNF and TrkB protein expressions determined by Western blot were similar to that of their mRNAs. On the other hand, the RPMI8226 activated HUVECs showed enhanced migration and net-like formation, being increased by 99% and 72% , respectively. Addition of anti-human BDNF antibody to the culture medium partly reduced these effects.</p><p><b>CONCLUSION</b>Multiple myeloma cells activated BDNF/TrkB autocrine loops in co-cultured endothelial cells and resulted in endothelial self-activating angiogenesis.</p>