Overexpression SPRY2 Suppresses Esophageal Squamous Cell Carcinoma Progression Via Inactivation of ERK/AKT Signaling / La Sobreexpresión SPRY2 Suprime la Evolución del Carcinoma de Células Escamosas de Esófago Mediante la Inactivación de la Señalización ERK/AKT

SUMMARY: Esophageal cancer is one of the most aggressive gastrointestinal cancers. Invasion and metastasis are the main causes of poor prognosis of esophageal cancer. SPRY2 has been reported to exert promoting effects in human cancers, which controls signal pathways including PI3K/AKT and MAPKs. However, the expression of SPRY2 in esophageal squamous cell carcinoma (ESCC) and its underlying mechanism remain unclear. In the present study, we aimed to investigate the detailed role of SPRY2 in the regulation of cell proliferation, invasion and ERK/AKT signaling pathway in ESCC. It was identified that the expression level of SPRY2 in ESCC was remarkably decreased compared with normal tissues, and it was related to clinicopathologic features and prognosis ESCC patients. The upregulation of SPRY2 expression notably inhibited the proliferation, migration and invasion of Eca-109 cells. In addition, the activity of ERK /AKT signaling was also suppressed by the SPRY2 upregulation in Eca-109 cells. Our study suggests that overexpression of SPRY2 suppress cancer cell proliferation and invasion of by through suppression of the ERK/AKT signaling pathways in ESCC. Therefore, SPRY2 may be a promising prognostic marker and therapeutic target for ESCC.

El cáncer de esófago es uno de los cánceres gastrointestinales más agresivos. La invasión y la metástasis son las principales causas de mal pronóstico del cáncer de esófago. Se ha informado que SPRY2 ejerce efectos promotores en los cánceres humanos, que controla las vías de señales, incluidas PI3K/AKT y MAPK. Sin embargo, la expresión de SPRY2 en el carcinoma de células escamosas de esófago (ESCC) y su mecanismo subyacente aún no están claros. En el presente estudio, nuestro objetivo fue investigar el papel detallado de SPRY2 en la regulación de la proliferación celular, la invasión y la vía de señalización ERK/AKT en ESCC. Se identificó que el nivel de expresión de SPRY2 en ESCC estaba notablemente disminuido en comparación con los tejidos normales, y estaba relacionado con las características clínico-patológicas y el pronóstico de los pacientes con ESCC. La regulación positiva de la expresión de SPRY2 inhibió notablemente la proliferación, migración e invasión de células Eca-109. Además, la actividad de la señalización de ERK/AKT también fue suprimida por la regulación positiva de SPRY2 en las células Eca-109. Nuestro estudio sugiere que la sobreexpresión de SPRY2 suprime la proliferación y la invasión de células cancerosas mediante la supresión de las vías de señalización ERK/AKT en ESCC. Por lo tanto, SPRY2 puede ser un marcador de pronóstico prometedor y un objetivo terapéutico para la ESCC.

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.

ERK mediated C-Myc/PD-L1 synergy to investigate tumor suppression effect of Shenqi Yiliu decoction combined with cisplatin in combination with H22 hepatocellular carcinoma tumor bearing mice / 中国免疫学杂志

Objective:To investigate the tumor suppressing effect of Shenqi Yiliu decoction combined with cisplatin via ERK-mediated C-Myc/PD-L1 phase-coordinated pathway on H22 hepatocellular carcinoma tumor-bearing mice and its mechanism.Meth-ods:In 60 SPF-grade male Kunming mice,10 mice were taken as blank group by random number table method,and the other 50 mice were replicated as H22 hepatocellular carcinoma tumor-bearing mouse model.After successful replication of the model,the model mice were randomly divided into model group,cisplatin group[2.5×10-3 g/(kg·3 d)],Shenqi Yiliu decoction low[13.515 g/(kg·d)],me-dium[27.03 g/(kg·d-1)],and high dose[27.030 g/(kg·d)]combined with cisplatin group[2.5×10-3 g/(kg·3 d)],10 mice in each group were treated for 13 d.After 24 h of the last dose,the mice were anesthetized and sacrificed,and the tumor inhibition rate,spleen index and thymus index of each drug group were determined;HE staining was performed to observe the histopathological changes of tumor in mice;ELISA kit was used to detect the contents of EGF and IFN-γ in tumor tissue homogenate;p-ERK1/2,C-Myc and PD-L1 protein expression in tumor tissue were detected by IHC and Western blot;ERK,C-Myc and PD-L1 mRNA expression levels in tumor tissue were detected by RT-PCR.Results:Compared with blank group,the average body mass and spleen index of mice in model group were decreased(P<0.05).Compared with model group,the tumor inhibition effect of each treatment group was obvious,and Shenqi Yiliu decoction combined with cisplatin group inhibited tumor growth in liver cancer mice in a dose-dependent way,im-proved the average body mass,spleen index and thymus index of mice,promoted the necrosis of tumor cells and increased the necrotic area.EGF and IFN-γ contents,P-ERK1/2,C-Myc,PD-L1 protein expressions and ERK,C-Myc,PD-L1 mRNA expression levels were decreased in tumor tissues(P<0.05).Compared with cisplatin group,the therapeutic effect of Shenqi decoction combined with cisplatin in medium and high dose groups was significant,and the difference was statistically significant(P<0.05).Conclusion:Shenqi Yiliu decoction combined with cisplatin effectively inhibited the tumor growth of H22 liver cancer tumor-bearing mice and significantly reduces the expression of C-Myc and PD-L1 proteins in the tumor tissues,which may be through the regulation of ERK signaling path-way-related protein expression to exert tumor suppressive effect.

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.

Effects of Electroacupuncture on Urodynamics and Expression of ERK/CREB/Bcl-2 Pathway in Spinal Cord of Rats with Neurogenic Bladder after Suprasacral Spinal Cord Injury / 中国中医药信息杂志

Objective To observe the effects of electroacupuncture at"Ciliao","Zhongji","Sanyinjiao"and"Dazhui"on urodynamics and expression of ERK/CREB/Bcl-2 pathway in spinal cord tissue of neurogenic bladder rats after suprasacral spinal cord injury.Methods Sixty female SD rats randomly selected 24 and divided into blank group and sham-operation group(12 rats in each group),the remaining 36 rats were subjected to surgical modeling.After modeling,rats were randomly divided into the model group and the electroacupuncture group,with 12 rats in each group.The electroacupuncture group received unilateral electroacupuncture stimulation at acupoints"Ciliao","Zhongji","Sanyinjiao",and"Dazhui"for 30 minutes each time,once a day,for 7 consecutive days.After administration,urodynamic testing was performed,HE staining was used to observe the morphology of bladder detrusor tissue,TUNEL method was used to detected apoptosis in spinal cord tissue,Western blot was used to detected expressions of p-ERK1/2,p-CREB,p-p90Rsk,CRE,Bcl-2,and Bax proteins in spinal cord tissue.Results Compared with the sham-operation group,the basal pressure,maximum pressure,and leakage point pressure of the bladder in the model group increased significantly(P<0.01),while the maximum capacity and compliance of the bladder decreased significantly(P<0.01);the structure of bladder smooth muscle cells was severely damaged and disorderly arranged,accompanied by a large amount of inflammatory cell infiltration;the apoptosis rate of spinal cord tissue cells significantly increased(P<0.01),and the expressions of p-ERK1/2,p-p90Rsk,p-CREB,CRE,and Bcl-2 proteins in spinal cord tissue were significantly decreased,while the expression of Bax protein significantly increased(P<0.01).Compared with the model group,the basal pressure,maximum pressure,and leakage point pressure of the bladder in the electroacupuncture group decreased significantly(P<0.05),while the maximum capacity and compliance of the bladder increased significantly(P<0.05,P<0.01);the integrity of bladder smooth muscle cells was enhanced,the degree of cell edema was reduced,and inflammatory cell infiltration was reduced;the apoptosis rate of spinal cord tissue cells was significantly reduced(P<0.05),and the expressions of p-ERK1/2,p-p90Rsk,p-CREB,CRE,and Bcl-2 proteins in spinal cord tissue significantly increased,while the expression of Bax protein was significantly decreased(P<0.05,P<0.01).Conclusion Electroacupuncture can promote the repair of bladder detrusor tissue in rats with neurogenic bladder model after suprasacral spinal cord injury,increase the maximum capacity and compliance of the bladder,alleviate the high pressure state in the bladder,and its mechanism is related to activating the ERK/CREB/Bcl-2 pathway,reducing secondary apoptosis of damaged neurons,effectively improving bladder innervation,and protecting bladder function.

Effects of Zishui Qinggan Decoction on the hippocampal protein expressions of ERK,GSK3β,CREB and BDNF in a mouse model of depression induced by chronic restraint stress / 中成药

AIM To explore the effects of Zishui Qinggan Decoction on the mouse model of depression induced by chronic restraint stress(CRS)via ERK/GSK3β/CREB/BDNF signaling pathway.METHODS Except for those of the blank group,the mice of other groups were induced into depression models by CRS,and divided into the model group,the fluoxetine hydrochloride group(10 mg/kg)and the low,medium and high dose Zishui Qinggan Decoction groups(8.835,17.670 and 35.340 g/kg)for the corresponding drug intervention and simultanous CRS treatment.The mice had their sugar water preference experiment and behavior experiment on the 7th and 14th day after administration;the observation of the hippocampal morphological changes by HE staining,the detection of the superoxide dismutase(SOD)activity and malondialdehyde(MDA)level in serum by kits,the detection of levels of serum 5-hydroxytryptamine(5-HT),tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)by ELISA,the detection of the hippocampal mRNA expressions of BDNF,TNF-α and IL-1β by RT-qPCR method,and the detection of the hippocampal protein expressions of ERK1/2,p-ERK1/2,GSK3β,p-GSK3β,CREB and BDNF by Western blot method 14 days after administration.RESULTS Compared with the model group,after 14 days of administration,both fluoxetine hydrochloride group and medium-dose Zishui Qinggan Decoction group displayed increased preference rate of sugar water(P<0.01),shortened immobility time of tail suspension and forced swimming(P<0.01),improved hippocampal damage of nerve cells,decreased levels of serum MDA,TNF-α and IL-1β(P<0.05,P<0.01),increased SOD activity and 5-HT level(P<0.05,P<0.01),decreased hippocampal mRNA expressions of TNF-α and IL-1β(P<0.01),and decreased expressions of BDNF mRNA and p-ERK1/2,p-GSK3β,CREB and BDNF proteins(P<0.05,P<0.01).CONCLUSION Zishui Qinggan Decoction can improve the depression-like behaviors in mice exposed to CRS,and its mechanism may be related to the regulation of hippocampal ERK/GSK3β/CREB/BDNF signaling pathway.

Effects of astragaloside Ⅳ on insulin resistance and MAPK/ERK pathway in obese rat model of polycystic ovary syndrome / 中成药

AIM To investigate the effects of astragaloside IV on improving insulin resistance(IR)in obese rat model of polycystic ovary syndrome(PCOS),and to analyze its effect on ovarian MAPK/ERK pathway as well.METHODS The obese PCOS rat models established by feeding of letrozole combined with high-fat and high-sugar diet were randomly divided into the model group,the metformin(135 mg/kg)group and the low-dose and high-dose astragaloside Ⅳ(25,50 mg/kg)groups,with 8 rats in each group in contrast to those of the control group.After 21 days oral administration,the rats had their body weight recorded;their ovarian index calculated;their levels of fasting blood glucose(FBG),serum triglyceride(TG),total cholesterol(TC),follicle-stimulating hormone(FSH),testosterone(T),estradiol(E2),luteinizing hormone(LH)and fasting insulin(FINS)measured;their HOMA-IR and LH/FSH values calculated;their ovarian expressions of MAPK/ERK pathway related proteins detected by Western blot;and their ovarian expression of vascular endothelial growth factor(VEGF)protein detected by immunofluorescence staining.RESULTS Compared with the control group,the model group showed increased polycystic pathological changes,levels of body weight,ovarian index,serum TG,TC,LH,FSH,T,FINS,and FBG,values of LH/FSH and HOMA-IR,and ovarian p-ERK1/2/ERK1/2,p-MEK1/2/MEK1/2,p-Raf/Raf,and VEGF protein expressions(P<0.01);and decreased serum E2 level(P<0.01).Compared with the model group,both astragaloside Ⅳ and metformin groups shared significantly alleviated ovarian polycystic lesions,and decreased body weight,levels of ovarian index,serum TG,TC,LH,FSH,T,FINS,and FBG,values of LH/FSH and HOMA-IR,ovarian p-ERK1/2/ERK1/2,p-MEK1/2/MEK1/2,p-Raf/Raf,and VEGF protein expressions(P<0.05,P<0.01),and increased serum E2 level(P<0.05,P<0.01).CONCLUSION Upon the obese PCOS rat models,astragaloside Ⅳ can antagonize their IR,improve their hormone levels and alleviate their ovarian lesions via inhibiting the activation of MAPK/ERK pathway.

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.

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.

Improvement effect and mechanism of sanguinarine on inflammatory pain in rats with lumbar disc herniation / 中国药房

OBJECTIVE To study the improvement effect and mechanism of sanguinarine （SG） on inflammatory pain in rats with lumbar disc herniation （LDH） and its mechanism. METHODS LDH model rats were established and divided into model group， SG low-dose， medium-dose and high-dose groups （1.00， 2.50， 6.25 mg/kg）， high-dose of SG+Anisomycin [mitogen-activated protein kinase （MAPK） activator] group （6.25 mg/kg SG+5 mg/kg Anisomycin）， with 10 rats in each group. Another 10 rats were included as the control group. Each group was given corresponding drugs intraperitoneally， while the control group and model group were given an equal volume of normal saline intraperitoneally， once a day， for 7 consecutive days. The general situation and neurological changes of rats in each group were observed， and the pain threshold [including paw withdrawal mechanical threshold （PWMT） and paw withdrawal thermal latency （PWTL）] of rats was determined； the histopathological changes of dorsal root ganglion （DRG） were observed in rats. The serum levels of inflammatory factors [tumor necrosis factor-α （TNF-α）， interleukin-1β （IL-1β）] and pain factors [neuropeptide Y （NPY）， 5-hydroxytryptamine （5-HT）] in rats were detected.The positive expressions of ionized-calcium binding adaptor molecule-1 （Iba-1） in spinal cord microglia and glial fibrillary acidic protein （GFAP） in astrocytes were observed. The expressions of proteins related to MAPK/extracellular signal-regulated kinase （ERK）/nuclear factor κB （NF-κB） signaling pathway， TNF-α and IL-1β proteins were detected in DRG tissue of rats. RESULTS Compared with the control group， the rats in the model group showed decreased appetite， hindlimb movement disorders， and disordered neuronal cell arrangement， the neurological score， the levels of TNF-α， IL-1β， NPY， the positive expressions of Iba-1 and GFAP， the phosphorylations of p38 MAPK， ERK1/2 and NF-κB p65， the protein expressions of TNF-α and IL-1β were significantly increased （P＜0.05）； PWMT， PWTL and the levels of 5-HT were significantly reduced （P＜0.05）. Compared with the model group， the rats of SG groups showed some relief in their mental appetite and hindlimb motor disorders， the intervertebral disc structure of DRG was restored， and the levels of the above quantitative indicators had significantly reversed （P＜0.05）. Anisomycin reversed the improvement effect of SG on inflammatory pain in LDH rats. CONCLUSIONS SG can improve inflammatory pain by inhibiting the activation of microglia in DRG tissue of LDH rats， reducing the release of inflammatory factors， and increasing pain threshold， and its mechanism of action may be related to the inhibition of MAPK/ERK/NF- κB signaling pathway.

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.

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.

Mechanism of beta-sitosterol on hypertrophic scar fibroblasts:an analysis based on network pharmacology / 中国组织工程研究

BACKGROUND:At present,effective preventive and therapeutic measures for hypertrophic scar are still limited.In contrast,most of botanical herbs have few side effects and abundant sources,offering new ideas and approaches for the prevention and treatment for hypertrophic scar. OBJECTIVE:To explore the potential molecular mechanism of plant-derived β-sitosterol on hypertrophic scar fibroblasts by network pharmacology and molecular docking techniques and to initially verify it by cytological experiments. METHODS:Through the network pharmacology,the relevant database and software were used to screen the drug targets of β-sitosterol and obtain the hypertrophic scar-related disease targets.The potential(intersection)targets of β-sitosterol on hypertrophic scar were obtained.Cytoscape software and STRING database were used to construct the"drug-target-disease"network and protein-protein interaction network,and screen out the core targets in the protein-protein interaction network.Gene ontology(GO)biological function and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analyses of intersection targets were conducted through the DAVID database,and the signaling pathways and core target genes closely related to the intersection targets were further identified through literature analysis.AutoDock software was used to perform the molecular docking of β-sitosterol and core target proteins.In vitro cellular assays were used to verify the effects of β-sitosterol on proliferation,apoptosis,cell cycle distribution and mRNA expression of core target genes in human hypertrophic scar fibroblasts. RESULTS AND CONCLUSION:There were 56 intersection targets of β-sitosterol and hypertrophic scar and 10 core targets were identified in the protein-protein interaction network,including tyrosine kinase,mitogen-activated protein kinase 3(MAPK3),cysteine protease 3(CASP3),apolipoprotein E,estrogen receptor 1,sterol regulatory element-binding transcription factor 1,peroxisome proliferator-activated receptor alpha,C-reactive protein,intercellular adhesion molecule 1,and catalase.Combined with the literatures and the functional analysis of the KEGG and GO,the MAPK signaling pathway was further identified to be closely related to the intersection targets,and MAPK3(ERK1-MAPK),CASP3,P53 and tumor necrosis factor were identified as the core targets.The molecular docking results indicated that β-sitosterol was well bound to the core target proteins.Cellular assays showed that 100 μmol/L β-sitosterol inhibited hypertrophic scar fibroblast proliferation,decreased mitochondrial membrane potential and induced apoptosis(P<0.01),increased the proportion of G1-phase cells and decreased the proportion of S-phase cells(P<0.05),upregulated the mRNA expression of CASP3,P53 and tumor necrosis factor(P<0.05),and downregulated the mRNA expression of MAPK3(P<0.001).To conclude,β-sitosterol may induce cell apoptosis in hypertrophic scar fibroblasts by activating the tumor necrosis factor pathway and upregulating the expression of CASP3 and P53,while inhibiting the ERK-MAPK pathway to arrest cell cycle and thus reduce the proliferation of hypertrophic scar fibroblasts.

Effects of biological amniotic membrane on tendon adhesion and healing in ruptured Achilles tendon rats / 中国组织工程研究

BACKGROUND:Achilles tendon adhesion after Achilles tendon injury can lead to decreased biomechanical properties,weakened healing ability,and ultrastructural changes of Achilles tendon,which further affects patients'daily life and work ability.Therefore,how to effectively deal with and prevent Achilles tendon adhesion has become a hot and difficult problem in clinical treatment. OBJECTIVE:To analyze the effects of biological amniotic membranes on postoperative Achilles tendon adhesion,biomechanics,and ultrastructural changes in rats with Achilles tendon rupture. METHODS:Sixty 6-week-old SD rats were selected to establish bilateral Achilles tendon rupture models and divided into two groups(n=30 per group)by the random number table method.In the model group,the severed end of the tendon was sutured directly.In the amniotic membrane group,the biological amniotic membrane was wrapped around the broken anastomosis and fixed by a suture.The adhesion,biomechanics,morphology,and structure of the Achilles tendon and the expression of p38 and ERK1/2 protein were evaluated 1,2,and 4 weeks after surgery. RESULTS AND CONCLUSION:(1)1 week after operation,the Achilles tendon and peritendinous tissues of the two groups were mildly edema,and the adhesion of the Achilles tendon tissues in the model group was more obvious.2 weeks after the intervention,the Achilles tendon and peritendinous tissues of the model group still had edema,and the adhesion degree between the Achilles tendon and the surrounding tissues was heavier than that of the amniotic membrane group.4 weeks after operation,there was no edema around the Achilles tendon in both groups,and the healing was well.The adhesion degree of the Achilles tendon in the amniotic membrane group was less than that in the model group.The maximum tension of Achilles tendons in the amniotic membrane group was higher than that in the model group at 2 and 4 weeks after operation(P<0.001).(2)Hematoxylin-eosin staining and transmission electron microscopy revealed that 1 week after operation,the tendon structure of rats of the two groups was disordered and the collagen fibers were sparsely arranged,in which the model group demonstrated obvious inflammatory reaction and adhesion to the Achilles tendon.Two weeks after operation,the model group still demonstrated obvious inflammatory response,adhesion of Achilles tendon,and irregular ordering of collagen fibers.The amniotic membrane group exhibited an orderly arrangement of collagen fibers and expansion of the endoplasmic reticulum of fibroblasts.At 4 weeks after operation,the collagen fibers of the Achilles tendon in the model group were thickened and disordered,and the rough endoplasmic reticulum was less in the fibroblasts,while the collagen fibers in the amniotic membrane group were ordered and thin,and the fibroblasts contained a large number of rough endoplasmic reticulum.(3)Four weeks after operation,western blot assay exhibited that the expressions of p38 and ERK1/2 protein in the Achilles tendon tissue of rats in the amniotic membrane group were lower than those in the model group(P<0.05).(4)The results confirm that the biologic amniotic membrane can promote the healing and inhibit the adhesion of Achilles tendon after the operation of the ruptured Achilles tendon,which may be associated with the regulation of the MAPK/ERK signaling pathway.

Effect of cell mechanics on morphogenesis of MDCK lobular organoid / 中国组织工程研究

BACKGROUND:The development of tissues and organs in the body is a precise and autonomously regulated process,and the function of biomechanical factors at this macroscale is a basic scientific question worth exploring. OBJECTIVE:To investigate the roles of cell mechanics in morphogenesis of the lobular organoid of 3D Madin-Darby canine kidney(MDCK). METHODS:The formation of MDCK lobular organoid was visualized by fluorescence resonance energy transfer technology,and the influence of different cellular mechanical signals and extracellular matrix environment on lobular organoid formation and corresponding changes in extracellular regulated protein kinases(ERK)activity were examined. RESULTS AND CONCLUSION:(1)Inhibition of ERK signaling pathway can inhibit the growth of MDCK lobular organoid.(2)Inhibition of cell contractile force signals such as ROCK pathway and Myosin Ⅱ activity,reduced ERK activity and lobular organoid size.(3)Selective inhibition of calcium channels in plasma membrane and endoplasmic reticulum led to reduced ERK activity and lobular organoid growth.(4)By inhibiting the mechanically-sensitive receptor Piezo ion channel or integrin signal on the cell membrane,the lobular organoid became smaller or MDCK cells could not generate tissue morphology.(5)Extracellular matrix compositions affected the morphogenesis of lobular organoid.The addition of type I collagen in Matrigel changed the lobular organoid to elongated shape.(6)The results of this study preliminarily show that mechanical signals in the cells and extracellular matrix environment play an important role in culturing MDCK lobular organoid,and provides certain molecular mechanisms.

Interleukin-1beta enhances migration and adhesion of mesenchymal stem cells in inflammatory environments / 中国组织工程研究

BACKGROUND:Mesenchymal stem cells possess characteristics such as rapid renewal,targeted homing,tissue repair,and immune regulation,which provide potential for the treatment of inflammatory diseases.In most inflammatory diseases,interleukin-1β is highly expressed.Both exogenous and endogenous mesenchymal stem cells unavoidably exist in an environment with high interleukin-1β concentration. OBJECTIVE:To study the interaction of interleukin-1β with mesenchymal stem cells in inflammatory environment and the mechanism of its influence on the migration and adhesion of mesenchymal stem cells to provide a theoretical basis for adjusting stem cell therapy strategies. METHODS:The first author searched for studies involving interleukin-1β enhancing migration and adhesion of mesenchymal stem cells by computer on CNKI,WanFang,VIP,PubMed,and Web of Science using search terms"interleukin-1β,mesenchymal stem cell,nuclear factor-κB,MAPK,ERK,p38,migration,adhesion"in Chinese and English.The literature tracing method was also used to search for some of the literature.Finally,65 articles were included in the review analysis. RESULTS AND CONCLUSION:(1)In the inflammatory environment,interleukin-1β can regulate the migration and adhesion ability of mesenchymal stem cells.This effect may be achieved by recruiting IRAK1 through interleukin-1RI and then activating TAK1 and IKK in turn.After IKK phosphorylation,nuclear factor-κB and ERK signaling pathways are activated or CXCR expression is upregulated through the p38 pathway to promote mesenchymal stem cell migration and adhesion.However,further standardized research needs to be carried out based on the genetic background of mesenchymal stem cells,the dose and processing time of interleukin-1β.(2)In vitro experiments using pre-stimulated mesenchymal stem cells with interleukin-1β can change the survival environment of mesenchymal stem cells and alter their secretion factors to make them develop towards a more anti-inflammatory direction.On the other hand,under the premise of producing higher levels of anti-inflammatory and pro-nutrient factors,extracted mesenchymal stem cell exosomes can exert anti-inflammatory effects.(3)It has been observed in various animal disease models that pre-stimulating mesenchymal stem cells with interleukin-1β regulates their immune regulation ability,thereby affecting the development and outcome of inflammation.However,this is limited to preclinical basic research only;further verification on efficacy and safety of stem cell therapy with interleukin-1β pre-treated mesenchymal stem cells is required in clinical settings.

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.