Objective To study the effects of L-arginine （L-Arg） on cell proliferation, inducible nitric oxide synthase (iNOS) expression and cell cycle in human colon carcinoma cell line LS174 through nitric oxide (NO) pathway. Methods LS174 cells were cultured in medium with L-Arg at different concentrations for different times. MTT method was employed to evaluate the level of the cell proliferation. The production of NO in culture supernatants of LS174 cell was detected with enzyme reduction of nitrate. The distribution of the cell cycle was detected with the flow cytometry (FCM). The expression level of iNOS in the cells was determined by Western blot and SP immunocytochemical staining method. Results The growth of LS174 was promoted by the L-Arg at low concentration (0.125 mmol/L) and inhibited at high concentrations (0.5， 2， 8 and 32 mmol/L). The level of NO was increased with the increasing concentration of L-Arg in culture medium. To compare with the control group, the ratio of cells at S phase was increased after 48 hours’ treatments with high concentrations (0.5, 2, 8 and 32 mmol/L) of L-Arg (P＜0.05, P＜0.01); while there was no obvious difference after treatments with low concentration (0.125 mmol/L) of L-Arg (Pgt;0.05). With the increase of the concentration of L-Arg, the expression of iNOS was increased as compared with control group. The higher the concentration of L-Arg was, the better the effect. Conclusion L-Arg can induce the expression of iNOS resulting in increase the production of nitric oxide (NO). Low concentration of L-Arg can promote the growth of LS174 cells, while high concentration ones can inhibit growth and proliferation. The high concentration of L-Arg could induce S phase arrestion in the cell cycle.
摘要:目的:探索槐耳清膏对体结肠癌SW480细胞增殖能力影响及其机制。方法:采用噻唑蓝(MTT)比色法检测槐耳清膏对SW480细胞增殖能力的作用,并探求最佳作用浓度;将体外培养细胞随机分为常氧组(NC组)、低氧组(HC组)和低氧槐耳组(HH组),逆转录聚合酶链反应（RTPCR）检测各组血管内皮生长因子(VEGF) mRNA表达水平,Western blot检测蛋白表达水平。结果:槐耳清膏对SW480细胞抑制率随药物浓度增加而上升,1 mg/mL时抑制率最大(66.7%),与氟尿嘧啶组(浓度为10 μg/mL)相比无统计学意义。HH组和HC组VEGF mRNA表达均显著高于NC组,分别为4.71±0.07,4.54±0.02和1.19±0.03(P＜0.05),但HH组与HC组比较差异无统计学意义。HC组VEGF蛋白表达显著高于NC组,分别为0.66±0.03和0.38±0.02(P＜0.05),HH组较HC组VEGF蛋白表达均显著下降,分别为0.37±0.03和0.66±0.03(P＜0.05)。结论:槐耳清膏可抑制SW480细胞增殖,1 mg/mL时抑制率最大。其机制为槐耳清膏下调细胞内VEGF蛋白表达,从而抑制肿瘤生长。Abstract: Objective: To investigate the effect of Huaier cream on proliferation of colon cancer cells SW480 and its mechanism. Methods: The proliferation was analyzed by MTT. SW480 cells were randomly divided into normoxic group (NC group), hypoxia group (HC group) and hypoxia group treated by Huaier (HH group). Levels of mRNA and protein expression of VEGF were detected by RTPCR and Western blot, respectively． Results: Huaier cream induced a dosedependent inhibition of SW480 cells. The maximum percentage of growth inhibition was 66.7% at a concentration of 1.0 mg/mL, but no significant difference was found compared to the positive control (5FU 10 μg/mL). VEGF mRNA levels were significantly higher in HC group and HH group than in NC group (4.71±0.07, 4.54±0.02 vs 1.19±0.03, all Plt;0.05), but not significantly different between HC group and HH group. VEGF protein expression was higher in HC group than NC group (0.66±0.03 vs 0.38±0.02, Plt;0.05). In HH group, VEGF protein was inhibited remarkably compared with HC group (0.37±0.03 vs 0.66±0.03, Plt;0.05). Conclusion: Huaier cream can significantly inhibit SW480 cells and the top inhibition concentration is 1.0 mg/mL. Huaier cream plays a role in inhibiting tumor through downregulating protein expression of VEGF.
ObjectiveThis study aims to study the effects and mechanism of resveratrol on hepatocellular carcinoma (HCC) cells through phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) axis.MethodsHepG2 cells at logarithmic growth stage were treated with different concentrations (0, 12.5, 25.0, and 50.0 μmol/L) of resveratrol, respectively. Then the proliferation of HepG2 cells was detected by the CCK8 method and real time cell anaIysis (RTCA) system, the expressions of signal molecules associated with PI3K/Akt axis was detected by the Western blot method, including PI3K p58, phosphorylation protein kinase B (p-Akt), total protein kinase B (t-Akt), and CyclinA2 protein.ResultsResveratrol had a significant inhibitory effect on the growth of HepG2 cells in a time and dosage dependent manner. After 48 h treatment of resveratrol to HepG2 cells, 50.0 μmol/L resveratrol inhibited the growth of HepG2 cells most significantly. Further, the RTCA system studies also found that resveratrol had a time and concentration dependent effect on the reduction of normalized cell index (NCI) in HepG2 cells. Flow cytometry results showed that, apoptosis rates of 12.5, 25.0, and 50.0 μmol/L group were higher than that of 0 μmol/L group. Compard with 0 μmol/L group, the expressions of PI3K p85, p-Akt, and CyclinA2 protein in HepG2 cells of 12.5, 25.0, and 50.0 μmol/L resveratrol group was significantly higher (P<0.05), although there was no significant effect of resveratrol on the expression of t-Akt in HepG2 cells (P>0.05).ConclusionsResveratrol might have anti-proliferation effects on HepG2 cells through PI3K p85/Akt signaling axis. This study could provide a novel idea for the treatment to HCC.
ObjectiveTo study the effect of tumor associated neutrophil (TAN) releasing a proliferation-inducing ligand (APRIL) on the proliferation of pancreatic cancer cells in microenvironment.Methods① The expressions of APRIL in neutrophils (differentiated by HL-60 cell) and TAN cells were detected by use ELISA. ② The expressions of APRIL receptors B cell maturation antigen (BCMA) and trans-membrane activator and CAML interactor (TACI) in pancreatic cancer cell line PANC-1 were confirmed by use Western blotting. ③ Pancreatic cancer PANC-1 cells were co-cultured with TAN, and divided into a PANC-1 control group (referred to as the control group), a PANC-1+TAN treatment group (referred to as the PANC-1+TAN group), PANC-1+TAN+APRIL antibody treatment group (referred to as PANC-1+TAN+APRIL group), and PANC-1+rtificial recombinant APRIL protein (rAPRIL) treatment group (referred to as PANC-1+rAPRIL group). The CCK8 method was used to determine TAN release of APRIL on PANC-1 effect of cell proliferation activity.Results① The APRIL content in the culture medium of TAN cell group was higher than that of neutrophil group [(556.20±84.38) pg/mL vs. (377.17±57.07) pg/mL, P=0.038]. ② PANC-1 cells express the receptors BCMA and TACI of APRIL. ③ PANC-1 cell activity of PANC-1+TAN group and PANC-1+rAPRIL group [(126.80±1.42)%, (168.95±12.54)%] were significantly higher than the control group [(100 ± 0.00)%, P<0.05, P<0.001], the activity of PANC-1 cells in the PANC-1+TAN group was significantly higher than that in the PANC-1+TAN+APRIL group [(86.29 ± 12.20)%, P=0.003] and significantly lower than that of PANC-1+rAPRIL group (P=0.002), the activity of PANC-1 cells in PANC-1+rAPRIL group was significantly higher than that in PANC-1+TAN+APRIL antibody group (P<0.001).ConclusionIn the microenvironment of pancreatic cancer, the release of APRIL from TAN increases, which promotes the proliferative activity of PANC-1 in pancreatic cancer cells, which provides a new idea for the mechanism research and treatment of pancreatic cancer progression.
Objective To investigate the effect of ginkgolide B (GB) on cysteinyl aspartate specific proteinase-3 (Caspase-3)/chromosome 10 deletion phosphatase-tension protein homologue (PTEN)/protein kinase B (Akt) pathway and cell proliferation and apoptosis in hypoxia/reoxygenation cardiomyocytes. Methods H9C2 cells were cultured in vitro. A control group was cultured in serum-free DMEM high glucose medium at 37°C and 5% CO2 for 28 hours. The remaining groups were prepared with hypoxia/reoxygenation models. A GB low-dose group and a GB high-dose group were treated with GB pretreatment with final concentration of 50 μmol/L and 200 μmol/L respectively at 1 h before hypoxia/reoxygenation. A carvedilol group was treated with carvedilol of a final concentration of 10 μmol/L at 1 h before hypoxia/reoxygenation. The proliferation and apoptosis of H9C2 cells were detected, and the levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), reactive oxygen species (ROS), PTEN, Akt, phosphorylated Akt (p-Akt) and Caspase-3 in H9C2 cells were also detected. Results Compared with the control group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in other groups decreased, and the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 increased (P<0.05). Compared with the hypoxia/reoxygenation group, the proliferation rate of H9C2 cell, and the levels of PTEN, Akt and p-Akt in all GB dose groups and the carvedilol group increased; the apoptosis rate, and the levels of LDH, MDA, ROS and Caspase-3 decreased, and the effect of GB was in a dose dependent manner; however, the effect of GB was not as strong as carvedilol (P<0.05). Conclusion GB can inhibit H9C2 cell apoptosis and promote H9C2 cell proliferation by activating Caspase-3/PTEN/Akt pathway.
Objective To investigate the feasibility of a dual-crosslinked injectable hydrogel derived from acellular musclar matrix (AMM) for promoting myoblasts proliferation and myogenic differentiation. Methods Firstly, hyaluronic acid was oxidized with NaIO4 and methylated to prepare methacrylamidated oxidized hyaluronic acid (MOHA). Then, AMM obtained by washing enzymatically treated muscle tissue was aminolyzed to prepare aminated AMM (AAMM). MOHA hydrogel and AAMM were crosslinked using Schiff based reaction and UV radiation to prepare a dual-crosslinked MOHA/AAMM injectable hydrogel. Fourier transform infrared spectroscopy (FTIR) was used to characterize MOHA, AAMM, and MOHA/AAMM hydrogels. The injectability of MOHA/AAMM hydrogel were evaluated by manual injection, and the gelation performance was assessed by UV crosslinking. The rheological properties and Young’s modulus of the hydrogel were examined through mechanical tests. The degradation rate of the hydrogel was assessed by immersing it in PBS. The active components of the hydrogel were verified using immunofluorescence staining and ELISA assay kits. The promotion of cell proliferation by the hydrogel was tested using live/dead staining and cell counting kit 8 (CCK-8) assays after co-culturing with C2C12 myoblasts for 9 days. The effect of the hydrogel on myogenic differentiation was evaluated by immunofluorescence staining and real time quantitative polymerase chain reaction (RT-qPCR). ResultsFTIR spectra confirmed the successful preparation of MOHA/AAMM hydrogel. The hydrogel exhibited good injectability and gelation ability. Compared to MOHA hydrogel, MOHA/AAMM hydrogel exhibited higher viscosity and Young’s modulus, a reduced degradation rate, and contained a higher amount of collagen (including collagen type Ⅰ and collagen type Ⅲ) as well as bioactive factors (including epidermal growth factor, fibroblast growth factor 2, vascular endothelial growth factor, and insulin-like growth factor 1). The live/dead cell staining and CCK-8 assay indicated that with prolonged incubation time, there was a significant increase in viable cells and a decrease in dead cells in the C2C12 myoblasts within the MOHA/AAMM hydrogel. Compared with MOHA hydrogel, the difference was significant at each time point (P<0.05). Immunofluorescence staining and RT-qPCR analysis demonstrated that the deposition of IGF-1 and expression levels of myogenic-related genes (including Myogenin, Troponin T, and myosin heavy chain) in the MOHA/AAMM group were significantly higher than those in the MOHA group (P<0.05). ConclusionThe MOHA/AAMM hydrogel prepared based on AMM can promote myoblasts proliferation and myogenic differentiation, providing a novel dual-crosslinked injectable hydrogel for muscle tissue engineering.
Objective To investigate the role of chemokine receptor CXCR7 in the development and progression of pancreatic carcinoma. Methods The short hairpin RNA (shRNA) targeting CXCR7 was designed and delivered into AsPC-1 pancreatic carcinoma cells to knock down CXCR7 expression. The cell proliferation, cell cycle, and apoptosis after CXCR7 knockdown was determined by MTT and flow cytometry, respectively. The invasive ability of pancreatic carcinoma cells was evaluated by using the Transwell system. Results Compared with the blank control group (BC group), transfection of AsPC-1 cells with CXCR7-shRNA resulted in a significantly decreased expression of CXCR7 at both mRNA and protein levels (P<0.05), and the ability of proliferation and invasion significantly decreased (P<0.05). Knockdown of CXCR7 also significantly increase apoptosis (P<0.05), induce cell cycle arrest at G0/G1 phase (P<0.05). Conclusions Taken together, the present study showes that the knockdown of CXCR7 expression may play an important role in pancreatic carcinoma development, invasion, and metastasis, CXCR7 may be a potential therapeutic target for the treatment of pancreatic carcinoma.
Pulmonary arterial hypertension(PAH) is a kind of pulmonary hypertension disease. Recently, the researches of its pathogenesis have reached more and more deeply. The treatment of pulmonary arterial hypertension is individual and systematic, not only relying on medicine treatment. The treatment of PAH is as follows: common treatment, non-specific medicine treatment, targeted medicine treatment, NO breath-in treatment, gene treatment, intervention and surgery treatment.The article reviews the main treatment of pulmanory arteral hypertesion to provide new thought and evidence in clinic.
ObjectiveTo investigate the effects of pipecolic acid oxidase (PIPOX) on the proliferation, apoptosis, migration and invasion of primary liver cancer cells. MethodsImmunohistochemical staining and analysis of The Cancer Genome Atlas (TCGA) database were used to examine the PIPOX expression levels in liver cancer tissues and paired adjacent normal tissues, and studied their relationship with patient prognosis. Liver cancer cell lines stably overexpressing or knocking out PIPOX were constructed to explore PIPOX’s impact on liver cancer cell proliferation, apoptosis, migration and invasion by conducting in vitro functional experiments such as CCK-8, EdU, apoptosis detection, and Transwell assays. In vivo, nude mice subcutaneous tumor models and lung metastasis models were used to verify PIPOX’s effect on liver cancer growth and metastasis. Real-time quantitative polymerase chain reaction (RT-qPCR) and western blot were both employed to detect the expression of epithelial-mesenchymal transition (EMT) markers in liver cancer cells. ResultsImmunohistochemical staining and TCGA database analysis revealed that PIPOX expression was significantly lower in liver cancer tissues compared to paired adjacent normal tissues (P<0.05). Prognostic analysis indicated shorter overall survival and disease-free survival in PIPOX low expression group (P<0.05). In vitro gain- and loss-of-function experiments showed that PIPOX significantly inhibited liver cancer cell migration and invasion (P<0.05), while having no significant effects on their proliferation and apoptosis (P>0.05). Animal experiments also confirmed that PIPOX significantly inhibited liver cancer lung metastasis (P<0.05), but had no significant effects on tumor growth (P>0.05). Finally, RT-qPCR and western blot results revealed that PIPOX promoted the expression of the epithelial marker E-cadherin (P<0.05) and inhibited the expression of mesenchymal markers (N-cadherin, vimentin, Snail) (P<0.05). ConclusionsPIPOX significantly inhibits liver cancer cell migration and invasion, potentially via suppressing the EMT process. However, PIPOX does not significantly affect liver cancer cell proliferation and apoptosis.
ObjectiveAfter using hyaluronic acid (HA) to modify curcumin (CUR), the effects of calcium phosphate cement (CPC) combined with HA/CUR on the proliferation and osteogenesis of osteoblasts were investigated.MethodsFirst, HA and CUR were esterified and covalently combined to prepare HA/CUR, and the characteristics were observed and the infrared spectrum was tested. Then, HA, CUR, and HA/CUR were mixed with CPC according to 5% (W/W) to prepare HA-CPC, CUR-CPC, and HA/CUR-CPC, respectively. Setting time detection, scanning electron microscope observation, injectable performance test, and compression strength test were conducted; and the CPC was used as a control. Osteoblasts were isolated and cultured from the skull of newborn Sprague Dawley rats, and the 2nd generation cells were cultured with the 4 types of bone cement, respectively. The effects of HA/CUR-CPC on the proliferation and osteogenesis of osteoblasts were estimated by the scanning electron microscopy observation, live/dead cell fluorescence staining, cell counting, osteopontin (OPN) immunofluorescence staining, alkaline phosphatase (ALP) staining,and alizarin red staining.ResultsInfrared spectroscopy test showed that HA and CUR successfully covalently combined. The HA/CUR-CPC group had no significant difference in initial setting time, final setting time, injectable rate, and compressive strength when compared with the other 3 groups (P>0.05); scanning electron microscope observation showed that HA/CUR was scattered on CPC surface. After co-culture of bone cement and osteoblasts, scanning electron microscopy observation showed that the osteoblasts, which had normal morphology and the growth characteristics of osteoblasts, clustered and adhered to HA/CUR-CPC. There was no significant difference in cell survival rate between HA/CUR-CPC group and other groups (P>0.05), and the number of cells significantly increased (P<0.05); the degrees of OPN immunofluorescence staining, ALP staining, and alizarin red staining were stronger than other groups.ConclusionHA/CUR-CPC has good biocompatibility and mechanical properties, which can promote the proliferation and osteogenesis of osteoblasts.