Chlamydia psittaci SINC protein inhibits host cell apoptosis through activating MAPK/ERK signaling pathway / 中华微生物学和免疫学杂志

Objective:To investigate the effects of SINC, a novel secreted protein of Chlamydia psittaci, on the apoptosis of host cells and the regulatory role of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway in it. Methods:HeLa cells were treated with recombinant SINC. The expression of Bax and Bcl-2 at protein level and the phosphorylation of ERK1/2 were analyzed by Western blot. Hoechst 33258 staining was used to detect the apoptosis of HeLa cells after SINC stimulation. Moreover, HeLa cells were pretreated with MEK1/2 inhibitor U0126 (50 μmol/L), and then stimulated with different concentrations of SINC for different time. Flow cytometry was used to detect the changes in cell apoptosis rates and Western blot was performed to detect the expression of Bax and Bcl-2 at protein level.Results:Treating HeLa cells with 10 μg/ml of SINC for 18 h resulted in down-regulated Bax and up-regulated Bcl-2 at protein level. Besides, the ratio of Bax/Bcl-2 was the lowest and a significant increase in the ratio of phosphorylated ERK1/2 (p-ERK1/2) to ERK1/2 was observed. Hoechst 33258 staining showed that the number of apoptotic bodies decreased significantly after stimulating HeLa cells with 5, 10 and 15 μg/ml of SINC. In the presence of MEK1/2 inhibitor U0126, the expression of Bcl-2 at protein level was down-regulated, while the expression of cleaved PARP was significantly up-regulated. Flow cytometry showed a significantly enhanced apoptosis of HeLa cells.Conclusions:SINC can inhibit the apoptosis of HeLa cells through activating the MAPK/ERK signaling pathway.

Hei Xiaoyaosan Regulates RAS/RAF/MEK/ERK Signaling Pathway to Ameliorate Oxidative Stress in Rat Model of AD / 中国实验方剂学杂志

ObjectiveTo investigate the role and mechanism of Hei Xiaoyaosan in intervening in oxidative stress in the rat model of Alzheimer's disease （AD） via modulating the rat sarcoma （RAS）/rapidly accelerating fibrosarcoma （RAF）/mitogen-activated protein kinase kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodOne hundred 4-month-old SPF-grade Wistar male rats were randomly grouped as follows： 10 in the blank group， 10 in the sham group （bilateral hippocampus injected with 1 μL normal saline）， and 80 in the modeling group ［bilateral hippocampus injected with 1 μL amyloid beta protein 1-42 （Aβ1-42） solution for the modeling of AD］. Fifty rats qualified for modeling were selected and randomized into the model， donepezil hydrochloride （0.5 mg·kg-1）， and high-， medium-， and low-dose （15.30， 7.65， 3.82 g·kg-1， respectively） Hei Xiaoyaosan groups. The rats were administrated with corresponding drugs by gavage once a day for 42 consecutive days. At the end of gavage， Morris water maze test was performed to examine the learning and memory abilities of the rats， and Nissl staining was used to observe the pathological changes of neurons in CA3 region of the hippocampus. The immunofluorescence assay was used to observe Aβ deposition and tau phosphorylation. Western blot was employed to determine the protein levels of RAS， RAF， phosphorylated （p）-RAF， MEK， p-MEK， ERK， and p-ERK in the hippocampal tissue. Biochemical methods were used to determine the levels of reactive oxygen species （ROS）， malondialdehyde （MDA）， and superoxide dismutase （SOD） in the hippocampal tissue. ResultCompared with the sham group， the model group showed prolonged escape latency （P<0.01）， shortened swimming distance in the target quadrant （P<0.01）， reduced and uneven stained Nissl bodies， enhanced fluorescence intensity of Aβ and p-tau （P<0.01）， up-regulated protein levels of RAS， p-RAF， p-MEK， and p-ERK in the hippocampal tissue （P<0.01）， increased ROS and MDA content （P<0.01）， and decreased SOD activity （P<0.01） on day 5. Compared with the model group， donepezil hydrochloride and high-， medium-， and low-dose Hei Xiaoyaosan shortened the escape latency （P<0.01）， increased the swimming distance in the target quadrant （P<0.01）， improved the arrangement， morphology， and structures of neurons and the number and distribution of Nissl bodies， decreased the fluorescence intensity of Aβ and p-tau （P<0.01）， up-regulated the protein levels of RAS， p-RAF， p-MEK， and p-ERK （P<0.05， P<0.01）， decreased the ROS and MDA content （P<0.01）， and increased the SOD activity （P<0.01） on day 5. ConclusionHei Xiaoyaosan may ameliorate oxidative stress， reduce Aβ and p-tau levels， and inhibit hippocampal neuronal damage by regulating the RAS/RAF/MEK/ERK signaling pathway， thus improving learning and memory abilities.

Echinacoside hampers malignant progression of breast cancer MCF-7 cells by modulating AKR1B10/ERK signal transduction / 中国中药杂志

This study analyzes the impact of echinacoside(ECH) in the proliferation, metastasis and adriamycin(ADR) resistance of breast cancer(BC) MCF-7 cells via the modulation of aldo-keto reductase family 1 member 10(AKR1B10)/extracellular signal-regulated kinase(ERK) pathway. The chemical structure of ECH was firstly confirmed. MCF-7 cells were treated with different concentration(0, 10, 20, 40 μg·mL~(-1)) of ECH for 48 h. Western blot was used to analyze expression of AKR1B10/ERK pathway-associated proteins and cell counting kit-8(CCK-8) assay to determine cell viability. MCF-7 cells were collected and classified into control group, ECH group, ECH + Ov-NC group, and ECH + Ov-AKR1B10 group. Then Western blot was employed to analyze the expression of AKR1B10/ERK pathway-associated proteins. CCK-8 and 5-ethynyl-2'-deoxyuridine(EdU) assay were used to examine cell proliferation. Cell migration was appraised with scratch assay, Transwell assay, and Western blot. Eventually, MCF-7 cells were treated with ADR for 48 h to induce ADR resistance. Cell viability was tested by CCK-8 assay and cell apoptosis was estimated based on terminal-deoxynucleoitidyl transferase mediated nick end labeling(TUNEL) assay and Western blot. Based on Protein Data Bank(PDB) and molecular docking, the binding affinity of ECH to AKR1B10 was assessed. Various doses of ECH decreased the expression of AKR1B10/ERK pathway-associated proteins in a dose-dependent manner and declined cell viability compared with the control group. Compared with the control group, 40 μg·mL~(-1) ECH blocked the AKR1B10/ERK pathway in MCF-7 cells and inhibited the proliferation, metastasis and ADR resistance of the cells. Compared with the ECH + Ov-NC group, ECH + Ov-AKR1B10 group showed the recovery of some biological behaviors of MCF-7 cells. ECH also targeted AKR1B10. ECH can inhibit the proliferation, metastasis, and ADR resistance of BC cells by blocking AKR1B10/ERK pathway.

Type III secretory protein SINC of Chlamydia psittaci promotes host cell autophagy by activating the MAPK/ERK signaling pathway / 南方医科大学学报

OBJECTIVE@#To investigate the effects of SINC, a secreted protein of Chlamydia psittaci, on autophagy of host cells and the role of MAPK/ERK signaling pathway in mediating SINC-induced autophagy.@*METHODS@#RAW 264.7 cells treated with recombinant SINC were examined for changes in expression levels of LC3-II, Beclin-1, phosphorylated and total ERK1/2 using Western blotting. The expression level of LC3 in the treated cells was detected using immunofluorescence analysis, and the formation of autophagosomes and autolysosomes was observed with transmission electron microscopy (TEM). The effect of pretreatment with U0126 (a specific ERK inhibitor) on the expression levels of LC3-II and Beclin-1 in RAW 264.7 cells exposed to different concentrations of SINC was examined using Western blotting, and LC3 puncta in the cells was detected with immunofluorescence analysis.@*RESULTS@#The expression levels of LC3-II and Beclin-1 were the highest in RAW 264.7 cells treated with 2 μg/mL SINC for 12h. Immunofluorescence analysis showed exposure to SINC significantly increased the number of cells containing LC3 puncta, where the presence of autophagosomes and autolysosomes was detected. Exposure to 2 μg/mL SINC for 15 min resulted in the most significant increase of the ratios of p-ERK1/2/ERK1/2 in RAW 264.7 cells. Pretreatment of the cells with U0126 prior to SINC exposure significantly decreased the ratio of p-ERK1/2/ERK1/2, lowered the expression levels of LC3-II and Beclin-1, and decreased LC3 aggregation in the cells.@*CONCLUSIONS@#SINC exposure can induce autophagy in RAW 264.7 cells by activating the MAPK/ERK signaling pathway.

Effect of Yanghetang-containing Serum on Breast Cancer 4T1 Cells Based on MEK/ERK Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo investigate the effects of Yanghetang （YHT） on breast cancer 4T1 cells and their mitogen-activated protein kinase （MEK）/extracellular signal-regulated kinase （ERK） signaling pathway. MethodThe YHT-containing serum was prepared from SD rats. The rats were randomly assigned into a blank group （normal saline） and low-， medium-， and high-dose （5.8， 11.6， 23.2 g·kg-1， respectively） YHT groups. The serum containing 10% YHT in each group was mixed with 90% RMPI 1640 complete medium， and the mixture was used to interfere with the cells. Cell counting kit-8 （CCK-8） method was used to detect the proliferation of the 4T1 cells treated with YHT for 24， 48， 72 h. The apoptosis， migration， and invasion of 4T1 cells were detected by flow cytometry， scratch test， and Transwell assay， respectively. Western blot was employed to determine the expression levels of MEK1/2， phosphorylation （p）-MEK1/2， ERK1/2， p-ERK1/2， and rat sarcoma virus （RAS） protein. ResultCompared with the blank group， the intervention with YHT-containing serum for 24， 48， and 72 h had significant inhibitory effect on 4T1 cell proliferation （P<0.05， P<0.01）. After intervention with YHT-containing serum for 48 h， the apoptosis rate of cells increased （P<0.01）. Compared with the blank group， the intervention with YHT for 24 h and 48 h decreased the healing ability of cells in the scratch test （P<0.01）. The invasive ability of cells treated with the low， medium， and high-dose YHT containing serum showed a decreasing trend （P<0.01）. Compared with the blank group， YHT-containing serum did not change the expression of MEK1/2 and ERK1/2 while down-regulating the expression of p-MEK1/2， p-ERK1/2， and RAS protein （P<0.01）. ConclusionYHT can inhibit the proliferation， migration， and invasion and promote the apoptosis of breast cancer 4T1 cells. In may promote the apoptosis by inhibiting the MEK/ERK signaling pathway and down-regulating the expression of p-MEK1/2， p-ERK1/2， and RAS protein.

LNX1 inhibits the proliferation,invasion and migration of renal clear cell carcinoma cells by regulating TIAM1/ERK signaling pathway / 肿瘤

Objective:To investigate the effect of ligand of numb-protein X1(LNX1)on the proliferation,invasion and migration of renal clear cell carcinoma cells and its underlying molecular mechanism. Methods:Gene Expression Profiling Interactive Analysis(GEPIA)database was used to analyze the mRNA expression level of LNX1 in renal clear cell carcinoma tissues and its relationship with the prognosis of patients with renal clear cell carcinoma.LNX1 gene specific shRNA(shLNX1)was delivered into renal clear cell carcinoma cell lines 786-O and ACHN by lentiviral infection,and flag-LNX1 plasmid was delivered into 786-O and ACHN cells by transient transfection.CCK-8 assay and colony formation assay were used to assess the effects of LNX1 silencing or overexpression on the proliferation of 786-O and ACHN cells.Transwell assay was used to evaluate the effects of LNX1 silencing or overexpression on the invasion and migration of 786-O and ACHN cells.Bioinformatics analysis was used to screen the downstream target genes of LNX1.Western blotting was used to examine the effects of LNX1 silencing or overexpression on the expression level of T-lymphoma invasion and metastasis-inducing protein 1(TIAM1)as well as the expression levels of total and phosphorylated ERK(phospho-ERK,p-ERK)in the ERK signaling pathway downstream of TIAM1 in 786-O and ACHN cells.786-O and ACHN cells overexpressing LNX1 were treated with proteasome inhibitor MG132,and the protein expression level of TIAM1 was analyzed by Western blotting.Finally,myc-TIAM1 recombinant plasmid was transfected into LNX1-overexpressing cells.Then,the expression levels of proteins in the ERK signaling pathway and the abilities of proliferation,invasion and migration of 786-O and ACHN cells were examined by Western blotting,colony formation assay and Transwell assay,respectively. Results:The mRNA expression level of LNX1 in renal clear cell carcinoma tissue was decreased(P＜0.05)and was positively correlated with the survival time of patients with renal clear cell carcinoma(P＜0.001).LNX1-silencing 786-O and ACHN cells and LNX1-overexpressing 786-O and ACHN cells were constructed successfully.After LNX1 silencing,the proliferation,invasion and migration of 786-O and ACHN cells were significantly enhanced(all P＜0.05).After LNX1 overexpression,the abilities of proliferation,invasion and migration of 786-O and ACHN cells were significantly decreased(all P＜0.05).Bioinformatics analysis identified TIAM1 as a potential target of LNX1.After silencing LNX1,the protein expression levels of TIAM1 and p-ERK were significantly increased(all P＜0.05),while the expression level of ERK remained unchanged.After LNX1 overexpression,the protein expression levels of TIAM1 and p-ERKwere significantly decreased(all P＜0.01),while the expression level of ERK was unchanged.Treatment with proteasome inhibitor MG132 increased the protein expression level of TIAM1 in LNX1-overexpressing 786-O and ACHN cells(P＜0.01 and P＜0.001).After LNX1-overexpressing cells were transfected with myc-TIAM1 plasmid,the protein expression level of p-ERK was increased,the abilities of cell proliferation,invasion and migration were enhanced(all P＜0.05),and the expression level of ERK protein remained unchanged. Conclusion:LNX1 inhibits the proliferation,invasion and migration of renal clear cell carcinoma cells by degrading TIAM1 which further regulates the phosphorylation of ERK.

Effect of Tongsai granules on airway epithelial barrier in a rat model of acute exacerbation of chronic obstructive pulmonary disease and role of EGFR/ERK signaling pathway / 中国病理生理杂志

AIM:To investigate the effect of Tongsai granules(TSG)on epithelial barrier dysfunction in acute exacerbation of chronic obstructive pulmonary disease(AECOPD)and the underlying mechanism.METHODS:Twenty-four Sprague-Dawley rats were randomly divided into control group,model group,TSG group,and moxifloxacin(MXF)+salbutamol(STL)group.Rat COPD model was established over 8 weeks.On day 3 of week 9,the rats with COPD were intratracheally administered Klebsiella pneumoniae to establish the AECOPD model.On days 1 to 2 and 4 to 7 in week 9,saline was administered via oral gavage to the rats in control and model groups,and the rats in TSG and MXF+ STL groups were treated daily with TSG and MXF+STL by gavage,respectively.Peak expiratory flow(PEF),histopatho-logical changes,and the expression levels of interleukin-1β(IL-1β),IL-6,tumor necrosis factor-α(TNF-α),matrix me-talloproteinase 2(MMP2),MMP9,zonula occludens-1(ZO-1),E-cadherin(E-Cad)and occludin(OCC)were deter-mined.Moreover,human bronchial epithelial BEAS-2B cells were exposed to cigarette smoke extract(CSE)and treated with different TSG fractions,and the protein levels of ZO-1,E-Cad,OCC,epidermal growth factor receptor(EGFR),phosphorylated EGFR(p-EGFR),extracellular signal-regulated kinase(ERK)and phosphorylated ERK(p-ERK)were determined.RESULTS:Treatment with TSG significantly reduced bronchial wall thickness,mean linear intercept,and the levels of IL-1β,IL-6,TNF-α,MMP2 and MMP9(P＜0.05 or P＜0.01),significantly increased mean alveolar number and PEF(P＜0.01),and up-regulated the ZO-1,E-Cad and OCC protein levels(P＜0.01)in the lungs of AECOPD rats.Treatment with TSG2,the second TSG fraction,increased the protein levels of ZO-1,E-Cad and OCC in a dose-dependent manner in CSE-exposed BEAS-2B cells(P＜0.05 or P＜0.01).Network pharmacology analysis of 328 targets of the com-pounds in TSG2 and 3 864 genes related to AECOPD suggested that TSG2 relieved AECOPD likely through the regulation of ERBB2,ERK,EGFR,IL and WNT signaling pathways.Treatment with TSG2 also inhibited CSE-induced increases in p-EGFR and p-ERK levels in BEAS-2B cells(P＜0.05 or P＜0.01).CONCLUSION:Treatment with TSG could maintain airway epithelial barrier function in AECOPD rats,likely through the inhibition of EGFR/ERK signaling pathway.

Research progress on function and molecular mechanism of transcription factor ELK1 of Ets / 中国生物制品学杂志

@#Ets transcription factor ELK 1,a member of the ternary complex factor(TCF) subfamily in the Ets family,is directly downstream signal of MAPK/ERK pathway and is activated by phosphorylation to execute the function of ERK signal.ELK1,which is highly expressed and phosphorylated in stem cells and tumor cells,plays a role in promoting proliferation,inhibiting apoptosis and differentiation in stem cells.In tumor cells,ELK1 has shown to promote proliferation,migration,and inhibit apoptosis.In neural cells,ELK1 is involved in long-term memory,learning,addiction and other functions.In this paper,the research progress on the main structure and basic biological characteristics of ELK1,the function and mechanism of ELK 1 in tumor cells,stem cells and nerve cells are reviewed in order to provide new ideas for the follow-up research.

Role of free radicals in cerebral ischemic tolerance of rats induced by limb ischemic preconditioning / 中国病理生理杂志

AIM: To explore whether free radicals participate in cerebral ischemic tolerance and the up-regula-tion of p38 MAPK and ERK signaling pathways in rats induced by limb ischemic preconditioning (LIP). METHODS: A total of 128 Wistar rats with permanent occlusion of bilateral vertebral arteries were randomly divided into sham group (n=16), cerebral ischemia (CI) group (n=16), LIP+CI group (n=16), DMTU (a free radical scavenger)+LIP+CI group (n=64) and DMTU+sham group (n=16). Six rats in each group were used to observe the delayed neuronal death (DND) in hippocampal CA1 region by thionin staining at 7 d after the end of operation. Other 10 rats in each group were used to de-tect the expression of p38 MAPK and ERK in hippocampal CA1 region by immunohistochemistry and Western blot. RE-SULTS: Lethal CI resulted in obvious DND in hippocampal CA1 region. However, LIP reversed the above injurious changes, represented by the decrease in histological grade and the increase in neuronal density compared with CI group (P<0. 01). Moreover, LIP significantly up-regulated the expression of p38 MAPK and ERK in hippocampal CA1 region com-pared with CI group (P<0. 01). Administration of free radical scavenger DMTU via femoral vein before LIP partially re-versed the neuroprotective effect of LIP, and blocked the up-regulation of p38 MAPK and ERK expression in hippocampal CA1 region in rats compared with LIP+CI group (P<0. 01). CONCLUSION: Free radicals are involved in the neuropro-tection and up-regulation of p38 MAPK and ERK expression induced by LIP in rats.

Licochalcone A inhibits osteosarcoma proliferation by Akt/ERK signaling pathway / 中国药理学通报

Aim To investigate the effect of licochalcone A (LCA) on proliferation and apoptosis of osteosarcoma HOS and U2OS cells and to explore its possible molecular mechanism. Methods The HOS and U2OS cells were cultured in vitro. MTT assay was used to detect the proliferation of the cells after being treated with different concentrations of LCA at different intervention time. Then HOS and U2OS cells were treated with 0. 1% DMSO, or different concentrations of LCA (5, 10, 20 μmol/L), and flow cytometry was used to assess the cell apoptosis. The expression of apoptosis-related protein cleaved PARP1, Bcl-2, Bax, and Akt, ERK were detected by Western blot. The antitumor effect of LCA was detected on U2OS xenograft mice in vivo. Results LCA could inhibit the proliferation of HOS and U2OS cells in a time-and dose-dependent manner. Flow cytometry showed that LCA treatment could induce cell apoptosis. Western blot results showed that LCA could inhibit the phosphorylation of Akt and ERK, increase the expression of cleaved PARP1 and Bax, and decrease the expression of Bcl-2. In the tumor-bearing mouse models, LCA significantly decreased the tumor volume (P < 0. 05) and weight (P < 0. 01). Conclusions LCA could inhibit the proliferation of HOS and U2OS and induce apoptosis possibly by inhibiting the Akt/ERK signaling pathway.

Leptin-mediated ERK Signaling Pathway Promotes the Transformation of Rat Alveolar Type II Epithelial Cells Induced by Yunnan Tin Mine Dust / 中国肺癌杂志

BACKGROUND@#Currently, a significant number of miners are involved in mining operations at the Gejiu tin mine in Yunnan. This occupational setting is associated with exposure to dust particles, heavy metals, polycyclic aromatic hydrocarbons, and radioactive radon, thereby significantly elevating the risk of lung cancer. This study aims to investigate the involvement of leptin-mediated extracellular regulated protein kinase (ERK) signaling pathway in the malignant transformation of rat alveolar type II epithelial cells induced by Yunnan tin mine dust.@*METHODS@#Immortalized rat alveolar cells type II (RLE-6TN) cells were infected with Yunnan tin mine dust at a concentration of 200 μg/mL for nine consecutive generations to establish the infected cell model, which was named R₂₀₀ cells. The cells were cultured normally, named as R cells. The expression of leptin receptor in both cell groups was detected using the Western blot method. The optimal concentration of leptin and mitogen-activated protein kinase kinase (MEK) inhibitor (U0126) on R₂₀₀ cells was determined using the MTT method. Starting from the 20th generation, the cells in the R group were co-cultured with leptin, while the cells in the R₂₀₀ group were co-cultured with the MEK inhibitor U0126. The morphological alterations of the cells in each group were visualized utilizing hematoxylin-eosin staining. Additionally, concanavalin A (ConA) was utilized to detect any morphological differences, and an anchorage-independent growth assay was conducted to assess the malignant transformation of the cells. The changes in the ERK signaling pathway in epithelial cells after the action of leptin were detected using the Western blot method.@*RESULTS@#Both the cells in the R group and R₂₀₀ group express leptin receptor OB-R. Compared to the R₂₀₀ group, the concentration of leptin at 100 ng/mL shows the most significant pro-proliferation effect. The proliferation of R₂₀₀ cells infected with the virus is inhibited by 30 μmol/L U0126, and a statistically significant divergence was seen when compared to the control group (P<0.05). Starting from the 25th generation, the cell morphology of the leptin-induced R₂₀₀ group (R₂₀₀L group) underwent changes, leading to malignant transformation observed at the 30th generation. The characteristics of malignant transformation became evident by the 40th generation in the R₂₀₀L group. In contrast, the other groups showed agglutination of P40 cells, and the speed of cell aggregation increased with an increase in ConA concentration. Notably, the R₂₀₀L group exhibited faster cell aggregation compared to the U0126-induced R₂₀₀ (R₂₀₀LU) group. Additionally, the cells in the R₂₀₀L group were capable of forming clones starting from P30, with a colony formation rate of 2.25‰±0.5‰. However, no clonal colonies were observed in the R₂₀₀LU group and R₂₀₀ group. The expression of phosphorylated extracellular signal-regulated kinase (pERK) was enhanced in cells of the R₂₀₀L group. However, when the cells in the R₂₀₀L group were treated with U0126, a blocking agent, the phosphorylation level of pERK decreased.@*CONCLUSIONS@#Leptin can promote the malignant transformation of lung epithelial cells infected by mine dust, and the ERK signaling pathway may be necessary for the transformation of alveolar type II epithelial cells induced by Yunnan tin mine dust.

Intervention effects of Jinkui shenqi pills on renal fibrosis model rats and its mechanism / 中国药房

OBJECTIVE To investigate th e intervention effect of Jinkui shenqi pills on renal fibrosis （RF）model rats and its mechanism based on transforming growth factor β1/Smads（TGF-β1/Smads）and TGF-β1/extracellular signal regulated kinase （ERK） signaling pathway. METHODS Male SD rats were given adenine suspension （250 mg/kg）to induce RF model. After modeling ， they were randomly divided into model group ，Colchicine tablet group （positive control ，0.45 mg/kg）and Jinkui shenqi pills low-dose，medium-dose and high-dose groups （0.5，1，2 g/kg），with 10 rats in each group. Other 10 healthy rats were selected as normal group. The rats in administration groups were given the corresponding drugs intragastrically ；normal group and model group were given 0.1％ sodium carboxymethyl cellulose solution ，once a day ，for consecutive 30 d. After last medication ，the serum levels of creatinine （Cr）and blood urea nitrogen （BUN），renal weight and body weight were detected. The ratio of BUN/Cr and renal coefficient were calculated. The pathological morphology of renal tissue in rats were observed. The protein and mRNA expressions of TGF-β1，Smad2，Smad3，ERK1 and ERK 2 were detected. RESULTS Compared with normal group ，serum levels of Cr and BUN and renal coefficient were all increased significantly in model group （P＜0.05），while the ratio of BUN/Cr was decreased significantly （P＜0.05）. The volume of the kidney was significantly increased ，and the surface was seriously granulated. Mesangial hyperplasia ，dilation or atrophy of renal tubules ，accompanied by large-area collagen deposition，could be found. Protein and mRNA expressions of TGF-β 1，Smad2，Smad3，ERK1 and ERK 2 were increased significantly in renal tissue （P＜0.05）. Compared with model group ，above indexes of Jinkui shenqi pills groups were all reversed significantly （P＜0.05）；dilation or atrophy of renal tubules was relieved ，and collagen deposition was reduced to different extents. CONCLUSIONS Jinkui shenqi pills can improve renal function of RF model rats ，the mechanism of which may be associated with inhibiting TGF-β1/Smads and TGF-β1/ERK signaling pathway.

Downregulation of cytokeratin 18 increases NMIIA expression and promotes breast cancer metastasis / 中国药理学通报

Aim To investigate the role of aberrant cytokeratin 18(CK18) expression in breast cancer metastasis, anrl to elucidate the mechanism by identif¬ying its target.Methods The expression of CK.18 in human breast cancer tissues and cells was determined using immunohistochemical staining and Western blot, respectively.CK18 expression in human breast cancer MCF-7 cells was effectively down-regulated by shRNA, and its effect on breast cancer metastasis was further determined by scratch wound healing assay.The co-lo- cation of CK18 and non-muscle II A ( NMIIA) in MCF-7 cells was examined using double immunofluo¬rescence staining.The effect of CK18 down-regulation on the levels of NMIIA and c-Abl-ERK signaling was quantified by Western blot.Results Lower CK18 lev¬els was found in metastatic than that in primary breast cancer tissues and in highly invasive MDA-MB-231 than that in MCF-7 cells.CK.18 down-regulation pro¬moted the wound repair ability of MCF-7 cells 72h after scratch.CK18 and NMIIA were shown to co-locate in cytoplasm of MCF-7 cells.Moreover, down-regulation of CK18 increased NMIIA expression and activated the c-Abl-ERK signaling pathway in MCF-7 cells.Con¬clusions Down-regulation of CK18 could promote me¬tastasis of breast cancer, which is related to increased NMIIA expression and the activation of c-Abl-ERK sig¬naling pathway.

Shikonin Affects Uterine Leiomyoma in Rats by Regulating ERK Signaling Pathway / 中国实验方剂学杂志

Objective:To investigate the effect of shikonin on uterine leiomyoma in rats and its molecular mechanism. Method:Sixty female SD rats， of SPF grade and weighing 200-250 g， were randomly divided into the control group， model group， low-， medium-， and high-dose （5， 10， 20 mg·kg^-1） shikonin groups， and mifepristone group. A rat model of uterine leiomyoma was established， and the changes in uterine wet weight， uterine coefficient， and smooth muscle thickness were detected after drug administration for four successive weeks. The pathological changes in uterine tissue were observed by hematoxylin-eosin （HE） staining. The contents of estrogen receptor （ER） and progesterone receptor （PR） in serum and uterus were measured by enzyme-linked immunosorbent assay （ELISA）， and the protein expression levels of ER， PR， phosphorylated extracellular signal-regulated kinase （p-ERK）， and ERK in the uterine tissue were assayed by Western blot. Result:Compared with the control group， the model group exhibited significantly increased uterine wet weight， uterine coefficient， and smooth muscle thickness （<italic>P</italic><0.01）， uterus deformity， focal smooth muscle cell necrosis and hyperplasia， neutrophil infiltration. elevated serum and uterine ER and PR （<italic>P</italic><0.01）， and up-regulated p-ERK protein expression in the uterine tissue （<italic>P</italic><0.01）. Compared with the model group， shikonin at the middle and high doses and mifepristone significantly reduced the uterine wet weight， uterine coefficient， and smooth muscle thickness （<italic>P</italic><0.01）， relieved the pathological changes in uterus，and lowered serum and uterine ER and PR， and down-regulated the p-ERK protein expression in the uterine tissue （<italic>P</italic><0.05）. In addition， the uterine wet weight， smooth muscle thickness， serum ER， and uterine PR and p-ERK protein expression in the low-dose shikonin group were significantly lower than those in the model group （<italic>P</italic><0.05）. Conclusion:Shikonin produces the anti-uterine leiomyoma activity possibly by inhibiting the activation of ERK pathway.

Mechanisms of Wenjing Huayu Zhitong Therapy for Primary Dysmenorrhea with Cold Coagulation and Blood Stasis Syndrome Based on MAPK/ERK Signaling Pathway / 中国实验方剂学杂志

Objective:To study the effect of Wenjing Huayu Zhitong therapy （Xiangyan Zhitong prescription， XZP） on the mitogen activated protein kinase （MAPK）/extracelluar regulated protein kinase （ERK） signaling pathway of primary dysmenorrhea （PD） rats， and explore the pathogenesis of PD and the mechanism of action of Wenjing Huayu Zhitong therapy. Method:Forty-eight female SPF-grade Wistar rats were randomly divided into blank group，model group， western medicine group， low-dose XZP group， medium-dose XZP group， and high-dose XZP group， with 8 rats in each group. In addition to the blank group， dysmenorrhea rat model with cold coagulation and blood stasis syndrome was established by cold stimulation combined with estradiol benzoate and oxytocin. The rats in the blank group，model group，western medicine group， low-dose XZP group， medium-dose XZP group， and high-dose XZP group were given distilled water， distilled water，0.06 g·kg^-1 ibuprofen， 6.55 g·kg^-1 XZP， 13.09 g·kg^-1 XZP， and 26.18 g·kg^-1 XZP， respectively， by gavage for 6 days. The writhing latency and writhing frequency of rats were recorded within 30 min after oxytocin injection.Western blot was used to detect the protein expression of B-Raf， mitogen activates extracellular regulated kinases1/2 （MEK1/2）， extracellular regulated kinases1/2 （ERK1/2）， p-MEK1/2， p-ERK1/2， c-Jun， and cyclooxygenase-2 （COX-2） in rat uterus. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） was employed to detect the mRNA expression of B-Raf， MEK1， MEK2， ERK1， ERK2， c-Jun， and cyclooxygenase-2 （COX-2） in rat uterus. Result:Compared with the model group，the treatment groups showed insignificantly prolonged writhing latency and significantly reduced writhing frequency （<italic>P</italic><0.01）. On the 6^th day of modeling， there was no significant difference in the quantitative scores of symptoms and signs among the treatment groups. On the 12^th day of modeling， the scores changed little in the western medicine group and the low-dose XZP group and decreased significantly in the medium- and high-dose XZP groups （<italic>P</italic><0.01） compared with those in the model group. Compared with those in the blank group， the protein and mRNA levels of p-MEK1/2， p-ERK， B-Raf， c-Jun， and COX-2 in the model group were significantly up-regulated （<italic>P</italic><0.01）. Compared with those in the model group， the protein and mRNA levels of p-MEK1/2， p-ERK1/2， B-Raf， c-Jun， and COX-2 in the western medicine group， medium-dose XZP group， and high-dose XZP group were significantly down-regulated （<italic>P</italic><0.05，<italic>P</italic><0.01）. Conclusion:The mechanism of Wenjing Huayu Zhitong therapy in treating PD with cold coagulation and blood stasis syndrome may be related to the down-regulation of MAPK/ERK signaling pathway.

Transgelin inhibits proliferation and invasion of human thyroid cancer cells by blocking the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway / 中华内分泌代谢杂志

Objective:To explore the role of transgelin(TAGLN) in the occurrence and development of papillary thyroid carcinoma (PTC) and its possible signal pathway.Methods:One hundred cases of PTC tissues and corresponding paracancerous normal thyroid tissues were collected. Realtime quantitative PCR (RT-qPCR), Western blotting, and immunohistochemistry were used to analyze the expression of TAGLN in PTC tissues and corresponding paracancerous normal thyroid tissues. PTC cells were transfected with plasmid and shRNA lentivirus vector respectively to up-regulate or down-regulate the expression of TAGLN in order to detect the effects of them on the proliferation, invasion, and migration by cell proliferation assay(cell counting kit-8, CCK-8)and cell invasion and migration assays (Transwell). The effects of TAGLN on mitogen-activated protein kinase (MAPK)/extracellular-signal regulating kinase (ERK) signal pathway was detected with Western blotting.Results:RT-qPCR showed that there was no difference in the expression of TAGLN mRNA between PTC and corresponding paracancerous normal thyroid tissues ( P>0.05); Western blotting demonstrated that the expression of TAGLN protein in PTC tissues was significantly lower than that in corresponding paracancerous normal thyroid tissues ( P<0.01). Immunohistochemical results revealed that the expression of TAGLN in PTC tissues was significantly lower than that in corresponding paracancerous normal thyroid tissues. Overexpression of TAGLN inhibited the proliferation, invasion, and migration of PTC cells ( P<0.01), but knockdown of TAGLN promoted the proliferation, invasion, and migration of PTC cells ( P<0.01). Overexpression of TAGLN decreased the expression of phosphorylated ERK ( P<0.05), whereas silencing TAGLN increased phosphorylated ERK level in PTC cells( P<0.01). Conclusion:The expression of TAGLN in PTC is significantly decreased. It is related to the occurrence and development of PTC, and its mechanism may be related to MAPK/ERK signal pathway.

Yishen Tonglong Decoction inhibits the epithelial-mesenchymal transition and Ras/ERK signaling pathway in human prostate cancer DU-145 cells / 中华男科学杂志

Objective@#To observe the effect of Yishen Tonglong Decoction (YTD) on the epithelial-mesenchymal transition (EMT) and Ras/ERK signaling pathway in human PCa DU-145 cells and explore its action mechanism.@*METHODS@#We treated human PCa DU-145 cells with normal plasma (the blank control) or plasma containing 5% (low-dose), 10% (medium-dose) and 15% (high-dose) YTD. After intervention, we examined the proliferation of the DU-145 cells in different groups with CCK-8 and their apoptosis by Annexin V/PI double staining. We detected the cell cycle by PI assay, the invasion and migration of the cells using the Transwell chamber and scratch test, and the expressions of the proteins and genes related to the EMT and Ras/ERK signaling pathways in the cells by Western blot and RT-PCR.@*RESULTS@#Compared with the blank control group, high-, medium- and low-dose YTD significantly inhibited the proliferation of the PCa DU-145 cells, decreased their adherence and growth (P < 0.05, P < 0.01), promoted their apoptosis (P < 0.01), regulated their cell cycles (P < 0.05, P < 0.01), and reduced their in vitro invasion and migration abilities (P < 0.05), all in a dose-dependent manner. The results of Western blot and RT-PCR revealed down-regulated protein and mRNA expressions of N-cadherin, zinc finger transcription factor (Snail), Ras, p-ERK1/2 and ERK1/2, but up-regulated protein and mRNA expressions of E-cadherin in the PCa DU-145 cells treated with YTD (P < 0.05, P < 0.01).@*CONCLUSIONS@#Yishen Tonglong Decoction can effectively inhibit the proliferation, promote the apoptosis, regulate the cell cycle and suppress the invasion and migration abilities and EMT process of human PCa DU-145 cells. The mechanism of Yishen Tonglong Decoction acting on PCa may be associated with its inhibitory effect on the EMT process and expression of the Ras/ERK signaling pathway in PCa cells./.

Regulatory effect of annexin A5 on glioma cell invasion andmigration and its mechanisms / 西安交通大学学报(医学版)

【Objective】 To investigate the regulatory effect of annexin A5 on glioma cell invasion and migration and its mechanism. 【Methods】 The expression of annexin A5 in 100 cases of glioma tissues and 20 cases of normal brain tissues was detected by immunohistochemistry. The expression of annexin A5 was downregulated by transfection with siRNA targeting annexin A5 (si-Annexin A5) in human glioma cell line (U251). The expression of annexin A5 was confirmed by RT-PCR and Western blot analysis. The proliferation ability of U251 cells was detected by MTT test and colony formation test, the apoptosis of U251 cells was detected by flow cytometry and Hoechst 33258 staining, and the migration and invasion ability of U251 cells was examined by wound healing test and Matrigel Transwell invasion test. The expressions of Raf, p-Raf, MEK1/2, p-MEK1/2, ERK1/2, p-ERK1/2, c-Myc and E-Cadherin in U251 cells were analyzed by Western blot. 【Results】 Compared with those of normal brain tissues, the mRNA and protein expression levels of Annexin A5 in glioma tissues increased by 2.45 times and 2.87 times, respectively (P<0.05). Pearson correlation analysis showed that with the increase of tumor grade, the positive rate of Annexin A5 gradually increased, and the tumor grade and positive rate were significantly positively correlated (r=1.000, P=0.000). The cell viability of U251 cells in the si-Annexin A5 group after 48 h and 72 h of culture was significantly reduced by 29.46% and 40.43%, respectively, compared with that in the control group (P<0.05). Compared with the control group, in the si-Annexin A5 group the colony formation rate was reduced by 68.58%, while the apoptosis rate was increased by 24.41 times (P<0.05); the cell migration rate and invasion rate were reduced by 65.35% and 68.80% (P<0.05). The protein expression of p-Raf, p-MEK1/2, p-ERK1/2 and c-Myc in the si-Annexin A5 group were significantly reduced by 54.67%, 70.37%, 60.26% and 54.95%, respectively, and that of E-Cadherin was increased by 3.58 times (P<0.05). 【Conclusion】 Downregulation of Annexin A5 inhibits the growth and motility of glioma cells and induces cell apoptosis by inhibiting the Raf/MEK/ERK signaling pathway.

Tanshinone I inhibited growth of human chronic myeloid leukemia cells via JNK/ERK mediated apoptotic pathways

Tanshinone I (Tan I) is one of the main bioactive ingredients derived from Salvia miltiorrhiza Bunge, which has exhibited antitumor activities toward various human cancer cells. However, its effects and underlying mechanisms on human chronic myeloid leukemia (CML) cells still require further investigation. This study determined the effects and mechanisms of anti-proliferative and apoptosis induction activity induced by Tan I against K562 cells. The cytotoxic effect of Tan I at varying concentrations on K562 cells was evaluated via MTT assay. Cell apoptosis was further investigated through DAPI staining and flow cytometry analysis. The expression levels of apoptosis-related proteins and activities of JNK/ATF2 and ERK signaling pathways were analyzed by western blot. Quantitative PCR was performed to further determine mRNA expression levels of JNK1/2 and ERK1/2 after Tan I treatment. The results indicated that Tan I significantly inhibited K562 cell growth and induced apoptosis in a concentration- and time-dependent manner. It induced significant cellular morphological changes and increased apoptosis rates in CML cells. Tan I promoted the cleavages of caspase-related proteins, as well as increased the expression levels of PUMA. Furthermore, Tan I significantly activated JNK and inhibited ATF-2 and ERK signaling pathways. The mRNA expression levels of JNK1/2 and ERK1/2 were up-regulated by Tan I, further confirming its regulatory effects on JNK/ERK signaling pathways. Overall, our results indicated that Tan I suppressed cell viability via JNK- and ERK-mediated apoptotic pathways in K562 cells, suggesting that it might be a promising candidate as a novel anti-leukemia drug.

The C-terminus of the amelogenin peptide promotes the proliferation of alc ameloblasts through accelerating cell cycle / 中国组织工程研究

BACKGROUND: C-terminus of the amelogenin peptide (AMG-CP) is a small molecular endogenous peptide that is highly shown that AMG-CP can regulate the proliferation and differentiation of cementoblasts, bone marrow mesenchymal stem cells and periodontal ligament fibroblasts, but the biological function of AMG-CP on ameloblasts has not been elucidated. O conserved among species. It is involved in important physiological processes during tooth development. Some studies have BJECTIVE: To investigate the effects of different concentrations of AMG-CP on the proliferation of ALC ameloblasts and its underlying mechanisms. METHODS: AMG-CP was successfully synthesized and determinated by liquid chromatography and mass spectrometry. The effects of AMG-CP at 0, 0.5, 1, 2 mg/L on the proliferation of ALC ameloblasts were observed by xCELLigence RTCA cell analysis system in real time. The effect of AMG-CP at 0, 1, 2 mg/L on cell cycle of ALC was detected by flow cytometry. Real-time PCR was used to detect the expression of cyclin D1, CDK4, MCM2, MCM5 mRNA in ALC cells treated with AMG-CP at 0, 1, 2 mg/L. Western blot was carried out to evaluate the effect of AGM-CP at 0, 1 mg/L on MAPK-ERK1/2 pathway by detecting the expression of phosphorylated ERK1/2 and total ERK1/2 in ALC cells. Pathway blockade assay was performed by using ERK1/2 blocker U0126 to pretreat ALC cells. Then cell proliferation ability as well as phosphorylated ERK1/2 expression was analyzed by xCELLigence RTCA cell analysis system and western blot. RESULTS AND CONCLUSION: Compared with the control group, AMG-CP promoted the proliferation of ALC cells, and decreased the population doubling time in a dose-depending manner. Flow cytometry detected the acceleration of cell cycle after treatment with AMG-CP. The results of Real-time PCR showed that AMG-CP upregulated cell cycle-related genes (cyclin D1, CDK4, MCM2, MCM5) expression. Western blot results showed that AMG-CP could upregulate the expression of phosphorylated ERK1/2 and activate MAPK-ERK1/2 signaling pathway in ALC cells. After U0126 was used to inhibit the MAPK-ERK1/2 pathway, the ability of AMG-CP promoting ALC proliferation was inhibited. These results suggest that AMG-CP has a potential to activate MAPK-ERK1/2 pathway, accelerate the process of cell cycle, and then promote the proliferation of ALC cells, all of which indicate that AMG-CP has the potential to promote the proliferation of ameloblasts.