Effect of MELK Inhibitor OTSSP167 on Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To investigate the effect of MELK inhibitor OTSSP167 against diffuse large B-cell lymphoma (DLBCL).@*METHODS@#The effect of OTSSP167 on activity, proliferation, and apoptosis of DLBCL cell line (SUDHL2 and HBL1) was detected by CCK-8 assay, 5-ethynyl-2'-deoxyuridine (EdU) staining, and Annexin V-FITC/PI double staining, respectively. DLBCL cells were inoculated into nude mice, after 4 weeks of OTSSP167 treatment, the effect of OTSSP167 on DLBCL growth in vivo was detected. Caspase-GloTM 3/7 enzyme activity assay kit was used to detect the effect of OTSSP167 on Caspase 3/7 enzyme activity of DLBCL cells. The expression levels of apoptosis and cycle-related proteins were detected by Western blot.@*RESULTS@#OTSSP167 significantly inhibited the activity of SUDHL2 and HBL1 cells in a dose-dependent manner (r =-0.61, r =-0.52). EdU staining showed that OTSSP167 could significantly inhibit the proliferation of SUDHL2 and HBL1 cells. Annexin V-FITC/PI result showed that OTSSP167 could significantly promote the apoptosis of SUDHL2 and HBL1 cells (P <0.001). The result of in vivo experiment showed that OTSSP167 could inhibit the growth of SUDHL2 cells in nude mice. The result of TUNEL staining of tumor further confirmed that OTSSP167 could promote the apoptosis of SUDHL2 cells. Caspase 3/7 enzyme activity test demonstrated that OTSSP167 could significantly increase caspase activity in SUDHL2 and HBL1 cells (r =0.98, r =0.87). Western blot showed that OTSSP167 could dose-dependently inhibit the expression of PARP, Bcl-xL, and Bcl-2 in apoptosis signaling pathway (r =-0.93, r =-0.66, r =-0.87), while p53 protein was significantly up-regulated (r =0.82). The expression of cell cycle-related proteins cdc2, Cyclin E1, Cyclin A2, and Cyclin B1 also showed a dose-dependent down-regulation (r =-0.89, r =-0.83, r =-0.61, r =-0.93).@*CONCLUSION@#The MELK inhibitor OTSSP167 can inhibit the proliferation and promote the apoptosis of DLBCL cells by inhibiting the expression of cycle-related proteins and anti-apoptosis-related proteins.

Effect of Manual and Machine Decocting on Chemical Constituents in Bazhentang Based on Non-targeted Metabolomics / 中国实验方剂学杂志

Objective: To evaluate the effect of manual decocting and machine decocting on the chemical constituents in Bazhentang based on non-targeted metabolomics, and to find the differential chemical constituents of these two decocting methods. Method: Bazhentang was boiled by standardized manual decocting and machine decocting methods,respectively. Orthogonal partial least square-discriminate analysis(OPLS-DA) and other multivariate statistical methods, combined with variable importance in the projection(VIP) value and t-test, were employed to analyze the effect of two decocting methods on the chemical constituents in Bazhentang. The differential chemical constituents were analyzed by UPLC-LTQ-Orbitrap MS under positive and negative ion modes,mobile phase was acetonitrile-0.1%formic acid aqueous solution for gradient elution,the scanning range was m/z 50-1 500. Result: Under the positive and negative ion modes of high-resolution mass spectrometry, a total of 87 differential components were found,40 of them were identified according to the mass spectrometry data and literature reports, including senkyunolide A, glycyrrhizin, ferulic acid, etc. Conclusion: Based on the analysis of color and chemical compositions of Bazhentang, there are obvious differences between the standardized manual decocting and machine decocting. If the advantages of these two methods are combined,a standardized decoction process can be established on the basis of maintaining the advantages of manual decocting, the effectiveness of traditional Chinese medicine decoction will be maximized and it will be convenient for patients to take it.

Effects of P 311 on the migration of epidermal stem cells in mice with superficial partial-thickness burn and injured cell model in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To study effects of P311 on the migration of epidermal stem cells (ESCs) in mice with superficial partial-thickness burn and injured cell model in vitro and to explore the mechanism.</p><p><b>METHODS</b>(1) Eighteen male C(57) BL/6 mice were used. Fifteen of them were inflicted with superficial partial-thickness burn on the back. In three injured mice wound tissue and skin of wound edge were obtained at post burn hour (PBH) 6, 12, 24, 48, 72 respectively. The rest three mice were used as normal control, and samples were harvested with the same method as above. The expressions of P311 in harvested samples were assessed with biotin-streptavidin-peroxidase (SP) staining. (2) Six newly born C(57) BL/6 mice were intraperitoneally injected with 50 µg/g BrdU (two times a day) for three days for ESCs-labelling. Seven weeks later, the mice were inflicted with superficial partial-thickness burn on the back. Serial slices of burn wound tissue were prepared at PBH 72 and immunohistochemically stained with SP for observation of the co-localization of BrdU-positive ESCs and P311-positive cells. (3) The empty vector pAdEasy-enhanced green fluorescence protein (EGFP) and the adenovirus P311-expressing vector named pAdEasy-EGFP-P311 were constructed and packed. Human ESCs were isolated by the method of rapid adhesion to collagen IV. After being divided into P311 high-expressing group (n = 3) and EGFP control group (n = 3), the ESCs in two groups were respectively infected by pAdEasy-EGFP-P311 and pAdEasy-EGFP. Scratching assay was performed on ESCs in both groups after they were treated by mitomycin C for 2 hours. The remaining area within the fixed range was measured at post scratching hour (PSH) 0, 24, 48, and 72, and the wound-area healing rate was calculated. Data were processed with independent samples t test.</p><p><b>RESULTS</b>(1) Expression amount of P311 was different in different parts of wound at different time points after burn. Expression amount of P311 in the newly formed epidermis and hair follicle of wound increased along with prolongation of time. Expression amount of P311 in the epidermis and hair follicle of wound edge peaked at PBH 12 and then decreased to normal levels at PBH 72. (2) Co-localization of BrdU-positive ESCs and P311-positive cells was observed in the new epidermal layer of wound tissue of mice, where ESCs were labeled by BrdU. (3) At PSH 48 and 72, wound-area healing rate was obviously higher in P311 high-expressing group [(69 ± 31)%, (89 ± 26)%] than in EGFP control group [(35 ± 12)%, (46 ± 31)%, with t values respectively -2.336, -2.611, P values all below 0.05].</p><p><b>CONCLUSIONS</b>P311 may promote the migration of ESCs both in rats with superficial partial-thickness burns and in injured cell model in vitro, and it may play an important role in wound healing.</p>

Biologic effect of nitric oxide on human epidermal stem cells in vitro / 中华烧伤杂志

<p><b>OBJECTIVE</b>To observe the effect of nitric oxide (NO) on adhesion, proliferation, and migration of human epidermal stem cells (ESC) in vitro.</p><p><b>METHODS</b>ESC were isolated and cultured by the modified method of rapid attachment to type IV collagen. (1) Morphology of cells was observed under inverted phase-contrast microscope. Expression levels of integrin β(1) and cytokeratin 19 (CK19) of cells were determined by Western blotting and immunofluorescence staining. (2) After being treated with scratching, ESC adhered to the wall was respectively treated with nitric oxide (NO) donor S-nitroso-N-acetylpenicillamine (SNAP) in the concentration of 1, 10, 100, 500 µmol/L. ESC without treatment of SNAP was used as control. The migration rate of ESC was detected at post scratching hour (PSH) 12 and 24. The chemotaxis of ESC (treated with SNAP in above-mentioned concentration) was tested by Transwell assay, and the transferred cell number was counted. (3) ESC was respectively treated with SNAP in the concentration of 10, 100, 500 µmol/L for 1 h. ESC without treatment of SNAP was used as control. The adhesion of ESC was detected with adhesion test, and the inhibition rate of adhesion was calculated. The proliferation of ESC (denoted as absorbance value) was determined by microplate reader at post-treatment hour (PTH) 0, 12, 24, 48. Data were processed with one-way analysis of variance and Dunnett t test.</p><p><b>RESULTS</b>(1) Small clone formed on post culture days (PCD) 5 to 9. On PCD 10 to 14, cell proliferation sped up. CK19 and integrin β(1) were detected to be expressed in the isolated cells. The cells were identified as ESC. (2) Compared with that of ESC without treatment of SNAP [(35.7 ± 0.3)%, (45.7 ± 5.0)%], migration of ESC treated with SNAP in the concentration from 1 to 100 µmol/L was promoted at PSH 12 and 24. Migration rates of ESC treated with 100 µmol/L SNAP were the highest [respectively (48.8 ± 2.7)%, (82.1 ± 15.8)%, with t value respectively 8.34, 5.10, P values both below 0.01]. The number of ESC transferred to membrane after being treated with 100 µmol/L SNAP was significantly larger than that of ESC without treatment of SNAP (t = 9.24, P = 0.00). (3) Absorbance values of ESC treated with 100, 500 µmol/L SNAP were obviously higher than that of ESC without treatment of SNAP (with t value respectively 4.30, 4.67, P values both equal to 0.00). Proliferation of ESC treated with 100, 500 µmol/L SNAP was obviously stronger than that of cells without treatment of SNAP at PTH 24, 48 (with t values from 2.84 to 8.17, P values all below 0.05).</p><p><b>CONCLUSIONS</b>Exogenous NO in suitable concentration can promote the migration of human ESC. Exogenous NO can inhibit the adhesion and promote the proliferation of human ESC in vitro.</p>