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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 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 study t he effects of bro mophenylcurcumin（GL63）on the apoptosis ，migration and invasion of human cholangiocarcinoma RBE cells ，and to investigate its mechanism based on JAK/STAT signaling pathway. METHODS ：MTT assay was used to detect the effects of different concentrations of GL 63 [0（blank control ，similarly hereinafter ），1.25，2.5，5，10， 20，40 μ mol/L] on the proliferation of RBE cells after 48 h treatment ；the IC 50 was calculated. The effects of different concentrations of GL 63（0，5，10，20 μmol/L）on colony formation were detected by crystal violet staining after 48 h treatment. Flow cytometry ，Hoechst 33342 staining，cell scratch test and Transwell chamber invasion test were used to detect the effects of different concentrations of GL 63（0，5，10，20 μmol/L）on cell cycle distribution ，apoptosis，migration and invasion ability after 24 h treatment. Western blotting assay was adopted to detect the effects of different concentration of GL 63（0，5，10，20 μmol/L） on the expression of JAK 2/STAT3 signal pathway associated proteins. RESULTS ：The proliferation inhibition rates of RBE cells in different concentrations of GL 63 groups（1.25-40 μmol/L）were significantly increase d，compared with blank control group （P＜ 0.01），and showed a dose-dependent trend ，with IC 50 of （8.46±1.30）μmol/L. Compared with blank control group， 85917439。E-mail：zhaoji-an-88@163.com inhibition rates of RBE cell colony formation were significantly decreased in different concentrations （5，10，20 μmol/L）of GL 63 groups（P＜0.01）. The percentage of RBE cells at G 0/G1 phase increased significantly ，while that at S phase decreased significantly （P＜0.01）. The apoptotic rate increased significantly（P＜0.01），and the nucleus showed dense pyknosis and apoptotic bodies. The rate of cell migration and healing was significantly decreased （P＜0.01），and the number of invasive cells through basement membrane was significantly decreased （P＜ 0.01）. The protein expression of p-JAK 2， p-STAT3， Bcl-2， MMP-2， MMP-9， Pro-caspase-9 and P ro-caspase-3 were down-regulated significantly while the expression of Bax ，Cyt-c，Cleaved-caspase-9 and Cleaved-caspase- 3 were up-regulated significantly（P＜0.01）. CONCLUSIONS ：GL63 may inhibit the proliferation ，migration and invasion of RBE cells and promote its apoptosis by inhibiting JAK 2/STAT3 signal pathway.
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Objective To explore the effect of luteolin on the proliferation of osteosarcoma stem cells.Methods CD133 +osteosarcoma stem cells were separated from MG63 cells by flow cytometer.MTT was used to investigate the effects of luteolin(0,0.01,0.02,0.04 mg/mL)on the proliferation of osteosarcoma stem cells.Western blot was used to detect the levels of Ki67 protein and components of JAK2/STAT3 signal pathway in osteosarcoma stem cells induced.Results After sorting,the content of the CD133 +fraction was enriched up to(87.60 ±5.06)%.MTT assay showed that,compared with the control group,luteolin(0.01,0.02,0.04 mg/mL)inhibited proliferation of CD133 + osteosarcoma stem cells(P <0.05).Western blot also showed that luteolin significantly decreased the level of Ki67 compared with the control group(P<0.05).In addition,the luteolin inhibited the expression of p-JAK2 and p-STAT3 in JAK2/STAT3 signal pathway of CD133 + osteosarcoma stem cells compared with the control group ( P <0.05 ) . Conclusion Luteolin might be a suppressor of osteosarcoma stem cells.
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Aim To investigate the inhibitory effect of Evodiamine on JAK2/STAT3 signal pathway in human colorectal cancer cell line HCT-116 . Methods Cells were cultured with 6. 0 μmol·L-1 Evodiamine for 2, 4 and 6 h, respectively. Cell nuclear morphology was detected by Hoechst staining and protein expression levels of JAK2 , p-JAK2 , STAT3 and p-STAT3 were examined by Western blot. Cells were treated with dif-ferent concentrations of AG490 for 48 h to select proper working concentration and cells treated with 6 μmol · L-1 EVO and 50 μmol · L-1 AG490 to compare the modulatory effect of EVO with AG490 on JAK2/STAT3 signal pathway. Results Hoechst staining revealed that Evodiamine could induce cells apoptosis, chroma-tin condensation gathered and typical apoptotic mor-phological changes in a time-dependent manner;West-ern Blot suggested that EVO could inhibit p-STAT3 significantly. After treatment with AG490, JAK2/STAT3 signal pathway was inactivated, the inhibitory effect of EVO on p-STAT3 was stronger than that of AG490 , while EVO combined with AG490 could fur-ther inhibit the expression of p-STAT3 significantly. Conclusions The anticancer effect of Evodiamine is mainly mediated by the modulation of JAK2/STAT3 signal pathway in HCT-116 cells.
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AIM: To investigate the correlation of hepatitis B virus X protein (HBx) with renal tubular epithelialcell apoptosis in hepatitis B virus-associated glomerulonephritis (HBVGN) and the possible signaling mechanism. METHODS: The activation of JAK2/STAT3 signal pathway and the expression of apoptosis -related proteins in humankindey proximal tubular epithelial cells (HK-2 cells) were determined by Western blotting after transfection with HBx eukaryoticexpression vector.The cell proliferation was observed by CCK-8 assay.The cell apoptosis was analyzed by the imagingof HO33342 staining, transmission electron microscopy and flow cytometry with Annexin V /PI double staining.RESULTS:After transfection of the target gene HBx, the expression levels of both p-JAK2 and p-STAT3 were significantly increased.At the same time, the cell proliferation was obviously inhibited, and the apoptotic rate was increased.After incubationwith AG490, the JAK2/STAT3 signal pathway was partially blocked, and the cell apoptosis induced by HBx was reduced. CONCLUSION: HBx up-regulates the activation of JAK2/STAT3 signal pathway to induce renal tubular epithelialcell apoptosis, which is possibly involved in the pathogenic mechanism that HBV directly damages nephridial tissue .
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Objective: To study the effects of serum containing Dahuang Fuzi (Rhubarb and Aconite) Decoction (DFD) on JAK2/STAT3 signal pathway in mice with severe acute pancreatitis (SAP). Methods: SAP model in mice was constructed, and then the peritoneal macrophages were vaccinated into the culture plate to set model group, serum containing DFD groups (2.5%, 5%, and 10%), AG490 (10 μmol/L ) positive group, and peritoneal macrophages of normal mice acted as normal control group. After an incubation of 2 h, cells were added with serum containing DFD at different concentration or AG490 0.5 mL, 24 h later, the concentration of TNF-α and IL-6 in supernatant were determined by quantitative sandwich enzyme-linked immunosorbent assay (ELISA) kits, mRNA and protein expression levels of JAK2 and STAT3 in cells were evaluated by Western blotting. Results: DFD could significantly decrease the levels of cytokines TNF-α and IL-6 in the supernatants, inhibit the mRNA and protein expression levels of JAK2 and STAT3 in peritoneal macrophages of SAP mice. Conclusion: DFD could inhibit the JAK2/STAT3 signal pathway and inflammatory responses in peritoneal macrophages of SAP mice.