ABSTRACT
The aim of this study was to explore the regulatory mechanism of Type Ⅲ domain-containing protein5 (FNDC5) on adipogenic differentiation in C3H10T1/2 cells. qRT-PCR and Western blot were used to detect the expression of FNDC5 during adipogenic differentiation of C3H10T1/2 cells. The lentivirus-coated overexpression and interference vector of FNDC5 were constructed and transfected into C3H10T1/2 cells. qRT-PCR was used to detect the expression of the key genes of adipogenic differentiation. Oil red O staining was used to detect the formation of lipid droplets; Western blot was used to detect the content of ERK1/2 and ERK1/2 phosphorylated protein (P-ERK1/2). After 8 days of adipogenic differentiation of C3H10T1/2 cells, the expression of Fndc5 increased significantly. After overexpression of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including peroxisome proliferator-activated receptor-酌 (PPAR酌), CCAAT enhancer binding protein beta (C/EBP茁), fatty acid binding protein 4 (FABP4) and CCAAT enhancer binding protein alpha (C/EBPα), all decreased significantly. The content of lipid droplets and P-ERK1/2 also decreased significantly. On the contrary, after interference of FNDC5 in C3H10T1/2 cells, the expression of key genes for adipogenic differentiation, including PPARγ, C/EBP茁, FABP4 and C/EBPα were significantly increased. Meanwhile, the content of lipid droplets and P-ERK1/2 also increased significantly. This study found that FNDC5 can inhibit the adipogenic differentiation of C3H10T1/2 cells by inhibiting the phosphorylation level of ERK1/2, which can provide reference data for the mechanism of FNDC5 in regulating fat deposition.
ABSTRACT
The aim of this study was to observe the effect of baicalin on the growth state of attention deficit hyperactivity disorder animal model and its regulation on Ca MKⅡand ERK1/2.In the present study,a total of 40 SHR rats were randomly divided into model group,methylphenidate hydrochloride group,and low,medium,and high dose baicalin groups,with 8 rats in each group.Eight WKYrats were selected as a normal control group.The methylphenidate hydrochloride group(0.07 g·L~(-1))and the low(3.33 g·L~(-1)),medium(6.67 g·L~(-1)),and high dose(10 g·L~(-1))baicalin groups received corresponding drugs by gavage administration according to the body weight(0.015 m L·g~(-1)),while the normal group and the model group received the same volume of normal saline by gavage.Thegavage administration lasted for 4 weeks,twice a day.The body weight of the rats and the amount of remaining feed were weighed daily,and the growth state of the rats was statistically evaluated weekly.Percoll density gradient centrifugation was used to prepare brain synaptosomes and an electron microscope was used to observe their structures.The Ca MKⅡand ERK1/2 protein and mRNA expression levels were detected with Western blot and Real-time PCR methods,respectively.RESULTS: showed that baicalin did not affect the normal eating and weight gain of rats,and the weight gain of rats was even more significant than that in the normal group(P<0.05).In the study of its effects on Ca MKⅡand ERK1/2 protein expression in rat synaptosomes,the expression of both proteins in each drug-administered group was higher than that in the model group(P<0.05);besides,the expression levels of Ca MKⅡand ERK1/2 protein were significantly increased in both baicalin high dose group and the methylphenidate hydrochloride group(P<0.05).The relative expression of Ca MKⅡand ERK1/2 mRNA in synaptosome was detected by PCR.The results showed that medium and high doses of baicalin and methylphenidate hydrochloride significantly increased the relative expression of Ca MKⅡand ERK1/2 mRNA in synaptosomes of SHR rats(P<0.05).In conclusion,baicalin does not affect the normal growth and development of SHR rats,so it is safe for administration.Both baicalin and methylphenidate hydrochloride could up-regulate the relative expression of Ca MKⅡand ERK1/2 in mRNA and protein,and the pharmacodynamic stability of baicalin is in a dose-dependent manner to certain extent.
Subject(s)
Animals , Rats , Attention Deficit Disorder with Hyperactivity , Disease Models, Animal , Flavonoids , Intracellular Signaling Peptides and Proteins , MAP Kinase Signaling System , Protein Serine-Threonine Kinases , Rats, Inbred SHR , Rats, Inbred WKYABSTRACT
Objective To detect the expression of extracellular-regulated kinase (ERK) and phosphatedextracellular-regulated kinase (P-ERK) in the hippocampus after pentylenetetrazoloe-induced status epilepsy and the effects of nimodipine on it.Methods Male Sprague-Dawley adult rats (200-250 g) were randomly divided into normal control group(NC,n =35),status epilepsy group (SE,n =40),nimodipine group (NIM,n =40).The rats were injected first with 40 mg/kg pentylenetetrazoloe(PTZ),followed 10 minutes later by 20 mg/kg PTZ,and subsequently,10 mg/kg PTZ ip every 10 minutes until SE occurred,apoint charactered by a loss of postural control and tonic-clonic seizures.Rats in control group received the same number of saline injections.Rats in NIM group were injected NIM(2.5 mg/kg) intraperitoneally 15 min before the injection of PTZ.Rats in every group were killed at 30 minutes,1 hour,3 hours,12 hours,24 horus,72 hours and 7 days after status epilepsy respectively and the hippocampus were dissected.The expression of ERK and P-ERK in the hippocampus were detected by Western blot.Results Nimodipine attenuated the convulsion of PTZ-induced status epilepsy.There was dynamic expression of P-ERK in SE group.In NIM group,the expression of P-ERK was markedly increased than that of SE group at 30 min,1h,3h,12h,24h,72h,and 7d (3.26 ±0.95 vs 2.56 ±0.82 at 30 min,P＜0.05).Conclusion Nimodipine attenuates the convulsion of PTZ-induced status epilepsy with increased expression of phosphated-ERK in the hippocampus of rats.
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Objective: To investigate the role of extracellular signal-regulated kinase 1/2 (ERK1/2) signal pathway in the pathogenesis of stress-induced gastric ulcer. Methods: Animal model of stress-induced gastric ulcer was established in rats with water-immersion restraint (WIR) stress. The mucosal activation of ERK1/2 was observed before and 5, 15 and 30 min, and 1, 2 and 3.5 h after WIR stress. Some animals were also treated with an intravenous injection of PD98059 (1 mg/kg), a specific ERK1/2 inhibitor, 1 h prior to WIR stress. Expression of total ERK1/2 and caspase-3 were detected by Western blot analysis; ERK1/2 activity was measured by kinase activity assay using myelin basic protein as a specific substrate. DNA-binding activities of the transcription factors activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) were determined by electrophoretic mobility shift assays (EMSA). Mucosal TNF-α and IL-1β mRNA expression was analyzed by Northern blot analysis. The degrees of the gastric mucosal lesions were expressed as ulcer index (UI) and pathological evaluation. Apoptosis in the gastric mucosa was examined by an in situ TdT-mediated dUTP nick-end labeling (TUNEL) method. Results: Activated ERK1/2 was very weakly expressed in the gastric mucosa of normal rats. ERK1/2 was rapidly activated in the gastric mucosa of rats 15 min after WIR stress and the activity reached the maximal after 3.5 h. Pretreatment with PD98059 significantly inhibited ERK1/2 activation, decreased AP-1 and NF-κB activities and TNF-α and IL-β mRNA expression, and obviously relieved gastric mucosal lesions, accompanied by caspase-3 activation and increased apoptosis. Conclusion: The present results indicate that ERK1/2 activation plays an important role in the development of stress-induced gastric ulcer.
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Objective To investigate the effects of 2 450 MHz microwave irradiation on the proliferation of cultured mouse fibroblasts in vitro, and the related gene and protein expressions. Methods Cells from mouse skin were directly radiated with microwaves of different intensities for different periods. The proliferation of fibroblasts was assayed by the MTT method, and the effects of microwave radiation on the fibroblast cell cycle were measured by flow eytometry. The mRNA expression of types Ⅰ and Ⅲ procollagen was detected by real-time RT-PCR. Anti-phosphoryl- ation extracellular-regulated kinase (ERK-1/2) antibody was introduced in immunofluorescence staining analysis to observe any changes in the phosphorylation of fibroblast protein ERK. Results ① 5 W/cm2 or 1.0 W/cm2 irradia- tion for 5 min, 15 min, or 30 min did not significantly decrease fibroblast proliferation, but irradiation at 5 W/cm2 for more than 5min caused a significant decrease in fibroblast proliferation. ②After 5 W/cm2 irradiation for 5 min, the percentage of cells in the G0/G1phase was significantly increased, and ERK was activated immediately after irradia- tion. ③The mRNA expression of type Ⅰ procollagen was down-regulated after microwave irradiation, and the magni- tude of the decreased expression correlated positively with the duration of irradiation. Thirty minutes of microwave ir- radiation at 1 W/cm2 or 5 minutes at 5 W/cm2 significantly down-regulated the mRNA expression of type Ⅰ procolla- gen and the ratio of types Ⅰ and Ⅲ. Conclusion High-intensity microwaves may inhibit the proliferation of fibro-blasts in a dose-dependent manner in vitro and down-regulate procollagen mRNA expression, which might be achieved by activating mitogen-activated protein kinase signal transduction gateways.
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BACKGROUND: Protease-activated receptor 2 (PAR2) belongs to a family of G protein- coupled receptors activated by proteolytic cleavage. Trypsin-like serine proteases interact with PAR2 expressed by a variety of tissues and immune cells. The aim of our study was to investigate whether PAR2 stimulation can lead to the activation of human macrophages. METHODS: PAR2-mediated proliferation of human macrophage cell line THP-1 was measured with MTT assay. We also examined the extracellular regulated kinase (ERK) phosphorylation and cytokine production induced by trypsin and PAR2-agonist using western blot and enzyme-linked immunosorbent assay (ELISA), respectively. RESULTS: Treatment of trypsin or PAR2-activating peptide increased cell proliferation in a dose-dependent manner, and induced the activation of ERK1/2 in THP-1 cells. In addition, trypsin-induced cell proliferation was inhibited by pretreatment of an ERK inhibitor (PD98059) or trypsin inhibitor (SBTI). Moreover, PAR2 activation by trypsin increased the secretion of TNF-alpha in THP-1 cells. CONCLUSION: There results suggest that PAR2 activation by trypsin-like serine proteases can induce cell proliferation through the activation of ERK in human macrophage and that PAR2 may play a crucial role in the cell proliferation and cytokine secretion induced by trypsin-like serine proteases.