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Objective To investigate the protective mechanism of MEK1/2 inhibitor PD98059 on ox-LDL induced injury of human umbilical vein endothelial cells (HUVEC),and its influence on the expression of LOX-1.Methods HUVEC damage models were established by using ox-LDL and were treated with PD98059 later,divided into the negative control group,the ox-LDL group,the positive control group and the PD98059+ox-LDL group.The effect of inhibition of MEK1/2 on ox-LDL induced HUVEC damage was measured.Results Compared with the negative control group,the levels in the ox-LDL group of LOX-1,pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were increased significantly,the proliferations of HUVEC and the productions of NO were decreased (P＜0.05).Compared with the ox-LDL group,the levels in the positive control group and the PD98059+ox-LDL group of pMEK1/2,RhoA,ROCK1,ROCK2,TNF-α and IL-6 were decreased,the proliferation of HUVEC and the production of NO were increased (P＜0.05).Conclusion PD98059 inhibit the MEK1/2 signaling pathway to suppress the ox-LDL induced damage of HUVEC by decreasing the expression of LOX-1.
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Aim To investigate the role and mecha-nism of exchange protein directly activated by cAMP (Epac) protein in the paraventricular nucleus(PVN) of the hypothalamus in the development of inflammatory pain in rats. Methods Adult SD male rats were cho-sen to establish the model of inflammatory pain through subcutaneous injection of complete Freund's adjuvant(CFA) on the center of left hind foot. Western blot was used to detect the changes of the expression of Ep-ac protein. Thermal withdrawal latency(TWL) was ob-served after the PVN injecting 8p-CPT-2′-O-Me-cAMP (8p-CPT),the agonist of Epac. Then activated down-stream MEK1/2 protein of Epac in PVN was detected using Western blot when the potency was the strongest.Results ① Compared with normal saline(control group),TWL decreased significantly on d 1, d 3, d 5, d 7,d 9 on the ipsilateral foot of CFA group rats(P<0.01),whereas it returned to normal level in d 13;the paw mechanical withdrawal threshold(PMWT) de-creased significantly on d 6,d 8,d 10,d 12 and d 14 (P<0.05);②Compared with the control,the Epac1 protein in CFA group rats began to decrease from d 3, and significantly decreased on d 3 and d 9(P<0.05), however the expression of Epac2 had no significant change, meanwhile p-MEK1/2 protein decreased sig-nificantly on d 3(P<0.05);③Compared with micro-injection of saline into the PVN(Saline group), the heat hyperalgesia of 20 min and 1h decreased signifi-cantly and TWL increased significantly after PVN ad-ministration of 8p-CPT(8p-CPT group)(P <0.05);paraventricular nucleus p-MEK1/2 protein expression increased significantly in 30 min(P <0.05) and re-covered to normal level 2 h after administration. Con-clusion The Epac1-MEK1/2 signaling pathway in the paraventricular nucleus of the hypothalamus may be in-volved in the development of chronic inflammatory pain induced by CFA.
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Objective The study is to investigate the role of miR-328 in endothelial mesenchymal transition (EMT)induced by high glucose in human umbilical vein endothelial cells (HUVECs)and its signaling mechanism.Methods HUVECs were cultured in high glucose environment to induce EMT;The recombinant lentiviruses were created by miR-328 and antagomiR- 328 transfection of HUVECs.The experiment was divided into seven groups: normal glucose;mannitol group;high glucose;miR-328;miR-328 virus negative control;high glucose + U0126;miR-328 + U0126.Double immunofluorescent staining was used to determine expression of EMT markers;Changes in miR-328 expression is examined by RT-qPCR;The expressions of type Ⅰ/Ⅲ collagen,p-MEK1/2 and p-ERK1/2 are examined by Western blot.Results 1)HUVECs showed positive staining for CD31 and α-SMA in high glucose group.2)The expression of miR-328 was up-regulated(P<0.05)in HUVECs treated by high glucose or miR-328.Compared with high glucose group or miR-328 group,miR-328 expression was less pronounced aftertreatment with U0126.3)The expressions of type Ⅰ/Ⅲ collagen increased in HUVECs treated by high glucose or miR-328(P<0.05) Compared with high glucose group or miR-328 group,typeⅠ/Ⅲ collagen expressions were less pronounced after treatment with U0126.4)The expressions of p-MEK1/2 and p-ERK1/2 were increased in HUVECs treated by high glucose or miR-328 in comparison to the control group (P<0.05);a lower expression of p-MEK1/2 and p-ERK1/2 were observed in U0126 group than in high glucose group or miR-328 group.Conclusions The phenomenon of EMT in HUVECs is induced by high glucose with increased expression of miR-328;overexpression of miR-328 induced EMT in HUVECs;miR-328 induced EMT is related with MEK1/2-ERK1/2 signaling pathway.
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Objective To observe the inhibitory effect of matrine on K-ras gene mutation colon cancer, and to clarify the inhibitory mechanism. Methods SW480 cells were treated with different concentrations of matrine. MTS method was used to detect the proliferation of SW480 cell lines. The apoptosis of SW480 cells was measured by flow cytometry. The migration of SW480 cells was examined by the scratch test. The expression of MEK1/2 protein was detected by Western blotting method. Results Compared with the blank control group, matrine (0.125-1 mg/mL) could inhibit the growth and proliferation of human colorectal cancer SW480 cell lines, promote the apoptosis, restrain the migration of SW480 cells, and inhibit the expression of MEK1/2 protein(P < 0.05), the effect showing a dose-dependent trend. Conclusion Matrine can effectively inhibit the proliferation and migration of SW480 cells, and promote SW480 cell apoptosis through the down-regulation of MEK1/2 protein expression in MAPK signal pathway system.
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MEK inhibition activates PI3K/AKT/mTOR pathway in triple negative breast cancer (TNBC) cell lines. Combination of PI3K inhibitor and MEK1/2 inhibitor is not appropriate for PI3K inhibitor insensitive TNBC cell lines. This study was designed to investigate the effects of dual treatments with mTOR1/2 inhibitor AZD8055 and MEK1/2 inhibitor PD0325901 in MDA-MB-435 cell line. MEK1/2 inhibition led to activation of AKT, which is the downstream signaling protein of PI3K pathway. The combination inhibited the phosphorylation of AKT and therefore abolished the feedback interaction of two pathways. Cell proliferation assay and DNA replication assay demonstrated that the dual treatments led to a significant synergistic inhibition of cell cycle progression and cell proliferation.
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Extracellular signal-regulated kinases 1 and 2 (ERK1/2) pathway, which was the first cell signal transduction pathway to be discovered, consisted of Ras, Raf, MEK1/2 and ERK1/2. After the activation of ERK1/2 pathway, extracellular signals can be transmitted from the cell membrane to the nucleus. It was involved in many physiological and pathological functions of cells, such as growth, proliferation, differentiation, apoptosis and etc. It was also related to the onset of many diseases, which included multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). The activation of ERK1/2 pathway can induce the activation of astrocyte, MG, T cell and macrophage, which released a variety of inflammatory cytokines. It caused myelin damage which induced MS/EAE onset. A number of studies indicated that inhibiting ERK1/2 pathway can reduce the releasing of inflammatory cytokines and myelin damage for MS/EAE alleviation. It provided an important target for the development of MS treatment medication.
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Objective To investigate the effects of extracellular signal-regulated kinase l/2(ERKl/2) pathway inhibitor U0126 on isopreterenol(ISO)-induced atrial fibrosis and connexin 40 (Cx40) remodeling in rats. Methods Thirty-two male SD rats were evenly randomized into control group, DMSO group, ISO (5 mg/[kg • d])+DMSO group (fibrosis group), and ISO (5 mg/[kg • d]) + U0126 (0. 5 mg/[kg • d]) + DMSO group (U0126-treated group). The corresponding reagents were given to each group once a day and the rats were killed and the myocardial tissues were collected after 7 d. The Ang IJ contents in the myocardial tissues were measured by radioimmunoassay; H-E staining and Masson staining were applied to measure the degree of atrial fibrosis; p-MEKl/2, p-ERKl/2, and Cx40 were detected by immunohistochemistry method. Results (1) The contents of Ang IJ were similar between control group ([242. 133 ± 4. 870] ng/L) and DMSO ([239. 412 ± 1. 795] ng/L) group (P>0. 05). Compared with the above two groups, Ang IJ contents in fibrosis group ([500. 250 ± 8. 869] ng/L)and U0126-treated group([498. 695 ± 9. 340]ng/L) were significantly increased (P alKO. 01). (2) Control group and DMSO group had no atrial fibrosis; the degree of atrial fibrosis in U0126-treated group was significantly lower than that in the fibrosis group (P0. 05), and those in the fibrosis group were significantly increased compared with control group and DMSO group(P0. 05), and was significantly decreased compared with the fibrosis group(P0. 05), and Cx40 was distributed in myocardial cell intercalated disc in a linear manner. The content of Cx40 was significantly reduced (P0. 05)and most of the Cx40 was linearly distributed in myocardial cell intercalated disc. Meanwhile, the reduce degree of Cx40 content in U0126-treated group was significantly decreased than that in the fibrosis group(P<0. 01), and some Cx40 was linearly distributed in myocardial cell intercalated disc. Conclusion Long-term Ang TJ elevation in myocardium may be involved in atrial fibrosis and Cx40 remodeling, and U0126 can efficiently improve atrial fibrosis and Cx40 remodeling by inhibiting the activation of ERK1/2 pathway.
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The mitogenic effects of periodic mechanical stress on chondrocytes have been studied extensively but the mechanisms whereby chondrocytes sense and respond to periodic mechanical stress remain a matter of debate. We explored the signal transduction pathways of chondrocyte proliferation and matrix synthesis under periodic mechanical stress. In particular, we sought to identify the role of the MEK1/2-ERK1/2 signaling pathway in chondrocyte proliferation and matrix synthesis following cyclic physiologic mechanical compression. Under periodic mechanical stress, both rat chondrocyte proliferation and matrix synthesis were significantly increased (P < 0.05) and were associated with increases in the phosphorylation of Src, PLCγ1, MEK1/2, and ERK1/2 (P < 0.05). Pretreatment with the MEK1/2-ERK1/2 selective inhibitor, PD98059, and shRNA targeted to ERK1/2 reduced periodic mechanical stress-induced chondrocyte proliferation and matrix synthesis (P < 0.05), while the phosphorylation levels of Src-Tyr418 and PLCγ1-Tyr783 were not inhibited. Proliferation, matrix synthesis and phosphorylation of MEK1/2-Ser217/221 and ERK1/2-Thr202/Tyr204 were inhibited after pretreatment with the PLCγ1 inhibitor U73122 in chondrocytes in response to periodic mechanical stress (P < 0.05), while the phosphorylation site of Src-Tyr418 was not affected. Inhibition of Src activity with PP2 and shRNA targeted to Src abrogated chondrocyte proliferation and matrix synthesis (P < 0.05) and attenuated PLCγ1, MEK1/2 and ERK1/2 activation in chondrocytes subjected to periodic mechanical stress (P < 0.05). These findings suggest that periodic mechanical stress promotes chondrocyte proliferation and matrix synthesis in part through the Src-PLCγ1-MEK1/2-ERK1/2 signaling pathway, which links these three important signaling molecules into a mitogenic cascade.