Dimethyl Sulfoxide Control Group Research Articles

Nifuroxazide induces the apoptosis of human non‑small cell lung cancer cells through the endoplasmic reticulum stress PERK signaling pathway.

The aim of the present study was to investigate the molecular mechanism of nifuroxazide (NFZ) in the induction of apoptosis of NCI-H1299 human non-small cell lung cancer (NSCLC) cells through the reactive oxygen species (ROS)/Ca2+/protein kinase R-like ER kinase (PERK)-activating transcription factor 4 (ATF4)-DNA damage inducible transcript 3 (CHOP) signaling pathway. Morphological changes of cells were observed by microscopy, and the apoptosis and intracellular ROS levels of cells were observed by inverted fluorescence microscopy. Cell viability after the addition of the PERK inhibitor, GSK2606414, were detected by Cell Counting Kit-8 assay. Annexin V-FITC was used to detect cell apoptosis, Brite 670 was used to detect intracellular ROS and Fura Red AM was used to detect Ca2+ content. Western blotting was used to detect PERK, phosphorylated (P)-PERK, ATF4, CHOP, P-Janus kinase 2 and P-signal transducer and activator of transcription 3 expression levels. Compared with the dimethyl sulfoxide control group, NFZ inhibited the survival activity in the H1299 NSCLC cell line, in a time- and dose-dependent manner. However, GSK2606414 inhibited the NFZ-induced apoptosis of H1299 cells. GSK2606414 also inhibited the increase in ROS and Ca2+ in H1299 cells induced by NFZ. Western blotting results demonstrated that NFZ significantly increased the expression levels of P-PERK, ATF4 and CHOP, whereas GSK2606414 significantly reduced the NFZ-induced increase in these protein expression levels. In conclusion, NFZ may induce the apoptosis of H1299 NSCLC cells through the ROS/Ca2+/PERK-ATF4-CHOP signaling pathway.

Effect of extracellular signal-regulated kinase inhibitor on calpain in rat cerebral cortex after cardiopulmonary resuscitation

To explore the effect of extracellular signal-regulated kinase (ERK) inhibitor PD98059 on calpain-related proteins in the brain, and to understand the pathophysiological changes of calpain in cerebral ischemia/reperfusion injury (CIRI). Forty-two rats were divided into sham operation (Sham) group (n = 6), model group (n = 12), dimethyl sulfoxide (DMSO) control group (n = 12), and PD98059 group (n = 12) by random number table. The rat model of CIRI induced by cardiac arrest-cardiopulmonary resuscitation (CA-CPR) was reproduced by transesophageal electrical stimulation to induce ventricular fibrillation. In the Sham group, only the basic operations such as anesthesia, tracheal intubation, and arteriovenous catheterization were performed without CA-CPR. The rats in the DMSO control group and PD98059 group were injected with DMSO or PD98059 0.30 mg/kg via femoral vein, respectively, 30 minutes after the restoration of spontaneous circulation (ROSC), and rats in the Sham group and model group were given the same amount of normal saline. The duration of CPR, 24-hour survival rate and neurological deficit score (NDS) after ROSC were recorded. Hematoxylin-eosin (HE) staining and Nissl staining were used to observe the pathological changes of the cerebral cortex. The expressions of phosphorylated ERK (p-ERK), ERK, calpastatin, calpain-1, and calpain-2 were detected by Western blotting. The co-expression of p-ERK and calpain-2 was detected by double immunofluorescence. There were no significant differences in the duration of CPR and 24-hour survival rate among all groups. In the model group, the nuclei of the cerebral cortex were obviously deformed and pyknotic, cells vacuoles and tissues were arranged disorderly, Nissl corpuscles were significantly reduced, NDS scores were also significantly reduced, level of ERK phosphorylation was increased, and calpain-2 protein was significantly up-regulated compared with the Sham group. There was no significant difference in the above parameters between the DMSO control group and the model group. After intervention with PD98059, the pathological injury of brain tissue was significantly improved, Nissl corpuscles were significantly increased, the NDS score was significantly higher than that in the model group [75.0 (72.0, 78.0) vs. 70.0 (65.0, 72.0), P < 0.05], the level of ERK phosphorylation and calpain-2 protein expression were significantly lower than those in the model group [p-ERK (p-ERK/ERK): 0.65±0.12 vs. 0.92±0.05, calpain-2 protein (calpain-2/GAPDH): 0.73±0.10 vs. 1.07±0.14, both P < 0.05], while there was no significant difference in the expressions of calpastatin and calpain-1 in the cerebral cortex among all the groups. Double immunofluorescence staining showed that p-ERK and calpain-2 were co-expressed in cytosol and nucleus, and the co-expression rate of p-ERK and calpain-2 in the model group was significantly higher than that in the Sham group [(38.6±4.3)% vs. (9.2±3.5)%, P < 0.05], while it was significantly lowered in the PD98059 group compared with the model group [(18.2±7.0)% vs. (38.6±4.3)%, P < 0.05]. ERK together with calpain-2 participated in CIRI induced by CA-CPR. PD98059 inhibited the expression of calpain-2 and ERK phosphorylation. Therefore, ERK/calpain-2 may be a novel therapeutic target for CIRI.

Effect of Taspine hydrochloride on the repair of rat skin wounds by regulating keratinocyte growth factor signal

ABSTRACT To explore the regulation of keratinocyte growth factor (KGF) in the process of repairing rat skin wounds by taspine hydrochloride (TA/HCl), 45 male Sprague-Dawley (SD) rats were purchased and divided into an experimental group, a dimethyl sulfoxide (DMSO) control group, and a basic fibroblast growth factor (bFGF) control group, each with 15 only. A back trauma model was innovatively adopted to prevent rats from biting and contaminating. The wound healing time and healing rate of the rat, and the Hydroxyproline (Hyp) and KGF expressions were observed. Morphological changes of wound tissue and the number of capillaries were observed after hematoxylin-eosin (HE) staining. The results showed that wound healing rate of experimental group and bFGF group was significantly higher than that of DMSO group (P < 0.05) after 2–15 days, and wound healing time of experimental group was 18 days, which was significantly lower than that of the DMSO group (P < 0.05). Expression levels of Hyp and KGF in the granulation tissue of rats in the experimental group were much higher than those in the DMSO control group after trauma (P < 0.05). In early stage of wound tissue repair, the number of new capillaries formed in experimental group was significantly higher than that in DMSO control group (P < 0.05). In summary, this study innovatively focused on KGF. The mechanism of TA/HCL promoting rat skin wound healing was closely related to KGF.

Animal Experimental Study of the Role of Menthol and Cineole, Two Tobacco Additives, on Nicotine Dependence

To establish a nicotine intravenous self-administration rat model, and to examine, with this model, the effects of two flavoring additives, menthol and cineole, on nicotine dependence. Thirty male Sprague-Dawley (SD) rats were included in the study. After jugular venous catheterization was performed, fixed concentration of nicotine was administered in order to train the rats and establish the rat model of intravenous self-administration groups, receiving intraperitoneal injection of menthol, cineole, and dimethyl sulfoxide (DMSO), the vehicle that was used for the control group. The rats were tested with different fixed-ratio (FR) schedules, including FR1 schedule, in which the rat received one nicotine infusion for every active nose poke, FR2 schedule, in which the rat received one nicotine infusion for every two active nose pokes, and FR5 schedule, in which the rat received one nicotine infusion for every five active nose pokes. The number of active and inactive poke responses and the number of nicotine infusion were documented accordingly. After 10 days of training in nicotine self-administration, the 30 rats demonstrated significant increase in the number of active poke responses and the number of nicotine infusion, which were maintained at a stable and relatively high level. The number of active poke responses was significantly higher that of inactive poke responses ( P< 0.001). The rat model of intravenous nicotine self-administration was successfully established. In the testing phase, under the FR2 schedule, the menthol group showed a reduced number of active poke responses ( P=0.020). Under the FR5 schedule, the groups showed obvious interaction between time and the number of active poke responses ( P<0.011), with the menthol group showing reduced number of active poke responses on day three ( P=0.011) and the cineole group showing rising number of active poke responses on day three ( P=0.003). The DMSO control group did not show any significant change. Menthol and cineole are shown to have an effect on nicotine dependence. When there is relative difficulty involved in obtaining nicotine, menthol suppresses nicotine dependence, whereas cineole enhances nicotine dependence.

NLRP3 inflammasome inhibitor CY-09 reduces hepatic steatosis in experimental NAFLD mice

AimsNon-alcoholic fatty liver disease (NAFLD) has become one of the most common chronic liver diseases. The NOD-like receptor protein 3 (NLRP3) inflammasome was suggested to be involved in the pathogenesis of NAFLD. A small-molecule named CY-09 is a new selective and direct inhibitor of the NLRP3 inflammasome. We aimed to investigate whether CY-09 is effective for the treatment of NAFLD in a high-fat diet (HFD)-induced mouse model. MethodsTwenty mice were fed by HFD for 14 weeks, and then were randomly assigned into two groups: (1) control group receiving dimethylsulfoxide (DMSO) solution; (2) CY-09 group receiving CY-09 injection. In an 8-week follow-up, oral glucose tolerance test (OGTT) and homeostasis model assessment of insulin resistance (HOMA-IR) were used to measure glucose metabolism. Liver steatosis was evaluated by the NAFLD activity score (NAS) and deemed as the primary outcome. ResultsThe body weight in CY-09 group was significantly lower than the DMSO control group on 27 weeks (41.0 ± 3.5 g vs. 49.7 ± 5.2 g, P = 0.014). The area under the curve (AUC) of OGTT was less in CY-09 group than that in DMSO group (35.81 ± 6.79 vs. 22.91 ± 2.58 mmol/L·hr, P = 0.004), as well as HOMA-IR (14.36 ± 3.89 vs. 8.82 ± 2.04 mmol.mIU.L-2, P = 0.023). Microscopically, liver lipid droplets dramatically improved and significantly lower NAS was observed in CY-09 group (8.25 ± 1.26 vs. 3.20 ± 0.45, P < 0.001). ConclusionCY-09 reduces hepatic steatosis in experimental NAFLD mice and CY-09 may be a potential therapeutic drug of NAFLD in clinical practice.

Study on mechanisms of interleukin-17A regulating the expressions of interleukin-1β and interleukin-23 in mouse keratinocytes

Objective: To investigate the mechanisms of interleukin-17A (IL-17A) regulating the expressions of IL-1β and IL-23 in mouse keratinocytes (KCs). Methods: Primary KCs were isolated from the skin of 400 newborn male and female wild type C57BL/6 mice and cultured in 24-well plates with Roswell Park Memorial Institute 1640 medium containing fetal bovine serum in the volume fraction of 10% for the following experiments. (1) The cells were divided into phosphate buffer solution (PBS) control group and IL-17A stimulation group according to the random number table (the same grouping method below), which were cultured with 10 μL PBS or 10 μL IL-17A in the mass concentration of 100 ng/mL for 6 hours, respectively. The expression levels of IL-1β and IL-23 mRNA in cells were detected by real-time fluorescence quantitative reverse transcription polymerase chain reaction (RT-PCR), with 3 samples in each group. (2) The cells were divided into dimethyl sulfoxide (DMSO) control group, IL-17A+ DMSO group, IL-17A+ nuclear factor κB (NF-κB) inhibitor group, IL-17A+ signal transduction and activator of transcription 3 (STAT3) inhibitor group, IL-17A+ extracellular signal-regulated kinase 1 (ERK1) inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ c-Jun N-terminal kinase (JNK) inhibitor group. The reagents were added to cells in corresponding groups respectively and cultured for 6 hours. The volume of each reagent was 10 μL, the mass concentration of IL-17A was 100 ng/mL, and the molarity concentrations of NF-κB, STAT3, ERK1, ERK2, JNK signal pathway inhibitors PDTC, S3I-201, SCH772984, SCH772984, SP600125 were 5 μmol/L, 100 μmol/L, 4 nmol/L, 1 nmol/L, and 10 μmol/L, respectively. The expression levels of IL-1β mRNA and IL-23 mRNA in cells were detected by real-time fluorescence quantitative RT-PCR, with 3 samples in each group. (3) The cells were grouped and treated the same as those in experiment (1). The levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation were detected by Western blotting, with 3 samples in each group. Data were statistically analyzed with two-tailed Student t test, one-way analysis of variance, t test, and Bonferroni correction. Results: (1) After culture of 6 hours, compared with those in PBS control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A stimulation group were significantly increased (t=13.46, 6.72, P<0.01). (2) After culture of 6 hours, the expression levels of IL-1β and IL-23 mRNA in cells in DMSO control group, IL-17A+ DMSO group, IL-17A+ NF-κB inhibitor group, IL-17A+ STAT3 inhibitor group, IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group were 1.00±0.11, 4.01±0.32, 0.32±0.06, 1.76±0.43, 3.62±0.24, 3.80±0.43, 4.26±0.74 and 1.03±0.29, 4.08±0.34, 4.76±0.38, 4.70±0.21, 1.06±0.42, 0.92±0.21, 0.39±0.05, respectively. Compared with those in DMSO control group, the expression levels of IL-1β and IL-23 mRNA in cells in IL-17A+ DMSO group were significantly increased (t=9.24, 12.60, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-1β mRNA was significantly decreased in cells in IL-17A+ NF-κB inhibitor group and IL-17A+ STAT3 inhibitor group (t=11.34, 6.91, P<0.01). Compared with that in IL-17A+ DMSO group, the expression level of IL-23 mRNA was significantly decreased in cells in IL-17A+ ERK1 inhibitor group, IL-17A+ ERK2 inhibitor group, and IL-17A+ JNK inhibitor group (t=12.44, 13.03, 15.21, P<0.01). (3) After culture of 6 hours, compared with those in PBS control group, the levels of NF-κB phosphorylation, STAT3 phosphorylation, ERK phosphorylation, and JNK phosphorylation in cells in IL-17A stimulation group were significantly increased. Conclusions: IL-17A promotes the transcription of IL-1β in mouse KCs through the phosphorylation of NF-κB and STAT3 pathways and IL-23 through the phosphorylation of ERK and JNK pathways.

Effects of salinomycin on proliferation and apoptosis of oral squamous cell carcinoma

To investigate the effects of salinomycin on the proliferation and apoptosis of oral squamous carcinoma cells and to further understand the mechanisms of these effects. The human oral squamous carcinoma cell line CAL-27 was cultured in different concentrations of salinomycin and cisplatin. After co-culture with 0, 1, 2, 4, 8, 16 and 32 μmol/L salinomycin or 0, 1.25, 2.5, 5, 10, 20, 40 and 80 μmol/L cisplatin for 24 hours and 48 hours, the proliferation of oral squamous carcinoma cells were detected by cell counting kit-8(CCK-8) assay. After being exposed to 0, 2, 4, 8 μmol/L salinomycin and 0, 5, 10, 20 μmol/L cisplatin for 48 hours, the cell cycle of oral squamous carcinoma cells was detected by flow cytometry assay, and Western blot analysis was performed to analyze the expressions of cysteine-containing aspartate-specific proteases-3(Caspase-3), cysteine-containing aspartate-specific proteases-9(Caspase-9), poly ADP-ribose polymerase (PARP), protein kinase B (Akt) and phosphorylated protein kinase B (p-Akt) protein in oral squamous carcinoma cells. Both salinomycin and cisplatin significantly inhibited the proliferation of oral squamous cell carcinoma CAL-27 cells in a time- and dose-dependent manner. However, compared with the first-line chemotherapeutic drug cisplatin, salinomycin showed stronger anti-proliferation activity in oral squamous carcinoma cells than cisp-latin (P < 0.001). After being exposed to 8 μmol/L salinomycin, CAL-27 cells exhibited markedly higher proportion in quiescent/ first gap phases (40.40%±1.99% vs. 64.46%±0.90%, P < 0.05), and had a significantly lower proportion in synthesis phases and second gap / mitosis phases (24.32%±2.30% vs. 18.73%±0.61%, P < 0.05; 35.01%±1.24% vs. 16.54%±1.31%, P < 0.05) compared with the dimethyl sulfoxide control group; moreover cisplatin didn't show cell-cycle specific effect on CAL-27. Western blot proved that salinomycin could up-regulate the expressions of Caspase-3 and Caspase-9 protein in oral squamous cell carcinoma CAL-27 cells (P < 0.05). At the same time, the levels of PARP, Akt and p-Akt protein were down-regulated (P < 0.05). Compared with cisplatin, salinomycin has a better inhibitory effect on the proliferation of oral squamous carcinoma cells and blocks the cell cycle process at the quiescent / first gap phase. At the same time, salinomycin could trigger apoptosis of oral squamous carcinoma cells and the mechanism is associated with the Akt/p-Akt signaling pathway.

Role of LINC00310 in the glycidyl methacrylate-induced malignant transformation of human bronchial epithelial cell

To investigate the expression and biological significance of LINC00310 in the malignant transformation of human bronchial epithelial cells(16 HBE) induced by glycidyl methacrylate(GMA). The 16 HBE cells recovered successfully used 1 μg/mL dimethyl sulfoxide(DMSO) as the solvent control group, and the final concentration was 8 μg/mL GMA as the treatment group, and were subcultured after repeated exposure 3 times for 72 hours each time. The 10 th, 20 th and 30 th generation cells of the GMA treatment group and corresponding DMSO control group were collected. The LncRNA microarrays was used to analyze the expression changes of LINC00310 in different periods, and the target gene and function prediction was performed by NCBI and cBioPortal bioinformatics database, and real-time quantification PCR(qPCR) was used to detect the relative expression levels of LINC00310 and predicted target genes. The result of the microarray showed that LINC00310 in the GMA-treated group was down-regulated by 2. 02-fold, up-regulated by 6. 17-fold, and up-regulated by 2. 03-fold in the pre-transformation, mid-term, and late, respectively. The result of qPCR confirmed that the expression of LINC00310 relative expression level of 10 th, 20 th and 30 th generation cells was consistent with the microarray result, which were down-regulated by 2. 76-fold, up-regulated by 2. 68-fold, and up-regulated by 3. 09-fold. Consistently, the relative expression of the target gene C-Myc was statistically significant in 20 th and 30 th generation cells. LINC00310 induced low expression in the early stage of malignant transformation of 16 HBE cells induced by GMA, and was highly expressed in the middle and late stages. It indicated that LINC00310 may play a cancer-promoting role in the process of cell malignant transformation through C-Myc.

Evaluation of the in vivo wound healing potential of the lipid fraction from activated platelet-rich plasma

Previous in vitro studies suggest a direct relevance for the peptide–free lipid fraction (LF) of platelet-rich plasma (PRP) in biological mechanisms related to wound healing. However, there are no scientific reports to date on the wound healing activities of this lipid component in vivo. Thus, the present study provides a scientific evaluation for the wound healing potential of the lipid portion of the activated PRP. For the wound healing activity assessment, in vivo full‐thickness excisional wounds were created on the dorsal skin of Sprague-Dawley rats. Lipid extract from pooled PRP was applied topically to the wounds on 0, 3, and 7 days after injury. Histological assessment of epidermal and dermal regeneration, granulation tissue thickness and angiogenesis by Sirius red and Masson’s trichrome staining, in addition to immunohistochemical staining for transforming growth factor beta-1 (TGF-β1), collagen type I (COL I), and collagen type III (COL III) were performed on skin biopsies at 3, 7 and 14 days. The total histological scores of the LF group were significantly higher than the 25% dimethylsulfoxide-control group. According to the immunohistochemical staining, the observed expression changes for TGF-β1, COL I and III at 3, 7, and 14 days after wounding were significantly better in the study group than the control group. Furthermore, COL I/III ratio in the lipid extract-treated group at day 14 was much higher than that of the control group. Meanwhile, analysis of the data also indicated that the LF has less positive effects on all evaluated parameters than PRP. From the present data, it could be concluded that the peptide–free LF of PRP has potent wound healing capacity in vivo for cutaneous wounds, although not as much as that of PRP. Strengthening our understanding of the wound healing potential of lipid components of PRP and platelet-derived lipid factors may provide new avenues for improving the healing process of a wound with elevated protease activity.

Effects of carbon monoxide release molecule-2 on sepsis-induced myocardial dysfunction in rats

To investigate the protective effect of carbon monoxide release molecule-2 (CORM-2) on sepsis-induced myocardial dysfunction in rats. 140 healthy male Sprague-Dawley (SD) rats were divided into sham operation (Sham) group, model group, CORM-2 pretreatment group, inactivated carbon monoxide release molecule-2 (iCORM) pretreatment group, and dimethyl sulfoxide (DMSO) control group by random number table, with 28 rats in each group. The rat sepsis model was reproduced by intraperitoneal injection of 10 mg/kg lipopolysaccharide (LPS). The rats in the Sham group were injected intraperitoneally with the same dose of normal saline (NS). The rats in the CORM-2 and iCORM-2 pretreatment groups were injected intraperitoneally with 8 mg/kg CORM-2 or iCORM-2 at 1 hour before LPS injection, respectively, and those in the DMSO group were injected intraperitoneally with the same dose of DMSO, but the rats in the Sham group and the model group were not treated after injection of NS or LPS. Twenty rats were randomly selected from each group to observe 10-day survival rate. Transthoracic echocardiography was performed on the remaining 8 rats at 12 hours after modeling, and the left ventricular ejection fraction (LVEF) and left ventricular fraction shortening (LVFS) were calculated to evaluate heart function. The blood of the inferior vena cava was harvested, then serum myocardial troponin I (cTnI) and brain natriuretic peptide (BNP) levels were measured by enzyme-linked immunosorbent assay (ELISA). Then the rats were sacrificed, and the myocardial tissues were harvested, the pathological morphology and ultrastructure of myocardium were observed. (1) Survival rates: all rats in the Sham group survived; compared with the Sham group, the survival rates of the model group, CORM-2 pretreatment group, iCORM-2 pretreatment group and DMSO control group were significantly decreased at 10 days [10% (2/20), 70% (14/20), 25% (5/20), 15% (3/20) vs. 100% (20/20), all P < 0.01]. However, the 10-day survival rate in the CORM-2 pretreatment group was significantly higher than those in the model group, iCORM-2 pretreatment group and DMSO control group (all P < 0.01). (2) Cardiac function: compared with the Sham group, LVEF and LVFS in the model group, CORM-2 pretreatment group, iCORM-2 pretreatment group and DMSO control group were significantly decreased, and left ventricular dilatation was obvious, indicating myocardial dysfunction in rats. However, LVEF and LVFS in the CORM-2 pretreatment group were significantly higher than those in the model group, iCORM-2 pretreatment group, and DMSO control group [LVEF: 0.760±0.029 vs. 0.634±0.021, 0.629±0.066, 0.673±0.023; LVFS: (39.32±2.38)% vs. (29.75±1.52)%, (29.61±4.15)%, (32.43±1.66)%, all P < 0.05], and the left ventricular dilatation in the septic rats was attenuated. (3) Myocardial injury markers: compared with the Sham group, serum cTnI and BNP levels were significantly higher in the model group, CORM-2 pretreatment group, iCORM-2 pretreatment group and DMSO control group. However, the levels of cTnI and BNP in the CORM-2 pretreatment group were significantly lower than those in the model group, iCORM-2 pretreatment group and DMSO control group [cTnI (ng/L): 3 283.54±803.50 vs. 6 449.18±1 105.10, 5 919.21±1 068.27, 6 349.80±1 153.08; BNP (ng/L): 3 456.62±905.85 vs. 6 070.18±1 287.62, 5 581.13±1 161.17, 5 974.89±988.89, all P < 0.05]. (4) Myocardial histopathological observation: optical microscope showed that the pathological changes in myocardial tissue of the model group, iCORM-2 pretreatment group and DMSO control group were severe. Transmission electron microscopy showed mitochondrial swelling, and some vacuoles changed. But the myocardial pathological morphology and mitochondrial ultrastructural integrity of the CORM-2 pretreatment group were significantly better than other groups of sepsis. CORM-2 can attenuate myocardial dysfunction and improve survival rate of septic rats, especially to protect myocardial mitochondrial integrity in sepsis.

Uptake of exogenous bovine GH\u2013pmKate2\u2013N expression vector by rams spermatozoa

BackgroundSperm-mediated gene transfer (SMGT) is a technique that utilizes the ability of the spermatozoa to take up exogenous DNA. Growth hormone is anabolic hormone that plays an important role in muscle-building process and milk production in all animals. High blood concentration of growth hormones (GH) was observed for animals that were genetically selected for high milk production or for low carcass fatness levels. The present study aimed to investigate and enhance the capacity of ovine spermatozoa to uptake exogenous growth hormone cDNA and its impact on sperm motility. The current study is an introduction for further future studies to produce transgenic Egyptian sheep characterized with high productive performance.MethodsThe growth hormone cDNA sequence was extracted from pituitary gland of Egyptian × Holstein (EH_GH) cattle and subcloned into the pmKate2–N vector to construct the EH_GH–pmKate2–N expression vector. The complete sequence of EH_GH mRNA was registered in GenBank (AC: KP221576). A total of three groups were assessed for the sperm uptake experiment, namely, negative control, positive control, and dimethyl sulfoxide (DMSO) groups; all treated groups were incubated with the EH_GH–pmKate2–N vector. The expression of EH_GH protein was detected in DH10B cells using a fluorescence microscope and the SDS polyacrylamide gel electrophoresis.ResultsThe EH_GH–pmKate2–N vector was expressed in cultured Escherichia coli cells, and the molecular weight of EH_GH protein was 24,558 Da. The EH_GH–pmKate2–N vector was introduced efficiently into the heads of the spermatozoa in the DMSO and positive control groups. Incubation of the spermatozoa with the vector caused a significant reduction in progressive motility compared to the negative control.ConclusionThe present results demonstrated the ability of ovine spermatozoa to take up the exogenous vector without notable deleterious effects on sperm motility. In subsequent studies, the successful introduction of the exogenous GH expression vector into the sperm head allows for the production of GH-transgenic sheep characterized by a high growth rate in order to reduce the meat shortage in Egypt.

A fetal mouse model of ventricular non-compaction using retinoic acid

ObjectiveTo develop a fetal mouse model of non-compaction of ventricular myocardium (NVM) using All-trans retinoic acid (ATRA). MethodsPregnant mice were divided into blank control group, dimethyl sulfoxide (DMSO) control group and ATRA group. The pregnant mice at 8.5 days after pregnancy were given 70 mg/kg ATRA in DMSO to induce fetal mouse model of NVM in ATRA group. All the hearts were acquired and sliced in short axis from the neonatal mice sacrificed after delivery. Pathological changes were visualized under 40- and 100-fold magnification with Hematoxylin-eosin (HE) staining at different ventricular levels. The criteria for pathological diagnosis of classical NVM were: prominent trabeculations on the endocardial surface and deep intertrabecular recesses communicating with the ventricular cavity and the thickness ratio of non-compacted layer (N) to compact myocardium layer (C) N/C > 1.4. Analysis of variance (ANOVA) and least significant difference (LSD) were used to analyze the differences of three groups, with P < 0.05 considered as significant. ResultsThe typical characteristics of NVM histopathological findings of ATRA fetal mouse were confirmed: compared to the hearts of blank control group (n = 20) and DMSO control group (n = 15), all the hearts of ATRA group (n = 17) showed the obviously thinner compacted layer and the much thicker non-compacted layer. The N/C ratio of left ventricles (LVs) in ATRA group was 2.735 ± 1.634, higher than those in DMSO control group 0.178 ± 0.119 and blank control group 0.195 ± 0.118 with significant difference (F = 32.550, P <0. 0001); N/C ratios of right ventricles (RVs) in the ATRA group were (6.068 ± 4.394), higher than those in the DMSO control group 0.459 ± 0.24 and in the blank control group 0.248 ± 0.182 with significant difference (F = 20.069, P <0.0001). LSD of LVs and RVs showed a significant difference between ATRA and blank control group (P < 0.0001), and between ATRA and DMSO control group (P < 0.0001). LSD showed no significant difference in two control groups of LVs (P = 0.963) and of RVs (P = 0.848) . ConclusionExcess ATRA could be used to induce NVM of fetal mice heart. This animal model might provide a platform for fundamental research of NVM pathogenesis and potential targeting treatment.

The effect of tanshinone on meibomian gland cell proliferation, differentiation and lipid synthesis

Objective To investigate the effects of cryptotanshinone and tanshinone ⅡA, two major monomer components of tanshinone, on the proliferation, differentiation and lipid synthesis of rat meibomian gland epithelial cells in vitro. Methods The eyelid meibomian gland tissue was isolated from 2-month-old SD rats and co-cultured with 3T3 trophoblasts for 5 days.Cryptotanshinone and tanshinone ⅡA were prepared with DMSO into different concentrations.The cells were grouped to 0.125 μmol/L, 0.250 μmol/L, 0.500 μmol/L, 1.250 μmol/L and 2.500 μmol/L drug groups and treated for 48 hours, respectively.Only dimethyl sulfoxide (DMSO) was added in medium in the DMSO control group.The expressions of keratin 14 (K14) and p63 in frozen sections of meibomian gland tissue and clones of meibomian gland cells were detected by immunofluorescence.Real-time fluorescence quantitative PCR was used to assay the relative expression of K16, cell proliferation related antigen 67 (Ki67) and CCAAT enhancer binding protein α (C/EBPα) in meibomian gland cell clones.Crystal violet staining and oil red staining were used to evaluate the colony formation rate and lipid synthesis of meibomian gland epithelial cells. Results Primary cultured meibomian gland cells were cloned on day 4-5 in vivo, and the cloning area was increased on day 7 after culture, p63 and K14 were positively expressed in clones.Compared with the DMSO control group, the relative expression levels of Ki67 mRNA were significantly elevated in the 0.500 μmol/L, 1.250 μmol/L and 2.500 μmol/L cryptotanshinone groups (all at P 0.05). No lipid drop was found in the tarsal gland cell clones; however, the accumulation of lipid was seen in the cell clusters at the margin of the clones by oil red O staining.The average clone formation rate of tarsal gland cells in the 1.250 μmol/L cryptotanshinone group was (2.55±0.20)%, which was significantly higher than (2.05±0.13)% in the DMSO control group (t=4.379, P 0.05). Conclusions Cryptotanshinone and tanshinone ⅡA promote the proliferation of meibomian gland epithelia cells, but play less impacts to lipid synthesis of meibomian gland epithelia cells in vitro.cryptotanshinone promote the clone tormation of meibomian gland epithelia cells. Key words: Tanshinone; Meibomian gland epithelial cells; Proliferation; Differentiation; Lipid metabolism/drug effects; Cell culture

Tumoral response and tumoral phenotypic changes in a rat model of diethylnitrosamine-induced hepatocellular carcinoma after salirasib and sorafenib administration.

BackgroundSeveral intracellular signaling pathways that are deregulated during hepatocarcinogenesis might constitute potential targets for hepatocellular carcinoma (HCC) therapy. The aim of this study was to test the potential synergic antitumor effect of salirasib and sorafenib in a diethylnitrosamine (DEN)-induced HCC model in rat. The hypothesis of tumor phenotype changes during treatment was also analyzed.Materials and methodsDEN was administered to Wistar rats during 9 weeks to induce cirrhosis and liver cancer. After tumor development, rats were treated with intraperitoneal injections of dimethyl sulfoxide (DMSO), or salirasib, and/or with oral sorafenib 5 days/week, during 4 weeks. At sacrifice, number and size of liver tumors as well as tumor burden were recorded, and all liver tumors were processed for histological and immunohistological analyses.ResultsMortality rate was significantly higher in rats treated with salirasib and/or sorafenib than in the control group (P=0.001). Tumor burden was smaller in the treated group compared with the DMSO control group (P=0.044), but a synergistic effect of the two chemotherapies could not be observed. In 62.5% of rats (10/16) treated with salirasib and/or sorafenib, a cytokeratin-7 and -19-positive hepatocholangiocellular carcinoma (HCC/CHC) was found vs 20% (5/25) developing such phenotype in the DMSO control group (P=0.018). Ki67 immunostaining showed significantly reduced tumor cell proliferation in treated rats (P=0.001), whereas apoptosis as assessed by caspase-3 activity in cell lysate was similar in all groups.ConclusionsThe addition of sorafenib to salirasib did not seem to provide any synergistic therapeutic effect in this study. Both chemotherapeutic agents, administered alone or in combination, induced tumoral phenotypic changes in the majority of rats, a finding not associated with an increased tumor cell proliferation or decreased apoptosis. The rat model described in this work constitutes the first experimental tool generating putatively more aggressive combined HCC/CHC tumors following chemotherapy. Further work is required to better characterize this clinically relevant phenomenon.

YC-1 Inhibits VEGF and Inflammatory Mediators Expression on Experimental Central Retinal Vein Occlusion in Rhesus Monkey

ABSTRACTPurposes: To investigate the therapeutic potential of YC-1 for experimental central retinal vein occlusion (CRVO) of rhesus monkey.Methods: Six adult rhesus monkeys were recruited in this study. Laser-induced CRVO was established in both eyes of all subjects. Intravitreal injection of YC-1 90 μl (200 μM with 0.01% dimethyl sulfoxide (DMSO) as vehicle) was administrated in right eye and 0.01% DMSO 90 μl in left eye respectively at 1 week after CRVO established. All eyes underwent routine examination at 1 day, 1 week, 2 week, and 1 month after intravitreal injection of YC-1 or DMSO. Meanwhile, vitreous fluid was collected at each time points to analyze concentration of VEGF, HIF-1α, IL-6, IL-8, and MCP-1 mediators by CBA or ELASA method.Results: The experimental CRVO was successfully established in six rhesus monkeys. As expected, the thickness of macular edema significantly decreased at 1 week and 2 weeks after YC-1 injection compared with that of DMSO injection. Subsequently, the central macular thickness in all eyes was recovered to the initial levels at 1 month after photocoagulation. Intraocular pressure (IOP) was not significantly different between two groups during all follow up. Meanwhile, the concentration of IL-6, IL-8, VEGF, and HIF-1α in vitreous fluid significantly decreased after YC-1 injection compared with that of DMSO injection, MCP-1 was not significantly different between both groups.Conclusions: Intravitreal injection of YC-1 significantly alleviated macular edema compared with that of DMSO control group. Meanwhile, both inflammatory factors and angiogenesis-related factors expression were inhibited in vitreous by YC-1 injection.

Potentiation of pentylenetetrazole-induced neuronal damage by dimethyl sulfoxide in chemical kindling model in rats.

OBJECTIVES:Dimethyl sulfoxide (DMSO) is commonly used as a vehicle for many hydrophobic drugs. This study aimed at evaluating the effect of low dose of DMSO (0.1%) on Pentylenetetrazole(PTZ) induced neuronal damage in rats.MATERIALS AND METHODS:Young male Wistar rats (n = 32) were divided into four groups as follows: saline control group, DMSO control group, PTZ group (35 mg/kg), and combination group (DMSO + PTZ). Animals were observed for seizure score, latency to develop kindling, percentage of animals kindled, and histopathological score of hippocampus.RESULTS:There was a significant increase in the seizure scores and histopathological scores in the combination group as compared to PTZ-treated group. The latency to develop kindling was, however, decreased in the combination group (4th week) as compared to PTZ (6th week) group.CONCLUSIONS:The present study has concluded that 0.1% DMSO in PTZ-induced rat model of epileptogenesis needs further optimization and should be used cautiously

Role of c-Jun N-terminal kinase in activation of astrocytes in midbrain periaqueductal gray of rats with neuropathic pain

Objective To evaluate the role of c-Jun N-terminal kinase(JNK)in activation of astrocytes in midbrain periaqueductal gray(PAG)of rats with neuropathic pain. Methods A total of 72 pathogen-free male Sprague-Dawley rats, aged 9 weeks, weighing 160-200 g, were divided into 4 groups using a random number table: control group(group C, n=8), neuropathic pain group(group NP, n=40), dimethyl sulfoxide control group(group DS, n=12)and JNK inhibitor SP600125 group(group SP, n=12). Neuropathic pain was produced by chronic constriction injury(CCI). At 14 days after CCI, 10 nmol JNK inhibitor SP600125 0.5 μl was intraperitoneally injected into the PAG in group SP, and 10% dimethyl sulfoxide 0.5 μl was given instead in group DS.Eight rats were selected in group C, before CCI and at 3, 7, 14 and 21 days after CCI in group NP, and in DS and SP groups, and the mechanical pain threshold was measured before CCI, before administration on 14 days after CCI and at 30, 45, 60, 75 and 90 min after administration.The rats in group C were sacrificed after the end of measurement of the mechanical pain threshold, and brains were removed for determination of phosphorylated JNK(p-JNK)and glial fibrillary acidic protein expression(by Western blot)in PAG region.The rats in group NP were sacrificed after the end of measurement of the mechanical pain threshold at each time point, and brains were removed for detection of p-JNK expression in PAG region.Four rats in DS and SP groups were sacrificed after the last measurement of the mechanical pain threshold at 45 min after administration, and brains were removed for determination of glial fibrillary acidic protein expression in PAG region. Results Compared with group C, the mechanical pain threshold was significantly decreased at each time point after CCI, and the expression of p-JNK was up-regulated at 7-21 days after CCI in group NP(P<0.01). Compared with group DS, the mechanical pain threshold was significantly increased at 30 min after administration, and GFAP expression was down-regulated at 45 min after administration in group SP(P<0.01). The mechanical pain threshold was significantly higher at 30-75 min after administration than before administration in group SP(P<0.01). Conclusion The mechanism underlying activation of astrocytes in PAG is related to activating JNK in the rats with neuropathic pain. Key words: Astrocytes; JNK mitogen-activated protein kinases; Cerebral aqueduct; Periaqueductal gray; Neuralgia

Curcumin inhibits endoplasmic reticulum stress induced by cerebral ischemia-reperfusion injury in rats.

The aim of the present study was to observe the dynamic changes of the growth arrest and DNA damage-inducible 153 (GADD153) gene and caspase-12 in the brain tissue of rats with cerebral ischemia-reperfusion injury (CIRI) and the impact of curcumin pretreatment. A total of 60 rats were randomly divided into the normal group (N), the sham operation group (S), the dimethyl sulfoxide control group (D) and the curcumin treatment group (C). For group D and C, 12 (T1), 24 (T2) and 72 h (T3) of reperfusion were performed after 2 h ischemia. The expression levels of GADD153 and caspase-12 in the brain tissue were detected and compared among the groups by immunohistochemistry, immunofluorescence double staining and western blotting. The expression levels of GADD153 and caspase-12 were increased at T1compared with groups N and S, and the expression of caspase-12 peaked at T2 in group D, while GADD153 was increased until T3 in group D. Compared with group D, the expression levels of GADD153 and caspase-12 in group C at T2 and T3 were significantly decreased (P<0.05). Endoplasmic reticulum stress is involved in the pathological process of CIRI. Curcumin may decrease the expression levels of the above two factors, thus exhibiting protective effects against CIRI in rats.

Effects of hypoxia inducible factor-1α on P311 and its influence on the migration of murine epidermal stem cells

Effects of hypoxia inducible factor-1α on P311 and its influence on the migration of murine epidermal stem cells

In vitro effects of tanshinone II A on autophagy of A375 melanoma cells and related signaling pathway

Objective To investigate in vitro effects of tanshinone ⅡA on the autophagy of A375 melanoma cells and related signaling pathway. Methods Some cultured A375 cells were divided into 5 groups to be treated with tanshinone ⅡA at concentrations of 0.5, 1, 2 and 4 mg/L, and DMEM containing 0.1% dimethyl sulfoxide (DMSO), respectively, for 24, 48, 72 hours. Methyl thiazol tetrazolium (MTT) assay was performed to estimate the proliferative activity of A375 cells. Some cultured A375 cells were divided into 4 groups to be treated with 1, 2 and 4 mg/L tanshinone ⅡA (1-, 2- and 4-mg/L tanshinone group), and DMEM containing 0.1% DMSO (control group), respectively, for 48 hours. Then, flow cytometry was conducted to count autophagosome-positive cells, and Western blot analysis to determine protein expression of autophagy-associated proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3) -Ⅱ, phosphatidylinositol 3-kinase (PI3K), protein kinase B (Akt), mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1 (p70S6K1). Results MTT assay showed that 24-, 48-, 72-hour treatments with tanshinone Ⅱ A at concentrations of 0.5, 1, 2 and 4 mg/L all could inhibit the proliferative activity of A375 cells, and the inhibitory effects increased in a dose- and time-dependent manner (F = 2 564.12, 1 235.25, both P < 0.05). The percentage of autophagosome-positive cells and protein expression of Beclin-1 and LC3-Ⅱ increased gradually and significantly in the 1-, 2- and 4-mg/L tanshinone groups (autophagosome-positive cells: 6.91% ± 0.35%, 13.11% ± 0.73%, 25.51% ± 0.83%, respectively; Beclin-1: 0.33 ± 0.01, 0.53 ± 0.04, 0.63 ± 0.02, respectively; LC3-Ⅱ: 0.41 ± 0.01, 0.52 ± 0.02, 0.64 ± 0.02, respectively), after 48-hour treatment, which were significantly different between the tanshinone groups (all P < 0.05), and higher in the tanshinone groups than in the control group (0.41% ± 0.02%; 0.09 ± 0.02; 0.21 ± 0.01, all P < 0.05). However, the protein expression of PI3K, phosphorylated Akt (p-Akt), p-mTOR and p-p70S6K1 in the PI3K-Akt-mTOR-p70S6K1 signaling pathway decreased gradually and significantly with the increase in tanshinone concentrations after 48-hour treatment, and were significantly lower in all the tanshinone groups than in the control group (all P < 0.05). Conclusion Tanshinone Ⅱ A can promote the auophagy of A375 cells, likely by blocking the PI3K-Akt-mtTOR-p70S6K1 signaling pathway. Key words: Melanoma; Tanshinone; Autophagy; A375 cells