Dual specificity phosphatases 8 directly interacts with mitogen-activated protein kinase 1 to regulate the proliferation and apoptosis of rheumatoid arthritis fibroblast-like synovial cells / 中华风湿病学杂志

Objective:To explore the interaction between dual specificity phosphatases 8 (DUSP8) and mitogen-activated protein kinase 1 (MAPK1) in rheumatoid arthritis fibroblast-like synovial (RA-FLS), and its effect on the proliferation and apoptosis of RA-FLSs.Methods:RA-FLS and normal fibroblast-like synovial cells (FLS) were cultured. Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) and Western blotting were used to detect the expression levels of DUSP8 mRNA and protein in the two groups of cells. DUSP8 overexpression cell lines and DUSP8 silencing cell lines were constructed using cell transfection technology and RNA interference technology, respectively. Cell counting kit 8 (CCK-8) method was used to detect cell proliferation in each group, and flow cytometry was used to detect cell apoptosis in each group. Western blotting was used to detect the expression levels of DUSP8, MAPK1, p-MAPK1, ki-67 and Bax protein in each group. The indirect immunofluorescence experiment was used to analyze the spatial co-localization of DUSP8 and MAPK1, and the co-immunoprecipitation experiment was used to analyze whether there was interaction between DUSP8 and MAPK1 protein. The t-test was used to compare the means of the two groups. One-way analysis of variance was used to compare the mean values of the three groups of samples, and then the LSD- t tests were used to compare the two groups. Results:In RA-FLS, both mRNA [(2.4±0.6) vs (11.2±0.8), t=21.63, P<0.001] and protein levels [(0.24±0.04) vs (0.74±0.08), t=9.45, P<0.001] of DUSP8 were significantly lower than FLS. Compared with the blank control group and the overexpression control group, RA-FLS cells transfected with pcDNA3.1-Myc-DUSP8 could inhibit the proliferation of RA-FLS cells [(90.5±5.6) vs (92.5±1.8) vs (56.4±4.4), F=138.60, P<0.001], increase the rate of apoptosis significantly [(12.7±1.4)% vs (12.6±1.3)% vs (27.5±3.0)%, F=16.98, P<0.001], increase the expression levels of DUSP8 [(0.49±0.05) vs (0.45±0.04) vs (0.73±0.07), Bax (0.39±0.06) vs (0.36±0.05) vs (0.89±0.10)] and down-regulate the expression levels of ki-67 [(1.07±0.12) vs (1.11±0.16) vs (0.70±0.08), and p-MAPK1/MAPK1 [(0.59±0.06) vs (0.65±0.07) vs (0.39±0.03) (all P<0.001). Compared with the blank control group and the silent control group, RA-FLS cells transfected with siRNA-DUSP8 could promote the proliferation of RA-FLS cells [(90.5±5.6) vs (91.1±2.9) vs (128.3±4.6), F=137.50, P<0.001) and decrease apoptosis rate [(12.7±1.4) vs (13.2±1.2) vs (5.4±0.7), F=16.98, P<0.001], down-regulate the expression levels of DUSP8 [(0.492±0.048) vs (0.432±0.051) vs (0.102±0.024)], Bax [(0.391±0.062) vs (0.411±0.058) vs (0.090±0.011)], and up-regulate the expression levels of ki-67 [(1.07±0.12) vs (1.11±0.15) vs (1.93±0.22)], p-MAPK1/MAPK1 [(0.59±0.06) vs (0.68±0.06) vs (0.93±0.11)] (all P<0.001). The results of indirect immunofluorescence tests showed that both DUSP8 and MAPK1 were ex-pressed in the cytoplasm and nucleus of RA-FLS. The co-immunoprecipitation study verified that DUSP8 and MAPK1 protein could interact with each other. Conclusion:DUSP8 can bind to MAPK1 and regulate the abundance of active phospho-MAPK1 through its phosphatase activity and by inhibiting the proliferation of RA-FLS and promoting apoptosis.

Noni (Morinda citrifolia L.) fruit juice delays immunosenescence in the lymphocytes in lymph nodes of old F344 rats / 中西医结合学报

<p><b>OBJECTIVE</b>Aging is associated with the development of diseases because of immunosuppression and altered functioning of the neuroendocrine system. The medicinal properties of Morinda citrifolia L. have been widely exploited for the treatment of age-associated diseases. This study aims to investigate the in vitro and in vivo effects of noni (M. citrifolia) fruit juice (NFJ) on neuro-immunomodulation in the lymph node lymphocytes of F344 rats.</p><p><b>METHODS</b>Lymphocytes isolated from axillary and inguinal lymph nodes of young (3-4 months) and old (18-21 months) rats were treated in vitro with different concentrations (0.0001%, 0.01%, and 1%) of NFJ for a period of 24 h. In the in vivo study, old (16-17 months) male F344 rats were treated with 5 mL/kg body weight of 5%, 10% and 20% of NFJ, twice a day, by oral gavage, and lymph node lymphocytes were isolated after 60 d. Concanavalin A (Con A)-induced lymphocyte proliferation, interleukin-2 (IL-2) and interferon-γ (IFN-γ) production and expression of intracellular markers, such as phospho-extracellular signal-regulated kinase (p-ERK1/2), phospho-cAMP response element-binding protein, phospho-protein kinase B (p-Akt), phospho-tyrosine hydroxylase (p-TH), phospho-nuclear factor of κ light polypeptide gene enhancer in B-cells inhibitor-α (p-IκB-α) and phospho-nuclear factor-κB (p-NF-κB p65 and p50) were examined in the lymphocytes of lymph nodes.</p><p><b>RESULTS</b>NFJ increased Con A-induced lymphocyte proliferation, IL-2 and IFN-γ production, and p-ERK1/2 expression both in vitro and in vivo. In in vivo NFJ-treated old rats, lymph node lymphocytes showed increased expression of p-TH and Akt, nitric oxide production and decreased expression of p-NF-κB p65 and p50.</p><p><b>CONCLUSION</b>These results suggest that the immunostimulatory properties of NFJ are facilitated through intracellular signaling pathways involving ERK1/2, Akt and NF-κB.</p>

Mechanisms of ERK1/2 signaling pathway participate in inflammatory reaction caused by coronary heart disease / 天津医药

Objective To investigate the effects of ERK1/2 signaling pathway on coronary atherosclerosis-associated inflammatory reaction in autopsy cases. Methods Forty-five autopsy cases were divided into three groups:coronary arterydisease (CHD)-associated death group, CHD group and control group (n=15 for each group). The inflammatory cell infiltration in myocardial tissues was observed through staining leucocyte common antigen (CD45) by HE and immunohistochemistry method. The protein expression level and distribution in extracellular signal-regulated kinase 1/2 (t-ERK1/2) and phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) of myocardial tissues were detected by immunohistochemistry and Western-blot assay. The expression level of tumor necrosis factor α (TNF-α) was determined using semiquantitative RT-PCR analysis. The activity of nuclear factor (NF)-κB was assessed using electrophoretic mobility shift assay (EMSA). Results Compared with CHD and control groups, myocardial inflammatory cell counts, phosphorylation of ERK1/2, TNF-α mRNA expression and NF-κB activation were significantly increased in CHD-associated death group (P < 0.05). Western blot analysis showed that the phosphorylation of ERK1/2 was positively correlated with expression of TNF-αmRNA and the number of inflammatory cells in CHD-associated death group (r=0.675, P<0.01;r=0.893, P<0.01). Conclusion Results reveal that the activation of ERK1/2 signaling pathway is considered as an important mechanism for coronary atherosclerosis caused myocardial inflammatory reaction, which indicates that the inhibition of ERK1/2 signal transduction pathway may become a potential new target for prevention and treatment of atherosclerotic coronary infarction.

The expression and significance of integrinαvβ8, p38 and ERK1/2 in the liver of children with biliary atresia / 天津医药

Objective To investigate the expression and significance of integrin αvβ8, p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) proteins, which are TGF-β1 pathway related regulatory protein, in liver fibrosis of children with biliary atresia (BA). Methods Fifteen cases of BA (Kasai group) and 10 cases of congenital biliary dilatation (CBD group) were collected in Tianjin Children’s Hospital. And liver biopsy specimens were collected in Tianjin first central hospital, including 10 cases of BA children who underwent liver transplantation due to liver failure after Kasai operation (liver transplantation group). The specimens of front part of the right lobe of the liver were taken for HE and immunohistochemistry (IHC) staining. The expressions ofαvβ8, p38 and ERK1/2 in liver were observed by IHC staining in three groups of liver tissues. Results HE staining showed fibroblast hyperplasia occasionally in CBD group, portal area expansion, fibrous tissue proliferation and wide spread bridging fibrosis with few pseudo lobules in Kasai group. In transplantation group, portal area was widened, the degree of fibrosis was severe and bridging fibrosis generally formed resulted in pseudo lobules widely. Imunohistochemistry showed that the expressions of αvβ8 and ERK1/2 were weakly positive, and the expression of p38 was negative in CBD group. In Kasai group, the expressions of αvβ8, p38 and ERK1/2 proteins were all strongly positive in liver cytoplasm, biliary epithelial cells and vascular endothelial cell cytoplasm. In liver transplantation group the expressions of αvβ8, p38 and ERK1/2 proteins were all strongly positive. The semi-quantitative analysis showed that the expressions levels of αvβ8, p38 and ERK1/2 were significantly higher in Kasai and liver transplantation groups than those of CBD group (P0.05). Conclusion The expressions ofαvβ8, p38 and ERK1/2 are gradually increased in liver of BA with the process of fibrosis, which indicate that they may be involved in the process of BA liver fibrosis.

Expressions of fibroblast growth factor-1, mitogen activated protein kinase-1 and CD34 in the lesions of patients with psoriasis vulgaris and their significance / 中华皮肤科杂志

Objective To estimate the relationship of fibroblast growth factor-1 (FGF-1),mitogen activated protein kinase-1 (MAPK-1) and CD34 expressions with clinicopathological findings in lesions of psoriasis vulgaris.Methods Skin specimens were collected from 30 patients with psoriasis vulgaris,whose general information and clinical findings were recorded.The protein expressions of FGF-1,MAPK-1 and CD34 in these specimens were detected by tissue chip technology and immunohistochemistry,and the mRNA expression of FGF-1 by reverse transcription-PCR.The correlations of FGF-1 protein expression with patients' gender,age,clinical stage,psoriasis area and severity index (PASI) score,angiogenesis and abnormal epidermal proliferation were statistically analyzed.Results The protein and mRNA expressions of FGF-1 were significantly higher in the lesions than in the control skin (both P ＜ 0.05).The protein expressions of CD34 and MAPK-1 were significantly increased,and positively associated with the protein expression of FGF-1 in psoriatic lesions.The up-regulation of FGF-1 protein was unrelated to the age or gender of patients,but associated with clinical stage and PASI score.The proportion of patients with increased FGF-1 expression was significantly larger in patients at active stage and with high PSAI score than in those at quiescent stage and with low PSAI score (P ＜ 0.05).Conclusions In psoriasis vulgaris,FGF-1 may participate in the abnormal epidermal proliferation and vascular dysplasia in dermal papilla,and the upregulation of FGF-1 appears to be associated with the progression of disease and aggravation of lesions.

A functional comparison between the HER2high/HER3 and the HER2low/HER3 dimers on heregulin-beta1-induced MMP-1 and MMP-9 expression in breast cancer cells

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2high/HER3 and the HER2low/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin-beta1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral-MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.

Inhibiting ERK1/2 pathway reduces brain edema and down-regulates matrix metalloproteinase-9 expression after subarachnoid hemorrhage in rats / 国际脑血管病杂志

Objective To study the effect of extracellular signal-regulated kinase1/2 (ERK1/2)inhibitor U0126 on matrix metalloproteinase-9(MMP-9)in brain tissue after subarachnoid hemorrhage(SAH)in rats and to investigate the action mechanisms of ERK1/2 and M M P-9 in blood-brain barrier(BBB)injury and brain edema after SAH.Methods Seventy-two male Sprague-Dawley rats were randomly divided into four groups:SAH model,sham operation,U0126 intervention,and vehicle groups.A SAH model was induced by injection of autologous blood into cisterna magna once.The dry-wet weight method was used to detected brain tissue water content in order to evaluate cerebral edema.BBB permeability was evaluated by the Evans blue extravasation method.The immunohistochemical method was used to detect the expression of MMP-9 and phosphorylated ERK1/2.Results The expression of phosphorylated ERK1/2 and MMP-9 was lower in the sham operation group.The expression of both was up regulated at 24 hours after SAH.The brain water content and Evans blue content also increased.U0126 treatment decreased the phosphorylation of ERK1/2 and the expression of MMP-9,improved the BBB permeability,and alleviated brain edema.Conclusions MMP-9 is involved in the pathophysiological processes of early BBB injury and brain edema aft er SAH.ERK1/2 pathway may play a vital role in the expression of MMP-9.U0126 may protect BBB and reduce brain edema after SAH by inhibiting the phosphorylation of ERK1/2.

Effects of GW5074 in the process of imDCs inducing differentiation of na(i)ve CD4+T cells into Treg cells in vitro / 中华器官移植杂志

Objective To establish a stable and efficient method of culturing imDCs in vitro,and to explore the effect of GW5074, which blocks ERK1/2 signal pathway in the process of imnature dentritic cells (imDCs) on inducing differentiation of the na(i)ve allogeneic CD4+ T cells into Treg cells in vitro. Methods The imDCs and mature DCs (mDCs) were isolated and cultured from the peripheral blood mononuclear cells (PBMC) derived from a healthy adult male volunteer, and they were identified by cell morphology, cell surface marker and cell functions respectively. Na(i)ve CD4+ T cells were isolated from newborn umbilical vein blood and were divided into 5 groups to be cultured: (1) Blank control group: Na(i)ve CD4+ T cells were cultured alone;(2) Positive control group: The irrDCs were Middle-concentration GW5074 group;(5) High-concentration GW5074 group. In the last three groups, imDCs and na(i)ve CD4+ T cells were co-cultured, the same as the positive control group, but these groups were added by GW5074 dilution at the concentrations of 8, 24, and 40μmol/Lrespectively. After co-culture for 5 days, the transformation ratio from naive CD4+T cells to Treg T cells was detected by flow cytometry. Results On the surface of imDCs, there was stronger pression of CD1a, but weaker expression of CD80 and CD83. On the contrary, on the surface of mDCs, there was weaker expression of CD1a, but stronger expression of CD80 and CD83. The stimulation index in imDCs group and mDCs group was 1.12±0.03 and 2.85±0. 07 respectively. The transformation ratio of Treg T cells in blank control group, positive control group, low-concentration GW5074 group, middle-concentration GW5074 group and high-concentration GW5074 group was (5. 81±1.36)%, (35.73±2.07)%, (22.53±2.11)%, (11.55±1.73)%, and (4.97±1.83)%respectively. One-way ANOVA analysis revealed that there was no significant difference between high-concentration GW5074 group and blank control group, P＞0. 05, but significant difference between the remaining groups, P＜0.01. Conclusion High purity of imDCs can be obtained from PBMC by induction with rhGM-CSF and rhIL-4. ERK1/2 signal pathway plays a role in inducing the immune tolerance. GW5074 can inhibit differentiation of na(i)ve CD4+ T cells into Treg T cells.

Thromboxane A2 modulates migration, proliferation, and differentiation of adipose tissue-derived mesenchymal stem cells

Prostanoid metabolites are key mediators in inflammatory responses, and accumulating evidence suggests that mesenchymal stem cells (MSCs) can be recruited to injured or inflamed tissues. In the present study, we investigated whether prostanoid metabolites can regulate migration, proliferation, and differentiation potentials of MSCs. We demonstrated herein that the stable thromboxane A2 (TxA2) mimetic U46619 strongly stimulated migration and proliferation of human adipose tissue-derived MSCs (hADSCs). Furthermore, U46619 treatment increased expression of alpha-smooth muscle actin (alpha-SMA), a smooth muscle marker, in hADSCs, suggesting differentiation of hADSCs into smooth muscle-like cells. U46619 activated ERK and p38 MAPK, and pretreatment of the cells with the MEK inhibitor U0126 or the p38 MAPK inhibitor SB202190 abrogated the U46619-induced migration, proliferation, and alpha-SMA expression. These results suggest that TxA2 plays a key role in the migration, proliferation, and differentiation of hADSCs into smooth muscle-like cells through signaling mechanisms involving ERK and p38 MAPK.

Endothelin 1 protects HN33 cells from serum deprivation-induced neuronal apoptosis through Ca2+-PKCalpha-ERK pathway

Endothelins (ETs), which were originally found to be potent vasoactive transmitters, were known to be implicated in nervous system, but the mode of mechanism remains unclear. ETs (ET-1, ET-2, and ET-3) were added to HN33 (mouse hippocampal neuron chi neuroblastoma) cells. Among the three types of ET, only ET-1 increased the intracellular calcium levels in a PLC dependent manner with the induction of ERK 1/2 activation. As the result of ET-1 exposure, the survival rate of HN33 cells and the PKCalpha translocation into the plasma membrane were increased. We suggest that ET-1 participated in the neuroprotective effect involving the calcium-PKCalpha-ERK1/2 pathway.

High expression of ERK5 in pancreatic acinus of diabetic rats and its significance / 第三军医大学学报

Objective To investigate the expression and significance of big mitogen-activated protein kinase 1(BMK1 or ERK5)in the pancreatic acinus of diabetic rats.MethodsTwenty SD rats were randomly divided into normal control group(n=10)and streptozotocin(STZ)-induced diabetic group(n=10).At the end of 14 weeks inducement,all rats were sacrificed and venous blood and pancreatic tissues were collected.The levels of fasting blood glucose,insulin and blood fat were determined to identify the establishment of diabetic rat model.The specimens of pancreas were fixed and embedded.Hematoxylin-eosin staining was performed to observe the morphological changes.Periodic acid-Schiff(PAS)staining was carried out for the vascular changes.The expressions of type Ⅰ and Ⅳ collagen were determined by inmmunohistochemical method.BMK1/ERK5-mRNA was detected by in situ hybridization.ResultsCompared with the control,in diabetic group the vascular wall was thickening,inflammatory cells were infiltrated in perivascular area,local acinus was atrophy and the expression of type Ⅳ collagen was mainly expressed in vascular basement membrane,while type Ⅰ collagen was in local atrophy acinus tissue with inflammation(P