Honokiol inhibits the proliferation of human adipose-derived mesenchymal stem cells / 基础医学与临床

Objective To investigate the effects of honokiol on proliferation and apoptosis of human adipose-derived mesenchymal stem cells(hADSCs),and to investigate the effect of the drug on the tumor microenvironment.Methods hADSCs were incubated with different concentrations of honokiol,the proliferation of hADSCs was detec-ted by MTS and Trypan blue staining,and cell apoptosis was assessed by annexin V/PI double staining.In the meantime,expression of mRNA and protein related to cell proliferation and apoptosis were detected by qPCR and Western blot,respectively.The expression of total MEK,phosphorylated MEK,total ERK and phosphorylated ERK proteins in the MEK-ERK1/2 signaling pathway were detected by Western blot.Results The effect of honokiol on inhibiting proliferation and promoting apoptosis of hADSCs was significantly enhanced with the increase of concen-tration.The expressions of proliferation-related genes CCND1,MKI67 and PCNA were down-regulated.The expres-sions of pro-apoptotic genes BAX and TP53 was up-regulated,and the expressions of anti-apoptotic gene BCL2 was down-regulated.Honokiol inhibited MEK and ERK1/2 phosphorylation in a concentration-dependent manner.Conclusions Honokiol inhibits proliferation and promotes apoptosis of hADSCs,and the specific mechanism is po-tentially related to the inhibition of MEK-ERK1/2 pathway.

Association of Polymorphisms in the 3'UTR of Genes in the ERK1/2 Signaling Pathway with Non-small Cell Lung Cancer / 昆明医科大学学报

Objective To investigate the association between four single nucleotide polymorphisms(SNP)(rs9340 in MAPK1,rs14804 in NRAS,rs712 and rs7973450 in KRAS)in the 3'UTR of ERK1/2 signaling pathway-related genes and non-small cell lung cancer(NSCLC).Methods A total of 478 NSCLC patients and 480 healthy controls were enrolled in this study.Four SNPs were genotyped by using TaqMan assays.The association between the four SNPs and NSCLC was analyzed.Results The distribution frequency difference of the allele of rs9340 was statistically significant between the control group and the non-small cell squamous cell carcinoma(SCC)group(P = 0.009),suggesting that the G allele of rs9340 may be a protective factor for non-small cell lung squamous cell carcinoma(OR = 0.67,95%CI:0.50～0.91).In addition,in the<50 years age group,the distribution frequency difference of the allele of rs9340 was statistically significant between the control group and the NSCLC group(P = 5.07×10-4),indicating that the G allele of rs9340 may be a protective factor for NSCLC(OR = 0.46,95%CI:0.29～0.72).Conclusion The SNP rs9340 in MAPK1 may be associated with the risk of NSCLC.

Role of TLR4/ERK1/2 signaling pathway in decidual tissue of unexplained spontaneous abortion / 中国免疫学杂志

Objective:To investigate the expression of Toll-like receptor 4(TLR4)and extracellular signal-regulated protein kinase 1/2(ERK1/2)in decidua tissue of patients with unexplained spontaneous abortion and their correlation.Methods:The expres-sions of TLR4,ERK1/2 and p-ERK1/2 in decidua tissues of 32 patients with unexplained spontaneous abortion(abortion group)and 32 normal pregnancy(control group)were detected by immunohistochemistry and Western blot,respectively.The correlation between TLR4 and p-ERK1/2 in abortion group were analyzed by Pearson hierarchical correlation analysis.Results:In immunohistochemical experiments,the cytoplasm of decidua cells in the two groups is the expression locus of TLR4,ERK1/2 and p-ERK1/2,the expression of the three proteins were different,and the expressions of TLR4 and p-ERK1/2 in abortion group were significantly higher than that in control group(P<0.01).There was no significant difference in ERK1/2 expression between abortion group and control group(P>0.05);In decidua tissue samples of abortion group,the protein level of TLR4 was higher than that of control group(P<0.05);the pro-tein level of p-ERK1/2 was significantly higher than that of control group(P<0.01),and the protein level of ERK1/2 in decidua tissue of abortion group was not statistically different from that of control group(P>0.05).TLR4 was positively correlated with p-ERK1/2 expression in abortion group(r=0.890,P<0.01).Conclusion:Abnormal activation of TLR4/ERK1/2 signaling pathway may be one of the mechanisms of unexplained spontaneous abortion.

WNK2 delays the proliferation and invasion of hepatocellular carcinoma by inhibiting the ERK1/2/ROS/SHP2 signaling pathway / 中国实验动物学报

Objective To investigate the effect of WNK2 on the ERK1/2/ROS/SHP2 signaling pathway in hepatocellular carcinoma(HCC)and to explore its role in cell proliferation and migration in HCC.Methods HepG2 cells were transfected with WNK2-mimic,sh-RNA WNK2,and corresponding negative control.The effect of WNK2 on the proliferation of HCC was examined by subcutaneous tumorigenesis assay in BALB/c nude mice.The expressions of WNK2,p40,gp90,p-SHP2,p-AKT,and p-ERK1/2 in tumor tissues were detected by Western Blot.After treatment with SHP2 inhibitor PHPS1,the expressions of WNK2,P40,gp90,p-SHP2,p-AKT,and p-ERK1/2 in HepG2 cells were detected by Western Blot.The migration ability and invasion ability of HepG2 cells were detected by cell scratch assay and Transwell.The proliferation ability of HepG2 cells was detected by monoclonal proliferation assay.Results Compared with the sh-NC group,the tumor volume of nude mice in the sh-RNA WNK2 group was significantly increased(P＜0.01);Compared with the NC-mimic group,the tumor volume of nude mice in the WNK2-mimic group was significantly reduced(P＜0.01).Western Blot result showed that compared with the sh-NC group,the expression of WNK2 in the sh-RNA WNK2 group was significantly decreased(P＜0.01),while the expressions of p40,gp90,p-SHP2,p-AKT and p-ERK1/2 were significantly increased(P＜0.01).Compared with the NC-mimic group,the expression of WNK2 was significantly increased in the WNK2-mimic group(P＜0.01),and the expressions of p40,gp90,p-SHP2,p-AKT,and p-ERK1/2 were significantly decreased(P＜0.01).In vitro experiment,compared with the sh-NC group,the expression of WNK2 was significantly decreased in the sh-RNA WNK2 group(P＜0.01),while the expressions of p40,gp90,p-SHP2,p-AKT and p-ERK1/2 were significantly increased in the sh-RNA WNK2 group(P＜0.01).Compared with the sh-NC+PHPS1 group,the expression of WNK2 was significantly decreased in the sh-RNA WNK2+PHPS1 group(P＜0.01),while the expressions of p40,gp90,p-SHP2,p-AKT,and p-ERK1/2 were reversed and had no significant differences compared with the sh-NC+PHPS1 group(P＞0.05).The cell scratch assay and Transwell result showed that the migration and invasion ability of HepG2 cells in the sh-RNA WNK2 group was significantly increased compared with the sh-NC group(P＜0.01).The migration and invasion ability of HepG2 cells in the sh-NC+PHPS1 group and sh-RNA WNK2+PHPS1 group were significantly decreased with no significant difference(P＞0.05).The result of the monoclonal proliferation experiment showed that the proliferation capacity of HepG2 cells in the sh-RNA WNK2 group was significantly increased compared with the sh-NC group(P＜0.01),while the proliferation ability of HepG2 cells in the sh-NC+PHPS1 group and sh-RNA WNK2+PHPS1 group was significantly decreased with no significant difference(P＞0.05).Conclusions WNK2 can inhibit the ERK1/2/ROS/SHP2 signaling pathway,thereby inhibiting ERK1/2/Akt signaling and delaying the proliferation and migration of HCC.

Effects of Jianpi Qutan Huayu Prescription on Inflammatory Response of Atherosclerosis in Mini-pigs Based on NOX5-ERK1/2 Signaling Pathway / 中国中医药信息杂志

Objective To observe the effects of Jianpi Qutan Huayu Prescription on oxidative stress and inflammatory response in mini-pigs with atherosclerosis(AS);To explore its mechanism based on the NOX5-ERK1/2 signaling pathway.Methods Twelve Bama mini-pigs were randomly divided into control group,model group,and Jianpi Qutan Huayu Prescription low-and high-dosage groups,with 3 pigs in each group.A high-fat diet was used to feed for 24 weeks to construct an AS model,and the treatment group was also supplemented with Jianpi Qutan Huayu Prescription in the feed.The general condition of mini-pigs(body length,abdominal circumference,body mass,food intake,and fecal water content)was measured at week 0,16,and 24 of administration,HE staining was used to observe the morphology of aortic tissue,while oil red O staining was used to observe lipid deposition in aortic and myocardial tissue,transmission electron microscopy was used to observe the ultrastructure of aortic tissue,and a fully automated biochemical analyzer was used to detect serum contents of TC,HDL-C,and LDL-C.ELISA was used to detect the contents of serum reactive oxygen species(ROS),interleukin(IL)-6,IL-10,tumor necrosis factor(TNF)-α,hypersensitivity-C-reactive protein(hs-CRP),vascular cell adhesion molecule-1(VCAM-1),and intercellular adhesion molecule-1(ICAM-1).Western blot was used to detect the expressions of NADPH oxidase 5(NOX5),extracellular signal regulated kinase 1/2(ERK1/2),p-ERK1/2,VCAM-1,and proliferating cell nuclear antigen(PCNA)proteins.Results Compared with the control group,the abdominal circumference,body mass,and food intake of mini-pigs in the model group increased at 16 and 24 weeks(P<0.01),there was significant thickening of the inner membrane of aorta,destruction of endothelial cells,lipid deposition,edema of smooth muscle cells,and significant swelling of mitochondria,serum TC,LDL-C contents and the contents of ROS,IL-6,IL-10,TNF-α,hs-CRP,VCAM-1,and ICAM-1 increased,while the content of HDL-C decreased(P<0.01);the expressions of NOX5,p-ERK1/2,VCAM-1,and PCNA proteins in aortic tissue increased(P<0.01).Compared with the model group,Jianpi Qutan Huayu Prescription low-and high-dosage groups showed a decrease in abdominal circumference,body mass,and food intake at 16 and 24 weeks(P<0.05,P<0.01),the plaque area and lipid deposition were reduced,and the damage to endothelial cells was alleviated,serum TC,LDL-C contents and the contents of ROS,IL-6,IL-10,TNF-α,hs-CRP,ICAM-1,and VCAM-1 decreased,and the content of HDL-C increased(P<0.01,P<0.05);the expressiond of NOX5,p-ERK1/2,VCAM-1,and PCNA proteins in aortic tissue decreased(P<0.01,P<0.05).Conclusion Jianpi Qutan Huayu Prescription can effectively alleviate AS in mini-pigs,and its mechanism may be related to inhibiting the activation of the NOX5-ERK1/2 signaling pathway and alleviating oxidative stress-induced inflammatory response.

Research Progress of Anti-cancer Strategies Based on ERK1/2 Post-translational Modification and Spatial Regulation / 肿瘤防治研究

ERK1/2 is a key protein that mediates cell signal transduction, and it is involved in regulating biological processes such as chromatin remodeling, nuclear disintegration, proliferation, survival, metabolism, and cell migration and differentiation. Its overactivation is closely related to the occurrence and progression of cancer, and the mechanism is manifested as the overactivation of ERK1/2 by gene mutations of upstream pathway molecules or regulators and the reactivation of ERK1/2 after inhibition against the above targets. ERK1/2 is a potentially valuable target. In this review, the mechanism of post-translational modification and spatial regulation of ERK1/2 and the application status of corresponding small-molecule inhibitors were discussed. The current antitumor strategy of targeting and regulating ERK1/2 was summarized, and the possibility of exploring potential targets was elucidated, thus providing new insights into the developmental research of ERK1/2 as an ideal anticancer target.

Anmeidan Regulates Hepatic Neurotransmitters and Circadian Rhythm in Rat Model of Insomnia via OX1R/PLCβ-1/PKCα/ERK1/2 Signaling Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effect and mechanism of the classic famous prescription Anmeidan （AMD） developed in the Qing Dynasty in regulating the hepatic neurotransmitters and circadian rhythm in the rat model of insomnia via the orexin-1 receptor （OX1R）/phosphatidylinositol-specific phospholipase Cβ-1 （PLCβ-1）/protein kinase Cα （PKCα）/extracellular signal-regulated kinase 1/2 （ERK1/2） signaling pathway. MethodSixty SPF-grade SD rats were randomized into blank， model， suvorexant （30 mg·kg-1·d-1）， and low-， medium-， and high-dose （4.55， 9.09， 18.09 g·kg-1·d-1， respectively） AMD groups， with 10 rats in each group. The rats in other groups except the blank group were modeled by intraperitoneal injection of p-chlorophenylalanine （PCPA） and administrated with corresponding drugs by gavage， and the blank group received an equal volume of normal saline. The general condition， body mass， and 24 h autonomic activity of each group were observed. The pathological changes of the liver tissue were observed by hematoxylin-eosin（HE）staining and Masson staining. The expression of gamma-aminobutyric acid （GABA）， 5-hydroxytryptamine （5-HT）， epinephrine （EPI）， norepinephrine （NE）， and acetylcholine （ACh） in the liver tissue was detected by enzyme-linked immunosorbent assay. The glutamate （Glu） expression in the liver tissue was detected by the biochemical method. The mRNA levels of biological clock genes Per1， Per2， Cry1， Cry2， Bmal1， and Bmal2 in the liver were determined by Real-time fluorescence quantitative polymerase chain reaction（Real-time PCR）. The protein and mRNA levels of factors in the OX1R/PLCβ-1/PKCα/ERK1/2 signaling pathway in the liver were determined by Western blot and Real-time PCR， respectively. ResultCompared with the blank group， the modeling decreased the body mass （P<0.05， P<0.01） and caused mania and disturbed resting rhythms （P<0.01）， hepatic muscle fiber fracture， and edema with inflammatory cell infiltration. In addition， the modeling decreased the GABA， 5-HT， EPI， NE， and ACh content， increased Glu content （P<0.01）， down-regulated the mRNA levels of Per1， Per2， Cry1， and Cry2 （P<0.01）， up-regulated the mRNA levels of Bmal1 and Bmal2 （P<0.01）， and promoted the expression of OX1R， PLCβ-1， PKCα， and ERK1/2 at both protein and mRNA levels （P<0.01）. Compared with the model group， suvorexant and AMD increased the body mass （P<0.05， P<0.01）， alleviated the mania， and increased the resting time and frequency （P<0.05， P<0.01）. Moreover， the medications elevated the levels of GABA， 5-HT， EPI， NE， and ACh， lowered the Glu level， up-regulated the mRNA levels of Per1， Per2， Cry1， and Cry2 （P<0.05， P<0.01）， down-regulated the mRNA levels of Bmal1 and Bmal2， and inhibited the expression of OX1R， PLCβ-1， PKCα， and ERK1/2 at both mRNA and protein levels （P<0.05， P<0.01）. ConclusionAMD can regulate hepatic neurotransmitters and improve circadian rhythm in insomniac rats by inhibiting the OX1R/PLCβ-1/PKCα/ERK1/2 signaling pathway， and high-dose AMD demonstrated the strongest effect.

Polysaccharide of Alocasia cucullata Exerts Antitumor Effect by Regulating Bcl-2, Caspase-3 and ERK1/2 Expressions during Long-Time Administration / 中国结合医学杂志

OBJECTIVE@#To study the in vitro and in vivo antitumor effects of the polysaccharide of Alocasia cucullata (PAC) and the underlying mechanism.@*METHODS@#B16F10 and 4T1 cells were cultured with PAC of 40 µg/mL, and PAC was withdrawn after 40 days of administration. The cell viability was detected by cell counting kit-8. The expression of Bcl-2 and Caspase-3 proteins were detected by Western blot and the expressions of ERK1/2 mRNA were detected by quantitative real-time polymerase chain reaction (qRT-PCR). A mouse melanoma model was established to study the effect of PAC during long-time administration. Mice were divided into 3 treatment groups: control group treated with saline water, positive control group (LNT group) treated with lentinan at 100 mg/(kg·d), and PAC group treated with PAC at 120 mg/(kg·d). The pathological changes of tumor tissues were observed by hematoxylin-eosin staining. The apoptosis of tumor tissues was detected by TUNEL staining. Bcl-2 and Caspase-3 protein expressions were detected by immunohistochemistry, and the expressions of ERK1/2, JNK1 and p38 mRNA were detected by qRT-PCR.@*RESULTS@#In vitro, no strong inhibitory effects of PAC were found in various tumor cells after 48 or 72 h of administration. Interestingly however, after 40 days of cultivation under PAC, an inhibitory effect on B16F10 cells was found. Correspondingly, the long-time administration of PAC led to downregulation of Bcl-2 protein (P<0.05), up-regulation of Caspase-3 protein (P<0.05) and ERK1 mRNA (P<0.05) in B16F10 cells. The above results were verified by in vivo experiments. In addition, viability of B16F10 cells under long-time administration culture in vitro decreased after drug withdrawal, and similar results were also observed in 4T1 cells.@*CONCLUSIONS@#Long-time administration of PAC can significantly inhibit viability and promote apoptosis of tumor cells, and had obvious antitumor effect in tumor-bearing mice.

Effect of Guipitang on ERK1/2 and p38 MAPK in Rats with Myocardial Ischemia / 中国实验方剂学杂志

ObjectiveTo explore the therapeutic effect and mechanism of Guipitang on rats with myocardial ischemia. MethodFifty SD rats were divided into five groups： a control group， a model group， low and high-dose Guipitang （7.52， 15.04 g·kg-1） groups， and a trimetazidine group （0.002 g·kg-1）. By intragastric administration of vitamin D3 and feeding rats with high-fat forage and injecting isoproterenol， the rat model of myocardial ischemia was established. After drug treatment of 15 d， an electrocardiogram （ECG） was performed to analyze the degree of myocardial injury. A fully automatic biochemical analyzer was used to detect the changes in the serum levels of total cholesterol （TC）， high-density lipoprotein cholesterol （HDL-C）， triglyceride （TG）， and low-density lipoprotein cholesterol （LDL-C）. Hematoxylin-eosin （HE） staining and Masson staining were used to observe myocardial histopathological changes. TdT-mediated dUTP nick end labeling （TUNEL） staining was used to detect cardiomyocyte apoptosis. Western blot was adopted to detect the protein levels of extracellular signal-regulated kinase 1/2 （ERK1/2）， phospho-ERK1/2 （p-ERK1/2）， p38 mitogen-activated protein kinase （p38 MAPK）， phospho-p38 MAPK （p-p38 MAPK）， B-cell lymphoma-2 （Bcl-2）-associated X （Bax）， Bcl-2， and cleaved cysteine aspartate proteolytic enzyme （cleaved Caspase-3）. ResultCompared with the control group， the ECG S-T segment decreased in the model group. The serum levels of TC， TG， and LDL-C were increased significantly （P<0.05）. The arrangement of myocardial tissue was disordered， and the proportion of cardiomyocyte apoptosis increased. The protein levels of cleaved Caspase-3， Bax， and p-p38 MAPK in the heart were increased， and the Bcl-2 expression was decreased （P<0.05）. Compared with the model group， the S-T segment downward shift was restored in the low and high-dose Guipitang groups and trimetazidine group， and the levels of TC， TG， and LDL-C were decreased. The protein expression of cleaved Caspase-3 and Bax in the heart dropped， and p-p38 MAPK and p-ERK1/2 protein expressions increased significantly （P<0.05）. The degree of myocardial injury was alleviated， and the proportion of cardiomyocyte apoptosis decreased. Bcl-2 protein expression was increased significantly in the low-dose Guipitang group （P<0.05）. ERK1/2 and p38 MAPK proteins had no significant difference among different groups. ConclusionGuipitang could alleviate myocardial injury and inhibit cardiomyocyte apoptosis in rats by activating the expression of ERK1/2 and p38 MAPK.

Morin Mediates Protective Effects on Bone in Aged Rats by Inhibiting ERK1/2-p38 Signaling Pathway / 中山大学学报(医学科学版)

ObjectiveTo investigate the effects of morin treatment on bone metabolism and bone mass in aged rats， and to clarify the possible mechanism. MethodsTen young female Sprague-Dawley rats （3 months old） and 20 old female Sprague-Dawley rats （24 months old） were randomly divided into three groups： Control group （CON， 10 young rats）； Model group （MOD， 10 young rats）； 10 old rats and SangHuangSu Group （SSS， 10 old rats）. During the experiment， the SSS group received intraperitoneal injection of morin （10 mg / kg） daily. The treatment lasted for 12 weeks. After treatment， Micro-CT， HE stained sections， serological tests and Western blot were used to observe the treatment effect and possible mechanism. ResultsAfter 12 weeks of treatment， compared with MOD group， the number and density of bone trabeculae in SSS group were significantly improved. The BMD， Conn. D， Tb. N， Tb.Th and Tb.Sp of the left femur in the SSS group were significantly better than those in the MOD group（P <0.05）. After 12 weeks of treatment， the levels of CTX-1， osteocalcin， TRACP-5b and PINP in SSS group were significantly lower than those in MOD group（P <0.05）. Compared with the MOD group， the ERK1/2-p38 signal pathway was significantly inhibited and the levels of ERK1/2 and p38 were significantly decreased in the SSS group（P <0.05）. ConclusionMorin pigment mediates the protective effect on the bones of aged rats by inhibiting the ERK1/2-p38 signaling pathway and reducing bone turnover.

Exploring the treatment of sepsis-associated acute lung injury with Liangge Powder via ERK1/2 and PI3K/AKT pathways: based on network pharmacology and whole animal experimentation / 中华劳动卫生职业病杂志

Objective: To investigate the therapeutic effect and mechanism of Liangge Powder against sepsis-induced acute lung injury (ALI) . Methods: From April to December 2021, the key components of Liangge Powder and its targets against sepsis-induced ALI were analyzed by network pharmacology, and to enrich for relevant signaling pathways. A total of 90 male Sprague-Dawley rats were randomly assigned to sham-operated group, sepsis-induced ALI model group (model group), Liangge Powder low, medium and high dose group, ten rats in the sham-operated group and 20 rats in each of the remaining four groups. Sepsis-induced ALI model was established by cecal ligation and puncture. Sham-operated group: gavage with 2 ml saline and no surgical treatment. Model group: surgery was performed and 2 ml saline was gavaged. Liangge Powder low, medium and high dose groups: surgery and gavage of Liangge Powder 3.9, 7.8 and 15.6 g/kg, respectively. To measure the wet/dry mass ratio of rats lung tissue and evaluate the permeability of alveolar capillary barrier. Lung tissue were stained with hematoxylin and eosin for histomorphological analysis. The levels of tumor necrosis factor-alpha (TNF-α), interleukin (IL) -6 and IL-1β in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The relative protein expression levels of p-phosphatidylinositol 3-kinase (PI3K), p-protein kinase B (AKT), and p-ertracellular regulated protein kinases (ERK) were detected via Western blot analysis. Results: Network pharmacology analysis indicated that 177 active compounds of Liangge Powder were selected. A total of 88 potential targets of Liangge Powder on sepsis-induced ALI were identified. 354 GO terms of Liangge Powder on sepsis-induced ALI and 108 pathways were identified using GO and KEGG analysis. PI3K/AKT signaling pathway was recognized to play an important role for Liangge Powder against sepsis-induced ALI. Compared with the sham-operated group, the lung tissue wet/dry weight ratio of rats in the model group (6.35±0.95) was increased (P<0.001). HE staining showed the destruction of normal structure of lung tissue. The levels of IL-6 [ (392.36±66.83) pg/ml], IL-1β [ (137.11±26.83) pg/ml] and TNF-α [ (238.34±59.36) pg/ml] were increased in the BALF (P<0.001, =0.001, <0.001), and the expression levels of p-PI3K, p-AKT and p-ERK1/2 proteins (1.04±0.15, 0.51±0.04, 2.31±0.41) were increased in lung tissue (P=0.002, 0.003, 0.005). The lung histopathological changes were reduced in each dose group of Liangge Powder compared with the model group. Compared with the model group, the wet/dry weight ratio of lung tissue (4.29±1.26) was reduced in the Liangge Powder medium dose group (P=0.019). TNF-α level [ (147.85±39.05) pg/ml] was reduced (P=0.022), and the relative protein expression levels of p-PI3K (0.37±0.18) and p-ERK1/2 (1.36±0.07) were reduced (P=0.008, 0.017). The wet/dry weight ratio of lung tissue (4.16±0.66) was reduced in the high-dose group (P=0.003). Levels of IL-6, IL-1β and TNF-α[ (187.98±53.28) pg/ml, (92.45±25.39) pg/ml, (129.77±55.94) pg/ml] were reduced (P=0.001, 0.027, 0.018), and relative protein expression levels of p-PI3K, p-AKT and p-ERK1/2 (0.65±0.05, 0.31±0.08, 1.30±0.12) were reduced (P=0.013, 0.018, 0.015) . Conclusion: Liangge Powder has therapeutic effects in rats with sepsis-induced ALI, and the mechanism may be related to the inhibition of ERK1/2 and PI3K/AKT pathway activation in lung tissue.

Acteoside Inhibits Epithelial Mesenchymal Transformation of Hepatoma Cells Through Regulation of ERK1/2 Signaling Pathway / 肿瘤防治研究

Objective To investigate the effect and mechanism of acteoside (ACT) in inhibiting epithelial-mesenchymal transition (EMT) in human hepatoma HCCLM3 cells by regulating the ERK1/2 pathway. Methods CCK-8 assay was used to detect the effect of hepatocellular carcinoma cell proliferation. The invasion and migration of HCC cells were detected by scratch and Transwell tests. The mRNA and protein expression levels of the ERK1/2 signaling pathway and EMT-related genes (E-cadherin and N-cadherin) were detected by real-time PCR and Western blot analyses. Results ACT reduced the activity of HCCLM3 cells and inhibited the proliferation of HCC cells, and the effects had certain correlation with drug concentration and time. ACT inhibited the migration and invasion process of HCCLM3 cells in a concentration-dependent manner. ACT downregulated the mRNA and protein expression of genes related to the ERK1/2 signaling pathway. It increased the mRNA and protein expression levels of the EMT-related gene E-cadherin but decreased those of N-cadherin. Conclusion ACT could inhibit EMT and the invasion and migration of HCCLM3 cells in human hepatoma, and the underlying mechanism is closely related to the downregulation of the ERK1/2 signaling pathway.

Quercetin exhibits neuroprotective effect by reduc-ing amyloid precursor protein expression and oxida-tive stress in Neuro2a/APPswe cell model / 中国药理学与毒理学杂志

OBJECTIVE The abnormal amyloid-β(Aβ)and oxidative stress assiociated with the progression of Alzheimer disease(AD).Quercetin has been reported to possess antioxidant and anti-inflammatory properties in neurodegenerative disorders.In this present study,we designed to characterize the mechanisms by which quer-cetin exerts neuroprotective effects in murine neuroblas-toma N2a cells stably expressing human Swedish mutant amyloid precursor protein(N2a/APP).METHODS N2a/APP cells were treated with quercetin at concentrations of 10,20 and 50 μ mol·L-1 for 24 h.Cell viability was examined with CCK-8 assays.The protein levels of ERK1/2 and Akt were detected by Western blotting.Intra-cellular reactive oxygen species(ROS)was detected by a fluorescent probe 2,7-dichlorofluorescein diacetate(DCFH-DA).The mitochondrial membrane potential(Δψ m)in N2a/APP cells was detected by using JC-1 staining method.Immunofluorescence was used to detect the generation of 8-hydroxy-2′-deoxyguanosine(8-OHdG)and 4-hydroxynonenal(4-HNE).RESULTS Quercetin attenuated the enhancement of p-ERK1/2,reductions of p-Akt,and decreased levels of APP expression.More-over,quercetin alleviated loss of mitochondria membrane potential(MMP)since it attenuates these oxidative stress,as reflected in the levels of ROS,4-HNE and 8-OHdG,was elevated in N2a/APP cells and these effects were again ameliorated by quercetin.CONCLUSION Neuroprotection by quercetin in N2a/APP cells involves normalizing the impaired mitochondrial function and reducing oxidative stress via inactivation of the ERK1/2 and activation of the Akt pathways.

Correlation Between ERK1/2 Signal Transduction and Ovarian Cancer / 肿瘤防治研究

Ovarian cancer is one of the common tumors in female reproductive organs and accounts for about 4% of all malignant tumors in women. It is also the leading cause of death among various gynecological tumors. Surgery combined with chemotherapy is the frequently used treatment for ovarian. Chemotherapy resistance is one of the main reasons for the recurrence and poor prognosis of ovarian cancer. Various components of the MAPK pathway, also known as the RAS-RAF-MEK-ERK signaling cascade, are related to cancer, and ERK1/2 is an indispensable key site in this pathway. Continuous research has found that ERK1/2 is a "ferryman" involved in regulating the occurrence, development, and drug resistance mechanisms of ovarian cancer. This article briefly introduces the activation mechanism and pathway of the ERK1/2 signaling pathway, summarizes its relationship to ovarian cancer, and emphasizes that ERK1/2 inhibitors may be a new treatment strategy with potential advantages in improving poor prognosis in patients with ovarian cancer.

Cytokine receptor-like factor 1 (CRLF1) promotes cardiac fibrosis via ERK1/2 signaling pathway / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Cardiac fibrosis is a cause of morbidity and mortality in people with heart disease. Anti-fibrosis treatment is a significant therapy for heart disease, but there is still no thorough understanding of fibrotic mechanisms. This study was carried out to ascertain the functions of cytokine receptor-like factor 1 (CRLF1) in cardiac fibrosis and clarify its regulatory mechanisms. We found that CRLF1 was expressed predominantly in cardiac fibroblasts. Its expression was up-regulated not only in a mouse heart fibrotic model induced by myocardial infarction, but also in mouse and human cardiac fibroblasts provoked by transforming growth factor-‍β1 (TGF‍-‍β1). Gain- and loss-of-function experiments of CRLF1 were carried out in neonatal mice cardiac fibroblasts (NMCFs) with or without TGF-‍β1 stimulation. CRLF1 overexpression increased cell viability, collagen production, cell proliferation capacity, and myofibroblast transformation of NMCFs with or without TGF‍-‍β1 stimulation, while silencing of CRLF1 had the opposite effects. An inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) signaling pathway and different inhibitors of TGF-‍β1 signaling cascades, comprising mothers against decapentaplegic homolog (SMAD)‍-dependent and SMAD-independent pathways, were applied to investigate the mechanisms involved. CRLF1 exerted its functions by activating the ERK1/2 signaling pathway. Furthermore, the SMAD-dependent pathway, not the SMAD-independent pathway, was responsible for CRLF1 up-regulation in NMCFs treated with TGF-‍β1. In summary, activation of the TGF-‍β1/SMAD signaling pathway in cardiac fibrosis increased CRLF1 expression. CRLF1 then aggravated cardiac fibrosis by activating the ERK1/2 signaling pathway. CRLF1 could become a novel potential target for intervention and remedy of cardiac fibrosis.

Mechanism of albiflorin in improvement of Alzheimer's disease based on network pharmacology and in vitro experiments / 中国中药杂志

This study aimed to explore the mechanism of albiflorin in the treatment of Alzheimer's disease(AD) based on network pharmacology, molecular docking, and in vitro experiments. Network pharmacology was used to predict the potential targets and pathways of albiflorin against AD, and molecular docking technology was used to verify the binding affinity of albiflorin to key target proteins. Finally, the AD cell model was induced by Aβ_(25-35) in rat pheochromocytoma(PC12) cells and intervened by albiflorin to validate core targets and pathways. The results of network pharmacological analysis showed that albiflorin acted on key targets such as mitogen-activated protein kinase-1(MAPK1 or ERK2), albumin(ALB), epidermal growth factor receptor(EGFR), caspase-3(CASP3), and sodium-dependent serotonin transporter(SLC6A4), and signaling pathways such as MAPK, cAMP, and cGMP-PKG. The results of molecular docking showed that albiflorin had strong binding affinity to MAPK1(ERK2). In vitro experiments showed that compared with the blank group, the model group showed decreased cell viability, decreased expression level of B-cell lymphoma 2(Bcl-2), increased Bcl-2-associated X protein(Bax), and reduced phosphorylation level of extracellular signal-regulated kinase 1/2(ERK1/2) and the relative expression ratio of p-ERK1/2 to ERK1/2. Compared with the model group, the albiflorin group showed potentiated cell viability, up-regulated expression of Bcl-2, down-regulated Bax, and increased phosphorylation level of ERK1/2 and the relative expression ratio of p-ERK1/2 to ERK1/2. These results suggest that the mechanism of albiflorin against AD may be related to its activation of the MAPK/ERK signaling pathway and its inhibition of neuronal apoptosis.

DUSP6: Potential interactions with FXR1P in the nervous system

Fragile X syndrome (FXS) is a leading genetic cause of autism intellectual disorder and autism spectrum disorder (ASD), with either limited treatment options or incurable. Fragile X-related gene 1 (FXR1) is a homolog of the Fragile X mental retardation gene 1 (FMR1), the causative gene of FXS, and both are highly homologous and functionally identical. In FXS, both PI3K (AKT/mTOR signaling pathway) and ERK1/2 (MAPK signaling pathway) expression levels were abnormal. Dual speci?city phosphatase 6 (DUSP6) is a member of the mitogen-activated protein kinases (MAPKs) that participates in the crosstalk between the two signaling systems of MEK/ERK and mTOR. By interacting with multiple nodes of MAPK and PI3K/AKT signaling pathways (including the mTOR complex), DUSP6 regulates cellular growth, proliferation, metabolism and participates in pathological processes of cancer and cognitive impairment. However, whether there is an interaction between FXR1P and DUSP6 and the effects of DUSP6 on the growth of SK-N-SH cells remains elusive. As demonstrated by our results, FXR1P was identified in the cytoplasm and nucleus of SK-N-SH cells co-localized with DUSP6, which might have regulated ERK1/2 signaling pathways in SK-N-SH cells. To a certain extent, FXR1P may reverse the negative regulation of ERK1/2 by DUSP6. Moreover, we discovered that not only does DUSP6 inhibit proliferation, but it also promotes the apoptosis of SK-N-SH cells.

CTHRC1 promotes the migration and invasion of bladder cancer 5637 cells through activating the FAK-ERK1/2 signaling pathway / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨胶原三螺旋重复蛋白1（CTHRC1）在膀胱癌组织和细胞中的表达及其对膀胱癌5637细胞迁移和侵袭的影响及其机制。方法：利用TCGA和Arrayexpress数据库中膀胱癌基因表达数据，分析CTHRC1转录和翻译水平。收集2014年9月至2020年12月重庆医科大学附属第一医院手术切除的144例膀胱癌组织和25例全膀胱切除的癌旁组织标本，以及人膀胱癌细胞RT4、5637、T24、UMUC-3、TCCSUP和输尿管上皮永生化细胞SV-HUC-1。采用免疫组织化学染色法、qPCR法和WB法检测膀胱癌组织和细胞中CTHRC1的表达水平，通过Kaplan-Meier曲线分析CTHRC1表达对总生存期（OS）的影响。运用RNAi技术，敲降5637细胞CTHRC1表达后，通过细胞划痕实验和Transwell实验检测CTHRC1表达下调对5637细胞迁移和侵袭的影响。利用基因集富集分析（GSEA）预测CTHRC1相关的潜在信号通路，WB法检测敲降CTHRC1表达对FAK-ERK1/2通路相关蛋白表达的影响。结果：CTHRC1的转录和翻译水平在肌层浸润性膀胱癌（MIBC）组织和细胞中表达显著上调（均P<0.05），CTHRC1高表达组患者5年OS较低表达患者缩短（P<0.05）。干扰CTHRC1表达后，膀胱癌5637细胞迁移及侵袭能力均显著降低（均P<0.01）。GSEA预测显示，CTHRC1高表达组主要富集在黏着斑激酶（FAK）、肌动蛋白细胞骨架调节、FAK和ERK1/2信号通路。WB法实验结果表明，重组CTHRC1蛋白促进膀胱癌5637细胞FAK-ERK1/2信号通路活化（P<0.05或P<0.01）。结论：CTHRC1在MIBC中表达上调，且与膀胱癌患者不良预后密切相关；CTHRC1促进膀胱癌细胞迁移和侵袭，该过程可能与FAK-ERK1/2信号通路的激活有关。

Lithocarpus polystachyus (Sweet Tea) water extract promotes human hepatocytes HL7702 proliferation through activation of HGF/AKT/ERK signaling pathway / 中草药·英文版

Objective: Sweet Tea (ST), derived from the leaves of Lithocarpus polystachyus, is a Chinese folk medicine with wide pharmacological activities. However, the promotive effects of ST water extract on hepatocytes proliferation and its underlying mechanism remains still unknown. In the present study, the beneficial effects of ST water extract on human hepatocytes and its possible mechanism were investigated. Methods: MTT assay was used to detect the safety range of ST; HL7702 cells were divided into four groups: control group, ST low- (50 μg/mL), medium- (200 μg/mL) and high-concentration (800 μg/mL) groups; BrdU ELISA and EDU staining were used to observe DNA content and cell proliferation; Moreover, flow cytometry was applied to analyze the distribution of cell cycle. Furthermore, the expression of cyclin D1, CDK4, HGF/c-Met, Akt, Erk1/2 were detected by Western blot. Results: It was found that ST water extract concentration-dependent promoted human hepatocytes HL7702 cell proliferation within 72 h through accumulating the cells in S phase and G2/M phase. Furthermore, ST water extract up-regulated expression of Cyclin D1 and CDK4 proteins. Moreover, ST water extract not only increased HGF expression and phosphorylation of c-Met level, but also activated the phosphorylation levels of AKT, ERK1/2. Interestingly, both of AKT inhibitor A6730 and ERK1/2 inhibitor U0126 reversed the promotive effects of ST water extract, which further confirmed that activation of AKT and ERK1/2 were involved. Conclusion: The findings reveal that ST water extract promoted HL7702 cells proliferation through the stimulation of cell cycle mediated by activating the AKT- and ERK1/2-related pathway.

Role of Nrf2 pathway in flutamide-induced mitochondrial biogenesis / 中国临床药理学与治疗学

AIM: To investigate the effect of flutamide on mitochondrial biogenesis and the regulating effect of anoxidative pathway Nrf2 on it. METHODS: Human hepatocyte HepG2 cells were treated with flutamide (0-50 μmol/L) for 24 h, then mtDNA copy number and protein expression of mitochondrial biogenesis were detected by RT-PCR and WB. The effects of ERK1/2 and the role of Nrf2 pathway in mitochondrial biogenesis were further observed by gene knockdown and protein activation/inhibition methods. RESULTS: Flutamide interfered mitochondrial biogenesis concentration-dependently, the mtDNA copy number, ERK1/2 and PGC-1α proteins increased with the dose. ERK1/2 inhibition and activation regulated flutamide-induced mtDNA copy number and PGC-1α expression, and inhibition of Nrf2 pathway also affected flutamide-induced mtDNA copy number and expression of PGC-1α, as well as ERK1/2 expression. CONCLUSION: Flutamide affects mitochondrial biogenesis, and the antioxidant pathway Nrf2 may be involved in the regulation of flutamine-induced mitochondrial biogenesis by regulating ERK1/2.