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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 investigate the effect of STIM1 on the survival and proliferation of breast cancer cells and its preliminary mechanism analysis.Methods:Normal mammary epithelial cells MCF-10A as control,the expression of STIM1 in MCF7, HCC1569,MDA-MB-231 and BT549 breast cancer cells were detected by Western blot;STIM1 siRNA sequence(STIM1-siRNA group) were transfected into MDA-MB-231 cells,and the negative control group and the blank control group were set up,the protein expression of STIM1 in each group were detected after cells were transfected for 48 h;MTT method was used to detect cell activity cells were transfected for 24 h,48 h and 72 h;cell apoptosis was detected after cells were transfected for 48 h by flow cytometry;the mRNA expression of IL-6 and TNF-α were detected by RT-PCR;the expression of PCNA,Bcl-2,Bax,Caspase3,STAT3 and p-STAT3 protein were detected by Western blot.Results:The expression of STIM1 protein in breast cancer cells was significantly higher than that in MCF-10A cells (P<0.05);the expression of STIM1 protein in MDA-MB-231 cells transfected STIM1-siRNA was significantly lower than the control group(P<0.05);compared with the control group,cell viability in STIM1-siRNA group decreased significantly in cells were transfected for 48 h and 72 h,the apoptosis rate in 48 h was significantly increased,the expression of IL-6 and TNF-α mRNA sig-nificantly decreased,the expression of PCNA,Bcl-2 and p-STAT3 protein were significantly decreased,the expression of Bax and Caspase3 protein increased significantly.Conclusion:STIM1 gene is highly expressed in breast cancer cells.Inhibition of STIM1 expression by RNA interference can down regulate the activity of cancer cells,induce apoptosis,enhance immunity and by down regulation STAT3 signal.
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Objective:To explore the effect and related mechanism on inflammatory response,proliferation and apoptosis of oxLDL-induced vascular smooth muscle cell of matrine.Methods:Theatherosclerotic model was conducted through treating human aort-icvascular smooth muscle cell with oxidized low density lipoprotein(oxLDL).Cell viability and proliferation was detected by CCK-8 as-say.The mRNA level of interleukin 1 beta(IL-1β),tumor necrosis factor alpha(TNF-α),IL-10 and IL-13 was tested by quantitative real-time reverse transcription PCR(qRT-PCR).Cell apoptosis was measured by flow cytometry.The expression of proliferation marker proteins antigen identified by monoclonal antibody(Ki-67) and proliferating cell nuclear antigen(PCNA),apoptosis marker proteins B cell lymphoma 2(Bcl-2) and Bcl-2-associated X protein(Bax),signal transducer and activator of transcription 3(STAT3) and signal transducer and activator of transcription 5(STAT5) was detected by Western blot.Results: Compared with control group,the mRNA level of IL-1β and TNF-α in model group was largely increased with decreased mRNA level of IL-10 and IL-13(P<0.01).Compared with model group,the mRNA level of IL-1β and TNF-α in treatment group was attenuated with enhancive mRNA level of IL-10 and IL-13(P<0.05).Cell proliferation and apoptosis in model group was higher than control group.Cell proliferation and apoptosis in treatment group was lower than model group(P<0.05).Compared with control group,the expression of Ki-67,PCNA and Bax in model group was augmented with decreased expression of Bcl-2(P<0.01).Compared with model group,the expression of Ki-67,PCNA and Bax in treatment group declined with elevated expression of Bcl-2(P<0.05).The expression of p-STAT3 and p-STAT5 in model group was higher than control group(P<0.01).The expression of p-STAT3 and p-STAT5 in treatment group was lower than model group(P<0.05).Compared with model group,the expression of Ki-67,PCNA and Bax in treatment group and model+Ruxolitinib group was decreased with enhancive expression of Bcl-2(P<0.05).The expression of Ki-67,PCNA and Bax in model+matrine+Ruxolitinib group was observably lower than model group and the expression of Bcl-2 in model+matrine+Ruxolitinib group was observably higher than model group(P<0.01).Compared with model group,the mRNA level of IL-1β and TNF-α in treatment group,model+Ruxolitinib group and model+matrine+Ruxolitinib group was attenuated with enhancive mRNA level of IL-10 and IL-13(P<0.05).Conclusion:Matrine represses inflammation,proliferation and apoptosis of vascular smooth muscle cell by inhibiting activation of JAK/STAT3 signal pathway in atherosclerosis.
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Objective To study the molecular mechanism of IL-17A induced the secretion of CXCL 12 in non-small cell lung cancer cell line A549.Methods Cultured non-small cell lung cancer cell line A549 in vitro with recombination cytokine IL-17A or STAT3 signal path-way inhibitor pre-incubated for 1 hour, and then the level of CXCL12 were detected by enzyme-linked immunosorbent assay .And chemotaxis assay was used to analyze the chemotactic movement of neutrophil .Results After IL-17A stimulation,the secretion of CXCL12 by non-small cell lung cancer cell line A549 was significantly increased(P<0.01),which is in a dose and time dependent manner .However,IL-17A in-duced the secretion of CXCL12 by A549 was significantly decreased after pre-incubated by the STAT3 inhibitor(P<0.01).In addition,neu-trophil could have chemotaxis by cell suspension obtained from IL-17A stimulated A549 cell line,but such chemotaxis would be declined while CXCL12 was neutralized.Conclusion IL-17A could induce the secretion of CXCL12 in non-small cell lung cancer cell line A549 through STAT3 signal pathway , so as to promote the chemotaxis of neutrophil .
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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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OBJECTIVE: To investigate the nephroprotective effect of danzhi jiangtang capsule(DJC) on diabetic rats induced by streptozotocin(STZ) and elucidate its mechanism. METHODS: Model of diabetic rats were established by combination of high-fat diet-fed and low-dose STZ injection in SD rats. The successful models were chosen and randomly divided into model group, low-dose and high-dose of DJC(600, 2 000 mg · kg-1, ig) groups, each consisting of 8 rats. Control and model groups were administered equal volume of distilled water. After 8 weeks of treatment, the rats were killed and serum was separated to detect blood lipid, renal function and oxidative stress. The kidneys were weighed, and the renal indexes were calculated. Renal tissue specimens were fixed for HE and PAS staining; the expressions of JAK2, STAT3, p-JAK2, p-STAT3 in the kidneys were observed by Western blot; the renal inflammatory factors TNF-α, IL-6 and MCP-1 were detected by ELISA. RESULTS: Fasting blood glucose (FBG), blood lipid, renal index, oxidative stress of model group were significantly increased, renal function was reduced, and the morphology of renal tissue changed significantly. The renal inflammatory cytokine TNF-α, IL-6 and MCP-1 and the expressions of p-JAK2, p-STAT3 increased significantly in diabetes mellitus rats. After 8 weeks of treatment, blood lipid and oxidative stress in DJC groups were decreased, but there was no obvious difference in FBG between DJC groups and model group, the renal function and the pathological changes of renal tissue were improved obviously. The expressions of inflammatory cytokine(TNF-α, IL-6 and MCP-1), p-JAK2 and p-STAT3 in renal cortex were significantly down-regulated. CONCLUSION: The nephroprotective effect of DJC is based on its antioxidant effect and anti-inflammatory effect via suppression of JAK2/STAT3 signal transduction.
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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.
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Aim To study the effect of Semen Sojae Preparatum isoflavone(SSPI)on proliferation and JAK2/STAT3 signal transduction pathway in rat vascular smooth muscle cell(VSMC)induced by Angiotensin Ⅱ(AngⅡ).Methods A cell proliferating model of VSMC induced by AngⅡ was established.The proliferation activity of VSMC was analyzed by MTT method.The expressions of angiotensin Ⅱ receptor 1(AT1R) were detected by RT-PCR method.The expressions of JAK2,STAT3 and phosphorylation protein were detected by Western blot.Results 100 ?g?L-1, 200 ?g?L-1 SSPI significantly inhibited the proliferation of VSMC induced by AngⅡ,and down-regulated the mRNA expression of AT1R.200 ?g?L-1 SSPI could significantly down-regulate the protein expressions of p-JAK2,p-STAT3.Conclusions The proliferation of VSMC induced by AngⅡcan be inhibited by SSPI.The mechanisms might be related to down-regulating the expressions of AT1R,and arresting the phosphorylation of JAK2/STAT3 signal transduction pathway.