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@#[摘 要] 目的：探讨溶瘤新城疫病毒（NDV）对IL-6诱导的人胶质母细胞瘤U87MG细胞增殖、迁移和侵袭的作用及其可能的机制。方法：将U87MG细胞分为对照组、IL-6组、NDV组、NDV+IL-6组，其中IL-6组与NDV+IL-6组用75 ng/mL IL-6预处理1 h，其余组用DMEM预处理1 h，后分别用DMEM、75 ng/mL IL-6、1 HU NDV、1 HU NDV+75 ng/mL IL-6处理24 h。MTT法、细胞划痕实验和Transwell侵袭实验分别检测IL-6、NDV对U87MG细胞增殖、迁移和侵袭的影响，WB法检测各组细胞JAK2、p-JAK2、STAT3、p-STAT3和MMP2蛋白的表达水平。结果：与对照组相比，IL-6组细胞迁移率显著升高（P<0.05），侵袭细胞数目显著增多（P<0.01）；与IL-6组相比，NDV+IL-6组U87MG细胞增殖率显著降低（P<0.05），细胞迁移率和侵袭细胞数目均显著降低（均P<0.01）。WB实验结果显示，与对照组相比，IL-6组p-STAT3/STAT3比值显著升高（P<0.01），NDV组p-JAK2/JAK2、p-STAT3/STAT3比值显著降低（P<0.05，P<0.01），MMP-2蛋白表达量显著降低（P<0.01）；与IL-6组相比，NDV+IL-6组p-STAT3/STAT3比值、MMP-2蛋白表达量均显著降低（均P<0.05）。结论：NDV能抑制IL-6对人脑胶质瘤U87MG细胞迁移和侵袭的诱导作用，其机制可能与JAK2/STAT3信号通路的参与调控有关。
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Objective:To explore the effects of Jianpi Bushen Jiedu Prescription on the proliferation and migration of hepatocellular carcinoma cells; To discuss its possible mechanism.Methods:Using human highly metastatic liver cancer cell line (HCCLM3) as the research object, they were randomly divided into control group and TCM group (100, 200, 400, 800, 1 600, 3 200 μg/ml Jianpi Bushen Jiedu Prescription) and Western medicine group (2.5, 5, 10, 20, 40 μmol/L sorafenib) using a random number table method. Cell viability was detected using cell counting reagent (CCK-8) method; HCCLM3 cells were divided into control group and TCM (Jianpi Bushen Jiedu Prescription 800 μg/ml) group and combined group (Jianpi Bushen Jiedu Prescription 800 μg/ml +sorafenib 20 μmol/L). Western blot method was used to detect the protein expressions of kinase/signaling transducer and transcriptional activator (JAK2/STAT3) pathway related proteins (p-JAK2, JAK2, p-STAT3, STAT3) in each group.Results:Compared with the control group, viability and mobility of HCCLM cell in TCM group and Western medicine group decreased ( P<0.01 or P<0.05); compared with the control group, the protein expressions of P-JAK2, JAK2, P-STAT3 and STAT3 in the TCM group and the combined group decreased ( P<0.05), and the JAK2 protein expression in the combined group was lower than that in the TCM group ( P<0.05). Conclusion:Jianpi Bushen Jiedu Prescription can inhibit the proliferation and migration of HCC cells by regulating JAK2/STAT3 pathway.
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Objective To observe the effects of acupoint catgut embedding therapy on body mass,lipid metabolism,serum leptin and mRNA and protein expressions of hypothalamic leptin receptor(LepR)-mediated Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)pathway in rats with diet-induced obesity(DIO).Methods Forty male SD rats were randomly divided into 10 in normal group and 30 in modeling group.A high-fat diet was used to establish the DIO rat model.After successful modeling,the modeled rats were randomly divided into the model group,the acupoint catgut embedding group and the acupoint catgut embedding + AG490(JAK2/STAT3 pathway blocker)group,with 10 rats in each group.The acupoint catgut embedding group and the acupoint catgut embedding + AG490 group were embedded on day(s)1,8,15 and 22 after successful modeling,the acupoints were selected from the Zhongwan(RN12),Shuidao(ST28),Tianshu(ST25),Pishu(BL20),Weishu(BL21),Sanjiaoshu(BL22)with a total of 4 treatments,and the acupoint catgut embedding + AG490 group was injected intraperitoneally with 1 mg/kg of AG490 every day during the treatment period;the normal group and the model group were only grasped and fixed.Body mass was measured before and after treatment.Lipid metabolism indexes of triglyceride(TG),total cholesterol(TC),low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C),and serum leptin levels were measured after treatment,and the mRNA expressions of hypothalamus LepR,JAK2 and STAT3 were detected by real-time quantitative polymerase chain reaction(RT-PCR),and the protein expressions of hypothalamus LepR,JAK2 and STAT3 were detected by Western Blot.Results Before treatment,compared with the normal group,the body mass of the model group,the acupoint catgut embedding group,and the acupoint catgut embedding+AG490 group were all elevated(P＜0.01),and compared with the model group,there was no significant difference in the body mass between the acupoint catgut embedding group and the acupoint catgut embedding+AG490 group(P＞0.05).After treatment,compared with the normal group,body mass,leptin and TG,TC,LDL-C levels were increased,and mRNA and protein expression levels of LepR,JAK2,STAT3 were decreased in the model group(all P＜0.01);compared with the model group,body mass,leptin and TG,TC,LDL-C levels were decreased in the acupoint catgut embedding group,and mRNA and protein levels of LepR,JAK2,STAT3 were increased in the acupoint catgut embedding + AG490 group(all P＜0.01);compared with the acupoint catgut embedding + AG490 group,the body mass,leptin and TG,TC,LDL-C levels were decreased,and mRNA and protein levels of LepR,JAK2,STAT3 were increased in the acupoint catgut embedding group(P＜0.05 or P＜0.01).Conclusion Acupoint catgut embedding has a good effect on weight loss and lipid reduction in DIO rats,and its central mechanism may be related to the down-regulation of serum leptin level and activation of hypothalamic LepR-mediated JAK2/STAT3 pathway.
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BACKGROUND:Shujin Jiannao Prescription is an empirical formula for the treatment of cerebral palsy in Dongzhimen Hospital,Beijing University of Chinese Medicine,with clear clinical efficacy,but the specific mechanism needs to be elucidated. OBJECTIVE:To explore the possible mechanism of Shujin Jiannao Prescription in treating cerebral palsy. METHODS:Sixty-four 7-day-old Sprague-Dawley rats were randomly divided into a normal group(n=12)and a model group(n=52).An animal model was established by the Rice-Vannucci method.After successful modeling,52 model rats were randomly divided into control model group(n=12),minocycline group,and the low-,medium-,and high-dose groups of the Shujin Jiannao Prescription(n=10 per group).Rats in the minocycline group were given 40 mg/kg·d minocycline by gavage;rats in the low-,medium,and high-dose groups were given 4,8,and 16 g/kg·d Shujin Jiannao Prescription granules by gavage,respectively;and rats in the normal group and control model group were given an equal dose of normal saline by gavage.Medication in each group was given once a day for 1 week.The rats in each group were evaluated behaviorally using suspension test,abnormal involuntary movement score,and Bederson score.The pathological changes of the cerebral cortex were observed by hematoxylin-eosin staining.The levels of tumor necrosis factor α,interleukin 1β,and interleukin 10 in the cerebral cortex were determined using ELISA.The positive expressions of Janus kinase 2(JAK2),phosphorylated Janus kinase 2(p-JAK2),phosphorylated signal transducer and activator of transcription 3(p-STAT3)in the cerebral cortex were detected using immunohistochemistry.The protein expression levels of JAK2,p-JAK2,and p-STAT3 were detected using western blot. RESULTS AND CONCLUSION:Compared with the normal group,the suspension test score and involuntary movement score were decreased in the control model group(P＜0.01 or P＜0.05).The pathological results showed structural disruption of nerve cells,formation of large numbers of vacuoles,cell swelling,and increased intercellular space in the control model group.In addition,the expressions of tumor necrosis factor α and interleukin 1β in the cerebral cortex were significantly increased(P＜0.01),the expression of interleukin 10 was decreased(P＜0.05),and the protein expressions of JAK2,p-JAK2,and p-STAT3 in the cerebral cortex were significantly increased(P＜0.01)in the control model group compared with the normal group.Compared with the model group,minocycline and Shujin Jiannao Prescription at each dose could improve the behavioral indexes of rats(P＜0.01 or P＜0.05)and ischemic-hypoxic pathological changes were attenuated,with only a small amount of necrotic nerve cells and a few vacuoles,and reduced intercellular space.Moreover,the expressions of tumor necrosis factor α and interleukin 1β in the cerebral cortex were decreased in each drug group compared with the control model group(P＜0.05),while the protein expressions of JAK2,p-JAK2,and p-STAT3 in the cerebral cortex were significantly decreased(P＜0.01).The most obvious improvement was observed in the high-dose Shujin Jiannao Prescription group.To conclude,Shujin Jiannao Prescription can inhibit inflammation in the cerebral cortex of rats with hypoxic-ischemic brain injury.The mechanism may be related to the regulation of the JAK2/STAT3 signaling pathway.
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Objective To observe the effects of electroacupuncture on central inflammatory response and neurotransmitter release in rats with post stroke spasticity(PSS);To exploring the mechanism in treating PSS based on IL-6/JAK2/STAT3 signaling pathway.Methods Totally 30 male SD rats were randomly divided into sham-operation group,model group and electroacupuncture group,with 10 rats in each group.The PSS model was prepared by the method of suture and N-methyl-D-aspartic acid receptor injection into internal capsule.The rats in the electroacupuncture group were electroacupulated on the affected side of the body at"Quchi"and"Yanglingquan"for 30 min/d for consecutive 7 d.The sham-operation group and the model group were only fixed without any interventions.The Zea Longa neurological function score and the modified Ashworth muscle tension score were evaluated before and after treatment in each group;the pathological changes of the cortex on the ischemic side were observed by HE staining;the contents of interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and γ-aminobutyric acid(GABA)in cortex on the ischemic side were detected by ELISA;the content of glutamate(Glu)was detected by biochemical kit;Western blot was used to detect the expressions of tyrosine kinase 2(JAK2),p-JAK2,signal transduction and transcription activating factor 3(STAT3)and p-STAT3 protein in ischemic cortex;RT-PCR was used to detect the mRNA expressions of JAK2 and STAT3 in ischemic cortex.Results Compared with the sham-operation group,neurological function score and muscle tension score significantly increased in the model group(P<0.01),with disorganized neurons in cerebral cortex,nucleus accumbens,the contents of IL-6,TNF-α and Glu significantly increased,the content of GABA significantly decreased(P<0.01),and p-JAK2,p-STAT3 proteins and JAK2 and STAT3 mRNA expression significantly increased(P<0.01,P<0.05).Compared with the model group,the neurological function score and muscle tension score of rats in the electroacupuncture group were significantly decreased(P<0.05),the degree of neuronal damage in cerebral cortex was reduced,the cell contour was clear,the content of IL-6,TNF-α and Glu were significantly decreased,and the content of GABA significantly increased(P<0.05,P<0.01),p-JAK2,p-STAT3 protein and JAK2,STAT3 mRNA expression significantly decreased(P<0.01).Conclusion Electroacupuncture may alleviate central inflammatory response and improve limb spasticity of PSS model rats by inhibiting the IL-6/JAK2/STAT3 signaling pathway.
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Objective To investigate the effect of folic acid–modified liposome quercetin (FLQ) on the proliferation and apoptosis of triple negative breast cancer (TNBC) cells and explore its underlying mechanism. Methods CCK-8 was used to detect the effect of FLQ on TNBC cell viability. Colony formation assay was conducted to detect the effect of FLQ on TNBC cell proliferation. Flow cytometry was performed to detect the effect of FLQ on TNBC cell apoptosis, the levels of intracellular ROS, and mitochondrial membrane potential. Western blot analysis was conducted to detect the expression levels of JAK2/STAT3 signaling pathway-related and apoptosis-related proteins. Results FLQ inhibited the proliferation and promoted the apoptosis of MDA-MB-231 cells (P=0.023, P<0.001). It promoted mitochondrial membrane potential collapse and increased the intracellular ROS levels of MDA-MB-231 cells (P=0.003, P=0.034); inhibited the phosphorylation levels of JAK2 and STAT3; upregulated the expression levels of the proapoptotic proteins Bax, Bak, cytochrome C, and Cleaved-Caspase-3 (P<0.001, P<0.001); and downregulated the expression levels of the antiapoptotic proteins Bcl2 and Bcl-xL (P=0.037, 0.028). Conclusion FLQ inhibits the proliferation and induces the apoptosis of MDA-MB-231 cells. These effects may be related to the activation of the mitochondrial apoptosis pathway through the inhibition of the JAK2/STAT3 signaling pathway.
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Abstract Objective Nasopharyngeal Carcinoma (NPC) is a malignancy of epithelium of epithelium of the nasopharynx, with the highest incidence of otolaryngeal malignancies. A growing number of studies confirm that Circular RNA (circRNA) plays an important role in tumor development, including Hsa_circ_0013561. This study aims to elucidate the process and mechanism of NPC regulation hsa_circ_0013561. Methods In this study, circRNA expression nodes and subcellular localization in NPC tissues were analyzed by fluorescence in situ hybridization. The expression of hsa_circ_0013561 in NPC cells was further clarified by RT-qPCR. At the same time, the lentivirus vector interfered by hsa_circ_0013561 was constructed and transfected. The cell proliferation was detected by CCK-8 method, EdU assay and plate cloning assay. The cell cycle and apoptosis were detected by flow cytometry, and the cell migration ability was detected by wound healing assay and Transwell assay. Western blotting examined the expression of apoptosis, Epithelial-Mesenchymal Transition (EMT)-associated proteins, and Janus Kinase/Signal Transductor and Activator of Transcription (JAK/STAT) signaling pathway-related proteins. Results The results showed that the expression of hsa_circ_0013561 in NPC samples was significantly upregulated and hsa_circ_0013561 localized in the cytoplasm. After down-regulating hsa_circ_0013561 expression, it significantly inhibited the proliferation and metastasis ability of NPC, inhibited EMT progression, and promoted apoptosis. Further studies showed that interference hsa_circ_0013561 significantly inhibited JAK2/STAT3 signaling pathway activation and induced the expression of apoptosis-related proteins. Conclusion In summary, we found that hsa_circ_0013561 is a pro-tumor circRNA in NPC, which can reduce the activation of JAK2/STAT3 pathway by knocking down hsa_circ_0013561, thereby slowing down the malignant progression of NPC. Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence Level 4.
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The goal of this study was to investigate the regulatory effect of angiotensin converting enzyme 2 (ACE2) on cellular inflammation caused by avian infectious bronchitis virus (IBV) and the underlying mechanism of such effect. Vero and DF-1 cells were used as test target to be exposed to recombinant IBV virus (IBV-3ab-Luc). Four different groups were tested: the control group, the infection group[IBV-3ab-Luc, MOI (multiplicity of infection)=1], the ACE2 overexpression group[IBV-3ab Luc+pcDNA3.1(+)-ACE2], and the ACE2-depleted group (IBV-3ab-Luc+siRNA-ACE2). After the cells in the infection group started to show cytopathic indicators, the overall protein and RNA in cell of each group were extracted. real-time quantitative polymerase chain reaction (RT-qPCR) was used to determine the mRNA expression level of the IBV nucleoprotein (IBV-N), glycoprotein 130 (gp130) and cellular interleukin-6 (IL-6). Enzyme linked immunosorbent assay (ELISA) was used to determine the level of IL-6 in cell supernatant. Western blotting was performed to determine the level of ACE2 phosphorylation of janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). We found that ACE2 was successfully overexpressed and depleted in both Vero and DF-1 cells. Secondly, cytopathic indicators were observed in infected Vero cells including rounding, detaching, clumping, and formation of syncytia. These indicators were alleviated in ACE2 overexpression group but exacerbated when ACE2 was depleted. Thirdly, in the infection group, capering with the control group, the expression level of IBV-N, gp130, IL-6 mRNA and increased significantly (P < 0.05), the IL-6 level was significant or extremely significant elevated in cell supernatant (P < 0.05 or P < 0.01); the expression of ACE2 decreased significantly (P < 0.05); protein phosphorylation level of JAK2 and STAT3 increased significantly (P < 0.05). Fourthly, comparing with the infected group, the level of IBV-N mRNA expression in the ACE2 overexpression group had no notable change (P > 0.05), but the expression of gp130 mRNA, IL-6 level and expression of mRNA were elevated (P < 0.05) and the protein phosphorylation level of JAK2 and STAT3 decreased significantly (P < 0.05). In the ACE2-depleted group, there was no notable change in IBV-N (P > 0.05), but the IL-6 level and expression of mRNA increased significantly (P < 0.05) and the phosphorylation level of JAK2 and STAT3 protein decreased slightly (P > 0.05). The results demonstrated for the first time that ACE2 did not affect the replication of IBV in DF-1 cell, but it did contribute to the prevention of the activation of the IL-6/JAK2/STAT3 signaling pathway, resulting in an alleviation of IBV-induced cellular inflammation in Vero and DF-1 cells.
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Animais , Humanos , Chlorocebus aethiops , Interleucina-6/genética , Janus Quinase 2/farmacologia , Vírus da Bronquite Infecciosa/metabolismo , Fator de Transcrição STAT3/metabolismo , Enzima de Conversão de Angiotensina 2/farmacologia , Receptor gp130 de Citocina/metabolismo , Células Vero , Transdução de Sinais , Inflamação , RNA MensageiroRESUMO
OBJECTIVE@#To investigate the efficacy of Bushen Huoxue Fang (BSHXF, a traditional Chinese medicine formula) for improving recurrent spontaneous abortion (RSA) in mice and the role of tyrosine kinase (JAK2) and transcriptional activator (STAT3) signaling pathway in its therapeutic mechanism.@*METHODS@#Female CBA/J mice were caged with male DBA/2 mice to establish RSA mouse models, which were randomly divided into model group, dydrogesterone group and BSHXF group, with the female mice caged with male BALB/c mice as the control group (n=6). From the first day of pregnancy, the mice were subjected to daily intragastric administration of BSHXF, dydrogesterone, or distilled water (in control and model groups) for 12 days. After the treatments, serum levels of antithrombin III (AT-III), activated protein C (APC), tissue plasminogen activator (t-PA), progesterone, human chorionic gonadotropin (HCG), and estradiol (E2) were detected in each group using ELISA. HE staining was used to observe the morphological changes of the endometrium of the mice. Western blotting was performed to determine the expressions of p-JAK2, p-Stat3 and Bcl-2 in the placenta of the mice.@*RESULTS@#Compared with the control mice, the mouse models of RSA showed a significantly increased embryo loss rate with decreased serum levels of AT-III, T-PA, progesterone, APC and HCG, increased placental expressions of p-JAK2, p-STAT3 and Bax, and decreased expression of Bcl-2 (P < 0.05). Treatments with BSHXF and dydrogesterone both increased serum levels of AT-III, t-PA and HCG in the mouse models; Serum APC level was significantly reduced in BSHXF group and serum progesterone level was significantly increased in dydrogesterone group (P < 0.05).@*CONCLUSION@#BSHXF can improve the prethrombotic state and inhibit cell apoptosis by downregulating the JAK2/STAT3 pathway to increase the pregnancy rate in mouse models of RSA.
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Animais , Camundongos , Aborto Habitual/prevenção & controle , Transdução de Sinais , Regulação para Baixo , Modelos Animais de DoençasRESUMO
OBJECTIVE To investigate the inhibitory effects of ursolic acid on interleukin-6 （IL-6）-mediated invasion and migration of breast cancer MDA-MB-231 cells （hereinafter referred to as “231 cells”）. METHODS The effects of 20， 40， 80， 160 and 320 µmol/L ursolic acid on the proliferation rate of 231 cells were measured by CCK-8 method. The breast cancer 231 cells were divided into control group， model group and administration group. The migration and invasion abilities of cells were detected by scratch assay and Transwell assay. Real-time quantitative polymerase chain reaction （q-PCR） assay and Western blot assay were used to detect the mRNA and protein expressions of epithelial-mesenchymal transition-related makers such as E cadherin （E-cad）， matrix metalloprotein 2 （MMP2）， MMP9， vimentin （Vim）， CD44 molecule （CD44） and aldehyde dehydrogenase 1 family member A1 （ALDH1A1）. The phosphorylation levels of JAK2 and STAT3 in the Janus kinase 2/signal transducer and activator of transcription 3 （JAK2/STAT3） signaling pathway （in terms of p-JAK2/JAK2 ratio and p-STAT3/STAT3 ratio） were detected by Western blot assay. RESULTS A low concentration of ursolic acid of 20 µmol/L （no significant inhibitory effect on cell proliferation ability） was selected as the subsequent administration concentration. Compared with the control group， the migration and invasion abilities of cells in the model group were significantly enhanced （P＜0.05）； compared with the model group， the migration and invasion abilities of cells in the administered group were significantly reduced （P＜0.05）. Compared with the control group， the relative mRNA and protein expressions of epithelial-mesenchymal transition-related markers MMP9， MMP2， Vim， ALDH1A1 and CD44 were all elevated to different extents， and the mRNA and protein expressions of E-cad were all decreased to different extents in the model group cells， and part of the differences had statistical significance （P＜0.05）， the p-JAK2/JAK2 ratio and p-STAT3/STAT3 ratio were significantly increased in the model group （P＜0.05）； compared with the model group， the expressions of the above indicators were reversed to some extent in the administration group. CONCLUSIONS Ursolic acid blocks the activation of JAK2/STAT3 signaling pathwby the inflammatory factor IL-6， which ultimately interrupts the invasion and metastasis of breast cancer cells.
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Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signaling pathway, this study investigated the effect of medicated serum of Sparganii Rhizoma(SR) and Curcumae Rhizoma(CR) on the proliferation, apoptosis, migration, and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs). Specifically, human ESCs were primary-cultured. The effect of different concentration(5%, 10%, 20%) of SR-, CR-, and SR-CR combination-medicated serum, and AG490 solution(50 μmol·L~(-1)) on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT) assay, and the optimal dose was selected accordingly for further experiment. The cells were classified into normal serum(NS) group, SR group(10%), CR group(10%), combination(CM) group(10%), and AG490 group. The apoptosis level of ESCs was detected by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-3(caspase-3), B-cell lymphoma(Bcl-2), and Bcl-2-associated X protein(Bax) and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot. The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01), especially the 10% drug-medicated serum, which was selected for further experiment. The 10% SR-medicated serum, 10% CR-medicated serum, and 10% CM-medicated serum could increase the apoptosis rate(P<0.01), up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01), down-regulate the expression of Bcl-2(P<0.01), decrease the cell migration rate(P<0.05 or P<0.01), and reduce the secretion levels of IL-1β, IL-6, and TNF-α(P<0.05 or P<0.01), and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01). Compared with the SR and CR groups, CM group showed low cell viability(P<0.01), high protein expression of caspase-3 and Bax(P<0.05 or P<0.01), and low protein expression of Bcl-2 and p-JAK2(P<0.05). After incubation with CM, the apoptosis rate was higher(P<0.05) and the migration rate was lower(P<0.01) than that of the CR group. The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05). The mechanism of SR, CR, and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway, inhibited ESC proliferation, promoted apoptosis, weakened cell migration, and reduced the secretion of inflammatory factors. The effect of the combination was better than that of RS alone and CR alone.
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Feminino , Humanos , Janus Quinase 2 , Caspase 3 , Proteína X Associada a bcl-2 , Interleucina-6/genética , Apoptose , Transdução de Sinais , Proliferação de Células , Fator de Transcrição STAT3/genéticaRESUMO
Objective:To explore the in vitro anticancer effect of bleomycin (Ble) combined with cisplatin (DDP) on human gastric cancer cells.Methods:CCK-8 experiment was conducted to detect the effect of Ble and DDP alone or in combination on the proliferation of gastric cancer cells; Flow cytometry was used to detect changes in cell apoptosis and changes in reactive oxygen species. Cell scratch test was employed to detect cell migration; Western blot experiment was conducted to detect protein expression.Results:The results of CCK-8 showed that 20 μM DDP could inhibit the proliferation of six types of gastric cancer cells by 40%, while the effect of 20 μM Ble was only about 20%. When used in combination, the inhibitory rate on six types of gastric cancer could reach over 60%, with the strongest inhibitory effect on AGS cells. When 10 μM Ble and 10 μM DDP were combined, the proportion of AGS apoptotic cells reached 75.68%, higher than that of the two drugs treated with 20%, respectively 20 μM processing effect. When 10 μM Ble and 10 μM DDP were used in combination, the number of migrating and invading cells was significantly reduced, and the inhibition rate reached 50%. Compared with single use, the difference was statistically significant ( P<0.05). Flow cytometry results showed that the combination of Ble and DDP promoted cancer cell apoptosis by up-regulating the level of reactive oxygen species in cells; Western blot results showed that the expression of p-JAK2 and p-STAT3 decreased after Ble and DDP were combined, indicating that the JAK2/STAT3 signal pathway was inhibited. Conclusions:The combination of Ble and DDP can inhibit the proliferation and migration of gastric cancer cells, promote the apoptosis of gastric cancer cells, and play a synergistic anti-cancer effect. Its mechanism may be related to the up-regulation of ROS levels in cells, thereby inhibiting the activation of the JAK2/STAT3 pathway.
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Objective To predict the potential mechanism of Yifei Sanjie prescription in the treatment of lung adenocarcinoma based on network pharmacology,and to verify one of the key signal pathways,Janus protein tyrosine kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3),by cell experiments in vitro.Methods To screen the main active components and potential action targets of Yifei Sanjie prescription,with traditional Chinese medicine system pharmacological database(TCMSP).To search and retrieve the main targets of lung adenocarcinoma,with human genetic database(GeneCards)and online human Mendelian genetic database(OMIM).To obtain the intersection targets by screening and apply Wayne diagram,then analysis the topology and establish the traditional Chinese medicine-active compound-target network diagram by using of Cytoscape 3.7.2 software.To construct the protein-protein interaction(PPI)network,with the protein-protein interaction platform(STRING)and Cytoscape3.7.2 software.To analyze the functional enrichment of gene ontology(GO)and Kyoto encyclopedia of genes and genomes(KEGG),with the Metascape database.To carry out the molecular docking verification by using of Vina1.2.3 software.Using CCK-8 method to detect the effect of Yifei Sanjie prescription on cell activity.Using the cell scratch test to observe the effect on cell migration.And using Western blot method to test the expression of p-STAT3,STAT3,p-JAK2,JAK2 and VEGF-A.Results 94 active components,329 related drug targets and 1358 lung adenocarcinoma targets were obtained from Yifei Sanjie prescription,among which,150 of them intersected.PPI network visualization analysis shows that the potential key targets of Yifei Sanjie prescription in the treatment of lung adenocarcinoma are protein kinase B1(AKT1),β-actin(ACTB),tumor suppressor gene p53(TP53),serum albumin(ALB),caspase-3(CASP3)and vascular endothelial growth factor A(VEGFA).KEGG enrichment analysis screened 138 related signal pathways,indicating that JAK/STAT signaling pathway may play a key role in the treatment of lung adenocarcinoma with Yifei Sanjie prescription.Molecular docking results showed that quercetin,luteolin,and ursolic acid had good binding activities with JAK2 and STAT3.The cell experiment showed that compared with the blank group,Yifei Sanjie prescription could significantly inhibit the activity of A549 cells,inhibit the migration of A549 cells,and decrease the expression of p-JAK2/JAK2,p-STAT3/STAT3 and VEGF-A protein.In addition,Colivelin,an activator of JAK2/STAT3 pathway,could reverse the effect of Yifei Sanjie prescription on the expression of A549 related proteins.Conclusion Yifei Sanjie prescription has the characteristics of multi-component,multi target and multi pathway in the treatment of lung adenocarcinoma,and its mechanism may be related to the down-regulation of p-JAK2,p-STAT3 and VEGF-A protein expression,thereby inhibiting cell proliferation and migration.
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Objective To observe the expression of tyrosine kinase 2(JAK2)and signal transducer and activator of transcription 3(STAT3)in myocardial tissue of rats with myocardial ischemia-reperfusion injury(MIRI)by electroacupuncture at"Neiguan"point,and explore the mechanism of electroacupuncture in up regulating JAK2/STAT3 signal pathway,promoting the expression of anti-inflammatory factors,and reducing MIRI injury.Methods Thirty male SD rats with normal echocardiography were randomly divided into sham operation group,model group and electroacupuncture group,with 10 rats in each group.MIRI models were prepared by ligation of left anterior descending coronary artery(LAD)in model group and electroacupuncture group,while in sham operation group,only threading was performed without ligation.In the electroacupuncture group,electroacupuncture(density wave,2 Hz/100 Hz,2 mA)at bilateral"Neiguan"points was performed on the 1st,3rd and 5th days after the modeling operation,once a day,20 minutes each time.The left ventricular ejection fraction(LVEF)of rats on the 1st,3rd and 5th day after operation was observed by echocardiography to evaluate cardiac function;HE staining was used to observe the pathological changes of rat myocardium;The myocardial fibrosis was observed by Masson staining;The expression of IL-4 and IL-6 in myocardium was detected by ELISA;The expressions of IL-1β and IL-10 in myocardial tissue were detected by immunohistochemistry;The expression of JAK2,p-JAK2,STAT3,p-STAT3 protein in myocardial infarction area was measured by Western blot.Results Compared with sham operation group,EF in model group decreased significantly(P<0.05),fibrosis area increased(P<0.05),IL-4,IL-6,IL-10,IL-1β The content of JAK2,p-JAK2,STAT3,p-STAT3 protein increased(P<0.05);Compared with the model group,the EF of rats in the electroacupuncture group increased(P<0.05),the fibrotic area of rats in the electroacupuncture group decreased(P<0.05),the content of IL-4 and IL-10 increased,and IL-6 and IL-1β The expression of JAK2,p-JAK2,STAT3,p-STAT3 protein increased(P<0.05).Conclusion Electroacupuncture at Neiguan point can significantly up regulate JAK2/STAT3 signal pathway,reduce the secretion of proinflammatory factors,increase the expression of anti-inflammatory factors,and alleviate myocardial ischemia reperfusion injury in rats.
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Objective:To observe any effect of electroacupuncture in the head motor area on motor impairment and JAK2/STAT3 signaling in rats modeling cerebral palsy.Methods:Thirty Sprague-Dawley rats were randomly divided into a sham operation group, a model group and an electroacupuncture group, each of 10. A model of cerebral palsy was induced in the model and electroacupuncture groups, while the sham operation group was not ligated in a sham operation. The motor functioning of all of the rats was evaluated 24h and 21d after the modeling using the Basso, Beattie Bresnahan locomotor rating scale (BBB scale). At 21d all of the rats were sacrificed and their brain tissues were collected. Hematoxylin-eosin staining was used to observe any pathological changes in the cerebral cortex, and fluorescence quantitative PCRs were employed to detect the mRNA expression of apoptotic gene B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2-associated x-protein (Bax), and cysteine-aspartate protein hydrolase-3 (Caspase-3). ELISA was used to detect the mRNA expression of tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interferon-γ(IFN-γ), and the oxidative stress indicators superoxide dismutase (SOD), total antioxidant capacity (T-AOC), and malondialdehyde (MDA). Western blotting was applied to detect the expression of JAK2, STAT3, p-JAK2 and p-STAT3.Results:The average BBB motor function score, Bcl-2 mRNA expression, SOD and T-AOC of the model group were all significantly lower than that of the sham-operated group at 21d. The average expression of Caspase-3 mRNA, TNF-α, IL-1β and IFN-γ, were significantly greater as were MDA content, p-JAK2, and p-STAT3 levels. The average BBB motor function score of the electroacupuncture group was significantly higher than the model group′s average, but caspase-3 mRNA expression, TNF-α, IL-1β, IFN-γ, MDA, p-JAK2 and p-STAT3 were all significantly lower.Conclusion:Electroacupuncture applied to the head motor area relieves motor function impairment in rats modeling cerebral palsy. That may be related to inhibition of the JAK2/STAT3 pathway′s activation resulting in less apoptosis, inflammation and oxidative stress.
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Objective To investigate the anti-inflammatory effect and mechanism of sappanone A(SA)on lipopolysaccharide(LPS)-induced RAW264.7 cell model based on JAK2-STAT3 signaling pathway.Methods MTT assay was used to detect the effects of sappanone A,LPS and AG490 on RAW264.7 cell viability.The LPS-induced inflammatory model in RAW264.7 cells was established,and the secretion level of interleukin-6(IL-6)in the supernatant was detected by ELISA.mRNA expressions of IL-6,Janus kinase 2(JAK2)and signal transducer and activator of transcription 3(STAT3)were tested by RT-PCR.The protein expressions of JAK2,phosphorylated JAK2(p-JAK2),STAT3 and phosphorylated STAT3(p-STAT3)were determined by Western Blot.Results Compared with control group,IL-6 secretion level was significantly increased,mRNA expressions of IL-6,JAK2 and STAT3 were up-regulated,and protein expressions of p-JAK2 and p-STAT3 were increased(all P＜0.01)in model group.Compared with model group,high-dosed sappanone A(5 μg·mL-1)was significantly decreased the secretion of IL-6,down-regulated the mRNA expressions of IL-6,JAK2 and STAT3,and inhibited the protein expressions of p-JAK2 and p-STAT3(all P＜0.01).Conclusion Sappanone A may play an anti-inflammatory role by inhibiting the JAK2-STAT3 signaling pathway and hence inhibiting the secretion of IL-6.
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Objective @#To investigate the imunomodulatory effects of umbilical cord mesenchymal stem cells(UC⁃MSCs) modified by miR⁃125b⁃5p through JAK2/STAT3 pathway on systemic lupus erythematosus(SLE) . @*Methods@#UC⁃MSCs were isolated and cultured under aseptic conditions ; Peripheral blood mononuclear cells ( PBMCs)were separated from SLE patients by density gradient centrifugation. The relative expressions of IL⁃17A , Foxp3 ,IFN⁃γ , IL⁃4 genes in PBMCs cultured with UC⁃MSCs for 48 h were detected by RT⁃qPCR; The relative expressions of JAK2 , p ⁃JAK2 , STAT3 , p ⁃STAT3 and IL⁃18 proteins in kidney tissues of MRL/lpr mice were detected by west⁃ern blot. @*Results @#Compared with the PBMCs culture group , the expression of IFN⁃γ and IL⁃17A and the propor⁃tion of Th17/Treg cells were significantly down⁃regulated in UC⁃MSCs + miR⁃125b⁃5p group , UC⁃MSCs + miR⁃NC group and UC⁃MSCs group(P < 0. 01) , the expression of the proportion of Th1/Th2 cells (P < 0. 05) was down⁃regulated in UC⁃MSCs + miR⁃125b⁃5p group and UC⁃MSCs + miR⁃NC group ; Compared with the untreated group of MRL/lpr mice , the relative expressions of JAK2 , p ⁃JAK2 , STAT3 , p ⁃STAT3 and IL⁃18 proteins in kidney tissues of MRL/lpr mice were significantly down⁃regulated in UC⁃MSCs + miR⁃125b⁃5p group (P < 0. 01) , and the rela⁃tive expressions of IL⁃18 proteins was significantly down⁃regulated in UC⁃MSCs + miR⁃NC group and UC⁃MSCs group (P < 0. 01) .@*Conclusion@#miR⁃125b⁃5p plays a synergistic role in UC⁃MSCs ,which regulates the differenti⁃ation of Th2 cells in PBMCs of SLE patients and the relative expressions of p ⁃JAK2/JAK2 , p ⁃STAT3/STAT3 , and IL⁃18 proteins in kidney tissues of MRL/lpr mice. UC⁃MSCs modified by miR⁃125b⁃5p may play immunomodulato⁃ry effect on SLE by JAK2/STAT3 signaling pathway.
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Objective To investigate the influence of limonin on the malignant biological behavior of non-small cell lung cancer (NSCLC) cells by regulating the protein tyrosine kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Methods CCK-8 method was applied to detect the survival rate of A549 cells treated with different concentrations of limonin (0, 5, 10, 25, 50, 75, 100 μmol/L). A549 cells were separated into normal culture (NC) group, low-dose limonin group (treatment with 10 μmol/L limonin for 24 h), medium-dose limonin group (treatment with 25 μmol/L limonin for 24 h), high-dose limonin group (treatment with 50 μmol/L limonin for 24 h), coumermycin A1 group (treatment with 10 μmol/L JAK2 activator coumermycin A1+50 μmol/L limonin for 24 h), and AG490 group (treatment with 10 μmol/L JAK2 inhibitor AG490+50 μmol/L limonin for 24 h). Clone formation assay was applied to detect the clones of each group of cells. Transwell assay was applied to detect cell migration and invasion, and flow cytometry was applied to detect apoptosis. Western blot analysis was applied to detect the protein expression levels of JAK2, p-JAK2, STAT3, p-STAT3, E-cadherin, N-cadherin, and vimentin in each group. Results The viability of A549 cells decreased significantly in a limonin concentration-dependent manner (P < 0.05), with IC50 of 45.16±1.66 μmol/L. Concentrations of 10, 25, and 50 μmol/L were selected for subsequent experiments. The numbers of clones, migration, and invasion of A549 cells and the protein expression levels of IL-6, p-JAK2, p-STAT3, N-cadherin, and vimentin in the low-, medium-, and high-dose limonin groups significantly decreased, compared with those in the NC group, and the apoptosis rate and E-cadherin protein expression significantly increased (P < 0.05). The JAK2 activator coumermycin A1 attenuated the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and attenuated the apoptosis ability. The JAK2 inhibitor AG490 enhanced the ability of limonin to inhibit the proliferation, migration, invasion, and other malignant biological behavior of A549 cells and enhanced the apoptosis ability. Conclusion Limonin can inhibit the malignant biological behavior of NSCLC cells, such as proliferation, migration, and invasion, by inhibiting the JAK2/STAT3 pathway.
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【Objective】 To investigate the regulatory effect of JAK2/STAT3 signaling pathway inhibitor AG490 on the functional phenotype of M2-like macrophages and its effects on proliferation, apoptosis, migration, and invasion of gastric cancer cells. 【Methods】 Human mononuclear cell line THP-1 was induced to differentiate into M0 macrophages by PMA in vitro. The M1-like phenotype was induced by LPS and IFN-γ, and M2-like phenotype was induced by IL-4 and IL-13, respectively, and identified by immunofluorescence labeling CD68, CD86 and CD206. The mRNA expressions of CD163, Arg1, CCL22, PPARγ, IL-10, IL-20 and TNF-α were determined by RT-qPCR. The expressions of key proteins in JAK2/STAT3 signaling pathway were detected by Western blotting. M2-like macrophages were treated with JAK2/STAT3 inhibitor (AG490) to observe the expression level of marker genes for M2 like phenotype. Macrophages were co-cultured with gastric cancer cells, and the effects of the macrophages on proliferation, migration, invasion, and apoptosis of gastric cancer cells were detected by CCK-8 method, healing assay, transwell intracellular Matrigel invasion assay, and flow cytometry. The xenograft tumor model of MKN45 gastric cancer in nude mice was prepared, and the tumor size and quality were observed for 20 days after the model was established. 【Results】 THP-1 cells were induced into M1-like macrophages and M2-like macrophages. M1-like marker (CD86) and M2-like marker (CD206) were identified by flow cytometry. The P38MAPK, JAK2, p-STAT3/STAT3 protein levels of M2-like macrophages treated with AG490 were significantly reduced. The mRNA expression levels of Arg1, CCL22, PPARγ and IL-10 were significantly reduced in the group of M2-like macrophages treated with AG490. Co-culture of M2-like macrophages with gastric cancer cells could promote gastric cancer cell viability, increase migration and invasion ability, and inhibit apoptosis. When the group of M2-like macrophages treated with AG490 was co-cultured with gastric cancer cells, the proliferation activity of MKN-45 cells and MGC823 was significantly lower than that in M2 group (1.047±0.062 vs. 1.426±0.076, 1.149±0.006 vs. 1.301±0.015). Compared to M2 group, the migration (100.0%±5.73% vs. 72%±3.85%) and invasion ability (100.0%±7.40% vs. 60%±6.54%) of MGC823 gastric cancer cells in AG490 treatment group were significantly reduced. The apoptosis rate of MGC823 cells in the AG490 treated group was significantly higher than that in M2 group (27.51%±0.70% vs. 20.82%±0.92%). In the nude mouse xenograft tumor model, the volume and weight of the transplanted tumor collected at day 20 were significantly lower in AG490 treated group than in M2 group (736.04±182.34 vs. 1 080.5±250.57)mm3, (0.64±0.11 vs. 0.87±0.17)g. 【Conclusion】 AG490 downregulates the activation level of JAK2/STAT3 signaling pathway in M2-like macrophages, inhibits M2-type polarization, partially reverses the cancer-promoting function of M2-like macrophages, inhibits proliferation, migration and invasion of gastric cancer cells, and induces apoptosis. The JAK2/STAT3 signaling pathway can be further studied as a potential therapeutic target for gastric cancer.
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Aim To investigate the mechanism of action of paeonol based on JAK2/STAT3 signaling pathway to ameliorate liver inflammation and oxidative stress injury induced by acute alcohol stimulation in mice. Methods C57BL/6 mice were randomly divided into normal group, model group, silibinin group (36. 8 mg • kg