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ObjectiveTo investigate the effect and mechanism of Scutellaria Baicalensis polysaccharides on intestinal immunity in mice with ulcerative colitis （UC）. MethodsC57BL/6 mice were randomly assigned to blank group， model group， mesalazine group and Scutellaria Baicalensis polysaccharides low-dose， medium-dose and high-dose groups （n=10 mice/group）. The daily status of mice was observed and the disease activity index （DAI） score was recorded. The expression of cytokines including IL-6， IL-17 and IL-23 in serum was detected by ELISA. After the mice were sacrificed， HE was used to observe the intestinal mucosal damage in mice， and the colonic tissue damage index （TDI） score was recorded. Western blot and immunohistochemistry were used to detect the expression levels of Janus kinase 2 （JAK2）， signal transducer and activator of transcription （STAT3）， p-JAK2 and p-STAT3 proteins. ResultsCompared with the blank group， the DAI score of the model group was significantly increased， the contents of IL-6， IL-17 and IL-23 in serum were significantly increased， the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 in colon tissue was significantly increased， and the expression levels of p-JAK2 and p-STAT3 were significantly increased. Compared with the model group， the DAI score of mice in each administration group was significantly decreased. The contents of IL-6， IL-17 and IL-23 in serum were decreased， the TDI score was decreased， the ratio of p-JAK2/JAK2 and p-STAT3/STAT3 in colon tissue was decreased， and the expression of p-JAK2 and p-STAT3 was decreased. ConclusionScutellaria Baicalensis polysaccharides may improve the inflammatory effect of UC mice through JAK2/STAT3 pathway and IL-23/IL-17 inflammatory axis.
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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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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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Animals , Humans , Chlorocebus aethiops , Interleukin-6/genetics , Janus Kinase 2/pharmacology , Infectious bronchitis virus/metabolism , STAT3 Transcription Factor/metabolism , Angiotensin-Converting Enzyme 2/pharmacology , Cytokine Receptor gp130/metabolism , Vero Cells , Signal Transduction , Inflammation , RNA, MessengerABSTRACT
Objective:To explore the alteration of JAK2/STAT3 pathway after carbon ion ( 12C 6+) irradiation and the difference in the infiltration of CD8 + T cells in lung cancer regulated by downstream protein FOXP3. Methods:Significantly altered JAK2/STAT3 pathway and related differentially-expressed genes and proteins such as FOXP3 in lung cancer after carbon ion irradiation were screened based on RNA sequencing analysis in the Lewis tumor model of C57BL/6 mice. The correlation between FOXP3 and major immune cell infiltration in the immune microenvironment of lung cancer was analyzed using the ssGSEA immune infiltration algorithm in the R software "GSVA" and CD8 + T cell infiltration in the immune microenvironment of lung cancer was evaluated based on the carbon ion combined with STAT3 inhibition pathway (niclosamide). Results:The JAK2/STAT3 pathway was inhibited and the expression of related genes and proteins was downregulated in lung cancer after carbon ion irradiation. Immune scoring based on the ssGSEA algorithm showed that FOXP3 expression was significantly negatively correlated with CD8 + T cell infiltration in the immune microenvironment of lung cancer. The role of targeting the JAK2/STAT3 pathway in increasing CD8 + T cell infiltration in lung cancer was further clarified by carbon ion irradiation combined with STAT3 inhibition (niclosamide). Conclusion:Carbon ion irradiation ( 12C 6+) can play a synergistic role with immunotherapy by targeting the JAK2/STAT3 pathway.
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Aim To explore the mechanism of Huoxue Rongluo reeipe in promoting angiogenesis after ischemic stroke based on the correlation between mir-370-3p and JAK2/STAT3 pathway.Methods Hats were randomly divided into six groups.MCAO/R method was used to establish the model.After seven days of intragastrie administration,the expressions of CD31 ,vWF and vascular endothelial growth factor ( VEGF) in brain tissue were observed by immunofluorescence stai- ning; the expression of JAK2 ,p-jak2,STAT3 and p- STAT3 in brain tissue was detected by Western blot; J the expressions of JAK2,STAT3 mRNA and mir-370- 3p in brain tissue were detected by real-time PCR f RT-PCR) ; the correlation between mir-370-3p and JAK2/STAT3 pathway was analyzed by Pearson correlation ; the expressions of lncma-hl9 and mir-370-3p were detected by real-time quantitative PCR ( RT qPCR) ; the targeting relationship between lncrna-hl9 and mir-370-3p was detected by luciferase reporter assay.Results Huoxue Rongluo decoction could increase the microvessel density and average fluorescence intensity of VEGF,up-regulate JAK2 and STAT3 mR- NA,down-regulate the expression of mir-37()-3p,and promote the expressions of JAK2,p-jak2 ,STAT3 and p- STAT3.Mir-370-3p was highly negatively correlated with JAK2 and STAT3 mRNA respectively,which could be reversed by stattic,an inhibitor of STAT3 SH2 domain.Conclusions Huoxue Rongluo recipe may stimulate angiogenesis after ischemic stroke by down- regulating the expression of mir-370-3p,activating JAK2 / STAT3 pathway and promoting the expression of downstream VEGF,so as to improve the symptoms of neurolo.
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Objective To explore the regulatory effeet of Qifu Yin ( QFY) on JAK2/STAT3 pathway in rats with type 2 diabetic cognitive impairment.Methods A small dose of STZ combined with high-fat and high- sugar feed was used to build the model.After success, they were divided into model group, QFY low-dose, high-dose group, and metformin group.After four weeks of intervention, fasting blood glucose ( FBG ) was measured; Morris water maze was used to detect spatial learning and memory ability in rats; Nissl staining and immunofluorescence staining were respectively used to detect the degree of brain injury and the expression of lba-1 , a marker of microglia .EL1SA was used to detect the expression of TNF-cx, IL6, ILK) and BDNF.Western blot was employed to detect the expression of JAK2/STAT3 pathway.Results Compared with the control group, the model group showed significant increase in blood glucose, decreased spatial learning and memory capacity, severely damaged hipp-ocampal neurons, increased activated microglia, significantly higher levels of TNF-cx, 1L6, p-JAK2/JAK2 and p-STAT3/STAT3, and signif icantly lower levels of ILK) and BDNF.Compared with the model group, QFY group effectively reduced FBG, inhibited the con-tinuous rise of FBG, improved learning and memory a- bility, improved hippocampal neuronal damage, reduced activated microglia, reduced TNF-cx, 1L6, p- JAK2/JAK2 and p-STAT3/STAT3 levels, and increased ILK) and BDNF levels.Conclusion QFY has been shown to improve type 2 diabetic cognitive impairment , and the mechanism may be related to the inhibition of blood glucose rise and regulation of the JAK2/STAT3 pathway, thereby inhibiting microglia activation.
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Objective To explore the effect and mechanism of ginsenoside Re on the intestinal mucosa of severe acute pancreatitis (SAP) mice by regulating the Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/ STAT3) pathway. Methods Totally 48 mice were divided into : control group, SAP group, SAP + ginsenoside Re group and SAP + ginsenoside Re + LY2784544 group (n = 12). The mice were intraperitoneally injected with caerulein solution (after fasting for 12 hours, 100 μg/kg, 6 times, injection interval 60 minutes) to establish SAP models. Mice in the SAP+ ginsenoside Re group were intraperitoneally injected with ginsenoside Re (4 mg/kg, 1 time a day for 7 consecutive days). Intraperitoneal injection of 12.7 mg/kg LY2784544 was used to inhibit the JAK2/STAT3 pathway. Pancreatic and intestinal mucosal injury were detected in each group. The wet to dry weight ratio of pancreas, serum amylase and inflammatory factor levels were detected in each group. The intestinal mucosal barrier function was analyzed by detecting the levels of D-lactic acid and fluorescein isothiocyanate dextran (FITC-D). The damage of pancreatic tissue and intestinal mucosa tissue was observed by HE staining. Western blotting was used to detect the levels of apoptotic proteins Bax and Bcl-2 in the intestinal mucosa. The JAK2/STAT3 pathway expression levels in pancreatic tissue and intestinal mucosa tissue were detected by Realtime PCR and Western blotting. Results The pancreatic index, serum amylase, interieukin 6 (IL-6) and tumor necrosis factor a (TNF-a), FITC-D, D-lactic acid, Bax protein levels, JAK2 and STAT3 mRNA and protein levels in the SAP group were significantly higher than those in the control group, while Bcl-2 protein was significantly lower than that in the control group (P<0.05). The Bcl-2 protein of SAP+ginsenoside Re group was significantly higher than that of SAP group, and other indexes were significantly lower than those in SAP group (P<0.05). The Bcl-2 protein of SAP+ginsenoside Re+ LY2784544 group was significantly higher than that of SAP + ginsenoside Re group, and other indexes were significantly lower than those in the SAP+ginsenoside Re group (P<0.05). Conclusion Ginsenoside Re may reduce the pancreatic injury in SAP model mice by inhibiting the JAK/STAT pathway to alleviate the inflammatory response, and may protect the small intestinal mucosal barrier by alleviating pancreatitis and inhibiting the intestinal mucosal JAK/STAT pathway to inhibit cell apoptosis.
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Dysregulated crosstalk between different signaling pathways contributes to tumor development, including resistance to cancer therapy. In the present study, we found that the mitogen-activated extracellular signal-regulated kinase (MEK) inhibitor trametinib failed to suppress the proliferation of PANC-1 and MGC803 cells by activating the Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, while the JAK2 inhibitor fedratinib failed to inhibit the growth of the PANC-1 cells upon stimulation of extracellular signal-regulated kinase (ERK) signaling. In particular, the most prominent enhancement of the anti-proliferative effect resulted from the concurrent blockage of the JAK2/STAT3 and ERK signaling pathways. Furthermore, the combination of the two inhibitors resulted in a reduced tumor burden in mice. Our evidence suggests novel crosstalk between JAK2/STAT3 and ERK signaling in gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC) cells and provides a therapeutic strategy to overcome potential resistance in gastrointestinal cancer.
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@#[Abstract] Objective: To investigate the effects of salidroside on the proliferation, invasion and apoptosis of cervical squamous cell carcinoma C33A cells and explore its possible mechanism. Methods: C33A cells were divided into 4 groups: control group, low-dose group (salidroside 50 μg/mL), high-dose group (salidroside 150 μg/mL), and AG490 group (inhibitor of JAK2/STAT3 signaling pathway, 50 μmol/L). Effects of salidroside and AG490 on the proliferation, invasion and apoptosis of C33A cells were detected by MTT method, EdU labeling experiment, Transwell assay, Rh123 staining and Flow cytometry, respectively. Western blotting was used to detect the effects of salidroside and AG490 on the expressions of JAK2/STAT3 pathway-related proteins (p-JAK2, p-STAT3) and apoptosis-related proteins (Bax, Bcl-2, caspase-3) in C33A cells. Result: Compared with the control group, the proliferation and DNA synthesis as well as the invasion of C33Acells in the low-dose group were significantly inhibited (all P<0.05), while the apoptosis was significantly enhanced (P<0.05); in the meanwhile, the fluorescence intensity of Rh123 was significantly reduced (all P<0.05) and the membrane structure of C33A cells were destroyed; moreover, the expressions of p-JAK2, p-STAT3 and Bcl-2 were significantly decreased while the expressions of Bax and caspase-3 were significantly increased (all P<0.05). Compared with the low-dose group, the effects of high-dose salidroside and AG490 on the proliferation, invasion, apoptosis and related protein expressions in C33A cells were more significant (all P<0.05), but there was no difference between the high-dose group and the AG490 group. Conclusion: Salidroside can inhibit the proliferation and invasion of C33A cells and promote cell apoptosis. Its mechanism may be related to inhibition of JAK2/ STAT3 signaling pathway.
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Objective:To investigate the effect of Huangqi Jianzhongtang on Janusprotein tyrosine kinase 2/signal transducers and transcriptional activator protein 3 (JAK2/STAT3) signal pathway in rats with spleen-stomach deficiency cold type gastric ulcer (GU). Method:A total of 60 SPF level Wistar rats were randomly divided into two groups: blank group and model group. Model rats were used to reconstruct the spleen-stomach deficiency cold type GU model by comprehensive modeling method. Model rats were divided into model group, Anweiyang group and high, medium and low-dose Huangqi Jianzhongtang groups according to the random number table, with 10 rats in each group. Rats in blank group and model group were given 10 mL·kg-1·d-1 distilled water, and 16, 8, 4 g·kg-1·d-1 Huangqi Jianzhongtang, respectively. Rats in the positive control group were given 0.14 g·kg-1·d-1 Anweiyang for 21 days. The gene expressions of JAK2 and STAT3 in the ulcer tissue were detected by Real-time fluorescent quantitative polymerase chain reaction (Real-time PCR), the protein expressions and phosphorylation levels of JAK2 and STAT3 in the ulcer tissue were detected by Western blot, and the contents of interleukin-10（IL-10）and interleukin-17（IL-17）in the gastric tissue of each group were detected by enzyme-linked immunosorbent assay (ELISA). Result:Compared with the blank group, the general survival condition of the model group was worse, the content of IL-10 in gastric homogenate was significantly reduced, while the content of IL-17 was significantly increased (P<0.05), the protein expressions of JAK2 and STAT3 in gastric tissue was not significantly increased, whereas the gene expressions and phosphorylation levels of JAK2 and STAT3 were significantly increased (P<0.05). Compared with the model group, the content of IL-10 increased, but the content of IL-17 decreased, the gene expressions of JAK2 and STAT3 and the level of protein phosphorylation decreased in the treatment group, especially in the high-dose Huangqi Jianzhongtang group, with statistically significant differences (P<0.05). Conclusion:Huangqi Jianzhongtang can improve the survival condition of rats with spleen stomach deficiency cold type gastric ulcer, and its mechanism may be related to the intervention of gastric mucosal immune barrier dysfunction mediated by JAK2/STAT3 pathway activation.
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Objective: To investigate the effect and mechanism of cinnamaldehyde on the angiogenesis of diabetic retinopathy, and the effect of cinnamaldehyde on vascular endothelial growth factor (VEGF) induced proliferation, migration, tube formation and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway of EA.hy 926 cells were observed. Method:EA.hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (60, 90, 120, 150 μmol·L-1). The methyl thiazolyl tetrazolium (MTT) assay and scratch test were used to observe the effect of cinnamaldehyde on the proliferation and migration of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (90, 150 μmol·L-1). The tube formation experiment was used to observe the effect of cinnamaldehyde on the tube formation of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), VEGF+AG490 group (50 μmol·L-1), VEGF+cinnamaldehyde group (90 μmol·L-1), VEGF+cinnamaldehyde group (150 μmol·L-1), and VEGF+cinnamaldehyde group (150 μmol·L-1)+AG490 group (50 μmol·L-1). Western Blot method was used to explore the effect of cinnamaldehyde on the JAK2/STAT3 signaling pathway in EA.hy 926 cells induced by VEGF. Result:Compared with the control group, model group obviously promoted the proliferation and migration of EA.hy 926 cells(P-1) significantly suppressed VEGF-induced proliferation and migration of EA.hy 926 cells (P-1) showed an obvious inhibitory effect on the number of nodes, junctions and meshes of tubules (PPPP-1) significantly reduced the expressions of P-JAK2, P-STAT3, STAT3 proteins (P-1) obviously reduced the expressions of p-STAT3 and STAT3 proteins (PPConclusion:Cinnamaldehyde showed a significantly inhibitory effect on the proliferation, migration and tube formation of VEGF-induced EA.hy 926 cells, which was related to the inhibition of the activation of JAK2/STAT3 pathway.
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Objective:To observe the effect of Wendantang on tumor necrosis factor-α (TNF-α),interleukin(IL)-6,IL-17,IL-22 and other related inflammatory factors in peripheral blood and the expression of STAT3[the key molecule of Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3) signal pathway in hypothalamus] mRNA and protein of obese rats with syndrome of phlegm-dampness,so as to explore the internal mechanism of Wendantang in interfering obesity with syndrome of phlegm-dampness. Method:A total of 100 rats were randomly divided into blank group(30 rats) and modeling group(70 rats),rats in the blank group was fed with basic feed and the modeling group was fed with high-fat feed for 6 weeks.Animal model of obesity with syndrome of phlegm-dampness was established by the method in literature.After successful modeling,16 obese rats were selected and randomly divided into the model group and Wendantang intervention group with 8 rats in each group,and another 8 rats in the blank group were randomly selected as the normal group.Rats in Wendantang intervention group were given 15 g·kg-1 of crude drug by gavage,while the model group and the normal group were given the same amount of distilled water for gavage once a day for 6 weeks.No eating but no prohibiting drinking before dealing with 12 h and then taking samples after anesthesia.The body weight,Lee's index and obesity rate of rats were measured,the levels of blood lipids[total cholesterol(TC),triglyceride(TG),low density lipoprotein cholesterol(LDL-C) and high density lipoprotein cholesterol(HDL-C)] of rats were detected with a full-automatic biochemical analyzer according to the requirements of the kit,the expression of TNF-α,IL-17,IL-22 and IL-6 in peripheral blood of rats was detected with enzyme-linked immunosorbent assay(ELISA),STAT3 mRNA expression in hypothalamus of rats was detected with real-time fluorescence quantitative polymerase chain reaction(Real-time PCR) and the expression of STAT3 protein in hypothalamus of rats was detected with Western blot. Result:The high-fat feed feeding could successfully replicate the obese rat model,and the obesity rate of rats in the modeling group was greater than 20%,and compared with the blank group,the body weight and Lee's index of rats in the modeling group were significantly increased(PPPPPPPPPPPConclusion:Wendantang has a good effect on improving phlegm-dampness in obese rats,and the mechanism may be related to regulating JAK2/STAT3 signal pathway then to improve the chronic inflammatory state of the body,and all of which provides a scientific basis for Wendantang in intervening obesity with syndrome of phlegm-dampness.
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Objective: To investigate the antagonistic cell injury effect and molecular mechanism of scutellarin (SCU) in hypoxia reoxygenation (HR) treated human cardiac microvascular endothelial cells (HCMECs). Methods: The method of 12 h hypoxia following by 12 h reoxygenation was used to culture HCMECs in vitro to built cell injury model. The groups were divided into control group, model (HR) group, and HR + SCU (0.1 µmol/L, 1 µmol/L, and 10 µmol/L) group. The cell viability was determined by MTT, and oxidative stress was detected by malondialdehyde (MDA) levels by biochemical assay kit. Protein expression of JAK2/p-JAK2 and STAT3/p-STAT3 were evaluated by Western blot. Results: The results of MTT and MDA showed that HR decreased the cell viability (P < 0.05) and increased MDA level significantly (P < 0.05), SCU played a contrary role in these processes. Western blot analysis indicates that, the expression of JAK2 and p-JAK2, STAT3, and p-STAT3 were increased in model group when compared with control group (P < 0.05); Compared with model group, their expression were reduced by SCU (P < 0.05). Conclusion: SCU took a protective effect on HR-treated HCMECs, and the molecular mechanism may be associated with the inhibition of JAK2/STAT3 signal transduction pathway.
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Objective:To observe the lipid-lowering effect of different transdermal absorption enhancers applied to the herbal cake-partitioned moxibustion in hyperlipidemia model rabbits, and to explore the possible mechanism. Methods:Forty New-Zealand rabbits were randomly divided into 5 groups using the random number table method, with 8 rats in each group. Rabbits in the blank group were fed routinely with normal diet; rabbits in the other groups were fed with high-fat diet for 12 weeks to establish the hyperlipidemia model. Rabbits in the blank and the model groups were not treated. After the model was prepared, rabbits in the non-transdermal absorption enhancer group received herbal cake-partitioned moxibustion without transdermal absorption enhancer; rabbits in the laurocapram group and the borneol group received herbal cake-partitioned moxibustion with laurocapram or borneol respectively. After 4 weeks of treatment, serum was collected for enzyme-linked immunosorbent assay (ELISA), and the liver tissues were isolated for immunohistochemistry, quantitative polymerase chain reaction (qPCR) and Western-blotting (WB) detection. Results: Serum ELISA results showed that leptin was significantly decreased in the model group compared with the blank group (P＜0.05); compared with the model group, leptin was significantly increased in the non-transdermal absorption enhancer, the laurocapram and the borneol groups (all P＜0.05); compared with the non-transdermal absorption enhancer group, leptin was significantly increased in the laurocapram group and the borneol group (both P＜0.05); there was no significant difference in leptin between the laurocapram and the borneol groups (P＞0.05). The qPCR results of rabbit liver tissues showed that the mRNA expressions of leptin, Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3) in the model group were significantly lower than those in the blank group (all P＜0.05); compared with the model group, the mRNA expressions of leptin, leptin receptor (LR), JAK2 and STAT3 in the non-transdermal absorption enhancer, the laurocapram and the borneol groups were significantly increased (all P＜0.05); compared with the non-transdermal absorption enhancer group, the mRNA expressions of leptin, LR, JAK2 and STAT3 in the laurocapram and the borneol groups were significantly increased (all P＜0.05); compared with the laurocapram group, the mRNA expressions of leptin, LR, JAK2 and STAT3 in the borneol group were significantly increased (P＜0.05). The trend of immunohistochemistry and WB detection results was basically consistent with the qPCR assay results. The immunohistochemistry and WB detection results of phosphorylated JAK2 (phospho-JAK2) and phosphorylated STAT3 (phospho-STAT3) were basically consistent with those of JAK2 and STAT3. Conclusion: The molecular expression of Leptin/JAK2/STAT3 pathway in the hyperlipidemia model rabbits was decreased. The molecular expression of Leptin/JAK2/STAT3 pathway was significantly increased after the herbal cake-partitioned moxibustion. The application of laurocapram and borneol, as transdermal absorption enhancers, in the herbal cake-partitioned moxibustion could more obviously up-regulate the factors of the Leptin/JAK2/STAT3 lipid-regulating pathway than the herbal cake-partitioned moxibustion alone.
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Objective: To observe the effect of electroacupuncture (EA) on Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway in knee joint synovial tissues of rats with rheumatoid arthritis (RA) and to explore the action mechanism of EA on RA. Methods: Twelve of the 48 SPF male Sprague-Dawley (SD) rats were assigned to a normal group by the random number table method. The remaining 36 rats were subjected to RA model preparation by intradermal injection of the Freund's complete adjuvant into the right hind foot pad of each rat under sterile conditions. After the model was successfully prepared, rats were then divided into a model group, a drug group and an EA group according to a random number table method (n=12). Rats in the drug group were treated with 2 mL aqueous solution of tripterygium glycosides [8.1 mg/(kg?bw)]; rats in the EA group were treated with EA at bilateral Yanglingquan (GB 34) and Zusanli (ST 36), for 30 min each time; rats in the normal group and the model group were placed in a special rat fixation tank for 30 min each time, and received the same dose of normal saline as those in the drug group. Rats in all groups received intervention once a day for 4 weeks. Diameter of rat ankle joint and rat arthritis index were measured before and after the intervention. At the end of the experiment, the expressions of phospho-JAK2 and phospho-STAT3 were determined by immunohistochemistry. Quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect JAK2 and STAT3 mRNAs expressions. Results: After the model was produced, the arthritis index >2 was considered successful in model preparation. Compared with the model group, the ankle joint diameters and arthritis indexes of rats in the drug group and the EA group were significantly lower (all P<0.01); immunohistochemical staining cells with phospho-JAK2 and phospho-STAT3 were significantly decreased (all P<0.01); the expression levels of JAK2 and STAT3 mRNAs were decreased with statistical differences (all P<0.01). There were no significant differences between the EA group and the drug group (all P>0.05). Conclusion: EA can alleviate the inflammatory response of RA rats, improve their pathological conditions, reduce the expressions of phospho-JAK2 and phospho-STAT3 in the synovial tissue of knee joint, and decrease the expressions of JAK2 and STAT3 mRNAs. The therapeutic effect of EA is comparable to that of the tripterygium glycosides. The mechanism of EA treatment may be related to the inactivation of the JAK2/STAT3 pathway.
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Objective@#To study the effect of Jak2-STAT3 pathway on cell proliferation, migration, mineralization and bone defect healing via simulating Jak2-STAT3 pathway inhibitor AG490 to bone marrow mesenchymal stem cells (BMSC) and bone defect mice models.@*Methods@#The effect of AG490 on BMSC proliferation was measured by MTT (methyl thiazolyl tetrazolium) assay. Regulation of AG490 on BMSC migration was tested by scratch assay and transwell assay. The BMSC migration related gene, matrix metalloproteinase (MMP)-7, MMP-9 and CXC subfamily receptor 4 (CXCR4), regulated by AG490 was studied by real-time PCR. Western blotting was adopted to analyze the regulation of Jak2-STAT3 phosphorylation through the simulation of AG490. The alizarin red staining and alkaline phosphatase (ALP) activity assay were performed to measure the effect of AG490 on BMSC mineralization and osteogenic differentiation. Mice femur bone defect models were built to analyzed the effect of AG490 on bone remodeling.@*Results@#AG490 significantly suppressed the migration rate of BMSC at 1 d and 2 d in the experiment group [(12.42±7.50) %, (41.8±2.6)%] compared with the control group [(55.5±9.9)%, (86.9±8.7)%] in scratch assay (P=0.000, P=0.000), the number of migrated BMSC in the experiment group (22.8± 5.9) was significantly suppressed compared with the control group (58.3±6.6) in Transwell assay (P=0.000). The expression of MMP-7, MMP-9 and CXCR4 were significantly downregulated in experiment group [(0.5± 0.1), (0.1±0.1) and (0.35±0.07)] compared with the control group [(1.1±0.1), (1.06±0.33), (1.08±0.13)] (P= 0.0003, P=0.000 and P=0.000). Also, the phosphorylation of Jak2-STAT3 was downregulated by AG490 in western blotting. After BMSCs were osteogenic induced for 14 days, the formation of mineralized nodule and the ALP activity of BMSC is significantly suppressed in experiment group (8.0±2.1) compared with the control group (35.7 ± 1.8) (P=0.0005). AG490 suppressed the bone defects healing,the expression level of phosphorylated Jak2 and phosphorylated STAT3, and the number of alkaline phosphatase positive cell at defect area in vivo is lower in experiment group than the control group. AG490 suppressed the relatively bone density at the defect area significantly (P=0.0004) at 5th week after the surgery.@*Conclusions@#AG490 could suppress proliferation, migration and mineralization to of BMSCs regulate bone defect healing via inhibiting Jak2-STAT3 pathway.
ABSTRACT
Objective: To observe the effects of Fufang Lishao Pills (FFLSP) on the proinflammatory factors, pain-relatedproteins and JAK2/STAT3 signaling pathways in nitroglycerin-induced chronic migraine model, and exploring thepharmacological target and mechanism of FFLSP on chronic migraine rats. Methods: Male Sprague-Dawley rats wererandomly divided into 6 groups: Control, Migraine, FFLSP-L (420 mg·kg-1), FFLSP-M (840 mg·kg-1), FFLSP-H (1680mg·kg-1) and Flunarizine Hydrochloride group (FH, 1 mg·kg-1) . Chronic migraine model was made by subcutaneousinjection of nitroglycerin (10 mg·kg-1) once every 3 days for 5 times. The rats were treated with FFLSP by intragastricadministration once a day for 30 days. Western blot was used to detect the protein expression of iNOS, COX-2, CGRPand c-Fos and the phosphorylation levels of JAK2 and STAT3 in cortex of rats. The production of IL-1β and TNF-αwere detected by enzyme linked immunosorbent assay (ELISA) . Results: Compared with Control rats, and the level ofiNOS, COX-2, CGRP, c-Fos expression (P ＜ 0.01) and IL-1β, TNF-α production remarkably up-regulated (P ＜ 0.05), and the phosphorylation of JAK2 and STAT3 also significantly increased in cotex of Migrainerats (P ＜ 0.01) . However, compared with Migrainerats, the levels of iNOS, COX-2, CGRP, c-Fos expression and IL-1β, TNF-α productionobviously declined (P ＜ 0.05; P ＜ 0.01), and the phosphorylation level of JAK2 and STAT3 showed a significantlydecrease in the cortex of Migraine rats with FFLSP treatment (P ＜ 0.01) . Conclusion: This study demonstrates that thepharmacological mechanism of FFLSP in improving chronic migraine may be achieved by inhibiting neuroinflammationthrough the blockage of JAK2/STAT3 pathwayin the cortex of rat with nitroglycerin induction.
ABSTRACT
ObjectiveTo investigate the effect of recombinant human growth hormone (rhGH) on JAK2-STAT3 signaling pathway and on the growth of human colon cancer cells at different growth hormone receptor (GHR) expression status.MethodsLOVO and HCT-8 cell lines were selected after the colon cancer cells were screened using flow cytometry according to the GHR expression status.The growth of human colon cancer cells after intervention with rhGH was detected by MTT assay.The proliferation index ( PI),cell cycle,and apoptosis were detected by flow cytometry.The expression of JAK2-STAT3 signaling pathway proteins was detected by Western blot.ResultsHCT-8 cell line showed positive GHR expression (59.6％),and LOVO cell showed negative GHR expression (3.5％).The growth rate of HCT-8 cells increased after rhGH treatment.Compared with the untreated HCT-8 cells,the rhGH-treated HCT-8 cells showed reduced apoptosis,elevated PI,and increased G2/Mphase cells ( P =0.0073).Western blot revealed that rhGH up-regulated the proteins of pJAK2,pSTAT3,VEGF,Cyclin D1,and Bcl-xL in HCT-8 cells.In contrast,no such changes were observed in LOVO cells treated with rhGH.ConclusionsrhGH may promote the growth of HCT-8,the cell line of high GHR expression,and up-regulate the expression of a number of key nodes in JAK2-STAT3 signaling pathway.However,rhGH may not affect LOVO,the cell line of low GHR expression.